Science Publications, 2005 OntoCell An ontology of Cellular Biology
海洋微生物产纤维素酶及其应用研究进展
海洋微生物产纤维素酶及其应用研究进展韩笑;闫培生;史翠娟;杨树燕;李永鹏;邹雪平【摘要】Cellulose is the most ancient, abundant and inexhaustible natural polymer resources.Cellulase widely exists in the organism including bacteria,fungi and animals.Microbial cellulase has been widely reported and utilized in industries of food, medicine, feed, detergent, textile, pulp and paper,etc..The ocean is a huge pool of resources,and cellulose-producing marine microorganisms have attracted a lot of attention.This paper briefly summarized the research progress of populations and source of marine microorganisms producing cellulase and genetic screening, the enzymatic properties and application fields of cellulase produced by marine microorganisms.Prospects of study on marine microbial cellulase were also discussed.%纤维素是地球上最古老、最丰富的天然高分子，是天然可再生资源。
纤维素酶广泛存在于自然界的生物体中，细菌、真菌和动物体内都能产生纤维素酶。
Editorial Board
European Journal of Scientific Research(ISSN: 1450-216X)Vol. 10, No: 2September, 2005© AMS Publishing, Inc. 2005Editor-In-Chief & Managing Editor: Adrian Marcus Steinberg, PhD Publisher: Lulu Press, Inc. Morrisville, USAEuropean Journal of Scientific ResearchEuropean Journal of Scientific Research is an international official journal of the AMS Publishing, Inc. (Austria) publishing high quality research papers, reviews, and short communications in the fields of biology, chemistry, physics, environmental sciences, business and economics, finance, mathematics and statistics, geology, engineering, computer science, social sciences, natural and technological sciences, linguistics, medicine, architecture, industrial, and all other applied and theoretical sciences. The journal welcomes submission of articles through EJSR@.Editorial BoardProf. Adrian M. Steinberg Wissenschaftlicher Forscher, AustriaDr. Parag Garhyan Auburn University, USAProf. Morteza Shahbazi Edinburgh University, UKProf. Raj Rajagopalan National University of SingaporeProf. Sang-Eon Park Inha University, KoreaDr. Said Elnashaie Auburn University, USADr. Subrata Chowdhury University of Rhode Island, USADr. Raj Rajagopalan National University of Singapore, SingaporeProf. Ghasem-Ali Omrani Tehran University of Medical Sciences, IranProf Ajay K. Ray National University of Singapore, SingaporeDr. Mutwakil Nafi China University of Geosciences, ChinaMr. Mete Feridun Cyprus International University, CyprusProf. Bansi Sawhney Dr. Jean-Luc Grosso Dr. Teresa Smith Prof. Ranjit Biswas University of Baltimore, USA University of South Carolina Sumter, USA University of South Carolina Sumter, USA Philadelphia University, JordanEUROPEAN JOURNALS INC.® realizes the meaning of fast publication to researchers, particularly to those working in competitive & dynamic fields. Hence, we offer an exceptionally fast publication schedule including prompt peer-review by the experts in the field and immediate publication upon acceptance. EUROPEAN JOURNALS INC.® pledges to review the submitted articles within three days of receipt and promptly include them in the forthcoming issue should they pass the evaluation process. Further information is available at: .European Journal of Scientific ResearchVol. 10, No: 2CONTENTSASPIRIN INDUCED CHANGES IN ENZYMES OF COLONIC ENERGY METABOLISM AND ATPASES OF RATS EXPOSED TO CYCAS AND FED ANIGERIAN-LIKE DIETG.E. ERIYAMREMU, E.C. ONYENEKE, N. J. ORHUE, S.I. OJEABURU, S. O.UANSEOJE, V.E OSAGIE, S.O. ASAGBAUSING MULTIQUADRIC METHOD IN THE NUMERICAL SOLUTION OF ORDINARY DIFFERENTIAL EQUATIONS WITH A SINGULARITY POINT AND PARTIAL DIFFERENTIAL EQUATIONS IN ONE AND TWO DIMENSIONSA. AMINATAEI, MAITHILI SHARANOPEN MASTOIDECTOMY AND TEMPORALIS FLAP IN THE CONTROL OFCHRONIC OTORRHOEALASISI, O.A., OLATOKE F, SANDABE M.B., KODIYA S.B.BLOOD OXYGENATION IN THE PULMONARY CIRCULATION: A REVIEWA. AMINATAEIEMBEDDING LINEAR ARRAY NETWORK INTO THE TREE-HYPERCUBENETWORKQATAWNEH MOHAMMEDX-EFFICIENCY ANALYSIS OF COMMERCIAL BANKS IN PAKISTAN:A PRELIMINARY INVESTIGATIONMOHAMMAD HANIF AKHTAR AND S. M. HUSNAIN BOKHARIMODEL PREDICTED PERCOLATION BELOW ROOTZONE UNDER RICE-WHEATIRRIGATED SYSTEMMUHAMMAD ARSHAD , M. RAFIQ CHOUDHRY, RIAZ AHMEDASPIRIN INDUCED CHANGES IN ENZYMES OF COLONIC ENERGY METABOLISM AND ATPASES OF RATS EXPOSED TO CYCAS AND FED ANIGERIAN-LIKE DIETG.E. Eriyamremu Ph.D1., E.C. Onyeneke Ph.D1., N. J. Orhue1, S.I. Ojeaburu1, S. O. Uanseoje1, V.E Osagie2 and S.O. Asagba Ph.D.31.Department of Biochemistry, University of Benin, P.M.B. 1154, Benin City, Nigeria.2.Department of Biochemistry, Ambrose Ali University, Ekpoma, Edo State, Nigeria.3.Department of Biochemistry, Delta State University, P.M.B. 1, Abraka, Delta State,Nigeria.ABSTRACTLong term administration of aspirin show efficacy in prevention of colon cancer and drug action is dependent on its interaction with nutrients. This study is aimed at assessing the role of the interaction between a Nigerian-like diet (NLD) or Western-like diet (WLD) and aspirin on energy metabolism and ATPases in early colon carcinogenesis. We observed that the WLD significantly (P<0.05) increased intestinal length of rats compared with the NLD and interaction with aspirin slightly reversed this effect. The study also reports that the WLD increased the activities of some enzymes of energy metabolism compared with the NLD and that this increase is more than tripled with the inclusion of cycas in the diet. Aspirin administration reduced the activities of these enzymes more in the rats fed with the WLD than the NLD. We also observed that like the WLD, cycas inclusion in the diet of rats significantly raised the activity of Mg2+ ATPase (9% in the NLD and 34% in the WLD) and decreased Ca2+ ATPase (30% in the NLD to 42% in the WLD). Aspirin reversed these effects of cycas on these ATPases (37% in the NLD and 40% in the NLD for Ca2+ ATPase). This study demonstrates that an increase in Mg2+ ATPase and a decrease in Ca2+ ATPase is associated with early colon carcinogenesis and that while the WLD promotes these enzyme changes, the NLD have the opposite effect. The study also reveal that aspirin effect on the enzymes of energy metabolism and ATPases supports its protective role against colon carcinogenesis and that the effect of the drug is dependent on the protein content of the diet. Key words: Colon carcinogenesis. Nigerian diet. Aspirin. Energy metabolism. ATPases. INTRODUCTIONIn North America, Oceania and Western Europe, colon cancer is common [1] and it is believed that the etiology of the disease is of both genetic and dietary origin. Colon cancer which originates from environmental causes (Sporadic colon cancer) represents about 95% of all cases of the disease and is mostly associated with dietary risk factors [2]. A variety of studies support the earlier finding of Burkitt [3] that the regular intake of carbohydrate; particularly of resistant starch and fibre, reduces the incidence of colon cancer [4], while populations on a high fat and high protein diet such as those in Western diets are at a high risk of developing colon cancer [5]. However, epidemiologic evidence supporting the fiber hypothesis has been rather controversial. A study of colon cancer rates in South Africa concluded that the low prevalence of colon cancer in black Africans cannot be explained by dietary "protective" factors, such as fiber, calcium, vitamins A, C and folic acid, but may be influenced by the absence of "aggressive" factors, such as excess animal protein and fat, and by differences in colonic bacterial fermentation [6]. On the other hand, a recent paperreported that the risk of colorectal adenoma (the precursor of colorectal cancer) decreased by 41% for every additional 5% unit of fiber intake/day [7]. Red meat fat increased the risk by 20%, and white meat fat decreased the risk by 67% for every additional 5% unit of respective intake/day. Experimental studies on the role of whole diet consumed by populations in colon carcinogenesis also agrees with some of these population studies though most have been carried out using semi-purified or purified diets.Several recent reviews [8, 9, 10] have also provided evidence that nonsteroidal anti-inflammatory drugs (NSAIDs) have promise as anticancer drugs as they reduced incidence of and mortality from colon cancer. NSAIDs have been shown experimentally to stimulate apoptosis and to inhibit angiogenesis, two mechanisms that help to suppress malignant transformation and tumor growth. Numerous experimental and epidemiologic (nonrandomized)studies [11, 12, 13, 14 15] have found that long-term users of aspirin or other NSAIDs have a lower risk of colorectal adenomatous polyps and colorectal cancer than nonusers, although some other studies show that NSAID have a deleterious effect [16, 17]. Little attention has been given to the interaction of drugs with certain foods particularly when such drugs have to be administered over a long period. Studies showing the beneficial effects of aspirin in colon carcinogenesis recommend long term intake of the drug thus making drug nutrient interaction important. Factors that can increase the potential for interactions include long-term drug administration, poor dietary intake, preexisting disease states (especially gastrointestinal disease). Nutritional status and diet can affect the action of drugs by altering absorption, distribution, metabolism and excretion of drugs and thus influence drug response [18]. In this context the aim of the present work was to further document the role of aspirin and diet interactions on aspects which relates to early carcinogenesis,with special attention placed on energy metabolism and ATPases.MATERIALS AND METHODSExperimental designSeventy two male albino rats (Wistar Strain) with an average weight of 100±2g were housed individually in stainless steel cages with wire mesh floor to prevent coprophagia. The rats were grouped into three diet classes with twenty-four animals each such that the weight difference between the groups was less than 0.2g. One group was fed with a normal wholly compounded diet (ND) and served as the diet control class; another class of animals was fed a wholly compounded Nigerian-like diet (NLD), which was low in protein and high in carbohydrate and fibre. The third class of rats was fed with a Western-like diet (WLD) which was high in protein and fat. Both the NLD and the WLD served as test diets (table1). The animals of each diet class were further distributed into three subgroups of eight rats each such that the mean weight difference between all the classes was less than 0.2g. In each class one group received the diet alone, another group received the diet and cycas and the third received the diet containing cycas and were administered with 60mg aspirin/kg body weight. All these animal treatment were carried out in accordance with the principles of laboratory animal care of the NIN guide for Laboratory Animal Welfare as contained in the NIN guide for grants and contracts, vol. 14, No. 3, 1985.The animals were given the food and water ad libitum. They were acclimatized with their respective diet for one week before the commencement of the study, which was for sixteen weeks. During this period, food intake and dry faecal output were measured daily, while weight gain was recorded weekly. At the end of the study period, the animals were fasted for18hrs and sacrificed after they were anaesthetized by intraperitoneal injection of 5ml/kg body weight of 25% urethane saline solution.Blood collection and treatmentWhile under anesthesia blood was collected from each rat via heart puncture and transferred into fluoride tubes, where they were allowed to coagulate. The serum from each blood sample was recovered by centrifugation at 3,000g. Serum glucose, total cholesterol, total lipid and total phospholipids levels were estimated using appropriate kits from Randox laboratories (England).Tissue treatmentThe colon (first 10cm of the proximal end of the large intestine) was recovered from each rat, flushed several times with ice-cold normal saline solution until free of debris. The intestine was inverted, and the mucosa was removed by scraping with a glass slide. The colonic tissue was homogenized, centrifuged at 4,000g for 30 minutes and both the supernatant and residue were frozen until needed for assay which generally was within 24hrs.Enzyme and Protein assayHexokinase, phosphofructokinase, lactate dehydrogenase, and glucose 6-phosphate dehydrogenase, activities were estimated by measuring the oxidation or reduction of NAD(P)(H) in straight or coupled reactions [19, 20].ATPase action was estimated by the method of [21] as modified by Takeo et al [22] and the inorganic phosphate released by the action of the enzyme was determined [23]. Total protein was estimated by the method of Lowry et al. [24].Statistical analysisThe results are expressed as Mean ± S.E.M. Analysis of variance was used to test for differences in the groups. Duncan’s multiple range test was used to test for significant differences between the means [25].RESULTSThe weight gain, food intake, dry faecal output and intestinal length of rats exposed to cycas and aspirin data are presented in table 2. Statistical evaluation of the data did not reveal any significant (P>0.05) difference in the weight gain of rats fed the ND and NLD, but in those fed with the WLD, there was a significant (P<0.05) increase in the weight gained. While the WLD fed to rats significantly decreased food intake and dry faecal output, the NLD significantly increased food intake and dry faecal output compared with the control ND fed rats. The inclusion of cycas to the various diets or the administration of aspirin did not significantly alter weight gain, food intake and dry faecal output data of the rats. Feeding rats with the WLD significantly (P<0.05) increased their intestinal length compared with the NLD and control ND. The administration of aspirin only slightly reduced intestinal length of the animals. This study indicates that weight, food intake, faecal output and intestinal length of rats are responsive to the type diet fed to the animals.Table 3 presents serum glucose and lipid profile in the experimental rats. Exposure of rats to cycas significantly increased (P<0.05) fasting serum glucose in the all the diet classes. Incontrast, aspirin reduced the level of glucose in the serum to levels comparable to those of the diet controls. The ND and the NLD fed rats had statistically similar levels (P>0.05) of glucose however these were significantly lower (P<0.05) than that observed in the WLD fed rats. The serum total cholesterol, total phospholipid and total lipid levels were significantly raised (P<0.05) by the WLD compared with the ND and NLD. Aspirin restored the levels of these lipids in the serum to levels comparable to those observed in the rats fed with the diet controls. Feeding of cycas significantly (P<0.05) increased serum total cholesterol in all the three diet classes. This study shows that WLD generally increased the serum glucose and lipids in rats.The specific activities of some representative enzymes of colonic energy metabolism are presented in table 4. Feeding rats with the WLD significantly raised the activities of hexokinase, phosphofructokinase, lactate dehydrogenase and glucose 6-phosphate dehydrogenase compared with the ND and NLD. When the activities of these enzymes in the NLD fed rats were compared with those of the control ND, no statistically significant (P>0.05) change was observed. In the all the diet classes, cycas inclusion in the diet resulted in significant (P<0.05) increases in the enzymes of energy metabolism assayed compared with their diet controls. The least increases in these enzymes were recorded in the NLD fed rats and the most in the WLD fed ones. The increases also varied from enzyme to enzyme. Hexokinase increased with about 71% in the NLD fed rats, but in the WLD class it was about 380% from the diet control. The increases observed in phosphofructokinase activity were much more than those observed in the other enzymes. It reached about 360%, 295% and 460% in the ND, NLD and WLD fed rats respectively. The administration of aspirin significantly (P<0.05) reversed the activities of the enzymes of energy metabolism though the degree of reversal varied. The degree of reversal was much more in the WLD fed animals then the ND before the NLD fed ones thus indicating the importance of protein and/or fat content in the diet in aspirin effect. The observed results demonstrate that some enzymes of energy metabolism are altered by diets and aspirin in ways which can affect colonic cell metabolism.Table 5 shows the colonic ATPases activity in the rats fed cycas and aspirin. The effect of the inclusion of cycas in the rat diets on the activity of Na+/K+ ATPase did not produce any clear cut effect. In the ND, it significantly (P<0.05) reduced Na+/K+ ATPase but increased the enzyme action in the NLD and WLD fed animals, though the increases failed to reached a statistically significant level (P>0.05). Administration of aspirin did not significantly alter the Na+/K+ ATPase activity in all the diet classes. Mg2+ ATPase and Ca2+ ATPase activities produced a clearer pattern as the inclusion of cycas significantly (P<0.05) raised Mg2+ ATPase in all the diet groups, it significantly reduced Ca2+ ATPase. The percentage increase in Mg2+ ATPase varied from 30% to 9% and 34% in the ND, NLD and WLD fed rats respectively. The decrease in Ca2+ ATPase varied from 28% in the ND to 30% in the NLD and 42% in the WLD fed rats. Aspirin administration caused both Mg2+ ATPase and Ca2+ ATPase activities to reverse to levels obtained in the diet controls, though the reversals were not total. In Ca2+ ATPases the reversal varied from about 43% to 37% to 40% in the ND, NLD and WLD fed rats respectively. This study reveals that ATPases are responsive to diet type and that changes in Mg2+ and Ca2+ ATPases occur with cycas treatment alone and with cycas and aspirin treatment of rats.DISCUSSIONMost of the experimental studies on the role of diet/dietary components on colon carcinogenesis investigate the progressional stage of the disease and few concentrate on the early events that lead to the development of tumor. Cycasin which is modified by the colonic microflora to dimethylhydrazine (DMH) (a potent carcinogen) is present in cycas. As the leaves of cycad plant contain less cycasin than the nuts and with the duration of the feeding protocol, the present study will therefore relate to early events in the initiation stage of colon carcinogenesis. Also nutrient type/intake can affect drug action in the long term, thus this study assessed the interaction between aspirin and NLD or WLD on some energy metabolizing enzymes and ATPases in early colonic carcinogenesis.The effect of the NLD, which is high in carbohydrate and fibre, on weight gain and dry faecal output observed in this study (Table 2) is consistent with those earlier observed in our laboratory [26, 27]]. Potential mechanisms by which dietary fibre can protect against the development of colorectal cancer include increases in stool bulk, dilution or binding of potential carcinogens and decrease in transit time [3]. Since the NLD increased faecal bulk, it will not only dilute out potential carcinogens/co-carcinogens, it will also increase the transit time of the stool thus may protect the rats from colorectal carcinogenesis. In comparison to the NLD, the WLD which had opposite effect on dry faecal output (Table 2) will predispose rats to colorectal carcinogenesis. The effect of stool bulk on transit time becomes all the more important as this study also reveal that the WLD increased the intestinal length of the rats. There will not only be more contact between the colonic contents and the colonic mucosa in these rats but also more pressure will be exerted on the colonic wall, a process that have been shown to improve proliferation [28]. Aspirin decreased intestinal length which will not only reduce transit time but will also reduce the pressure exerted by the diet on the colon and may be an important adaptive feature by which this NSAID decreases tumourigenesis. From the calculated metabolizable energy, the WLD contains more energy than the NLD and so animals consuming the WLD do not need to consume large quantities of the diet to meet their energy demand and this would have accounted for the low food intake observed in the animals on this diet (Table 2).The observed effect of the feeding of the NLD to rats on fasting serum glucose level compared with the WLD fed rats is not surprising (Table 3). Though the NLD is rich in starch and is expected to increase blood glucose level, but it can also pass into the colon largely undigested when one considers that it decreased intestinal length (Table 2) and thus may decrease digestion and absorption. Earlier studies have shown that diets rich in fibre and resistant starch are fermented in the colon into short chain fatty acids (SCFA) [29] which are rapidly absorbed [30] and they affect different metabolic processes. Propionate inhibits gluconeogenesis and cholesterol synthesis and stimulates glycolysis. An earlier study had shown that the NLD produced more propionate and butyrate than the WLD [31]. Reduced production of propionate would in part account for the high glucose level observed in the WLD fed rats. The effect of propionate on cholesterol and triglyceride synthesis may also help explain the observed effects of the diets on serum cholesterol and triglyceride levels. Earlier studies have shown that there is an inverse relationship between serum cholesterol and cancer [32, 33], therefore the observed effect of cycas on serum cholesterol in all the diet classes (Table 3) is not surprising.The WLD increased the activities of enzymes of glycolysis and of a key enzyme in the pentose phosphate pathway (Table 4) which would be needed to provide energy as well as ribose sugars to support the proliferation. These observations imply an improved utilizationof glucose. Earlier studies have shown that an increase in glucose uptake are associated with cell transformation from a normal to a neoplastic state [34] and that changes occur in some enzymes of energy metabolism in the progressional stages of cancer to support the proliferation process [35]. The improved chance of transformation in the colon of rats fed the WLD from a normal to a neoplastic one is heightened because the WLD contains high level of fat and will thus increase enterohepatic circulation of bile salts which increase the chance of loss of these bile salts to the colon where they increase cell proliferation and promote carcinogenesis [36]. On the other hand, the NLD which contains low level of fat will decrease losses of bile salts to the colon and decrease colonic cell proliferation and would account for the low levels of the enzymes of energy metabolism assayed in these rats compared with those fed with the WLD. As cycas is modified in the colon into carcinogens, the high activity of the enzymes of energy metabolism observed in the cycas fed animals would be the result of excessive proliferation in these animals. Recently, glycolytic bioenergetic changes have been suggested as a useful marker for colon and other carcinomas [37, 38] therefore implying that the WLD may indeed support carcinogenesis as it increased the activities of glycolytic enzymes. Glucose supplies only a part of the energy utilized by the colon and butyrate nourishes the colon and may be used preferentially to glucose in colonic energy metabolism [39], bypassing the glycolytic sequence. As the NLD had earlier been shown to produce more butyrate than the WLD, it may also in part contribute to the low levels of enzymes of glycolysis observed in the NLD fed rats compared with those observed in the WLD fed animals (Table 4). Also, butyrate has been shown to induce apoptosis [40] and may in part also contribute to the low percentage increase in enzymes of glucose metabolism in the NLD and cycas fed rats compared with the WLD and cycas fed ones (Table 4).The mechanisms underlying the chemopreventive effects of aspirin are less well understood and are a matter of ongoing debate. One potential mechanism of aspirin action involves inhibition of cyclo-oxygenase activity,which limits tumorigenesis by reducing the production of mutagens that result from arachidonic acid metabolism [41]. Protection might occur through several other pathways, including cell cycle arrest and induction of apoptosis [42, 43]. These may explain the reduction of the enzymes of energy metabolism to levels comparable to those observed in the diet controls (Table 4). Data on the influence of aspirin on enzymes of energy metabolism are lacking and data comparison is difficult and also the data provided in this study are far from been conclusive, but suffice to say that aspirin do exert indirect effects which may have arisen from its effect on cell proliferation.This study reveals that the WLD increases Mg2+ ATPase and decreases Ca2+ ATPases activities, while the NLD had the opposite effect and that