Pyrroline-5-carboxylate reductase from Cucurbita cotyledons

Pyrroline-5-carboxylate reductase, which required reduced pyridine nucleotide and Δ′-pyrroline-5-carboxylate for proline synthesis, was isolated from pumpkin cotyledons. The enzyme was found in the soluble fraction and had a 4.5-fold greater activity with NADH than NADPH. The enzyme was inhibited by NH₂OH, NADP, ATP and slightly by proline. Glutathione or pyridoxal-5-phosphate had little effect on enzyme activity. The enzyme had a pH optimum between 7·0 and 7·6 and was not inhibited by high concentrations of NADH or Δ′-pyrroline-5-carboxylate.     

A Novel Optimization of Water-Soluble Compound Polysaccharides from Chinese Herbal Medicines by Quantitative Theory and Study on Its Characterization and Antioxidant Activities

The present study optimized the extraction characterization and antioxidant activities of water-soluble compound polysaccharides (CPs) from hawthorn, lotus leaf, Fagopyrum tataricum, semen cassiae, Lycium barbarum, and Poria cocos Chinese herbal medicines that have mass ratios of 4 : 2 : 2 : 1.5 : 1 : 1. The CPs yield equation was predicted using quantitative theory, to which a maximum CPs yield of 7.18±0.24 % under the following optimal extraction conditions: a water-to-raw material ratio of 30 mL/g, an extraction temperature of 65 °C, an extraction time of 45 min, and extraction mode ultrasonic-assistant extraction. CPs were consisted of Ara, Gal, Glc, Xyl, Man, GalA and GlcA in a molar ratio of 3.1 : 2.6 : 50.6 : 1.7 : 20.4 : 17.2 : 4.2. The HPGPC profiles and FT-IR spectra implied that CPs were heterogeneous acidic polysaccharides and possessed the β-d -pyranose configuration. Congo red test, CD spectrum and SEM revealed that CPs with three helix conformation showed a flocculent, granulous or sheet-like appearance. Furthermore, the relationships between antioxidant activity and concentration of CPs displayed significant positive correlation, and the scavenging abilities for DPPH, hydroxyl radical, ABTS, superoxide-anion radical and reducing power of CPs were 93.56±2.51 %, 84.03±1.69 %, 83.29±1.93 %, 37.49±1.93 % and 0.467±0.006 at a concentration of 4.0 mg/mL. Therefore, CPs could be applied as a potential natural antioxidant in pharmaceutical or functional food fields.     

Isolation of phosphorylated polysaccharides from algae: the immunostimulatory principle of Chlorella pyrenoidosa

The major immunostimulatory principle in the hot aqueous extract of Chlorella pyrenoidosa has been isolated by a sequence of ethanol precipitation, precipitation with a cationic surfactant (CTAB), size exclusion chromatography, and anion exchange chromatography. A series of phosphorylated polysaccharides were obtained having different molecular masses but with similar structures. The higher molecular mass fractions showed considerable activity in the stimulation of mouse peritoneal macrophages to synthesize nitric oxide. The structure of the major polysaccharide was established by sugar analysis, configurational analysis, and 1D and 2D NMR experiments at 500 and 800 MHz on the parent polysaccharide, the de-O-acetylated polysaccharide, and on the components obtained after hydrolysis of the phosphate diesters. It had a β-d-Galp-(1→3)-β-d-Galp-(1→3)-backbone with half of the Galp units substituted at O-6 by terminal β-d-Glcp units. The remaining Galp units were substituted on O-6 by about equal amounts of α-d-Manp-1-phosphate and 3-O-Me-α-Manp-1-phosphate diesters. The substituents were not located in a regularly alternating fashion on the backbone. The O-acetyl groups were largely located on O-2 and O-4 of Galp and 35% of the Galp residues were O-acetylated. This is the second observation of a phosphorylated polysaccharide in an alga and the first where it is present to a significant extent.     

Interaction between nucleic acids and glycans: In silico and in vitro

Quantum-chemical (PM3) calculations prove the possibility of hydrogen bonding between all nucleic bases and carboxyls of acidic or hydroxymethyls of neutral sugars, with energy in the former case comparable to that for canonical GC or AT pairing. However, there is appreciable energy preference for carboxyl H-bonding with purines, and for hydroxymethyl H-bonding with pyrimidines. Simulation reveals that the H-bonds formed in purine-uronide and pyrimidine-hexose pairs can give rise to double-stranded nucleic acid-polysaccharide complexes. Indeed, dot hybridization with radioactive probes and hypochromic effects in solution testify that a hexose homoglycan (amylose) selectively forms a complex with polypyrimidine, whereas uronic acid homoglycans selectively complex with polypurine. Complexing is also observed between heteropolymers such as thymus DNA and hyaluronic acid. These results are consistent with the idea of template-directed synthesis of the polysaccharide moiety of glycosaminoglycans involving nucleic acids.     

