Structural and immunological studies of an exopolysaccharide from Acinetobacter junii BB1A

Structural and immunological studies of an exopolysaccharide from Acinetobacter junii BB1A
Structural and immunological studies of an exopolysaccharide from Acinetobacter junii BB1A

Carbohydrate Polymers 101 (2014) 188–195

Contents lists available at ScienceDirect

Carbohydrate

Polymers

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c a r b p o

l

Structural and immunological studies of an exopolysaccharide from Acinetobacter junii BB1A

Ipsita Kumar Sen a ,Amit Kumar Mandal b ,Ranadhir Chakraborty b ,Birendra Behera c ,Krishna Kant Yadav b ,Tapas Kumar Maiti c ,Syed Sirajul Islam a ,?

a

Department of Chemistry and Chemical Technology,Vidyasagar University,Midnapore 721102,West Bengal,India b

Omics Laboratory,Department of Biotechnology,University of North Bengal,Siliguri 734013,West Bengal,India c

Department of Biotechnology,Indian Institute of Technology (IIT)Kharagpur,Kharagpur 721302,West Bengal,India

a r t i c l e

i n f o

Article history:

Received 19July 2013

Received in revised form 6September 2013Accepted 8September 2013Available online xxx

Keywords:

Acinetobacter junii BB1A Heteropolysaccharide NMR

Structural characterization Immunoactivation

a b s t r a c t

A water-soluble heteropolysaccharide (PS)was isolated from extracellular polymeric substances (EPS)produced by a novel metal tolerant bacterium,Acinetobacter junii BB1A.Sugar analysis showed that the PS was composed of mannose,galactose and arabinose in a molar ratio of nearly 3:1:1.Struc-tural characterization of the PS was carried out using methylation analysis,periodate oxidation,Smith degradation and 1D/2D NMR experiments.Methylation analysis revealed that the PS was consisted of 2,4-linked-mannopyranosyl,3,4-linked-mannopyranosyl,2-linked-galactopyranosyl,ter-minal mannopyranosyl and arabinopyranosyl residues in a relative proportion of nearly 1:1:1:1:1.Smith degradation of the PS showed the presence of hydrated glyceraldehyde containing disaccharide unit con-sisting of ?-d -Man p -(1→and →4)-?-d -Man p -(1→residues where the later was directly attached to a hydrated glyceraldehyde moiety.This polysaccharide showed signi?cant in vitro splenocyte,thymocyte,and macrophage activations with optimum dose of 100?g/mL for macrophage and 25?g/mL both for the splenocyte and thymocyte.

? 2013 Elsevier Ltd. All rights reserved.

1.Introduction

Extracellular polymeric substances (EPS)are metabolic prod-ucts which accumulate on the bacterial cell surface (Morgan,Forster,&Evison,1990)and serve as a protective layer for the cells against harsh environmental condition.EPS serve as carbon as well as energy source during the starvation of bacteria (Liu &Fang,2002)and are found to compose of organic substances like polysaccharide,protein,DNA,humic acid (Wang,Liu,Yao,&Cai,2006)out of which polysaccharide was identi?ed as the predom-inant constituent (Liu &Fang,2002).Polysaccharides in EPS show variations in sugar compositions and linkages,chain length,repeat-ing units and substitutions in the side chains (Sutherland,1972).Bacterial exopolysaccharides are signi?cant because of their appli-cation as bio?occulant,bioadsorbents,heavy metal removal and drug delivery agents (Wang,Ahmed,Feng,Li,&Song,2008)and also for their biological activities like antitumor,immunostimula-tory,and anti-in?ammatory activities (Arena et al.,2006;Weiner,Langille,&Quintero,1995).Polysaccharides activate macrophages,

?Corresponding author.Tel.:+9103222276558x437;fax:+9103222275329;mobile:+919932629971.

E-mail address:sirajul 1999@https://www.360docs.net/doc/e66852091.html, (S.S.Islam).

T-helper,NK,and other effector cells and thereby activate the var-ious cytokines (IL-2,IL-6,IL-10,and IL-12),interferon (IFN-?),and chemokines which ultimately stimulate host’s immune system.It has been observed that the molecular mass,degree of branch-ing,conformation and chemical modi?cation of polysaccharides signi?cantly affect their anti-tumor and immunomodulatory activ-ities (Bohn &BeMiller,1995;Okazaki,Adachi,Ohno,&Yadomae,1995).Three species of the genus Acinetobacter were earlier reported to produce emulsi?ers made up of polysaccharides.RAG-1emulsan produced by A.venetianus was reported to compose of d -galactosamine,l -galactosaminuronic acid,and a diamino,2-deoxy N -acetylglucosamine (Dams-Kozlowska,Mercaldi,Panilaitis,&Kaplan,2008).The BD4emulsan produced by Acinetobacter cal-coaceticus consists of a polysaccharide containing l -rhamnose,d -glucose,d -glucuronic acid,and d -mannose in molar ratio of 4:1:1:1(Kaplan,Rosenberg,Jann,&Jann,1985).The bioemulsan produced by A.radioresistens KA53is a complex anionic polysac-charide (Navon-Venezia et al.,1995).Acinetobacter junii BB1A,a novel metal-tolerant bacterium isolated from Torsa River of North-ern West Bengal,India,produces extracelluar polymeric substances (EPS)(Yadav et al.,2012).Chemical analysis revealed that the EPS was composed of both polysaccharide and protein.The average molecular weight of the polysaccharide (PS)fraction of EPS was ~2×105Da and GC analysis of PS revealed the presence of three