cycas further extends these effects of the WLD (Table 5). An earlier study had shown that ATPases activities change with diet types [44]. ATPases are membrane bound enzymes that transport ions from one compartment to another and from extracellular fluid into the cell. As the source of the ATPases assayed in the present study is whole tissue homogenate, the study does not provide the activity in different organelles and therefore limits its findings. Dietary fibre can bind cations and make them unavailable for absorption and could trigger a compensatory rise in ATPases to maximize absorption [44]. This would account for the improved activities of ATPases in the NLD fed rats compared with the ND and the WLD fed ones (Table 5).The increase in Mg2+ ATPase observed in the cycas fed rats (Table5) may be related to improved energy production in the colon of these rats as Mg2+ ATPase is also closely associated with mitochondria and ATP formation. It has been observed that in carcinogenesis, there is de-regulation of cell-type-specific programmes that controlmitochondria biogenesis and function in different mammalian tissues [45]. So if indeed the increase in Mg2+ ATPase also reflects changes in the mitochondria, then in the cycas fed animals there would be increase in energy production to meet the increased proliferation. This reflects the relevance of both the glycolytic sequence and mitochondria oxidative phosphorylation in colon carcinogenesis. As aspirin lowers Mg2+ ATPase it may improve the regulation of mitochondria function and lower ATP formation. Also if the aspirin decrease cell proliferation, there would be a corresponding drop in energy requirement and a decrease in Mg2+ ATPase activity.Ca2+ ATPase activity was reduced in the cycas fed rats in all the diet groups compared with the diet controls (Table 5). This infers that a reduction in this ATPase action is an early event in improved colon proliferation and carcinogenesis. Ca2+ ions are known to mediate diverse array of physiological processes including gene expression and regulation [46, 47]. Its subcellular concentrations is essential in these processes, but in this study however, the subcellular levels of Ca2+ ion or the ATPase responsible for its transport were not measured. Even with these drawbacks, the study provides information on the effect of cycas, aspirin and diet on the cellular level of activity of the ATPase. The influence of diet becomes more evident when the percentage reduction in the enzyme activity is considered. Whilst the ND caused about 28% reduction, the NLD caused about 30.3% reduction and the WLD about 42% reduction in the activity of Ca2+ ATPase. Thus if the reduction of the enzyme action is related to colonic cell proliferation, then the WLD may promote colonic tissue transformation to a cancerous state. It is also pertinent to note that the high percentage reduction in Ca2+ ATPase activity observed in the NLD fed animals compared with the controls may in part be as a result of improved binding of ions by fibre which may then affect the ATPase action [44]. It was observed that the improvement of Ca2+ ATPase by aspirin administration (Table 5) was also diet related. Reduced Ca2+ ATPase action improved from 42% to 24% in the WLD and from 30% to 19% in the NLD fed rats representing about 43% and 36% improvement in the WLD and NLD respectively. This emphasizes the importance protein on aspirin effect on ATPases action in colon carcinogenesis.The effect of aspirin on the degrees of restoration of the activities of enzymes of energy metabolism (Table 4) and ATPases (Table 5) also seems to vary with protein content of the diets. The percentage restoration increase from the NLD to the ND to the WLD thus suggesting that aspirin/protein interaction is important for its action. High protein intake, particularly of animal sources has in itself been associated with colon carcinogenesis [6, 7]. Despite these findings, a fine balance as regards the amount and type of protein consumed is thus important. Generally, fish as a source of protein have been shown to be of more benefits in colon carcinogenesis than diary sources. In the Southern part of Nigeria, the major source of protein is fish while diary products constitute the major source of protein for the population of Northern Nigeria. Thus on balance a typical meal in Southern Nigeria with improved fish protein would be more beneficial than the Northern Nigerian diet in relation to aspirin and colon carcinogenesis. Nutrient/aspirin interaction in colon carcinogenesis needs to be further investigated particularly as a lack of histopathological investigations in this study limits its claims.In summary, the NLD and WLD affect colon carcinogenesis by inducing changes in the intestinal length and affecting the activities of enzymes of energy metabolism and Mg2+ and Ca2+ ATPases. The study suggests that an increase in Mg2+ ATPase and a decrease in Ca2+ ATPase are associated with early carcinogenesis and that these events are reversed by aspirin in a manner dependent on the protein content of the diet.。
Atg8, a ubiquitin-like protein required for autophagosome formation
Atg8,a Ubiquitin-like Protein Required for Autophagosome Formation,Mediates Membrane Tethering and HemifusionHitoshi Nakatogawa,1,2Yoshinobu Ichimura,1,3and Yoshinori Ohsumi1,*1Department of Cell Biology,National Institute for Basic Biology,Okazaki444-8585,Japan2PRESTO,Japan Science and Technology Agency,Saitama332-0012,Japan3Present address:Department of Biochemistry,Juntendo University School of Medicine,Bunkyo-ku,Tokyo113-8421,Japan. *Correspondence:yohsumi@nibb.ac.jpDOI10.1016/j.cell.2007.05.021SUMMARYAutophagy involves de novo formation of double membrane-bound structures called autophagosomes,which engulf material to be degraded in lytic compartments.Atg8is a ubiq-uitin-like protein required for this process in Saccharomyces cerevisiae that can be conju-gated to the lipid phosphatidylethanolamine by a ubiquitin-like system.Here,we show using an in vitro system that Atg8mediates the teth-ering and hemifusion of membranes,which are evoked by the lipidation of the protein and reversibly modulated by the deconjugation enzyme Atg4.Mutational analyses suggest that membrane tethering and hemifusion ob-served in vitro represent an authentic function of Atg8in autophagosome formation in vivo.In addition,electron microscopic analyses indicate that these functions of Atg8are in-volved in the expansion of autophagosomal membranes.Our results provide further insights into the mechanisms underlying the unique membrane dynamics of autophagy and also in-dicate the functional versatility of ubiquitin-like proteins.INTRODUCTIONAutophagy is an evolutionally conserved protein degrada-tion pathway in eukaryotes that is essential for cell survival under nutrient-limiting conditions(Levine and Klionsky, 2004).In addition,recent studies have revealed a wide variety of physiological roles for autophagy(Mizushima, 2005)as well as its relevance to diseases(Cuervo,2004). During autophagy,cup-shaped,single membrane-bound structures called isolation membranes appear and expand,which results in the sequestration of a portion of the cytosol and often organelles.Eventually,spherical, double membrane-bound structures called autophago-somes are formed(Baba et al.,1994),and then delivered to and fused with lysosomes or vacuoles to allow their contents to be degraded.Studies in S.cerevisiae have identified18ATG genes required for autophagosome formation,most of which are also found in higher eukary-otes(Levine and Klionsky,2004).Recent studies have shown that Atg proteins constitutefive functional groups: (i)the Atg1protein kinase complex,(ii)the Atg14-contain-ing phosphatidylinositol-3kinase complex,(iii)the Atg12-Atg5protein conjugation system,(iv)the Atg8lipid con-jugation system,and(v)the Atg9membrane protein recycling system(Yorimitsu and Klionsky,2005).The mechanisms by which these units act collaboratively with lipid molecules to form the autophagosomes,how-ever,are still poorly understood.Atg8is one of two ubiquitin-like proteins required for autophagosome formation(Mizushima et al.,1998;Ichi-mura et al.,2000).Because it has been shown that Atg8 and its homologs(LC3in mammals)localize on the isola-tion membranes and the autophagosomes,these proteins have been used in various studies as reliable markers for the induction and progression of autophagy(Kirisako et al.,1999;Kabeya et al.,2000;Yoshimoto et al.,2004). In S.cerevisiae,Atg8is synthesized with an arginine resi-due at the C terminus,which is immediately removed by the cysteine protease Atg4(Kirisako et al.,2000).The resulting Atg8G116protein has a glycine residue at the new C terminus and can serve as substrate in a ubiqui-tin-like conjugation reaction catalyzed by Atg7and Atg3, which correspond to the E1and E2enzymes of the ubiq-uitination system,respectively(Ichimura et al.,2000). Remarkably,unlike other ubiquitin-like conjugation sys-tems,Atg8is conjugated to the lipid phosphatidylethanol-amine(PE),thereby Atg8is anchored to membranes (Ichimura et al.,2000;Kirisako et al.,2000).Immunoelec-tron microscopy revealed that Atg8,probably as a PE-conjugated form(Atg8-PE),is predominantly localized on the isolation membranes rather than on the complete autophagosomes(Kirisako et al.,1999),suggesting that Atg8-PE plays a pivotal role in the process of autophago-some formation.The precise function of Atg8-PE,how-ever,has remained unknown.The conjugation of Atg8to PE is reversible;Atg4also functions as a deconjugation enzyme,resulting in the Cell130,165–178,July13,2007ª2007Elsevier Inc.165release of Atg8from the membrane(Kirisako et al.,2000). This reaction is thought to be important for the regulation of the function of Atg8and/or the recycling of Atg8after it has fulfilled its role in autophagosome formation.We reconstituted the Atg8-PE conjugation reaction in vitro with purified components(Ichimura et al.,2004). Here,we show using this system that Atg8mediates the tethering and hemifusion of liposomes in response to the conjugation with PE.These phenomena observed in vitro are suggested to reflect a bonafide in vivo function of Atg8 in the expansion of the isolation membrane.Based on mutational analyses and structural information,the mech-anisms of Atg8-mediated membrane tethering and hemi-fusion as well as its regulation are discussed.This study sheds light on the molecular basis of unconventional membrane dynamics during autophagy,which is gov-erned by the Atg proteins.RESULTSLipidation of Atg8Causes Clustering of LiposomesIn VitroAs reported previously(Ichimura et al.,2004),when puri-fied Atg8G116(hereafter,referred to as Atg8),Atg7,and Atg3were incubated with liposomes containing PE in the presence of ATP,Atg8-PE was efficiently formed (Figure1A,lanes1–6).Intriguingly,the reaction mixture became turbid during the incubation(Figure1B),which under a light microscope,was found to be a result of grad-ually forming aggregates(Figure1C).Both the degree of turbidity and the size of the aggregates appeared to corre-late with the amount of Atg8-PE produced in the mixture. Size-distribution analyses using dynamic light scattering (DLS)clearly showed that the aggregates formed in an Atg8-PE dose-dependent manner(Figure1D).These aggregates disappeared when the samples were treated with the detergent CHAPS(Figure1E,+CHAPS).In addi-tion,if a small amount of PE modified with thefluorescent dye7-nitro-2,1,3-benzoxadiazol-4-yl(NBD)was included in the liposome preparation,the aggregates became uniformlyfluorescent(Figure1E,NBD-PE).These results suggest that the aggregates generated during the produc-tion of Atg8-PE were clusters of liposomes.When the proteins were denatured with urea,the clus-ters of liposomes dissociated,although Atg8remained conjugated to PE(Figure1E,+urea and Figure1F,lane 2),indicating that the liposomes aggregated due to some function of the Atg8protein rather than an artifact caused by Atg8-PE as the lipid with the extraordinarily large head group.When the aggregates were sedimented by centrifugation,Atg8-PE co-precipitated with the lipo-somes(Figure1G,lane2),whereas Atg7,Atg3,and unconjugated Atg8did not(Figure1G,lane3).The sedimented liposomes containing Atg8-PE remained clustered even if they were briefly sonicated(Figure1H, ppt.).These results suggested that Atg8-PE molecules function to tether together membranes to which they are anchored.Atg8-PE Also Mediates Liposome FusionWe also examined if membrane fusion occurred between the liposomes connected