The identity of the O-specific polysaccharide structure of Citrobacter strains from serogroups O2, O20 and O25 and immunochemical characterisation of C. youngae PCM 1507 (O2a,1b) and related strains

Serological studies using SDS-PAGE and immunoblotting revealed that from five strains that are ascribed to Citrobacter serogroup O2, four strains, PCM 1494, PCM 1495, PCM 1496 and PCM 1507, are reactive with specific anti-Citrobacter O2 serum. In contrast, strain PCM 1573 did not react with anti-Citrobacter O2 serum and, hence, does not belong to serogroup O2. The LPS of Citrobacter youngae O2a,1b (strain PCM 1507) was degraded under mild acidic conditions and the O-specific polysaccharide (OPS) released was isolated by gel chromatography. Sugar and methylation analyses along with (1)H- and (13)C-NMR spectroscopy, including two-dimensional (1)H,(1)H COSY, TOCSY, NOESY and (1)H,(13)C HSQC experiments, showed that the repeating unit of the OPS has the following structure: [structure: see text]. NMR spectroscopic studies demonstrated that Citrobacter werkmanii O20 and C. youngae O25 have the same OPS structure as C. youngae O2. Sugar and methylation analyses of the core oligosaccharide fractions demonstrated structural differences in the lipopolysaccharide core regions of these strains, which may substantiate their classification in different serogroups.     

β-D-Glycan synthases and the CesA gene family: lessons to be learned from the mixed-linkage (1→3),(1→4)β-D-glucan synthase

Cellulose synthase genes (CesAs) encode a broad range of processive glycosyltransferases that synthesize (14)-D-glycosyl units. The proteins predicted to be encoded by these genes contain up to eight membrane-spanning domains and four `U-motifs' with conserved aspartate residues and a QxxRW motif that are essential for substrate binding and catalysis. In higher plants, the domain structure includes two plant-specific regions, one that is relatively conserved and a second, so-called `hypervariable region' (HVR). Analysis of the phylogenetic relationships among members of the CesA multi-gene families from two grass species,Oryza sativa and Zea mays, with Arabidopsis thaliana and other dicotyledonous species reveals that the CesA genes cluster into several distinct sub-classes. Whereas some sub-classes are populated by CesAs from all species, two sub-classes are populated solely by CesAs from grass species. The sub-class identity is primarily defined by the HVR, and the sequence in this region does not vary substantially among members of the same sub-class. Hence, we suggest that the region is more aptly termed a `class-specific region' (CSR). Several motifs containing cysteine, basic, acidic and aromatic residues indicate that the CSR may function in substrate binding specificity and catalysis. Similar motifs are conserved in bacterial cellulose synthases, the Dictyostelium discoideum cellulose synthase, and other processive glycosyltransferases involved in the synthesis of non-cellulosic polymers with (14)-linked backbones, including chitin, heparan, and hyaluronan. These analyses re-open the question whether all the CesA genes encode cellulose synthases or whether some of the sub-class members may encode other non-cellulosic (14)-glycan synthases in plants. For example, the mixed-linkage (13)(14)-D-glucan synthase is found specifically in grasses and possesses many features more similar to those of cellulose synthase than to those of other -linked cross-linking glycans. In this respect, the enzymatic properties of the mixed-linkage -glucan synthases not only provide special insight into the mechanisms of (14)-glycan synthesis but may also uncover the genes that encode the synthases themselves.     

