Sequence and functional analysis of the cloned Neisseria meningitidis CMP-NeuNAc synthetase

The CMP-N-acetylneuraminic acid (CMP-NeuNAc) synthetase gene of Neisseria meningitidis group B is located on a 2.3-kb EcoRI fragment within the cps gene cluster. Nucleotide sequence determination of the gene encoding the CMP-NeuNAc synthetase revealed a 515-bp open reading frame that can encode a 18.9-kDA protein. A computer data base scan revealed a 59.4% identity to the CMP-NeuNAc synthetase gene of E. coli K1. Enzymatic activity was confirmed in vitro and in vivo. Transformation of the CMP-NeuNAc defective E. coli K1 strain EV5 with the meningococcal CMP-NeuNAc synthetase could complement the defect in E. coli.     

Optical activity of the cellulose acetate-mesophase-generating solvent system in the formation of a lyotropic liquid-crystalline phase

It was demonstrated experimentally that the vapors of a mesophase-generating solvent, i.e., a solvent forming a lyotropic liquid-crystalline phase with a polymer, changed the spatial structure and optical density of natural polysaccharides (cellulose acetates). In this process, the value of specific optical rotation of the polymer modified by the vapors varied in a wide range up to sign inversion. The action of vapors on the polymer follows a peculiar dose-effect pattern, with lower doses producing a stronger effect. Application of this approach to the study into specific structural characteristics of biopolymers, such as DNA, is proposed.     

Unusual rheological properties of a new associative polysaccharide in salt media

New amphiphilic polysaccharides based on alginate-grafted-Poly (ε-caprolactone) or alg-g-PCL bearing two kinds of PCL chains with different molar masses (1250 and 530 gmol⁻¹) with various amounts from 3% to 15% were prepared. Rheological properties in aqueous solutions of such systems have been investigated as a function of polymer concentration, added salt and temperature in semi-dilute regime. Strong hydrophobic intermolecular associations were clearly demonstrated in pure water whatever the PCL chain length and extend of modification. Increasing polymer concentration, grafting rate and/or PCL chains length can lead to a structured liquid behaviour. Rheological properties of the most organized system have been found independent to the temperature (until 60 °C). In salt media, a strong dependence of hydrophobic interactions to the length of PCL chains was observed. For M_PCL = 1250 g.mol⁻¹ the screening of charges promotes the establishment of intermolecular interactions and leads to a strong physical gel for the highest grafting rates. For M_PCL = 530 g mol⁻¹, ionic strength leads to a decrease of rheological properties when increasing grafting rate. This result may indicate an increase of hydrophobic clusters even in the entangled regime. This unusual behaviour opens the ways for the preparation of suitable hydrogels for drug release.     

Crystal Structures of Wzb of Escherichia coli and CpsB of Streptococcus pneumoniae, Representatives of Two Families of Tyrosine Phosphatases that Regulate Capsule Assembly

Many Gram-positive and Gram-negative bacteria utilize polysaccharide surface layers called capsules to evade the immune system; consequently, the synthesis and export of the capsule are a potential therapeutic target. In Escherichia coli K-30, the integral membrane tyrosine autokinase Wzc and the cognate phosphatase Wzb have been shown to be key for both synthesis and assembly of capsular polysaccharides. In the Gram-positive bacterium Streptococcus pneumoniae, the CpsCD complex is analogous to Wzc and the phosphatase CpsB is the corresponding cognate phosphatase. The phosphatases are known to dephosphorylate their corresponding autokinases, yet despite their functional equivalence, they share no sequence homology. We present the structure of Wzb in complex with phosphate and high-resolution structures of apo-CpsB and a phosphate-complexed CpsB. We show that both proteins are active toward Wzc and thereby demonstrate that CpsB is not specific for CpsCD. CpsB is a novel enzyme and represents the first solved structure of a tyrosine phosphatase from a Gram-positive bacterium. Wzb and CpsB have completely different structures, suggesting that they must operate by very different mechanisms. Although the mechanism of Wzb can be inferred from previous studies, CpsB appears to have a tyrosine phosphatase mechanism not observed before. We propose a chemical mechanism for CpsB based on site-directed mutagenesis and structural data.     

