Structures of the O-polysaccharide chains of the lipopolysaccharides of Xanthomonas campestris pv phaseoli var fuscans GSPB 271 and X campestris pv malvacearum GSPB 1386 and GSPB 2388

O-polysaccharides of phytopathogenic bacteria Xanthomonas campestris were isolated by mild acid degradation of the lipopolysaccharides and studied by sugar and methylation analysis, along with 1H and 13C NMR spectroscopy. The following structures of the repeating units of the polysaccharides of X. campestris pv. phaseoli var. fuscans GSPB 271 (1). and X. campestris pv. malvacearum GSPB 1386 and GSPB 2388 (2). were established:The O-polysaccharides of X. campestris are structurally similar to those of some Pseudomonas syringae strains.     

Structure of the O-chain polysaccharide of the lipopolysaccharide of Xanthomonas campestris pv. manihotis GSPB 2755 and GSPB 2364

The O-chain polysaccharide of the lipopolysaccharide of Xanthomonas campestris pv. manihotis strains GSPB 2755 and GSPB 2364 was studied by sugar and methylation analyses and 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC) experiments. The polysaccharide was found to contain L-rhamnose and L-xylose in the ratio 3:1, and the following structure of the tetrasaccharide repeating unit was established: [formula: see text]     

Structure of the O-polysaccharide of Xanthomonas cassavae GSPB 2437

The following structure of the O-polysaccharide of the phytopathogenic bacterium Xanthomonas cassavae GSPB 2437 was determined by sugar analysis along with 1H and 13C NMR spectroscopy: [structure: see text].     

Structure of the O-polysaccharide of Erwinia carotovora ssp. carotovora GSPB 436

The O-polysaccharide of a phytopathogenic bacterium, Erwinia carotovora ssp. carotovora GSPB 436, was studied by sugar and methylation analysis, along with 1H and 13C NMR spectroscopy. The following structure of the branched tetrasaccharide repeating unit of the O-polysaccharide was established: [carbohydrate structure in text] The O-polysaccharide contains a minor proportion of 4-O-methylrhamnose, which is suggested to terminate the polymer main chain.     

The O-chain polysaccharide of the lipopolysaccharide of Xanthomonas campestris pv. begoniae GSPB 525 is a partially L-xylosylated L-rhamnan.

The O-chain polysaccharide (OPS) of the lipopolysaccharide of Xanthomonas campestris pv. begoniae GSPB 525 was found to contain L-rhamnose and L-xylose in the ratio 1:0.6. The OPS lacked strict regularity because of nonstoichiometric xylosylation of the main rhamnan chain. Based on methylation analysis, Smith degradation, and 1H and 13C NMR spectroscopy, including COSY, TOCSY, NOESY, and H-detected 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments, the following structure of the OPS was established: [formula: see text].     

Synthesis of an xylosylated rhamnose pentasaccharide, the repeating unit of the O-chain polysaccharide of the lipopolysaccharide of Xanthomonas campestris pv. begoniae GSPB 525

A xylosylated rhamnose pentasaccharide, alpha-L-Rhap-(1-->3)-[beta-L-Xylp-(1-->2)-]-alpha-L-Rhap-(1-->3)-[beta-L-Xylp-(1-->4)]-L-Rhap, the repeating unit of the O-chain polysaccharide (OPS) of the lipopolysaccharides of Xanthomonas campestris pv. begoniae GSPB 525 was synthesized by a highly regio- and stereoselective way. Thus coupling of 1,2-O-ethylidene-beta-L-rhamnopyranose (1) with 2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl trichloroacetimidate (2) to give (1-->3)-linked disaccharide (3), subsequent benzoylation, deethylidenation, acetylation, 1-O-deacetylation, and trichloroacetimidation afforded the disaccharide donor 11. Condensation of 11 with 1 yielded 2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->3)-2-O-acetyl-4-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->3)-1,2-O-ethylidene-beta-L-rhamnopyranose (12), and selective deacetylation of 12 yielded the trisaccharide diol acceptor 15. Coupling of 15 with 2,3,4-tri-O-benzoyl-alpha-L-xylopyranosyl trichloroacetimidate (16), followed by deprotection, gave the target pentasaccharide 19.     