0144-8617/$–see front matter ? 2013 Elsevier Ltd. All rights reserved.https://www.360docs.net/doc/e66852091.html,/10.1016/j.carbpol.2013.09.018

I.K.Sen et al./Carbohydrate Polymers101 (2014) 188–195189

constituent sugars,mannose,galactose,and arabinose in a molar ratio of3:1:1as reported earlier(Yadav et al.,2012).Although there are some reports on the sugar composition of the exopolysaccha-rides produced by different Acinetobacter species,report regarding their detailed structural characterization is still absent in the liter-ature.In the present study,detailed structural characterization of the exopolysaccharide isolated from A.junii BB1A was carried out along with some in vitro immunoactivation studies and reported herein for the?rst time.

2.Materials and methods

2.1.Isolation and puri?cation

A.junii BB1A,a novel metal tolerant bio?lm forming bacteria was reported earlier to produce extracellular polymeric substances (EPS)in liquid brain heart infusion(BHI)medium(Yadav et al., 2012).EPS was isolated from culture broth through centrifuga-tion,precipitation in double volume of cold95%ethanol followed by dialysis in a dialysis tubing cellulose membrane(D9652, Sigma–Aldrich,retaining MW>12,400Da)against distilled water for24h.The dialyzed material was again centrifuged at9587.5×g for30min at4?C and the supernatant was freeze-dried to obtain crude EPS(500mg).Protein fraction of EPS was removed by adding trichloroacetic acid(30%)followed by centrifugation and residue was discarded.Two volumes of95%ethanol was added to the supernatant and centrifuged at9587.5×g for20min to obtain 400mg of protein-free crude polysaccharide(Zhang,Liu,Wang,& Jiang,2002).This crude polysaccharide(30mg)was passed through Sepharose6B gel permeation column(90×2.1cm)using water as the eluant with a?ow rate of0.4mL min?1as described in the earlier report(Yadav et al.,2012).A total of95test tubes (2mL each)were collected using Redifrac fraction collector and monitored spectrophotometrically(Shimadzu UV–vis spectropho-tometer1601)at490nm with phenol–sulfuric acid reagent(York, Darvill,McNeil,Stevenson,&Albersheim,1986).A single fraction of puri?ed polysaccharide(22mg)was obtained.

2.2.Determination of optical rotation

Optical rotation of PS was measured on a Jasco Polarimeter model P-1020at31.1?C.

2.3.Absolute con?guration of the monosaccharide

The method used was based on Gerwig,Kamerling,and Vliegenthart(1978).After the hydrolysis of PS(1mg)by TFA, the acid was removed by co-distillation with water.A solution of 250?L of0.625M HCl in R-(?)-2-butanol was added into it,and the mixture was heated at80?C for16h.After butanolysis,the solu-tion was neutralized with Ag2CO3and the supernatant solution was concentrated under reduced pressure at45?C.The residue was dried for12h in vacua over P2O5,and then treated with hexamethyldisilazane–chlorotrimethylsilane–pyridine(0.1ml, 1:1:5)for30min at room temperature.The products were ana-lyzed by GC using a capillary column(SPB-1,30m×0.26mm) with a temperature program(3?C min?1)from150to210?C. The(?)-2-butyl-2,3,4,6-tetra-O-TMS-glycosides obtained were identi?ed by comparison with those prepared from the d-and l-enantiomers of the monosaccharide.

2.4.Methylation analysis

PS(4.0mg)was methylated using the procedure described by Ciucanu and Kerek(1984)and the product was isolated by mak-ing a partition between CHCl3and water(5:2,v/v).The methylated biopolymer was hydrolyzed with90%HCOOH(1mL)at100?C for 1h and excess HCOOH was evaporated by co-distillation with dis-tilled water.The hydrolyzed product was reduced with NaBH4 (9mg)followed by acidi?cation with dilute CH3COOH,and then co-distillation with CH3OH to remove excess boric acid.The reduced sugars(alditol)were acetylated with1:1pyridine–Ac2O in a boiling water bath for1h to give alditol acetates.The alditol acetates of the methylated sugars were analyzed by GC–MS.GC–MS analysis was performed on Shimadzu GC–MS Model QP-2010Plus automatic system,using ZB-5MS capillary column(30m×0.25mm).The pro-gram was isothermal at150?C;hold time5min,with a temperature gradient of2?C min?1up to a?nal temperature of200?C.