The siaA gene involved in capsule polysaccharide biosynthesis of Neisseria meningitidis B codes for N-acylglucosamine-6-phosphate 2-epimerase activity

The capsule polysaccharide of Neisseria meningitidis serogroup B is composed of a homopolymer of alpha-2-->8 linked N-acetyl-neuraminic acid (sialic acid). The enzymes required for sialic acid biosynthesis and polymerization are encoded in region A of the capsule gene complex. We here describe the enzymatic activity of the siaA gene product as determined by biochemical analysis. siaA was overexpressed in Escherichia coli and the SiaA protein was purified to homogeneity. Enzymatic assays revealed that SiaA did not accept N-acetyl-glucosamine as substrate, but only N-acetyl-glucosamine-6-phosphate (EC 5.1.3.9). SiaA catalyzes the isomerization of N-acetyl-glucosamine-6-phosphate to form N-acetyl-mannosamine-6-phosphate. This reaction represents the first step in capsule biosynthesis of N. meningitidis B.     

Characterisation of the polysaccharide produced by Acetobacter xylinum strain CR1/4 by light scattering and atomic force microscopy

The molecular weight of the extracellular polysaccharide (CR1/4) produced by Acetobacter xylinum strain CR1/4 has been shown to be dependent upon growth conditions. Under normal growth conditions a high molecular weight polysaccharide (>1×10⁶ Da) is produced. Maintaining the pH at 5 results in an order of magnitude increase in the total yield of polysaccharide, but also an order of magnitude decrease in molecular weight. Analysis of the CR1/4 polysaccharides by the techniques of atomic force microscopy and static light scattering suggests that they are double helices. In solution the molecules behave as stiff coils with a Kuhn statistical segment length of 325 nm.     

Influences of milk components on biofilm formation of Cronobacter spp. (Enterobacter sakazakii)

Aim:  To determine the critical component(s) of skim milk for biofilm formation of Cronobacter species.
Methods and Results:  Biofilm forming ability of 72 Cronobacter strains in skim milk preparation was assayed by crystal violet staining. The results revealed that whey protein and casein are more important determinants of skim milk for biofilm formation than lactose, although there was a wide variation in biofilm forming ability. Biofilm structure and capsular material of six strains exhibiting different biofilm forming ability was investigated via electron microscopes. Scanning electron microscopy showed visually that while the strong biofilm formers (E27B, FSM 30 and 2·82) resulted in almost complete coagulation of skim milk, the weak biofilm formers (55, FSM 290 and 2·84) caused less coagulation. No capsule was clearly delineated in transmission electron micrographs of either strong or weak biofilm formers.
Conclusion:  These results indicate that, for biofilm formation of Cronobacter species in skim milk, nitrogen source is probably a more important determinant than carbohydrate, and that strong biofilm formers are responsible for substantial coagulation of skim milk.
Significance and Impact of the Study:  This study provides information for better understanding of the underlying mechanisms by which Cronobacter species form biofilm in infant formula milk.     

芦荟多糖含量测定及芦荟膏体外透皮吸收的实验研究

目的:研究芦荟膏中各功能成分体外透皮吸收的能力。方法:以Wistar大鼠的背部皮肤为透皮实验原料,每隔一定时间通过分光光度法和高效液相色谱法测定透皮后接收池内芦荟多糖及蒽醌类含量。结果:随着芦荟膏剂量的增加,渗透量逐渐增加,芦荟膏中芦荟多糖、芦荟大黄素、芦荟苷的渗透量随时间延长逐渐增加,但是渗透速率逐渐降低。结论:芦荟膏有较强的体外透皮吸收能力,芦荟膏经皮给药能充分发挥其作用。     

Structural characterization and bioactivity evaluation of an acidic proteoglycan extract from Ganoderma lucidum fruiting bodies for PTP1B inhibition and anti‐diabetes

A water‐soluble PTP1B inhibitor, named FYGL‐a, was fractionated for structure investigation and bioactivity evaluation. FYGL‐a is an ingredient of a reported antihyperglycemia extract from Ganoderma Lucidum fruiting bodies. Composition analysis indicated that FYGL‐a was a 100.2 kDa acidic proteoglycan, consisting of 85 ± 2% heteropolysaccharide chain with rhamnose, galactose, glucose, and glucuronic acid residues in a mole ratio of 1.0:3.7:3.9:2.0, and the 15 ± 2% protein moiety of FYGL‐a was covalently bonded to the polysaccharide chain in O‐linkage type via threonine residues. The complete sequence of FYGL‐a was characterized systematically by periodate oxidation, Smith degradation, methylation analysis, ¹H & ¹³C 1D NMR, and 2D NMR (HSQC, HMBC, NOESY, COSY, & TOCSY). The chemical structure of FYGL‐a was determined as following, which may play special role in the competitive inhibition of PTP1B and antihyperglycemia potency. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 613–623, 2014.