Proteomic analysis of kidney and protective effects of grape seed procyanidin B2 in db/db mice indicate MFG-E8 as a key molecule in the development of diabetic nephropathy

Diabetic nephropathy, as a severe microvascular complication of diabetic mellitus, has become the leading cause of end-stage renal diseases. However, no effective therapeutic strategy has been developed to prevent renal damage progression to end stage renal disease. Hence, the present study evaluated the protective effects of grape seed procyanidin B2 (GSPB2) and explored its molecular targets underlying diabetic nephropathy by a comprehensive quantitative proteomic analysis in db/db mice. Here, we found that oral administration of GSPB2 significantly attenuated the renal dysfunction and pathological changes in db/db mice. Proteome analysis by isobaric tags for relative and absolute quantification (iTRAQ) identified 53 down-regulated and 60 up-regulated proteins after treatment with GSPB2 in db/db mice. Western blot analysis confirmed that milk fat globule EGF-8 (MFG-E8) was significantly up-regulated in diabetic kidney. MFG-E8 silencing by transfection of MFG-E8 shRNA improved renal histological lesions by inhibiting phosphorylation of extracellular signal-regulated kinase1/2 (ERK1?2), Akt and glycogen synthase kinase-3beta (GSK-3β) in kidneys of db/db mice. In contrast, over-expression of MFG-E8 by injection of recombinant MFG-E8 resulted in the opposite effects. GSPB2 treatment significantly decreased protein levels of MFG-E8, phospho-ERK1/2, phospho-Akt, and phospho-GSK-3β in the kidneys of db/db mice. These findings yield insights into the pathogenesis of diabetic nephropathy, revealing MFG-E8 as a new therapeutic target and indicating GSPB2 as a prospective therapy by down-regulation of MFG-E8, along with ERK1/2, Akt and GSK-3β signaling pathway.     

Grape seed procyanidin B2 inhibits advanced glycation end product-induced endothelial cell apoptosis through regulating GSK3β phosphorylation

To investigate the effects of GSPB2 (grape seed procyanidin B2) on the apoptosis of HUVECs (human umbilical endothelial cells) induced by AGEs (advanced glycation end products), HUVECs were treated with AGEs (200 μg/ml) in the presence or absence of GSPB2 (2.5, 5.0 and 10.0 μmol/l). Our findings showed that (i) AGEs induced HUVEC apoptosis and up-regulated the expression of caspase-3 activation and lactadherin and reduced the phosphorylation of GSK3β (glycogen synthase kinase 3β) at baseline. (ii) Treatment of HUVEC with GSPB2 significantly inhibited the cell apoptosis and the expression of caspase-3 activation and lactadherin induced by AGEs. Moreover, GSPB2 inhibited intracellular reactive oxygen species in a dose-dependent manner in AGEs-treated cells as determined by flow cytometry. (iii) GSPB2 increased the phosphorylation of GSK3β of HUVEC in response to AGEs. These findings suggest that the signalling pathway involving phosphorylation of GSK3β and lactadherin might play a key role in the endothelial apoptosis. GSPB2 therapy could become an effective approach to battling AGEs-induced endothelial apoptosis.     