2.5.Periodate oxidation and Smith degradation study

PS(25mg)was added to7.5mL0.1M sodium metaperiodate solution and the mixture was kept for48h in the dark at4?C.The excess periodate was destroyed by adding ethylene glycol(5.0mL) and the solution was dialyzed against distilled water for2h.The volume of the dialyzed material was concentrated to2–3mL.This material was reduced with NaBH4,12h,neutralized with50% AcOH,and dialyzed with distilled water and?nally freeze dried (14.0mg).The periodate-reduced material was divided into three portions.One portion(1.5mg)was hydrolyzed with2M CF3COOH (1mL)at100?C for18h,used for alditol acetate preparation and analyzed by GC.The second portion(2.0mg)was methylated by the method of Ciucanu and Kerek(1984),followed by preparation of alditol acetates which were analyzed by GC–MS.Smith degrada-tion experiment was performed with the third portion(10.0mg). The mild hydrolysis of the periodate oxidized–reduced material was performed by the addition of0.5M CF3COOH for15h at25?C to destroy the residues of oxidized sugars attached to the polysaccha-ride chain.The excess acid was removed by repeated freeze drying. The material was further puri?ed by passing through a Sephadex G-25column,kept over P2O5in vacuum for several days and?nally used for13C NMR studies.

2.6.NMR studies

PS was dried over P2O5in vacuum for several days and then deuterium exchanged?ve times,followed by lyophilization with D2O(99.96%.atom2H,Aldrich)(Due?nas-Chaso et al.,1997).Then 1H NMR and13C NMR experiments were performed with a Bruker Avance DPX-500instrument at30?C.The1H NMR spectrum was recorded by suppressing the HOD signal(?xed at?4.70)using the WEFT pulse sequence(H?rd,Zadelhoff,Moonen,Kamerling, &Vliegenthart,1992)using acetone as internal standard?xing methyl proton signal at?2.225.13C NMR experiment of PS was carried out taking acetone as the internal standard,?xing the methyl carbon signal at?31.45.The2D-(DQF-COSY)NMR experi-ment was carried out using standard pulse sequence at30?C.The TOCSY experiment was recorded at a mixing time of60–300ms. The NOESY and ROESY mixing delay were300ms.Delay time in the HMBC experiment was80ms.

2.7.Study of immunoenhancing activities of PS

2.7.1.Preparation of LPS free PS

Prior to the immunoactivation studies,LPS free PS was prepared in order to rule out the contribution of LPS which may contam-inate in the course of isolation and puri?cation process.The PS was passed through polymyxin-B agarose matrix(P1411,Sigma and Aldrich,USA)packed in2mL column(1cm×2cm)with a?ow rate of0.5mL min?1.It was equilibrated with10mM phosphate buffer,pH7.4.The bacterial lipopolysaccharides(LPS)were bound

190I.K.Sen et al./Carbohydrate Polymers101 (2014) 188–195

to the matrix and the unbound LPS free PS(LFPS)were eluted and collected for immunoactivation studies.

2.7.2.Limulus amebocyte lysate(LAL)test

Limulus amebocyte lysate(LAL)test was carried out in vitro for detection of bacterial endotoxin.The test was performed using gel clot technique(Liu et al.,2009).Limulus amebocyte lysate (LAL)(G2125,sensitivity:0.125EU/mL)was purchased from Quan-tum Biotech,Mumbai,India.The control standard endotoxin(CSE) (code E0125)and water(code W1004)for the bacterial endotoxin test(BET)were provided by Quantum Biotech,Mumbai,India. Four tubes were taken,each containing0.1mL of LAL reagent. In two tubes,0.1mL LFPS aqueous solution were added,mean-while0.1mL BET water and0.1mL CSE were added to the rest two tubes as negative control and positive control,respectively. All tubes were incubated for1h at37?C.After the test tube was inverted180?slowly,it is positive(+)if the gel in tube is not deformed and does not slip from the wall and a negative (?)test is characterized in absence of a gel or by the forma-tion of a viscous mass which does not hold when the tube is inverted.Test is invalid when positive control is(?)or negative control is(+).

2.7.