     

Two‐dimensional chromatography method applied to the enantiomeric determination of lansoprazole in human plasma by direct sample injection

A two‐dimensional HPLC method based on the direct injection of biological samples has been developed and validated for the determination of lansoprazole enantiomers in human plasma. The lansoprazole enantiomers were extracted from the biological matrix using an octyl restricted access media bovine serum albumin column (C₈ RAM BSA) and the enantioseparation was performed on an amylose tris(3,5‐dimethoxyphenylcarbamate) chiral column using acetonitrile:water (35:65 v/v) and UV detection at 285 nm. Analysis time was 25 min with no time spent on sample preparation. The method was applied to the analysis of the plasma samples obtained from nine Brazilian volunteers who received a 30 mg oral dose of racemic lansoprazole and was able to quantify the enantiomers of lansoprazole in the clinical samples analyzed. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Experimental and computational studies of enantioseparation of structurally similar chiral compounds on amylose tris(3,5‐dimethylphenylcarbamate)

The enantioseparation of 14 structurally similar chiral solutes, with one or two chiral centers, are studied for a commercially important polysaccharide‐based chiral stationary phase, amylose tris(3,5‐dimethylphenylcarbamate) (ADMPC). Among these solutes, only two solutes show significant enantioresolutions of 2 to 2.5 in n‐hexane/2‐propanol (90/10, v/v) at 298 K. The retention factors of the chiral solutes vary significantly from 0.7 to 7.0, and they are compared with those of simpler nonchiral solutes having similar but fewer functional groups. The sorbent–solute H‐bonding interactions between the solute functional groups and the polymer C?O and NH functional groups are probed with attenuated total reflection infrared spectroscopy (ATR‐IR). The H‐bonding interactions of the polymer C?O and NH groups with the solutes result in changes in the IR amide band wavenumbers of ADMPC upon solute adsorption. The nanostructure of an ADMPC cavity and the potential interactions with the chiral solutes are proposed based on the sorbent–solute–solvent HPLC data, the sorbent–solute IR data, and the sorbent–solute molecular dynamics (MD) simulations. The results are consistent with the three point attachment hypothesis and indicate that a significant enantioresolution in ADMPC requires at least three different interaction sites for simultaneous H‐bonds and phenyl–phenyl interactions for phenylpropylamine (PPA) and various structurally similar chiral solutes. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

淡紫拟青霉胞外多糖的分离、纯化及结构分析

淡紫拟青霉NH-PL-03菌株的胞外多糖粗提物对枯萎病病原菌-尖孢镰刀菌具有较好的抑制效果,文中对淡紫拟青霉胞外多糖进行了分离纯化和结构分析,以期为其构效关系研究奠定基础。采用乙醇沉淀法从淡紫拟青霉发酵液中提取粗多糖,经Sevage法脱蛋白后,过Superdex-G75凝胶层析柱分离得到胞外多糖EP-1。紫外分光法和Sephacryl S-200 HR凝胶层析柱检测EP-1为均一多糖,Sephacryl S-200柱层析测得EP-1的分子量为35.2 kDa,完全酸水解后纸层析检测EP-1的单糖组成中仅有葡萄糖,红外光谱、高碘酸氧化和Smith降解结果表明EP-1的化学结构是以β-(1,3)糖苷键连接而成的无分枝的葡聚糖。刚果红络合试验表明EP-1在稀的碱溶液中以3股螺旋构象存在。