by Atg8-PE.To this end,we took advantage of a well-characterized lipid mixing assay (Struck et al.,1981).This method is based on energy transfer from NBD to lissamine rhodamine B(Rho),each of which is conjugated to PE.Because the amino group of the ethanolamine moiety is modified with the dyes, these lipids cannot be conjugated with Atg8.If both of the conjugated dyes are present at appropriate concen-trations in the same liposome,thefluorescence of NBD is effectively quenched by Rho(Figure2A,compare col-umns1and4).If a‘‘NBD+Rho’’liposome is fused with a‘‘nonlabeled’’liposome,which results in an increase of the average distance between the two dyes on the membrane,the NBDfluorescence will be dequenched.A mixture of the nonlabeled and NBD+Rho liposomes were subjected to the conjugation reaction.The resulting liposome clusters were dissociated by proteinase K treat-ment,followed byfluorescence measurements.Remark-ably,a significant ATP-dependent increase of thefluores-cence was observed(ATP is required for the production of Atg8-PE;Figure2B,column6).This increasedfluores-cence was not observed with samples of nonlabeled lipo-somes alone,NBD+Rho liposomes alone,or a mixture of nonlabeled liposomes and liposomes containing NBD-PE but not Rho-PE(Figure2B,columns1-3).These results suggest that membrane fusion occurred between the lipo-somes tethered together by Atg8-PE.The increasedfluo-rescence was only observed if the reaction mixture was treated with proteinase K(Figure2B,columns4and6). This appeared to be due to the presence of Atg7and/or Atg3rather than Atg8or some effect of the clustering, because the NBDfluorescence was not increased by the addition of Atg4(Figure2B,column5),which detached Atg8from the membranes and dissociated the clusters of liposomes(see below).Instead,decreasing the con-centrations of the conjugation enzymes allowed the dequenching of the NBDfluorescence to be detected without proteinase K digestion(Figure2B,column7). The fusion of the liposomes was examined with various amounts of Atg8(Figure2C).The level of fusion increased Atg8dose-dependently and reached maximum at2m M (Figure2C).In contrast,a larger amount of Atg8produced an inhibitory effect(data not shown).This suggested that formation of the large aggregates resulted from excessive tethering by Atg8-PE,which no longer lead to fusion. We also carried out time-course experiments to roughly estimate the fusion rate using the lower concentrations of the conjugation enzymes(Figure2D),which eliminated the need for the proteinase K treatment(Figure2B).It should be noted that the incubation time includes the times re-quired for the formation of Atg8-PE and the subsequent tethering and fusion reactions.Under these conditions, the band of Atg8-PE could be seen on an SDS-PAGE gel after a10min incubation,and the reaction was completed within30min(Figure S1in the Supplemental Data available with this article online).It appeared that166Cell130,165–178,July13,2007ª2007Elsevier Inc.Figure1.Membrane Tethering Function of Atg8-PE In Vitro(A–C)Purified Atg8(10m M),Atg7(1m M),and Atg3(1m M)were incubated with liposomes(350m M lipids)composed of55mol%DOPE,30mol% POPC,and15mol%blPI in the presence(lanes1–6)or absence(lanes7–12)of1mM ATP at30 C for the indicated time periods,followed by urea-SDS-PAGE and CBB-staining(A),measurement of the absorbance at600nm(B),or observation under a light microscope(Nomarski images)(C).(D)Conjugation reactions with the various amounts of Atg8were performed as described in(A).After incubation for60min,the size distribution of the aggregates was examined using DLS measurements.d.nm,apparent diameter(nm).(E and F)The conjugation reactions were carried out as described in(A).They were further incubated at30 C for30min in the presence of either 6M urea or1%CHAPS and were then subjected to microscopy(E)or urea-SDS-PAGE and CBB-staining(F).The reaction was also performed with liposomes containing1mol%NBD-labeled DOPE(thus containing54mol%unlabeled DOPE),followed byfluorescence microscopy.Afluo-rescence image with afilter for YFP(NBD-PE,FL)and a Nomarski image(NBD-PE,DIC)are shown.(G and H)Atg8(30m M),Atg7(2m M),and Atg3(2m M)were incubated with liposomes(350m M lipids)consisting of70mol%DOPE and30mol% POPC in the presence of1mM ATP at30 C for45min(total).The mixture was microcentrifuged at15,000rpm for10min to generate the pellet (ppt.)and the supernatant(sup.)fractions.The fractions were briefly sonicated and were analyzed by urea-SDS-PAGE(G)or observed under a light microscope(H).In this experiment,blPI was omitted to prevent Atg7and Atg3from tightly binding to the liposome.We showed that Atg8could also cause hemifusion of liposomes with this lipid composition.Cell130,165–178,July13,2007ª2007Elsevier Inc.167the liposomes began to fuse shortly after the formation of Atg8-PE.The fusion reaction proceeded concurrently with the conjugation reaction and continued for 30min after the completion of the Atg8-PE production (Figure 2D,filled circles).Small liposomes <100nm in diameter tend to sponta-neously fuse (Chen et al.,2006),and the liposomes we used in the above experiments were 70nm in diameter (Figure 1D).However,we also showed that Atg8-PEcaused a significant level of fusion between larger lipo-somes in spite of their stability against spontaneous fusion (Figure S1).Taken together,these results suggest that not only tethering but also fusion of the liposomes is mediated by Atg8-PE.The Atg8-Mediated Membrane Fusion Is Hemifusion Recent in vitro studies on membrane fusion mediated by SNARE proteins and a class of viral proteinsrevealedFigure 2.Membrane Hemifusion Occurs between Liposomes Tethered by Atg8-PE(A and B)Nonlabeled (55mol%DOPE,30mol%POPC,and 15mol%blPI),NBD-labeled (55mol%DOPE,29mol%POPC,15mol%blPI,and 1mol%NBD-DOPE),and NBD+Rho-labeled (55mol%DOPE,27.5mol%POPC,15mol%blPI,1mol%NBD-DOPE,and 1.5mol%Rho-DOPE)liposomes were mixed in the differ-ent combinations and ratios indicated.Their relative intensities of the NBD fluorescence ob-served are shown (the value obtained with a 4:1mixture of the nonlabeled and NBD+Rho lipo-somes was defined as 1)(A).These mixtures of liposomes were incubated with Atg8(4m M),Atg7(0.5or 1.0m M),and Atg3(0.5or 1.0m M)in the presence (filled columns)or absence (open columns)of 1mM ATP for 60min,and were then treated with 1unit/ml apyrase.The mixtures were further incubated for 30min with the buffer (columns 4and 7),1m M Atg4(columns 5and 8),or 0.2mg/ml proteinase K (columns 1-3,6and 9),followed by measure-ment of the NBD fluorescence.The experi-ments were repeated three times and the average fluorescence values divided by those obtained from the original liposome samples (F/F 0)are presented with error bars for the stan-dard deviations (B).(C)A 4:1mixture of the nonlabeled and NBD+Rho liposomes was incubated with various amounts of Atg8,1.0m M Atg7,and 1.0m M Atg3in the presence (open circles)or absence (filled circles)of ATP,and the samples were then treated with proteinase K,followed by measuring the NBD fluorescence.(D)The conjugation reactions were performed with the mixed liposomes used for the lipid mixing assay,0.5m M Atg7,and 0.5m M Atg3in the presence or absence of Atg8(4m M)and ATP.After incubation for the indicated time periods,an aliquot of the samples was immediately subjected to the fluorescence measurements.The values that were obtained by subtracting the signals observed in the absence of ATP from those observed in the presence of ATP are presented.(E)The lipid mixing assay was performed with 4m M Atg8,1m M Atg7,and 1m M Atg3in the presence or absence of ATP (white bars in columns 3and 2,respectively)as described in(C).For PEG-induced fusion reactions,the mixed liposomes were incubated at 37C for 30min in the presence or absence of 12.5%PEG 3350(white bars in columns 5and 4,respectively).These samples as well as the original liposomes (column 1)were then incubated with 20mM sodium dithionite on ice for 20min in the presence (black bars)or absence (gray bars)of 0.5%Triton X-100,followed by the NBD fluorescence measurement.168Cell 130,165–178,July 13,2007ª2007Elsevier Inc.that fusion proceeds through an intermediate state called hemifusion,in which outer(contacting)leaflets of two apposed lipid bilayers merge,while inner(distal)leaflets remain intact(Chernomordik and Kozlov,2005).It was also reported that fusion can be arrested or delayed at the hemifusion state under some conditions.Therefore, we investigated whether the liposome fusion caused by Atg8in vitro was complete fusion(the merger of both inner and outer leaflets)or hemifusion(Figure2E).This can be examined using the membrane impermeable reductant sodium dithionite that selectively abolishes thefluores-cence of NBD conjugated to the lipid head group in the outer leaflet(Meers et al.,2000).Accordingly,when so-dium dithionite was added to the original liposomes,the background level of the NBDfluorescence was decreased by about50%,whereas it was hardly detected in the pres-ence of the detergent(Figure2E,column1).Strikingly,the NBDfluorescence increased by the Atg8-mediated fusion was totally eliminated by addition of sodium dithionite to the same level as those observed in the original lipo-somes and the reaction mixture incubated without ATP (Figure2F,columns1-3).Whereas,we confirmed that in liposome fusion induced by polyethylene glycol(PEG), which causes complete fusion(Akiyama and Ito,2003), about half of the increasedfluorescence was retained af-ter sodium dithionite treatment(Figure2F,columns4and 5).Taken together,membrane fusion mediated by Atg8 in vitro was suggested to be hemifusion.To obtain direct evidence of hemifusion,we analyzed the morphology of liposomes by electron microscopy (Figures3A–3E),in which liposomal membranes were observed as double white lines that correspond to the outer and inner leaflets.When the clusters of liposomes formed by Atg8-PE were analyzed,tight junctions between the liposomes were observed(Figures3B and 3C,arrowheads).Consistent with the biochemical results suggesting that complete fusion does not occur,the size of the individual liposomes did not appear to significantly increase(compare Figures3A and3B).Instead,hallmarks of hemifusion,trifurcated structures formed by one contin-uous outer leaflet and two separate inner leaflets,could be observed at the junction between the liposomes(Figures 3C–3E,arrows).These results strongly support our con-clusion that Atg8-PE causes hemifusion of liposomes. Atg8Forms a Multimer in Responseto the Conjugation with PEWe also performed immunoelectron microscopy of the liposomes clustered by Atg8-PE(Figures3F–3I).Intrigu-ingly,Atg8-PE tended to be enriched at the junction be-tween the liposomes(Figures3G–3J).While,if the mixture incubated without ATP was similarly analyzed,the signal was rarely observed on the liposome(Figure3F;the gold particles observed should represent unconjugated Atg8 adsorbed onto the grid).These results indicate that Atg8-PE is directly involved in the tethering and hemifu-sion of liposomes.We observed that‘‘naked’’liposomes do not associate with liposomes carrying Atg8-PE(data not shown), suggesting that tethering should be achieved due to interactions between Atg8-PE molecules on different membranes.We therefore examined the intermolecular interaction of Atg8-PE by crosslinking experiments(Fig-ure4).The reaction mixture containing Atg8-PE or unconjugated Atg8was incubated with the lysine-to-lysine reactive crosslinker DSS.We found that a crosslink adduct with a molecular weight of 24kDa on a SDS-PAGE gel specifically appeared in the sample containing Atg8-PE(Figure4A,lane5).Considering the molecular weights of the proteins included,this adduct should represent an Atg8-PE homodimer.Immunoblotting anal-yses with anti-Atg8revealed that two additional crosslink adducts of about37and100kDa were also specifically produced in the Atg8-PE-containing sample(Figure4B, lanes2–4and Figure S2).These products were immuno-stained neither with anti-Atg7nor anti-Atg3(data not shown),suggesting that they represent a trimer and a larger multimer of Atg8-PE,respectively,and thus that Atg8multimerizes in response to PE conjugation. We also showed that this multimerization correlates with the membrane tethering ability of Atg8(see below), indicating that interactions between Atg8-PE molecules on different membranes are responsible for the tethering of the membranes.The Membrane-Tethering and HemifusionFunctions of Atg8Are Modulatedby the Deconjugation Enzyme Atg4Our results suggest that the