3.Test for macrophage activity by nitric oxide assay

RAW264.7growing in Dulbecco’s modi?ed Eagle’s medium (DMEM)at37?C was seeded in96well?at bottom tissue cul-ture plate at5×105cells/mL concentration(180?L)(Maiti et al., 2011).Cells were left overnight for attachment and treated with different concentrations(12.5,25,50,100or200?g/mL)of LFPS. After48h of treatment,culture supernatant of each well was col-lected and NO production was estimated using Griess Reagent (1:1of0.1%in1-napthylethylenediamine in5%phosphoric acid and1%sulfanilamide in5%phosphoric acid)(Green et al.,1982). Lipopolysaccharide(LPS)(L6511of Salmonella enterica serotype typhimurium,Sigma,St.Louis,USA,4?g/mL)was used as positive control and soluble starch(Merck,India,100?g/mL)as negative control(Maity et al.,2013).

2.7.4.Splenocyte and thymocyte proliferation assay

A single cell suspension of spleen and thymus were prepared from the normal mice under aseptic conditions by homogeniza-tion in Hank’s balanced salt solution(HBSS).The suspension was centrifuged to obtain cell pellet.The contaminating red blood cells (RBC)were removed by hemolytic Gey’s solution.After washing two times in HBSS the cells were resuspended in complete RPMI (Roswell Park Memorial Institute)medium.Cell concentration was adjusted to1×106cells/mL and the viability of splenocytes and thymocytes(as tested by trypan blue dye exclusion)was always over90%.The cells(180?L)were plated in96-well?at-bottom tissue culture plates and incubated with20?L of various concentrations of LFPS(12.5,25,50,100,and200?g/mL).PBS (Phosphate Buffer Saline,10mM,pH7.4)was taken as carrier control and soluble starch(Merck,India,100?g/mL)as negative control(Maity et al.,2013).LPS(L6511of S.enterica serotype typhimurium,Sigma,St.Louis,USA,4?g/mL)served as positive control for splenocytes and Concanavalin A(Himedia,10?g/mL) for thymocytes.All cultures were set up at37?C for72h in a humidi?ed atmosphere of5%CO2.Proliferation of splenocytes (%Splenocyte Proliferation Index or%SPI)and thymocytes(% Thymocyte Proliferation Index or%TPI)were checked by MTT assay method(Sarangi,Ghosh,Bhutia,Mallick,&Maiti,2006). The data are reported as the mean±standard deviation of seven different observations and compared against PBS control(Green et al.,1982;Maiti et al.,2008

).Fig.1.1H NMR spectrum(500MHz,D2O,30?C)of heteropolysaccharide(PS)iso-lated from EPS produced by A.junii BB1A.Acetone was taken as the internal standard,?xing the methyl proton signal at?2.225.

3.Results and discussion

3.1.Puri?cation and chemical analysis of PS

A single fraction was obtained after fractionating water solu-ble crude polysaccharide(30mg)through Sepharose6

B column. The fraction(test tubes8–18)was collected and freeze dried, yielding22mg of puri?ed polysaccharide(PS).The isolation and puri?cation steps are summarized in Scheme1.The PS showed a speci?c rotation of[?]D31+15.8(c0.1,water)and was com-posed of mannose,galactose and arabinose in a molar ratio of nearly3:1:1as reported earlier(Yadav et al.,2012).Determina-tion of absolute con?guration of the monosaccharides showed that mannose and galactose were present in D and arabinose in L con?guration.The GC–MS analysis of partially methylated alditol acetates revealed the presence of2,4-linked-mannopyranosyl, 3,4-linked-mannopyranosyl,2-linked-galactopyranosyl,terminal mannopyranosyl and arabinopyranosyl residues in a relative pro-portion of approximately1:1:1:1:1(Table1a).G

C analysis of alditol acetates of the periodate-oxidized(Goldstein,Hay,Lewis, &Smith,1965;Hay,Lewis,&Smith,1965),NaBH4-reduced PS was found to contain d-mannose only.GC-MS analysis of periodate-oxidized,reduced,and methylated PS showed the presence of 1,2,4,5-tetra-O-acetyl-3,6-di-O-methyl-mannitol,1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl-mannitol in a molar ratio of nearly1:1 (Table1b),indicating the2,4-and3,4-linked-mannopyranosyl residues remained unaffected during periodate-oxidation.This observation further con?rmed the mode of linkages present in the PS.

3.2.Structural analysis of PS

The1H NMR spectrum(Fig.1)of PS at30?C showed the pres-ence of?ve signals in the anomeric region at?5.27,5.13,5.09, 5.02,and5.01in a ratio of nearly1:1:1:1:1.In13C spectrum(Fig.2) four signals were found at?102.2,102.1,100.1,and98.3in a ratio of nearly1:2:1:1.The?ve anomeric proton signals corresponded to?ve sugar residues designated as A,B,C,D and E according to their decreasing proton chemical shift values.In the HSQC spectrum (Fig.3),the anomeric proton signal of A at?5.27was correlated to the carbon signal at?100.1.The anomeric proton signal at?5.13was correlated to the carbon signals at?102.2and assigned to anomeric carbon of residue B.The anomeric proton signal of C at?5.09was correlated to the carbon signal at?98.3.Again,the anomeric proton signal at?5.02and5.01were correlated to the car-bon signal at?102.1corresponding to the anomeric carbon of both residues D and E.All the1H and13C signals(Table1c)were assigned from DQF-COSY,TOCSY,and HSQC experiments.The proton cou-pling constants were measured from DQF-COSY experiment.