Quantitative proteomics study of protective effects of grape seed procyanidin B2 on diabetic cardiomyopathy in db/db mice

Diabetic cardiomyopathy is one of the major complications of diabetes mellitus. Oxidative stress appears to play a substantial role in cardiomyopathy. Grape seed procyanidin B2 (GSPB2) has been known as an anti-oxidant in treating diabetes mellitus; however, little is known about its effects and underlying mechanisms on diabetic cardiomyopathy. The present study is to explore the molecular targets of GSPB2 responsible for the anti-oxidative effects in db/db mice by quantitative proteomics. GSPB2 (30?mg/kg body weight/day) were intragastric administrated to db/db mice for 10?weeks. Proteomics of the heart tissue extracts by isobaric tags for relative and absolute quantification analysis was obtained from db/db mice. Our study provides important evidence that GSPB2 protect against cardiomyopathy in diabetes mellitus, which are believed to result from regulating the expression of key proteins involving cardiac fibrosis and proliferation. GSPB2 could be expected to become novel clinical application in fighting against diabetic cardiomyopathy.     

PIMT Prevents the Apoptosis of Endothelial Cells in Response to Glycated Low Density Lipoproteins and Protective Effects of Grape Seed Procyanidin B2

Background

The development of diabetic angiopathy is associated with profound vascular endothelial cells (VEC) dysfunction and apoptosis. Glycated low density lipoproteins (gly-LDL) continuously produced in the setting of diabetic patients play an important role in causing VEC dysfunction and apoptosis. However, the underlying molecular mechanism remains largely elusive. Protein L-isoaspartyl methyltransferase (PIMT) is a widely expressed protein repair enzyme by multiple cell types of arterial wall including VEC. Our previous proteomic studies showed that the expression of PIMT was significantly decreased in the aorta of diabetic rats as compared with control rats and treatment with grape seed procyanidin extracts significantly increased the PIMT expression in diabetic rats. We hypothesized that PIMT plays a critical role in gly-LDL induced VEC apoptosis; grape seed procyanidin B2 (GSPB2) protect against gly-LDL induced VEC apoptosis through PIMT regulation.

Methods and Results

HUVEC transfected negative control and PIMT siRNA were treated with or without GSPB2 (10 µmol/L) for 48 h. Moreover, HUVEC of PIMT overexpression were stimulated by gly-LDL (50 µg/ml) in the presence or absence of GSPB2 (10 µmol/L) for 48 h. Our results showed that gly-LDL downregulated PIMT expression and PIMT overexpression or GSPB2 significantly attenuated gly-LDL induced VEC apoptosis. PIMT siRNA increased VEC apoptosis with up-regulation of p53, cytochrome c release, caspase-9 and caspase-3 activation. Mechanistically, overexpression of PIMT or GSPB2 increased the phosphorylation of ERK1/2 and GSK3β in the gly-LDL induced VEC.

Conclusion

In summary, our study identified PIMT as a key player responsible for gly-LDL induced VEC apoptosis and GSPB2 protect against gly-LDL induced VEC apoptosis by PIMT up-regulation. Targeting PIMT including use of GSPB2 could be turned into clinical application in the fighting against diabetic vascular complications.     

Grape seed procyanidin b2 inhibits human aortic smooth muscle cell proliferation and migration induced by advanced glycation end products

Advanced glycation end product (AGE)-induced vascular smooth muscle cell (VSMC) proliferation is vital to the progression of diabetic vasculopathy. A grape seed procyanidin extract has been reported to possess anti-oxidative and anti-inflammatory properties and to display a significant cardiovascular protective effect, but little is know about the underlying mechanism. The objective of this present study was to determine whether GSPB2 (grape seed procyanidin B2), which is a dimeric procyanidin and more biologically active, could inhibit AGE-induced VSMC proliferation by affecting the production of ubiquitin COOH-terminal hydrolase 1 (UCH-L1), the degradation of IκB-α and nuclear translocation of NF-κB in human aortic smooth muscle cells (HASMCs). Our data show that GSPB2 preincubation markedly inhibited AGE-induced proliferation and migration of HASMCs in a dose-dependent manner and upregulated the protein level of UCH-L1. Further studies revealed that the GSPB2 pretreatment markedly attenuated the degradation of IκB-α and nuclear translocation of NF-κB by modulating ubiquitination of IκB-α in AGE-exposed HASMCs. These results collectively suggest that AGE-induced HASMC proliferation and migration was suppressed by GSPB2 through regulating UCH-L1 and ubiquitination of IκB-α. GSPB2 may therefore have therapeutic potential in preventing and treating vascular complications of diabetes mellitus.     