membrane-tethering and hemifusion functions of Atg8are evoked by the conjuga-tion with PE,whereas Atg4functions as a deconjugase that cleaves the linkage between Atg8and PE(Kirisako et al.,2000).We reconstituted this reaction in vitro.After producing Atg8-PE using the conjugation reaction,the re-action was terminated by adding apyrase to deplete the remaining ATP.When purified Atg4was then added, Atg8-PE was rapidly and almost completely deconjugated (Figure4C,lanes1-6).In contrast,when the Atg4was pretreated with the cysteine protease inhibitor N-ethylma-leimide(NEM),the deconjugation reaction did not occur (Figure4C,lanes7–12).These results clearly show that Atg4is sufficient for the deconjugation of Atg8-PE.Upon deconjugation,the liposome aggregates immediately dissociated(Figure4D).In addition,we found that multi-merization of Atg8is also reversible;the crosslink adducts corresponding to the Atg8-PE dimer(Figure4A,lane6)as well as the trimer and the multimer(data not shown)were hardly formed when DSS was added after the deconjuga-tion reaction.We also showed that the presence of Atg4in the conjugation reaction retarded the accumulation of Atg8-PE and accordingly interfered with the tethering and hemifusion of the liposomes(data not shown).It was indicated that membrane tethering and hemifusion by Atg8can be regulated by the balance between the conjugation and deconjugation reactions.Cell130,165–178,July13,2007ª2007Elsevier Inc.169Identification of Mutations that Impair the Postconjugational Function of Atg8In VivoIf the function of Atg8-PE we observed in vitro was involved in autophagosome formation in vivo,Atg8mutants defi-cient for this function should result in defective autophagy.To examine this idea,we performed structure-based and systematic mutational analyses of Atg8(Figure 5).The structures of mammalian homologs revealed that Atg8family proteins consist of two domains:an N-terminal heli-cal domain (NHD)and a C-terminal ubiquitin-like domain (ULD)(Paz et al.,2000;Coyle et al.,2002;Sugawara et al.,2004;Figures 5E–5H).Among the highly conserved residues in the ULD,we selected those with side chains that were exposed on the domain surface (Figure 5A),and individually replaced them with alanine,except that serine was substituted for Ala75.Consequently,we didnot mutate residues suggested to be important for interac-tions with the conjugation enzymes,because these resi-dues are conserved only for their hydrophobic nature (Sugawara et al.,2004).The Atg8variants were expressed from centromeric plasmids in D atg8yeast cells,and their autophagic activities were biochemically assessed (see Supplemental Experimental Procedures ).In nutrient-rich media,the autophagic activity was low in all of the mutant cells as well as in the wild-type cells (data not shown).In contrast,in nitrogen starvation conditions,which strongly induced autophagy,a number of mutants were found to have defective autophagic phenotypes (Figure 5B).Ala-nine replacement of seven residues,Ile32,Lys48,Leu50,Arg65,Asp102,Phe104,and Tyr106,significantly impaired the autophagic activity to 30%–60%of that of the wild-type (Figure 5B).Immunoblotting analyses showedthatFigure 3.Electron Microscopic Analyses of the Liposomes Tethered and Hemi-fused by Atg8-PEConjugation reactions were performed with 4m M Atg8,1m M Atg7,and 1m M Atg3in the presence (B–E and G–I)or absence (A and F)of ATP for 60min and subjected to phospho-tungstic acid-staining and electron micros-copy (A–E).The junctions between the lipo-somes and the structures suggested to represent hemifusion are indicated with arrow-heads and arrows,respectively.The same samples were also subjected to immunostain-ing using purified anti-Atg8-IN-13and anti-rab-bit IgG conjugated with 5nm gold particles,fol-lowed by phosphotungstic acid-staining and electron microscopic observation (F–I).To as-sess the enrichment of Atg8-PE at the junction of the liposomes (J),images of two contacting liposomes as shown in G and H were randomly picked up (n =41).The lengths of contacting (CR)and noncontacting regions (non-CR)of the liposomal membranes were measured (white bars),thereby the number of gold parti-cles on each region (gray bars)was divided,in which the length of the contacting region was doubled,to calculate the linear density (black bars).The average values are presented with error bars for the standard deviations.170Cell 130,165–178,July 13,2007ª2007Elsevier Inc.a substantial amount of each of the Atg8mutant proteins accumulated in the cells (Figure 5D),although there were some differences in their mobilities in SDS-PAGE analysis;for instance the PE-conjugated and unconjugated forms of the D102A mutant exhibited almost the same mobility.None of the mutations significantly affected the formation of Atg8-PE (Figure 5D),suggesting that the mutations impaired a function of Atg8that was exerted after the conjugation with PE.Notably,these mutants accumulated different levels of unconjugated Atg8under the starvation conditions (Figure 5D,starvation),which allowed us to classify them into three groups.For the class I mutants K48A and L50A,the levels of the unconjugated forms were similar to that of the wild-type (Figure 5D,denoted in purple).On the other hand,compared to the wild-type,lower levels of the unconjugated forms were detected in the class II mutants I32A,D102A,F104A and Y106A (Figure 5D,denoted in red),whereas a larger amount of the unconju-gated class III mutant R65A accumulated (Figure 5D,denoted in orange).We then mapped the mutated resi-dues onto the three-dimensional structure of LC3(Suga-wara et al.,2004),which revealed that class of the mutant corresponded to the location of the mutation.All the class II residues were clustered in a specific region on the ULD (hereafter,referred to as the class II region),and the two neighboring class I residues were located close to the class II region (Figure 5E).In contrast,the class III residue was located away from the other mutated residues (Fig-ures 5G and 5H).The NHD of Atg8contains two helices:a 1and a 2(Figure 5A).We constructed two mutants,one with a dele-tion of a 1(D N8)and a second bearing deletions of both helices (D N24).It was shown that the NHD is involved in autophagy partially but significantly;the D N8and D N24mutations decreased the autophagic activity by about 30and 40%,respectively (Figure 5C).We also showed that the deletions did not affect the stability of the proteins or the formation of the PE conjugates (Figure S3).Effects of the Atg8Mutations on the Membrane-Tethering FunctionWe next examined whether the mutations affected the liposome-clustering ability of Atg8in vitro (Figure 6).Figure 4.The Membrane-Tethering Function and Multimerization of Atg8Are Reversibly Regulated in Response to Conjugation with PE(A)Conjugation reactions were performed as described in Figure 3in the presence (lanes 2,3,5,and 6)or absence (lanes 1and 4)of ATP.They were mixed with 1unit/ml apyrase,and then incubated with (lanes 3and 6)or with-out (lanes 1,2,4,and 5)purified Atg4(0.5m M)at 30 C for 30min.These samples were further incubated with (lanes 4–6)or without (lanes 1–3)100m M DSS for 30min,and then analyzed by urea-SDS-PAGE and CBB-staining.(B)The reaction mixture including ATP was in-cubated with different concentrations of DSS as indicated,followed by urea-SDS-PAGE and immunoblotting with anti-Atg8-IN13.We also identified a crosslink product that reacted with anti-Atg3(Atg8xAtg3).(C and D)The conjugation reactions performed as described in Figure 1A were mixed with 1unit/ml apyrase.Atg4(0.5m M)pretreated with (lanes 7–12)or without (lanes 1–6)10mM NEM was then added,and the samples were incubated for the indicated time periods and subjected to urea-SDS-PAGE and CBB-stain-ing (C).The same samples were also observed under a light microscope (D).Cell 130,165–178,July 13,2007ª2007Elsevier Inc.171。
2024届湖北省武汉市高三下学期4月调研(二模)英语试题
2024届湖北省武汉市高三下学期4月调研（二模）英语试题一、听力选择题1．What is the man busy with?A．A visit.B．A project.C．A video.2．What do the speakers plan to do tomorrow?A．Go camping.B．Do some shopping.C．Find a blanket. 3．Where are the speakers going to?A．A station.B．Another country.C．Their hometown. 4．What does the woman think of Jimmy?A．Silent.B．Caring.C．Hard-working. 5．What are the speakers talking about?A．The new laws.B．A healthy lifestyle.C．Profitable industries.听下面一段较长对话，回答以下小题。
6．What is the probable relationship between the speakers?A．Mother and son.B．Brother and sister.C．Husband and wife. 7．Why does the man apologize?A．For missing a chance.B．For being late.C．For doing Lisa a damage.听下面一段较长对话，完成下面小题。
8．Who is the woman expressing thanks to?A．The man.B．The fans.C．The sponsor.9．What is probably the woman?A．An online celebrity.B．A travel enthusiast.C．A magazine editor.听下面一段较长对话，回答以下小题。
四大发明的印刷术,英语作文
四大发明的印刷术,英语作文Printing: A Monumental Invention of Ancient China.In the annals of human ingenuity, few innovations have had such a profound and lasting impact as the invention of printing. This transformative technology emerged from the ingenuity of ancient China, where it played a pivotal role in shaping the course of civilization. The Chinese invention of printing, one of the Four Great Inventions along with gunpowder, the compass, and papermaking, revolutionized the dissemination of knowledge, ideas, and culture.The origins of printing can be traced back to the 2nd century CE, when artisans began carving characters onto stone tablets for use in official inscriptions and record-keeping. This technique, known as "stone rubbings," marked the earliest form of printing in China. However, it was relatively labor-intensive and impractical for mass production.The breakthrough came in the 7th century CE with the development of woodblock printing. This method involved carving characters onto wooden blocks and using them to transfer ink onto paper or cloth. Woodblock printing allowed for greater flexibility and speed in producing multiple copies of texts. The technique was particularly useful for printing religious scriptures, government documents, and popular literature.The next significant advancement occurred in the 11th century CE, when the inventor Bi Sheng developed movable type printing. This revolutionary concept involved creating individual characters from ceramic or metal that could be assembled and rearranged to form different words and sentences. Movable type printing significantly increased the efficiency and accuracy of printing, paving the way for mass production of books and other printed materials.The invention of movable type printing had a profound impact on Chinese society. It led to an explosion in the production of books and other printed materials, makingknowledge and education more accessible to a wider population. The widespread dissemination of printed texts fostered intellectual exchange, stimulated cultural development, and played a crucial role in the emergence of the Song Dynasty as a period of unprecedented scientific and technological advancements.The Chinese invention of printing did not remain confined within the borders of China. Through trade and cultural exchange, the technology gradually spread to other parts of Asia, Europe, and the rest of the world. In the15th century, Johannes Gutenberg, a German goldsmith, adapted the Chinese movable type printing technique to create the first printing press in Europe. This momentous event marked the dawn of a new era in communication and knowledge dissemination in the West.The printing press revolutionized the production of books in Europe, making them more affordable and accessible to the masses. It played a pivotal role in the Renaissance and Reformation, facilitating the spread of new ideas and challenging the authority of the Church. The printing pressalso enabled the widespread dissemination of scientific knowledge, paving the way for the Scientific Revolution and the Enlightenment.The invention of printing had a profound impact on virtually every aspect of human society. It facilitated the preservation and transmission of knowledge, ideas, and culture across generations. It fostered the development of education, science, literature, and the arts. It promoted intellectual exchange and stimulated the growth of new knowledge and ideas.Printing remains a cornerstone of modern society. From books and newspapers to magazines and online publications, printed materials continue to play a vital role in informing, educating, and entertaining people around the world. The invention of printing in ancient China stands as a testament to human ingenuity and its enduring legacy on the course of civilization.。