The residue A was assigned for the galacto con?guration for its large J H-2,H-3coupling constant(~8Hz)and relatively small J H-3,H-4coupling constant(~3Hz).The?-con?guration of residue

I.K.Sen et al./Carbohydrate Polymers 101 (2014) 188–195

191

for 20 min at 4 °C

(Rejected) Precipitated with double volume of cold EtOH, kept for 12 h at 4oC Residue Dissolved in water, dialyzed through dialysis

(Rejected)

tubing cellulose membrane, dialysate was freeze dried

EPS (500 mg)

9587.5 × g , 20 Crude Polysaccharide (400 mg)

Purified through Sepharose-6B column (30 mg)

Purified Polysaccharide, PS (22 mg)

Scheme 1.Flow diagram of isolation and puri?cation of the heteropolysaccharide (PS)from EPS produced by A.junii BB1A.

Table 1a

GC–MS analysis of methylated heteropolysaccharide (PS)isolated from EPS produced by A.junii BB1A.

Residue

Methylated sugars

Molar ratio

Linkage type

Major mass fragments (m /z )

A 3,4,6-Me 3-Gal 1→2)-d -Gal p -(1→43,57,74,87,99,129,145,173,200

B 2,3,4,6-Me 4-Man 1d -Man p -(1→43,59,73,87,101,117,129,145,161,205

C 2,3,4-Me 3-Ara 1l -Ara p -(1→

43,58,71,87,101,117,129,131,161

D 3,6-Me 2-Man 1→2,4)-d -Man p -(1→43,59,74,87,99,129,143,173,189,203,233

E

2,6-Me 2-Man

1

→3,4)-d -Man p -(1→

43,58,74,87,101,117,129,143,159,173,189,233

Table 1b

GC–MS analysis of periodate oxidized methylated heteropolysaccharide (PS)isolated from EPS produced by A.junii BB1A.

Residue

Methylated sugars

Molar ratio

Linkage type

Major mass fragments (m /z )

F 3,6-Me 2-Man 1→2,4)-d -Man p -(1→43,59,74,87,99,129,143,173,189,203,233

G

2,6-Me 2-Man

1

→3,4)-d -Man p -(1→

43,58,74,87,101,117,129,143,159,173,189,233

A was assigned from J H-1,H-2coupling constant (~3Hz)and J C-1,H-1of (~170Hz)and from its anomeric proton (?5.27)and carbon (?100.1)signals.The down?eld shift of C-2(?77.8)with respect to standard values of methyl glycosides (Agrawal,1992;Rinaudo &Vincendon,1982)indicated that residue A was a (1→2)-linked-d -galactopyranosyl residue.

Residue B was assigned to Man p which showed large coupling constants of J H-3,H-4(~7Hz)and J H-4,H-5(~9Hz).The anomeric pro-ton signal at ?5.13and the coupling constant values of J H-1,H-2(~1.6Hz),J H-2,H-3(~3.5Hz)and J C-1,H-1(~170

Hz)clearly indicated that the residue B was ?-linked mannopyranosyl moiety.The car-bon chemical shifts of residue B from C-1to C-6corresponded

Fig.2.13C NMR spectrum (125MHz,D 2O,30?C)of heteropolysaccharide (PS)isolated from EPS produced by A.junii BB1A.Acetone was taken as the internal standard,?xing the methyl carbon signal at ?31.45.

192I.K.Sen et al./Carbohydrate Polymers 101 (2014) 188–

195

Fig.3.Part of HSQC spectrum of heteropolysaccharide (PS)isolated from EPS produced by A.junii BB1A.The annotation A1refers to AH1/AC1cross peak,B1refers to BH1/BH2cross peak and so on.Inset shows the HSQC spectrum of the anomeric region.

nearly to the standard values of methyl glycoside of ?-d -mannose indicating residue B was terminal ?-d -mannopyranosyl moiety.Residue C was assigned to Ara p which showed a large coupling constant J H-2,H-3(~8.2Hz),J H-3,H-4(~5Hz),relatively small coupling constant J H-1,H-2and two H-5signals (?3.80and 3.93).The anomeric proton chemical shift for residue C at ?5.09and carbon chemical shift at ?98.3,J H-1,C-1~170Hz (measured from proton coupled 13C spectra)indicated that l -arabinose is ?con?gured at the anomeric center.The carbon chemical shifts of residue C from C-1to C-5corresponded nearly to the standard values of methyl glycosides of ?-l -arabinose indicating that the residue C was non-reducing end Ara p .