A Novel Gene Encoding an Enzyme That Degrades a Polysaccharide from the Sheath of Sphaerotilus natans

A gene encoding an enzyme that is able to depolymerize the basic polysaccharide prepared from the sheath of Sphaerotilus natans was identified in a sheath-degrading bacterium, Paenibacillus koleovorans. The gene was constructed from 2217 bp coding for 738 amino acids, including the signal sequence of 34 amino acids. No closely related protein or gene was indicated by a homology search. The gene was expressed in Escherichia coli as a glutathione S-transferase fusion protein. The fusion protein depolymerized the sheath polysaccharide into an oligosaccharide, introducing an unsaturated sugar residue, suggesting that the gene codes for a polysaccharide lyase acting on a basic polysaccharide.     

Microencapsulation of Linoleic Acid with Low- and High-molecular-weight Components of Soluble Soybean Polysaccharide and Its Oxidation Process

Soluble soybean polysaccharide (SSPS) was fractionated into its low- (LMW) and high-molecular-weight (HMW) components to test their antioxidative and emulsifying properties. Linoleic acid was emulsified with an aqueous solution of SSPS, HMW, a mixture of LMW or HMW with maltodextrin, or maltodextrin alone. The emulsions prepared with SSPS, HWM and the mixture of HMW with maltodextrin were stable. These emulsions were spay-dried to produce microcapsules. The encapsulated linoleic acid was oxidized at 37°C and at various levels of relative humidity. Linoleic acid encapsulated with the mixture of LMW with maltodextrin or HMW was stable to oxidation, and this stability increased as the weight fraction of LMW in the mixture was increased. The LMW components also had high DPPH-radical scavenging activity. These results indicate that LMW played an important role in suppressing or retarding the oxidation of linoleic acid encapsulated with SSPS. The oxidative stability of linoleic acid encapsulated with a mixture of the LMW and HMW components was high at low and high relative humidity, but not at intermediate levels of relative humidity.     

Turnover of cell surface-bound capsular polysaccharide in Staphylococcus aureus

The capsular polysaccharide (CPS) of Staphylococcus aureus strain Smith was labelled by growth of bacteria in the presence of radioactive N-acetylglucosamine and was separated from labelled cell wall components by affinity chromatography on wheat germ agglutinin following dissolution of the cells by lysostaphin. The products were partially characterised chemically and immunochemically. Similar labelled components were found in the culture fluid during growth. In a pulse-chase experiment, cell-bound CPS was released continuously into the culture fluid at the same rate as cell wall turnover and there was no evidence of direct excretion of CPS.     

Alleviation of Liver Dysfunction,Oxidative Stress,and Inflammation Underlines the Protective Effects of Polysaccharides from Cordyceps cicadae on High Sugar/High Fat Diet-Induced Metabolic Syndrome in Rats

This study investigated the protective effects of two polysaccharides (CPA-1 and CPB-2) from Cordyceps cicadae against high fructose/high fat diet (HF/HFD) induced obesity and metabolic disorders in rats. Rats were either fed with normal diet or HF/HFD and treated with CPA-1 and CPB-2 (100 and 300 mg/kg) for 11 weeks. Administration of CPA-1 and CPB-2 significantly and dose dependently reduced body and liver weight, insulin and glucose tolerance, serum insulin and glucose levels. Furthermore, serum and hepatic lipid profiles, liver function enzymes and proinflammatory cytokines (TNF-α, IL-1β and IL-6) were markedly reduced. Additionally, CPA-1 and CPB-2 treatment alleviated hepatic oxidative stress by reducing lipid peroxidation level (MDA) and upregulating glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) activities as well as ameliorated histological alterations through the reduction of hepatic lipid accumulation. These results suggested that the polysaccharides from C. cicadae showed protective effects against HF/HFD induced metabolic disturbances and may be considered as a dietary supplement for treating obesity.