J Cell Physiol
J Cell PhysiolJ Cell Physiol is a scientific journal that focuses on the field of cellular physiology. It covers a wide range of topics related to the functioning and behavior of cells, with a particular emphasis on their physiological processes. The journal publishes high-quality original research papers, reviews, and commentaries that contribute to our understanding of cell physiology.Cell physiology is an interdisciplinary field that studies the functions and activities of cells, including their metabolism, communication, and response to environmental stimuli. Understanding cell physiology is crucial for understanding the basic processes that underlie complex biological systems and for developing new approaches for the diagnosis and treatment of diseases.Research published in J Cell Physiol encompasses a variety of topics, including cellular metabolism, cell signaling, cell cycle regulation, cell growth and differentiation, cellular responses to stress, and cell communication. The journal also welcomes studies investigating the impact of various factors on cell physiology, such as genetic and epigenetic changes, environmental factors, and therapeutic interventions.One area of interest in J Cell Physiol is cellular metabolism. This field aims to understand the complex network of metabolic pathways that cells utilize to generate energy and synthesize biomolecules. Elucidating the regulation of cellular metabolism can provide insights into various diseases, such as cancer and metabolic disorders, and can help identify potential therapeutic targets.Cell signaling is another key area covered in J Cell Physiol. Cells communicate with each other through a variety of signaling pathways, including cell surface receptors, intracellular signaling cascades, and gene expression regulation. Understanding how cells transmit and process these signals is essential for deciphering the molecular mechanisms underlying normal cellular functioning and for identifying aberrant signaling events associated with disease.The regulation of the cell cycle is vital for cell growth and tissue homeostasis. J Cell Physiol publishes studies investigating the control and coordination of the cell cycle, including the role of key regulatory proteins and the mechanisms involved in cell cycle checkpoints. Dysregulation of the cell cycle is a hallmark of many diseases, including cancer, and understanding its regulation can lead to the development of novel therapeutic strategies.Cell growth and differentiation are also areas of interest in J Cell Physiol. These processes are fundamental for tissue development, repair, and regeneration. Research published in the journal examines the molecular and cellular mechanisms governing cell growth and differentiation, as well as the factors that influence these processes, such as growth factors, signaling pathways, and the cellular microenvironment.Cellular responses to stress, both physiological and pathological, are investigated in J Cell Physiol. Cells have evolved intricate mechanisms to respond to various forms of stress, including oxidative stress, DNA damage, and proteotoxic stress. Understanding these adaptive responses can provide insights into the pathogenesis of diseases and may lead to the development of therapeutic interventions.Finally, J Cell Physiol also covers studies on cell communication. Cells communicate through various mechanisms, including direct cell-cell contact, secretion of signaling molecules, and extracellular vesicles. The journal publishes research on the molecular mechanisms underlying cell communication and its role in physiological and pathological processes, such as immune responses, tissue regeneration, and cancer metastasis.In conclusion, J Cell Physiol is a scientific journal dedicated to advancing our understanding of cellular physiology. It covers a wide range of topics related to the functioning and behavior of cells, with the aim of unraveling the intricate mechanisms underlying normal cellular processes and disease pathology. By publishing high-quality research papers, reviews, and commentaries, the journal contributes to the field of cell physiology and provides a platform for scientific exchange and collaboration.。
干细胞研究及其应用概况
17
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三、胚胎干细胞
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1998年美国有两个小组分别培养出了人多能( pluripotent )干细胞：
James A. Thomson在 Wisconsin大学领导一个研究小组从人胚胎组 织中培养出了干细胞株。他们使用的方法是：人卵体外受精后， 将胚胎培育到囊胚阶段，提取 inner cell mass细胞，建立细胞株。 经测试这些细胞株的细胞外表 marker 和酶活性，证实它们就是胚 胎干细胞。用这种方法，每个胚胎可取得15－20个细胞用于培养。 John D. Gearhart在 Johns Hopkins大学领导另一个研究小组也从 人胚胎组织中建立了干细胞株。他们的方法是：从受精后5－9 周人工流产的胚胎中提取生殖母细胞( primordial germ cell )。由 此培养的细胞株，证实具有全能干细胞的特征。
分化为神经胶质细胞，
*聚集培养的ES细胞诱导分化那么可分化为有节律性收缩
的心肌细胞。
*将ES细胞注射到同源动物皮下，可形成组织瘤，其细胞
组成可代表3个胚层细胞。 *用ES细胞作核供体进行细胞核移植，可以得到可发育重
构胚和动物个体。
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碱性磷酸酶的表达
许多资料说明，小鼠、大鼠的桑椹胚细胞和 囊胚细胞均有碱性磷酸酶〔AKP〕表达，小鼠 的EC细胞和ES细胞中均含有丰富的AKP。而在 已分化的EC细胞和ES细胞中AKP呈弱阳性或阴 性。猪、兔的桑椹胚和早期囊胚AKP呈阳性。 因此，AKP常用来作为鉴定EC细胞或ES细胞分 化与否的标志之一。
干细胞 或分化为特定细胞。
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有关旧书籍收集和捐赠的英语作文
有关旧书籍收集和捐赠的英语作文全文共3篇示例，供读者参考篇1My Love for Old Books and Why You Should Donate YoursI'm what you might call a book nerd - the kind of person who loves the musty smell of aged paper and the feel of a weathered hardcover in my hands. While e-books are convenient, there's just something magical about holding a physical book that has been passed down through generations of readers before me.I started my collection of vintage books when I was about 10 years old. My mom's childhood copy of Alice's Adventures in Wonderland, its cover tattered and pages yellowed, sparked my fascination. I loved thinking about who else had turned those thin leaves before me over the decades. Whose eyes had soaked in the words? Whose hands had gently brushed the illustrations?From then on, I was hooked on hunting for other discarded literary treasures. I started scouring garage sales, library book swaps, and used bookstores for affordable vintage finds. With each newfound gem, be it a cloth-bound edition of Jane Eyrefrom the 1920s or a Ray Bradbury sci-fi paperback from the 1960s, my little collection grew.To me, these well-loved books are almost like portals to earlier eras. Just seeing the dated cover art and classic typography is enough to whisk me away to another time and place. And then there are the personal touches former owners left behind - marginalia, inscriptions, pressed flowers used as bookmarks. These little glimpses into past readers' lives make the books feel imbued with sentimental value and human connection.As my collection expanded over the years, so did my reasons for loving old books. There's the environmentally-friendly aspect of reusing existing items rather than consuming new resources. The hunt for fresh finds exercises perseverance and an appreciation for humble odds and ends. Best of all, saving these literary works from the landfill preserves ideas and stories that might otherwise be lost to time.Which brings me to why you should consider donating your old books instead of tossing them: it keeps the cycle going for book lovers like me while benefitting others as well.For starters, many organizations collect pickle books to help strengthen literacy efforts and get reading materials tounderserved communities. A single donated book may seem insignificant, but it can spark a lifelong love of reading in a child who doesn't normally have access to books at home. Or it might be a low-income student's only reading material as they work toward their education goals. Just one book really can make a difference.Other groups distribute gently used books to hospitals, shelters, prisons, and troops stationed overseas to provide educational resources as well as psychological comfort through literature's transformative power. Older publications also stock libraries in developing nations and fill the halls of underfunded school systems. In all these cases, your old books can make a positive impact on countless lives around the world.Finally, there are organizations that sell donated books to raise funds for literacy initiatives, libraries, battered women's shelters, and other great causes. So even if the books themselves can't be directly circulated, your donation still winds up helping those in need.Honestly, I try not to judge others for seeing books as disposable items to clear off their shelves and out of their homes.I get that not everyone shares my sentimental attachment to printed works. To each their own. But I'd still encourageeveryone to look into book donation options in their area before trashing any unwanted volumes.It's so easy to box up your old paperbacks, hardcovers, textbooks - whatever you don't need anymore - and drop them off for someone else who could use them or benefit from their sale. You'll prevent books from becoming landfill waste while supporting education, literacy, and libraries. All it takes is a little effort to put your gently used reading materials back into circulation instead of sending them to the curb.As for me, I'll keep scouring used bookstores and rummaging through the book nook at garage sales, always on the hunt for the next addition to my treasured collection. Not only does it satisfy my nerdy, nostalgic soul, but it keeps books out of landfills too. I have a hard time parting with any of my vintage finds, but I do donate duplicates and books I don't have a personal attachment to so they can go to a new reader and home.So hang onto your beloved favorites, but consider passing along the books you don't love anymore instead of discarding them. Your unwanted reads could ignite a new reader's passion, provide a low-income classroom with resources, deliverentertainment and comfort to someone in difficult circumstances, or raise funds for a good cause.And who knows? The book you donate today could itself become a well-loved vintage treasure for someone like me to discover decades from now. Just let that idea warm your bibliophilic heart.篇2The Forgotten Treasures: My Journey into Book Collection and DonationAs a student, I've always been drawn to the world of books, their musty scent, and the promise of untold stories waiting to be discovered within their well-worn pages. However, it wasn't until I stumbled upon a dusty old bookshop tucked away in a forgotten alley that my passion for collecting and donating old books truly blossomed.It was during my first year of college when I found myself wandering aimlessly, trying to escape the overwhelming stress of midterms. That's when the quaint little shop caught my eye, its faded sign barely visible amidst the towering skyscrapers and bustling streets. Curiosity piqued, I stepped inside, and what greeted me was a bibliophile's paradise.The shop was crammed with towering shelves, each one groaning under the weight of thousands upon thousands of books. From leather-bound tomes to tattered paperbacks, every inch of space was occupied by these forgotten treasures. As I ran my fingers along their spines, a sense of wonder and excitement washed over me.It was then that I realized the true value of these books, not just as objects of entertainment or education, but as vessels carrying the collective wisdom, experiences, and imaginations of countless authors throughout history. Each book was a portal, waiting to transport me to different realms, different eras, and different perspectives.From that day on, I became a regular at the shop, spending countless hours scouring the shelves, unearthing hidden gems, and engaging in lively discussions with the proprietor, an elderly gentleman whose encyclopedic knowledge of literature never ceased to amaze me.My collection grew steadily, and with each addition, my appreciation for these forgotten treasures deepened. I delved into classic novels, immersing myself in the timeless tales of love, tragedy, and triumph. I explored obscure texts on philosophy,science, and history, expanding my understanding of the world and the human condition.But as my collection grew, so did my realization of the vast number of books that languished in obscurity, gathering dust on forgotten shelves or facing the threat of being discarded and forgotten forever. It was then that I decided to embark on a mission to not only collect these literary gems but also to ensure their preservation and dissemination.I began scouring second-hand bookstores, yard sales, and even dumpsters, rescuing books destined for