The manno con?guration of residues D and E was supported from large coupling constants of J H-3,H-4(~7Hz)and J H-4,H-5(~9Hz).The anomeric proton signals at ?5.02and 5.01and the coupling constant values of J H-1,H-2(~1.6Hz),J H-2,H-3(~3.5Hz)and J C-1,H-1(~170Hz)clearly indicated that both D and E residues were ?-linked mannopyranosyl moieties.The down?eld shift of C-2(?78.0)and C-4(?76.6)with respect to standard values of methyl glycosides (Agrawal,1992;Rinaudo &Vincendon,1982)indicated that residue D was a 2,4-linked-d -mannopyranosyl residue.The down?eld shift of C-3(?81.8)and C-4(?75.4)of residue E with respect to standard values of methyl glycosides (Agrawal,1992,Rinaudo &Vincendon,1982)indicated that it was a 3,4-linked-d -mannopyranosyl residue.

The sequence of glycosyl residues was established from ROESY as well as NOESY (not shown)experiments.In ROESY experiment (Fig.4a,Table 1in supplementary data),the inter-residual contacts from A H-1/D H-1and E H-4;B H-1/D H-2;C H-1/E H-3;D H-1/A H-1,A H-2and E H-1/D H-4along with other intra-residual contacts were observed.A long range HMBC experiment was carried out to con?rm the connectivites obtained from ROESY experiment.Inter-residual cross-peaks,A H-1/E C-4,A C-1/E H-4;B H-1/D C-2;B C-1/D H-2;C H-1/E C-3,C C-1/E H-3;D H-1/A C-2,D C-1/A H-2;E H-1/D C-4,E C-1/D H-4along with some intra-residual cross peaks were also observed (Fig.4b,Table 2in supplementary data).Thus,the HMBC and ROESY connectivities clearly supported the presence of the pentasaccharide repeating

unit in the heteropolysaccharide (PS)isolated from EPS produced by A.Junii BB1A as:

D A E

→4)-αα-D -Man p -(1→2)-α-D -Gal p -(1→4) -α-D -Man p -(1 →

↑ ↑ 1 12 3

-D -Man p β-L -Ara p

B C

3.3.Smith degradation study

Smith degradation was carried out with the PS where a disaccharide containing hydrated glyceraldehyde moiety is pro-duced (Scheme 1in supplementary data)and the product was analyzed by 13C NMR spectroscopy (Table 2,Fig.1in supple-mentary data)to con?rm further the sequence of the sugar residues present in the repeating unit.The 13C NMR spectrum of Smith-degraded PS showed two anomeric carbon signals at ?103.2and 103.1corresponding to ?-d -Man p -(1→(F )and →4)-?-d -Man p -(1→(G )residues,respectively indicating a disaccharide unit produced during Smith degradation.The carbon signal at ?76.2clearly indicated the presence of 4-linked ?-d -Man p unit (G )which was generated from →2,4)-?-d -Man p -(1→residue (D)and the →3,4)-?-d -Man p -(1→residue (E )was converted to nonreduc-ing end ?-d -Man p unit (F )during degradation.During oxidation two aldehyde groups were generated at C-3and C-4positions of →2)-?-d -Gal p -(1→residue (A )which on reduction followed by hydrolysis produced free glycerol and disaccharide containing glyc-eraldehyde which in water may exist in hydrated form (H).The carbon signals at ?90.5,72.1,and 62.6were corresponded to C-1,C-2,and C-3of the hydrated glyceraldehyde moiety (H).Hence,the structure of hydrated glyceraldehyde containing disaccharide unit obtained from heteropolysaccharide (PS)after Smith degradation was established as:

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193

Table 1c

The 1H a and 13C b NMR chemical shifts of the sugar residues of heteropolysaccharide (PS)isolated from EPS produced by A.junii BB1A in D 2O at 30?C.

Glycosyl residue

H-1/C-1

H-2/C-2

H-3/C-3

H-4/C-4

H-5/C-5

H-6a,H-6b/C-6→2)-?-d -Gal p -(1→ 5.27 4.10 3.91 3.89 3.77 3.78c ,3.86c A

100.177.870.070.071.061.1

?-d -Man p -(1→ 5.13 4.04 3.79 3.63 3.80 3.72c ,3.87c B

102.270.070.266.773.0

61.1

?-l -Ara p -(1→ 5.09 4.15 3.96 3.69 3.80c ,3.93d C

98.369.369.170.963.2→2,4)-?-d -Man p -(1→ 5.02 4.01 3.81 3.83 3.74 3.74c ,3.88c D

102.178.070.176.673.161.1

→3,4)-?-d -Man p -(1→ 5.01 4.04 3.90 3.93 3.74 3.74c ,3.88c E

102.1

70.0

81.8

75.4

73.1

61.1

a Values of the 1H chemical shifts were recorded with respect to the HOD signal ?xed at ?4.7at 30?C.

b Values of the 13C chemical shifts were recorded with reference to acetone,?xing the methyl carbon signal at ?31.45and proton signal at ?2.225using D 2O as the solvent.c

Interchangeable.