Ornithine transaminase from Cucurbita maxima cotyledons

During germination a marked increase in both soluble and particulate ornthine transaminase occurs in pumpkin cotyledons. Both enzymes had a pH optimum of 8.3 and a requirement for ornthine and α-ketoglutarate. Other keto acids or amino donors showed little activity. The enzymes required an active sulphydryl group for maximum activity. Exogenous pyridoxal phosphate was not required, but hydroxylamine inhibited the reaction and added pyridoxal phosphate overcame this inhibition. Proline inhibited the reaction and may play a role in the fate of ornithine in pumpkin cotyledons.     

Structural analysis and antioxidative properties of mutan (water-insoluble glucan) and carboxymethyl mutan from Streptococcus mutans

Streptococcus mutans (MTCC 497) was grown anaerobically in acidic Brain heart infusion (BHI) medium with 15 % sucrose to produce cell-bound and extracellular water-insoluble polysaccharide mutan. Fourier transformed infrared (FTIR) and ¹³C NMR studies revealed a mixed linkage of α-1−3 and α-1–6 mutan with a production yield of 1.8 g/L. Mutan has a branched structure with a molecular weight (M_w) of 5654 Da. Water-insoluble mutan was carboxymethylated at 0.93 degrees of substitution. FTIR spectra with characteristic peaks at 1603 cm⁻¹ and 1418 cm⁻¹ due to symmetric and asymmetric vibrations of the COO- group confirmed carboxymethylation. Thermal gravimetric analysis showed that native mutan and carboxymethyl mutan exhibited higher thermal stability. Carboxymethylation enhanced solubility and antioxidative radical-scavenging activity. The in-vitro antioxidative radical scavenging analysis revealed 52 % and 47 % inhibition of DPPH and ABTS radicals.     

Cellulose depolymerization to glucose and other water soluble polysaccharides by shear deformation and high pressure treatment

The simultaneous action of shear deformation and high pressure (SDHP) creates changes in the structure of wood and its main components (cellulose, hemicelluloses, lignin). The formation of water and alkali soluble polysaccharides under SDHP action, proceeds in seconds in the solid state, without the use of any reagents and solvents. Therefore, SDHP seems to be a technologically safe method and friendly to the environment. The amorphization of cellulose crystallites and depolymerization of cellulose chains were observed under a wide range of pressures (1–6 GPa), both for cellulose samples and the cellulose part of wood. Similar depolymerization occurs in the hemicellulose part of wood. The decomposition of polysaccharides under SDHP causes the formation of the water soluble part, whose content increases with pressure and the applied shear deformation. A maximum solubility of 40% and 55% was registered at 6 GPa following treatment of cellulose and birch wood samples. A higher output in the case of wood can be explained by a specific role of lignin under SDHP, which acts as a ‘grinding stone’ during cellulose and hemicelluloses destruction. As shown by high-performance size exclusion chromatography, the water soluble fraction obtained from cellulose contained glucose (2.6%), cellobiose (9.6%), cellotriose (16.6%) and other higher water soluble oligomers (71%). Almost complete dissolution (98%) of the treated cellulose sample can be achieved by extraction with 10% NaOH solution. The SDHP treated birch wood was subjected to submerged fermentation (with Trichoderma viride), and a 13% output of proteins was obtained. In this case, the water soluble part played the role of the so called ‘start sugars’. Abbreviations: ASF, alkali soluble fraction; DP, degree of polymerization; EC, energy consumption; HP, high pressure; LMWS, low molecular weight sugars; MC, moisture content; MCC, microcrystalline cellulose; SD, shear deformation, SDHP, shear deformation under high pressure; SS, shear strength; WSF, water soluble fraction This revised version was published online in November 2006 with corrections to the Cover Date.     

Radioprotective activity of neutral polysaccharides isolated from the fruiting bodies of Hohenbuehelia serotina

In this study, the radioprotective effect of neutral polysaccharides from Hohenbuehelia serotina (NTHSP) against the damages induced by ⁶⁰Co-γ radiation was investigated. The results showed that NTHSP could significantly improve the activity of glutathione peroxidase (GSH-Px) and increase the contents of glutathione (GSH) and ceruloplasmin in plasma after treated with 6 Gy-radiation compared with the radiation controls (p < 0.05). Furthermore, administration with NTHSP could effectively increase the quantity of marrow DNA (p < 0.05) and reduce the rates of chromosome aberration and micronuclei (p < 0.01) in bone marrows of mice. In addition, NTHSP could markedly inhibit the expressions of Bax protein and promote the expressions of Bcl-2 protein, accordingly inhibit the releases of cytochrome c and expressions of activated Caspase-3, and therefore block the mitochondrial apoptotic pathway of splenocytes in mice induced by ⁶⁰Co-γ radiation. These results suggested that NTHSP might be a natural radioprotective agent against the injuries induced by radiation.