the trash heap. With each rescued tome, I felt a sense of accomplishment, knowing that I had saved a piece of history, a window into another time and place, from oblivion.However, my collection soon outgrew my modest dorm room, and I knew that I had to find a way to share these treasures with others. That's when I discovered the joy of book donation.I started small, donating a few books to my local library, but as my passion grew, so did the scope of my donations. I reached out to schools, community centers, and even prisons, offering them the opportunity to enrich their libraries with these literary gems.The reactions I received were overwhelming. I witnessed the pure joy on the faces of children as they eagerly flipped through the pages of a beloved classic, their imaginations ignited by the words on the page. I saw the spark of curiosity in the eyes of adults as they delved into subjects they had never explored before, their minds expanded by the knowledge contained within these forgotten tomes.But perhaps the most profound impact came from the inmates at the local prison. As I distributed books among them, I witnessed the transformative power of literature firsthand. Men and women who had once felt forgotten and cast aside by society found solace and escape within the pages of these books. Their eyes lit up with newfound hope and determination, as they realized that knowledge and education could be the keys to their redemption.It was in those moments that I truly understood the significance of my mission. These books were not mere objects, but vessels of human experience, capable of transcending boundaries, breaking down barriers, and fostering understanding and empathy.As I neared the end of my college years, my collection had grown exponentially, and my passion for preserving and sharingthese forgotten treasures had become an integral part of who I was. I knew that my journey had only just begun, and that there were countless more books waiting to be discovered, cherished, and shared.Today, as I look back on my journey, I am filled with a sense of gratitude and pride. Gratitude for the opportunity to have played a small role in preserving these literary gems, and pride in the knowledge that my efforts have touched the lives of countless individuals, igniting their curiosity, expanding their horizons, and fostering a love for the written word.To those who share my passion, I say this: never underestimate the power of a book. Within its pages lie the keys to unlocking the human experience, transcending time and space, and connecting us to the shared tapestry of our existence. Embrace these forgotten treasures, for they are not mere objects, but portals to a world of wonder, knowledge, and enlightenment.And to those who have yet to discover the magic of these literary gems, I extend an invitation: open your mind, crack open a book, and let the words within transport you to realms beyond your wildest dreams. For in the end, it is not the number of bookswe possess that matters, but the impact they have on our lives and the lives of those around us.篇3The Allure of Old Books and the Joy of Sharing ThemAs an avid reader and bibliophile, there's something special about holding an old book in my hands. The worn pages, the distinctive smell of aged paper, and the weight of history contained within each volume captivate me in a way that brand-new books simply can't match. Every old book tells a story beyond its printed words – a tale of the lives it has touched, the journeys it has taken, and the hands that have lovingly turned its pages over the years.My passion for collecting old books began in childhood, when I would spend hours scouring the shelves of local thrift stores and flea markets, hunting for literary treasures. There was something exhilarating about unearthing a dog-eared copy of a classic novel or a tattered volume of poetry, each one bearing the marks of its previous owners. I would carefully examine the inscriptions, marginalia, and other personal touches left behind, imagining the lives of those who had read these books before me.As I grew older, my collection expanded, becoming a reflection of my diverse interests and the eras that fascinated me. From weathered volumes of Shakespeare's plays to crumbling issues of National Geographic from the 1950s, each addition to my library held a unique charm. I relished the opportunity to preserve these physical embodiments of knowledge and creativity, rescuing them from obscurity and giving them a new home on my ever-growing bookshelves.However, as my collection grew, so did my realization that these books deserved more than just a place on my shelves. They deserved to be read, shared, and appreciated by others who could find joy and inspiration within their pages. That's when I began to explore the idea of book donation – a way to pay forward the wonder and wisdom contained within these aging tomes.My first book donation was a humbling experience. I carefully selected a box of well-loved volumes, each one holding a special place in my heart, and delivered them to a local library. The librarian's grateful smile and the knowledge that these books would soon find new readers filled me with a sense of purpose. From that moment on, I was hooked on the idea of sharing my literary treasures with the world.Over the years, I have donated books to schools, community centers, hospitals, and even prisons – anywhere I could find people eager to explore the realms of knowledge and imagination contained within the printed word. With each donation, I feel a sense of satisfaction knowing that these books, which once gathered dust on my shelves, now have the opportunity to ignite the minds and hearts of new readers.One of my most memorable donation experiences was when I stumbled upon a small rural school in need of books for their library. The sight of those bare shelves tugged at my heart, and I knew I had to do something. I gathered as manyage-appropriate books as I could from my collection, carefully selecting titles that I knew would captivate young minds and inspire a love of reading. The looks of pure joy on the students' faces when they received those books will forever be etched in my memory.Beyond the act of donation itself, I have found immense fulfillment in sharing the stories behind the books I donate. Each volume carries its own unique history, and I take great pleasure in recounting the tales of how I acquired them, the adventures they have been on, and the impact they have had on my life. By doing so, I hope to inspire others to see these books not just asobjects, but as living entities imbued with the power to transport us through time and across cultures.In a world increasingly dominated by digital media, the preservation and sharing of physical books has become a vital endeavor. These tangible vessels of knowledge and imagination hold a magic that can never be fully replicated on a screen. By donating old books, we not only ensure their survival but also foster a sense of connection and continuity between generations of readers.To those who share my passion for collecting and donating old books, I offer this advice: cherish each volume as a unique treasure, but remember that true joy comes from sharing those treasures with others. Seek out organizations and individuals who can benefit from the wisdom and wonder contained within those pages. Engage with the communities you donate to, sharing the stories behind the books and encouraging a love of reading that transcends age and social boundaries.Every book has the potential to change a life, to open a mind, or to ignite a spark of curiosity that burns brightly for years to come. By collecting and donating old books, we become stewards of that potential, ensuring that the voices of the pastcontinue to resonate in the present and shape the minds of future generations.So, fellow book lovers, let us embrace the allure of old books and revel in the joy of sharing them with the world. For in doing so, we not only preserve the written word but also cultivate a deeper appreciation for the transformative power of literature and the enduring bonds that bind readers across time and space.。
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Journal of Computer Science 1 (3): 445-449, 2005ISSN 1549-3636© Science Publications, 2005OntoCell: An ontology of Cellular BiologyLynda DIB and Mohamed T LaskriUniversity Badji Mokhtar Annaba, AlgeriaAbstract:This article presents OntoCell, a cellular ontology that we constructed and edited under Protege2000. It regroups and unifies the main concepts and relations related to the cell's structure and behavior. OntoCell has been validated by experts in biology (by the UMRS INSERM 514). Moreover, it will be validated in the context of the development of a multi-agent system simulating the behavior of a cellular population.Keywords: Ontology, cellular biology, cell, concepts, relations.INTRODUCTIONThis work is inscribed in project which aims is the multi-agent modelisation and simulation of cellular populations. The aim is to propose a multi-agent platform in order to facilitate the modeling of cellular populations and the observation of their behaviors. The observed collective behaviors will be then confronted by the biologists to the observations and to the quantitative data results obtained in video-microscopy. This step allows to refine the biology models existed notably in tumoral invasion.The multi-agent simulation of cellular populations offer a very powerful tool to study the behavior of cells and their interactions. However, the necessary models for the development of this simulation require the knowledge in biology which are very difficult to identify and to structure.The first reflex when we speak about structuration and representation of data and knowledge is to think about data bases and their usual models (entity-association model, relational model, oriented object model…) and to thesaurus. Ontologies come to enrich and emphasis this modelisation of the domain and to offer the powerful tools of research and manipulation.The cellular biology, which is a part of experimental sciences domain, is characterized by a biologic language using an extremely rich and very difficult vocabulary to manipulate by the none biologists. In order to automate the treatment of biologic information, firstly, it must conceive a formal model. This model must unify the vocabulary, define clearly concepts and underlying relations and offer a research mechanisms and an easy manipulation to use. The ontologies are a very powerful formalisms of representation knowledge domains as complex and rich that the cellular biology. The aim of this article is to present OntoCell, a cellular ontology that we constructed in order to represent knowledge of cellular biology. Hence, OntoCell represents the concepts of cellulars bases, their components their behaviors and their interactions. It regroups and unify most concepts and relations linked to the cell and the milieu in which it evolves. It's constructed and edited under Protege2000 [17]. OntoCell is very rich, it regroups about fifty concepts. Otherwise, it is very easy to enrich it basing on more detailed study of cells and by exploiting the Protege2000 tool.This article is organized as follows : In the first section, we present an art state of ontologies for the cellular biology domain. In the second section, we describe the methodology followed by the OntoCell development which is summarized by the four steps : the requirements analysis, conceptualization, formalization and maintenance.Ontologies in Biology: The last years, many ontologies appeared in the biology domain. They have a common objective: to facilitate the division and the exchange of knowledge.The first category regroups Gene Ontology[2]. This ontology is very pragmatic. It is dedicated to the product’s annotation of gene among a vocabulary reference. It objective is to palliate terminology heterogeneity problems by furnishing a common control vocabulary facilitating the exchange of information. The terms of GO were defined from “Oxford Dictionary Biology” [26] and from “Swissprot” [3]. GO [2, 24] is a system combining three independent ontologies describing biological processes, molecular functions and cellular components.The conception of ontologies of this first category doesn't always respect the definition of the ontology’s reference (T.Gruber defines the ontology as “descriptions structured and formal of concepts of domain and their interrelations”), but