3 1 -D -Man p -(14)--D -Man p -(12) -CH(CH 2OH)CH(OH) 2

F G H

Therefore,Smith degradation results further con?rmed the repeating unit present in the heteropolysaccharide (PS)isolated from EPS produced by A.junii BB1A.

3.4.Immunostimulating properties of LFPS

A negative (?)LAL test indicated that LFPS which was obtained after passing the PS through polymixin-

B matrix was free from bacterial endotoxin.Macrophage activation by LFPS was observed in vitro .Upon treatment with different concentrations of LFPS,enhanced production of NO was observed in a dose dependent manner with optimum production of 20.00?M NO per 5×105

Table 2

The 13C NMR a chemical shifts of disaccharide unit obtained after Smith-degradation of heteropolysaccharide (PS)isolated from EPS produced by A.junii BB1A in D 2O at 30?C.

Sugar residue

C-1

C-2

C-3

C-4

C-5

C-6

?-d -Man p -(1→

103.270.870.266.773.261.2F

→4)-?-d -Man p -(1→

103.1

70.8

69.8

76.2

73.2

61.2

G

1 2 3

(HO)2HC-CH-CH 2OH

H

90.5

72.1

62.6

a

Values of the 13C chemical shifts were recorded with reference to acetone,?xing the methyl carbon signal at

?31.45.

Fig.4.(a)Part of ROESY spectrum of heteropolysaccharide (PS)isolated from EPS produced by A.junii BB1A.The ROESY mixing time was 300ms.(b)Part of HMBC spectrum of heteropolysaccharide (PS)isolated from EPS produced by A.junii BB1A.The delay time in the HMBC experiment was 80ms.

194I.K.Sen et al./Carbohydrate Polymers 101 (2014) 188–

195

Fig.5.(a)In vitro activation of macrophage stimulated with different concentrations of LPS free PS (LFPS)in terms of NO production.Effect of different concentrations of LPS free PS (LFPS)on proliferation of (b)splenocyte and (c)thymocyte (*statistically signi?cant differences compared to PBS control).

macrophages at an effective dose of 100?g/mL (Fig.5a).Hence,the effective dose of LFPS for macrophage activation was 100?g/mL.Proliferation of splenocytes and thymocytes is an indicator of immunoactivation.The splenocyte and thymocyte activation tests were carried out in mouse cell culture medium with LFPS by the MTT assay method (Sarangi et al.,2006).LFPS was tested to stim-ulate splenocytes and thymocytes and the results are shown in Fig.5b and c,respectively.Both splenocyte and thymocyte pro-liferation index were found to be maximum at 25?g/mL of LFPS,above or below which it decreases.Hence,it can be concluded that 25?g/mL is the optimum concentration of LFPS both for spleno-cyte and thymocyte proliferation.Recently,an exopolysaccharide produced by Bacillus licheniformis 8-37-0-1was found signi?cantly to stimulate the proliferation of splenocytes in a dose-dependent manner at concentrations from 50?g/mL to 800?g/mL (Liu et al.,2010).

From the above results it is noted that optimum concentration of LFPS for NO production was 100?g/mL whereas,for spleno-cyte and thymocyte proliferation,the optimum concentration of LFPS was 25?g/mL.It has been reported that polysaccharides

interact with cell surface receptor molecules like dectin-1,CR3,and TLRs (Gantner,Simmons,Canavera,Akira,&Underhill,2003)and activate macrophages,T-helper,NK,and other effector cells.The cellular responses like proliferation and NO production depend on intensity of signal produced in upstream at cell membrane receptor level which further depends on cell type,the number of receptor molecules present at the cell surface and nature of the stimulant (here polysaccharide)(Bohn &BeMiller,1995;Okazaki et al.,1995;Ohno,Miura,Nakajima,&Yadomae,2000).These factors may in?u-ence on the results obtained with PS,which induces maximum NO production at four times higher concentration than requires for splenocyte and thymocyte proliferation.However,further studies on mechanism of action of PS are needed to get more insight of its action.

4.Conclusions

A heteropolysaccharide (PS)was isolated from extracellular polymeric substances (EPS)produced by a novel metal tolerant bacterium,A.junii BB1A.On the basis of chemical analysis and NMR

I.K.Sen et al./Carbohydrate Polymers 101 (2014) 188–195

195

studies the structure of the repeating unit of this heteropolysaccha-ride was established as:

→4)-αα-D -Man p -(1→2)-α-D -Gal p -(1→4) -α-D -Man p -(1 →

↑ ↑ 1 1

2 3

-D -Man p

β-L -Ara p The PS showed signi?cant in vitro macrophage,splenocyte and

thymocyte activations.