Grape Seed Procyanidin B2 Inhibits Human Aortic Smooth Muscle Cell Proliferation and Migration Induced by Advanced Glycation End Products

Advanced glycation end product (AGE)-induced vascular smooth muscle cell (VSMC) proliferation is vital to the progression of diabetic vasculopathy. A grape seed procyanidin extract has been reported to possess anti-oxidative and anti-inflammatory properties and to display a significant cardiovascular protective effect, but little is know about the underlying mechanism. The objective of this present study was to determine whether GSPB2 (grape seed procyanidin B2), which is a dimeric procyanidin and more biologically active, could inhibit AGE-induced VSMC proliferation by affecting the production of ubiquitin COOH-terminal hydrolase 1 (UCH-L1), the degradation of IκB-α and nuclear translocation of NF-κB in human aortic smooth muscle cells (HASMCs). Our data show that GSPB2 preincubation markedly inhibited AGE-induced proliferation and migration of HASMCs in a dose-dependent manner and upregulated the protein level of UCH-L1. Further studies revealed that the GSPB2 pretreatment markedly attenuated the degradation of IκB-α and nuclear translocation of NF-κB by modulating ubiquitination of IκB-α in AGE-exposed HASMCs. These results collectively suggest that AGE-induced HASMC proliferation and migration was suppressed by GSPB2 through regulating UCH-L1 and ubiquitination of IκB-α. GSPB2 may therefore have therapeutic potential in preventing and treating vascular complications of diabetes mellitus.     

The protective effects of grape seed procyanidin B2 against asporin mediates glycated low‐density lipoprotein induced‐cardiomyocyte apoptosis and fibrosis

The progression of diabetic cardiomyopathy is related to cardiomyocyte dysfunction and apoptosis. Our previous studies showed that asporin (ASPN) was significantly increased in the myocardium of db/db mice through proteomics, and grape seed procyanidin B2 (GSPB2) significantly inhibited the expression of ASPN in the heart of db/db mice. We report here that ASPN played a critical role in glycated low‐density lipoproteins (gly‐LDL) induced‐cardiomyocyte apoptosis. We found that gly‐LDL upregulated ASPN expression. ASPN increased H9C2 cardiomyocyte apoptosis with down‐regulation of Bcl‐2, upregulation of transforming growth factor‐β1, Bax, collagen III, fibronectin, and phosphorylation of smad2 and smad3. However, GSPB2 treatment reversed ASPN‐induced impairments in H9C2 cardiomyocytes. These results provide evidence for the cardioprotective action of GSPB2 against ASPN injury, and thus suggest a new target for fighting against diabetic cardiomyopathy.     

Sucrose synthase is involved in the conversion of sucrose to polysaccharides in filamentous nitrogen-fixing cyanobacteria

Higher plants and cyanobacteria metabolize sucrose (Suc) by a similar set of enzymes. Suc synthase (SuS, UDP-glucose: D: -fructose 2-alpha-D: -glucosyl transferase, EC 2.4.1.13) catalyses the synthesis and cleavage of Suc, and in higher plants, it plays an important role in polysaccharides biosynthesis and carbon allocation. In this work, we have studied the functional relationship between SuS and the metabolism of polysaccharides in filamentous nitrogen-fixing cyanobacteria. We show that the nitrogen and carbon sources and light regulate the expression of the SuS encoding gene (susA), in a similar way that they regulate the accumulation of polysaccharides. Furthermore, glycogen content in an Anabaena sp. mutant strain with an insertion inactivation of susA was lower than in the wild type strain under diazotrophic conditions, while both glycogen and polysaccharides levels were higher in a mutant strain constitutively overexpressing susA. We also show that there are soluble and membrane-bound forms of SuS in Anabaena. Taken together, these results strongly suggest that SuS is involved in the Suc to polysaccharides conversion according to nutritional and environmental signals in filamentous nitrogen-fixing cyanobacteria.     