they answer to the division and to the exchange problematic of Gene knowledge. These first ontologies demonstrated their utility, the essential concepts having been defined, related to each other and serving to the annotation of the partners data bases. Actually, they were in restructuration in order to be formalized, it is the case of Gene Ontology [10].A second ontologies categories, such as EcoCyc[15, 16, 18] and OMB [20, 21, 22, 23], treat the key points in problematic related on the number and heterogeneity of biologic resource : definitions of the common vocabulary, consensual description of knowledge basis scheme proper to a theme, extraction information distributed on different sources, and definition of Web bioinformatics services. The EcoCyc ontology is described to cover the general knowledge. It is not adapted to the molecular biology because is too general. EcoCyc serves to visualize the biochemical reactions and the gene’s disposition with the chromosomes. The treated concepts by this ontology are : the gene, the proteins, the small molecules and biochemical functions. It is presented to the biologists by using encyclopedic metaphor. It covers "E.coli genes", metabolism, regulation and traduction signal [5] . The OMB ontology (Ontology for the Molecular Biology) is elaborated by Schulze-Kremer, It models the molecular biology. In OMB the biological concepts are regrouped in three categories : the biological objects, the experimental procedures and the aspects in silico of biological molecular [19]. OMB is not rich (less deeper) because it treats less the biological concepts. The ontologies of the last category combine two or several existing ontologies. For example, RiboWeb [6, 1] is a set of four ontologies allowing the stake in common of the Web resources dedicated to the ribosomes. This basis allows the comparison and the interpretation of data in order to establish new distributed models. Otherwise, TAO the ontology of the Tambis system [4, 5] has the objective to answer to the problematic raising the extraction of information from different sources. This ontology has a central role in the TAMBIS system. The interrogation of set of data extern basis is realized by using only one constructed request, by the user, via an interface, from defined concepts in Tao. Hence, the biologists don't need to learn the languages of the specific request, nor the scheme of all susceptible basis to be interrogated.Since, these ontologies are well specific to their provided use (representation of biological knowledge for the study of the gene or of the protein), they treat the key points in the problematic linked to the number and the heterogeneity of the biological resources : definitions of the common vocabulary, consensual description of the scheme of the basis knowledge proper to a theme, extraction information dispatched on different sources, and definition of Web bioinformatics services, we propose, so, a new ontology OntoCell. OntoCell is different to the other ontologies because it doesn't interest itself to the survey of the gene or proteins nor to give the detail of experiences employees to study the structure of the RNA/proteins complex, but it concerns the study of the behavior of cell’s population. it is about the modeling of the cell, its components (its structure), its behavior in the environment, its adaptation and its change of state in one hand, and their interactions with other cells of its population on the other hand. This ontology is a hierarchical concepts, rich and full concerning the biological domain. These concepts were determined from different meetings of work and documents furnished by the biologists of the UMRS INSERM 514. OntoCell :It exists a great number of methodologies for the development and the maintenance of the ontologies such as Tove [11, 12, 13], Methodology [8], One-To-knowledge [25] and KBSI IDEF5 [14]. A comparative study of these different methodologies has been realized by Fernández-Lopez, Gómez-Pérez and Juristo [9] who show that these methodologies adopt the same cycle of development:•requirements analysis•Conceptualization•Formalization•Maintenance.OntoCell has been created while basing on the cycle of the development. The different steps are described in the following paragraphs.Requirements Analysis :Beginning a development of an ontology, means to define its domain and its reach. OntoCell is conceived in the aim to represent the biological basis knowledge cellular. It represents the structures of the cells, their environment and their behaviors. Hence, the domain is clearly defined. One of the proposed methods to determine the reach of the ontology, consists to write a list of questions which the final knowledge’s basis can answer, called questions of competence [12].In the case of OntoCell, the objective is to modelize the cell (healthy or cancerous) in its environment. In fact, we have elaborated many questions of competences such as :•What are the cell characteristics?•What are the necessary resources to its survival? •What are the characteristics of a cancerous cell? •What are the differences between a healthy cell and a cancerous cell?•What is the difference between a benign tumoral cell and a malignant cancerous cell?• When the cancerous cell can migrate from its origin tissue?• How cell passes from first phase of cancer to 4th phase of cancer?• When cancerous cells carry a metastasis?From these questions and the different interactions with biologists, we have acquired the knowledge of basis in cellular biology and collected the most information and necessary documents to the conceptualization step. Conceptualization:In this step, we begin the real creation of OntoCell. It is to introduce the concepts and the different relations. Hence, we were based on classical approach of information systems analysis ‘Merise’ [7]. We started to define the dictionary of the data basing on the different discussions with biologists also the documents that they furnished to us. Mostly, the dictionary contains the properties of the cellular biology domain. Table 1 gives the examples of properties of the OntoCell dictionary data.Table 1. Example of OntoCell data.Property of OntoCell Significance Example of values CatCellCategory of Cell Cell of thecrystalline lens StateCell State of Cell Cancerous, Quiescent LifeCellLength of Cell’s life One day NameDeplComp Name of Displacement Components Lamellipode StateJonc State JunctionDestroyed CatMol Category of Molecule Occludine, Catenine SizeMol size of Molecule (fuzzy value)Large Molecule CatFact Category of Factor Creatinin, Flumazenil FactType Factor TypeLiposoluble Factor CatMolAdhCategory of Adherence MoleculeIntegrine, Cadherine StateAdhMol State of AdherenceMoleculeActivate CatDissMol Category of DissociationMoleculeProtease StateDissMol State of DissociationMoleculeActivate TypeActiDissMol Type of activation ofDissociation MoleculeActivate with neutral pHThe second step consists to search the functional dependences between the different properties listed in the dictionary. Table 2 represents few functional dependences of data in Table 1.The functional dependences (FD) permit to construct the theoretical access structure (TAS). This TAS schematizes the links (FD) that exist between all the properties. Fig 1 gives the TAS a part of the dictionary OntoCell (see Table 1).The conceptual model of data is easily deduced from the TAS. This model offers a representation of data, easily comprehensible, describing the system with the help of entities and their relations.Fig. 2 gives the relation entity model corresponding to TAS of Fig. 1.Table 2: Example of Functional Dependences. CatCell StateCellCatCell NameDeplComp CatCell LifeCell CatCell CatFact CatMol SizeMol CatMol CatMolAdh CatMol CatMolDiss CatMol CatFactCatAdhMol StateAdhMol CatDissMol StateDissMolCatMolDiss TypeActi DissMol CatFact FactTypeCatDissMol + CatCell StateJoncFig. 1. TAS of Table 2.An analysis of the different relations of relation-entities model allowed us to identify two categories of relations:• General relations,• Specific relations to cellular biology.In what follows, we give two examples of relations: a general relation (isa) and specific relation (modified).The relation “isa”The isa relation conduct to the factorization of certain concepts in one generic concept and/or to specialization of generic concept (Fig.2). In the example of Fig 3, the Receptor is generic concept, but the Intracellular Receptor and Membranaire Receptor are a specific concepts. The is a relation represented by the arrows defines the hierarchical relation. Thus, Membranaire Receptor is a Receptor and Intracellular Receptor isa ReceptorSpecific relations to cellular biologyA specific relation expresses the biologic dependence bond between concepts. Relations of dependences of the example given in Fig 4 are:• Transforms "the Cell Transforms the Ligand", • Secretes "the Cell secretes a Factor", • Is in "the Cell is in the Position",• Gives out "the Cell Gives out Components of displacement",• Modifies properties "the Ligand Modifies properties of the Receptor",Fig. 2. The Conceptual Model of the TAS..Fig. 3: Example Extracted from OntoCell representing a Hierarchical relation.Fig. 4. Example extracted from OntoCell representingthe relations specific to cellular biology.Formalization: DAML (DRAPAAgent Markup Language) (/) is a language of an ontology description based on XML. It allows the division of the semantic. DAML can be associated to OIL (Ontology Inference Layer) (/oil/) which is another language of description and inference on the ontologies and which takes support on the logics of description. DAML+OIL is language chosen for the formalization of OntoCell. This formalization is realized by using the ontology’s editor ‘Protege-2000’ [17]. This editor allow to construct an ontology for a given domain, to define the input forms of data, and to reach data with the help of these forms under the form of instances of this ontology.Maintenance: The step of conception validation allowed to construct a core of OntoCell (Fig 5). This core can be easily putted at point, when the changes were suggested by the evaluation (with experts of the domain) or by the introduction of new objectives. This step allows to enrich and to correct the OntoCell ontology.Since the cell lives in environment, we enriched the first core by adding the presence of molecule (Fig 6).Fig. 5. OntoCell describing the cell. Here we have few examples of OntoCell enrichment.Fig. 6. OntoCell enriched in its environment.Fig 7: OntoCell enriched according to a more precise objectives.An important problem for the biologists, in different domain of study and specially that of tumoral invasion and that of cellular migration. The OntoCell has been enriched in order to incorporate this particular aspect (Fig 7).The enrichment of OntoCell in this example is illustrated by a description more detailed of Abnormal Cell: Abnormal cell is either a Quiescent Cell or Tumoral Cell and Tumoral Cell is Malignant Tumoral Cell or Benign Tumoral Cell.Actually, OntoCell contains fifty concepts of cellular biology domain. It can be enriched according to future objectives such that the communication between different types of cellular populations, the union of two different cellular populations,…etc.CONCLUSIONIn this article, we presented the OntoCell ontology that we constructed. It allows to regroup the most of the concepts used in the domain of cellular biology. The objectives is to offer an ontology as complete as possible, coherent and easy to manipulate can beexploited in the development of system of previsionsimulation of the cellular behavior.We described the methodology of OntoCell development which is passed by the requirements analysis, the conceptualization, the formalization, and the maintenance. The explicit and formal representation of OntoCell given by the ontological language DAML+oil is important in order to establish and to exploit the system of previewed simulation. This formal representation is edited by the Protege-2000 tool . Actually, OntoCell contains fifty concepts. We judge that those accepted are the most important for the cell and its behavior in its milieu of life.It has been validated by experts of the biological domain (by the UMRS INSERM 514), nevertheless it can be enriched by other concepts specific to biologic and specific objectives waited in function of the needs of our project.REFERENCES1. R. Altman, M. 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