Acknowledgements

The authors are grateful to Dr.S.Roy,Director,IICB,Kolkata,for providing instrumental facilities.Mr.Barun Majumdar of Bose Institute,Kolkata,is acknowledged for preparing NMR spectra.Two of the authors I.K.S.and A.K.M,were provided with independent fellowship (CSIR-09/599(042)/2011-EMR-I)and (NBU-JRF/3726/R-2007)respectively.This work was supported by a research grant from University Grant Commission,India [F.No.41-558/2012(SR);dated:18July 2012].

Appendix A.Supplementary data

Supplementary data associated with this article can be found,in the online version,at https://www.360docs.net/doc/e66852091.html,/10.1016/j.carbpol.2013.09.018.

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使用有止痒作用的洗剂、膏霜等,如炉甘石洗剂、苯海拉明软膏、皮质醇类软膏等。 2.局部封闭或穴位注药 如皮质醇激素、维生素B12、非那根等。 3.针对病因治疗。 4.预防1. 注意经期卫生,勤清洗。 2.不要冲洗阴道,因为阴道有自清的功能,如果刻意冲洗反而不利 3.忌乱用、烂用药物,忌抓搔及局部摩擦。 4.忌酒及辛辣食物,不吃海鲜等及易引起过敏的药物 6 .久治不愈者应作血糖检查。少吃糖类可避免常常感染霉菌,如少吃淀粉类、糖类以及刺激性的食物(例如酒、辛辣物、油炸类),多吃蔬菜水果类,水份要充足。 5、不穿紧身兜裆裤,内裤更须宽松、透气,并以棉制品为宜。 6.就医检查是否有霉菌或滴虫,如有应及时治疗,而不要自己应用“止痒水”治疗。 8.保持外阴清洁干燥,尤其在经期、孕期、产褥期,每天用女性护理液清洗外阴更换内裤。 9.不穿化纤内裤、紧身裤,着棉织内衣裤。局部坐浴时注意溶液浓度、温度及时间、注意事项。 10.外阴瘙痒者应勤剪指甲、勤洗手,不要搔抓皮肤,以防破溃感染从而继发细菌性感染。 11.上完厕所请记得由前往后擦,因为肛门可能会带来不少细菌,所以如厕后请不要由肛门擦到阴部,才能减少感染的机会。 12.内裤要和其他的衣物分开洗,最好暴晒,可以减少细菌的滋生。如果患有霉菌性阴道炎的话,最好内裤都有热水煮 外阴溃疡外阴溃疡是发生于外阴部的皮肤黏膜发炎、溃烂、缺损。病灶多发生于小阴唇和大阴唇内侧,其次为前庭黏膜及阴道口周围。病程有急性及慢性。 大小阴唇、阴道口周围、阴蒂等处(外阴疾病发展中出现的一个过程,不是一个独立的疾病,有急性和慢性)急性外阴溃疡:非特异性外阴炎病情较轻,多在搔抓之后出现一般比较表浅,但疼痛比较厉害 慢性外阴溃疡:持续时间较长,或者反复发作 癌症引起的溃疡,与结核性溃疡很难鉴别,需做确诊

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员会),负责对脐带血造血干细胞库设置的申请、验收和考评提出论证意见。专家委员会负责制订脐带血 造血干细胞库建设、操作、运行等技术标准。 第八条脐带血造血干细胞库设置的申请者除符合国家规划和布局要求,具备设置一般血站基本条件之外, 还需具备下列条件: (一)具有基本的血液学研究基础和造血干细胞研究能力; (二)具有符合储存不低于1 万份脐带血的高清洁度的空间和冷冻设备的设计规划; (三)具有血细胞生物学、HLA 配型、相关病原体检测、遗传学和冷冻生物学、专供脐带血处理等符合GMP、 GLP 标准的实验室、资料保存室; (四)具有流式细胞仪、程控冷冻仪、PCR 仪和细胞冷冻及相关检测及计算机网络管理等仪器设备; (五)具有独立开展实验血液学、免疫学、造血细胞培养、检测、HLA 配型、病原体检测、冷冻生物学、 管理、质量控制和监测、仪器操作、资料保管和共享等方面的技术、管理和服务人员; (六)具有安全可靠的脐带血来源保证; (七)具备多渠道筹集建设资金运转经费的能力。 第九条设置脐带血造血干细胞库应向所在地省级卫生行政部门提交设置可行性研究报告,内容包括:

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精神分裂症的发病原因是什么

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外阴白色病变的检查诊断方法是什么

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