Proteomic Analysis of Aorta and Protective Effects of Grape Seed Procyanidin B2 in db/db Mice Reveal a Critical Role of Milk Fat Globule Epidermal Growth Factor-8 in Diabetic Arterial Damage

Background

Atherosclerosis is one of the major complications of type 2 diabetic patients (T2DM), leading to morbidity and mortality. Grape seed procyanidin B2 (GSPB2) has demonstrated protective effect against atherosclerosis, which is believed to be, at least in part, a result of its antioxidative effects. The aim of this study is to identify the target protein of GSPB2 responsible for the protective effect against atherosclerosis in patients with DM.

Methods and Results

GSPB2 (30 mg/kg body weight/day) were administrated to db/db mice for 10 weeks. Proteomics of the aorta extracts by iTRAQ analysis was obtained from db/db mice. The results showed that expression of 557 proteins were either up- or down-regulated in the aorta of diabetic mice. Among those proteins, 139 proteins were normalized by GSPB2 to the levels comparable to those in control mice. Among the proteins regulated by GSPB2, the milk fat globule epidermal growth factor-8 (MFG-E8) was found to be increased in serum level in T2DM patients; the serum level of MFG-E8 was positively correlated with carotid-femoral pulse wave velocity (CF-PWV). Inhibition of MFG-E8 by RNA interference significantly suppressed whereas exogenous recombinant MFG-E8 administration exacerbated atherogenesis the db/db mice. To gain more insights into the mechanism of action of MFG-E8, we investigated the effects of MFG-E8 on the signal pathway involving the extracellular signal-regulated kinase (ERK) and monocyte chemoattractant protein-1 (MCP-1). Treatment with recombinant MFG-E8 led to increased whereas inhibition of MFG-E8 to decreased expression of MCP-1 and phosphorylation of ERK1/2.

Conclusion

Our data suggests that MFG-E8 plays an important role in atherogenesis in diabetes through both ERK and MCP-1 signaling pathways. GSPB2, a well-studied antioxidant, significantly inhibited the arterial wall changes favoring atherogenesis in db/db mice by down-regulating MFG-E8 expression in aorta and its serum level. Measuring MFG-E8 serum level could be a useful clinical surrogate prognosticating atherogenesis in DM patients.     

Elaboration of both the group W135 and group Y capsular polysccharides by a single strain of Neisseria meningitidis

A single strain (8021) of Neisseria meningitidis, isolated from a child with disseminated meningococcal disease, was found to elaborate two serogroup-specific capsular polysaccharides-Y and W135. The original isolate as well as the progeny of ten single colony sub-isolates each agglutinated with both group Y and group W135 serogrouping antisera. The capsular polysccharide of strain 8021 contained the chemical constituents of both the W135 and Y capsular polysaccharides in a ratio of about 2.5:1. The patient responded immunologically to both capsular polysaccharides with haemagglutinating antibodies. Analysis by double diffusion in agar revealed that the capsular polysaccharide of strain 8021 contained individual molecules of group W135 and group Y capsular polysaccharides as well as a mosaic molecule containing both antigenic determinants.     

Niches of two polysaccharide-degrading Polaribacter isolates from the North Sea during a spring diatom bloom

Members of the flavobacterial genus Polaribacter thrive in response to North Sea spring phytoplankton blooms. We analyzed two respective Polaribacter species by whole genome sequencing, comparative genomics, substrate tests and proteomics. Both can degrade algal polysaccharides but occupy distinct niches. The liquid culture isolate Polaribacter sp. strain Hel1_33_49 has a 3.0-Mbp genome with an overall peptidase:CAZyme ratio of 1.37, four putative polysaccharide utilization loci (PULs) and features proteorhodopsin, whereas the agar plate isolate Polaribacter sp. strain Hel1_85 has a 3.9-Mbp genome with an even peptidase:CAZyme ratio, eight PULs, a mannitol dehydrogenase for decomposing algal mannitol-capped polysaccharides but no proteorhodopsin. Unlike other sequenced Polaribacter species, both isolates have larger sulfatase-rich PULs, supporting earlier assumptions that Polaribacter take part in the decomposition of sulfated polysaccharides. Both strains grow on algal laminarin and the sulfated polysaccharide chondroitin sulfate. For strain Hel1_33_49, we identified by proteomics (i) a laminarin-induced PUL, (ii) chondroitin sulfate-induced CAZymes and (iii) a chondroitin-induced operon that likely enables chondroitin sulfate recognition. These and other data suggest that strain Hel1_33_49 is a planktonic flavobacterium feeding on proteins and a small subset of algal polysaccharides, while the more versatile strain Hel1_85 can decompose a broader spectrum of polysaccharides and likely associates with algae.     

Chemical composition of extracellular polysaccharides of cowpea rhizobia

The extracellular polysaccharides (EPS) of six strains of cowpea rhizobia were examined. The strains (MI50A, M6-7B, IRC253) produced polysaccharides containing glucose, galactose and mannose in a molar ratio of 2:1.1:1, 1:1.3:3.1 and 1:1.3:3.5 respectively. Two strains (513-B and Ez-Aesch) produced polysaccharides containing galactose and mannose in a molar ratio of 2:3. Mannose was the only sugar detected in the EPS of strain IRC291. Pyruvate, acetate, glucuronic acid and galacturonic acid were not detected in any strain.Abbreviations EPS Extracellular polysaccharide - YEMA yeast-extract mannitol agar - YEMB yeast extract mannitol broth     

Location of the O-methyl groups in the O polysaccharide of Pseudomonas syringae pv. phaseolicola

The O-methylation pattern of the O polysaccharide (OPS) of the lipopolysaccharide of Pseudomonas syringae pv. phaseolicola GSPB 1552 was revealed by methylation (CD₃I) analysis, Smith degradation, and NMR spectroscopy. Together with the major O repeats consisting of -rhamnopyranose ( -Rhap) and -fucofuranose ( -Fucf), there are minor repeats (30%) containing 3-O-methyl- -rhamnose ( -acofriose), which is 2-substituted in the interior repeats and occupies the terminal non-reducing end of the OPS. It was suggested that the methylated O repeats are linked to each other nearby the non-reducing end of the OPS and that the ‘biological’ O repeat of the OPS has the following structure:

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The O-acetylation patterns in the O-antigens of Hafnia alvei strains PCM 1200 and 1203, serologically closely related to PCM 1205

Serological tests revealed immunochemical similarities between the lipopolysaccharides of Hafnia alvei strains PCM 1200, 1203 and 1205. Immunoblotting and ELISA showed cross-reactions between the strains. NMR spectroscopy showed that the O-deacetylated O-specific polysaccharides isolated from lipopolysaccharides of H. alvei strains PCM 1200 and 1203 possessed the same composition and sequence as the O-deacetylated O-specific polysaccharide of H. alvei strain PCM 1205, that is a glycerol teichoic-acid-like polymer with a repeating unit of the following structure: [carbohydrate structure: see text] NMR spectroscopic studies of the polysaccharides concluded that O-3 of the side chain beta-D-GlcpNAc is partially O-acetylated (50-80%) in both investigated strains. In strain PCM 1203 an additional O-acetyl group (50-80%) is linked to O-6 of the chain -->3)-alpha-D-GlcpNAc-(1--> residue. The structural features of the isolated O-specific polysaccharides were also the same as those of the O-specific polysaccharides on the bacterial cells directly observed by the HR-MAS NMR technique.