Structural investigation of a water-soluble glucan from an edible mushroom, Astraeus hygrometricus

A water-soluble glucan, Fraction I, was isolated from the aqueous extract of the fruit bodies of the mushroom Astraeus hygrometricus. On the basis of total hydrolysis, methylation analysis, periodate oxidation, and NMR studies ((1)H, (13)C, 2D-COSY, TOCSY, NOESY, and HSQC), the structure of the repeating unit of the glucan is determined as:This glucan shows strong splenocyte activation.     

Fibrinogenolytic enzymes of Trimeresurus mucrosquamatus venom

By means of CM-Sephadex C-50 column chromatography, Trimeresurus mucrosquamatus venom was separated into twenty fractions. The fibrinogenolytic activity was concentrated in Fractions 8, 10, 12, 13 and 14. Fractions 8 and 13 had the highest ratio of fibrinogenolytic and caseinolytic activities. Fraction 8 possessed tosyl-l-arginine methyl esterase activity, while the others did not. The caseinolytic activities of Fractions 10, 12, 13 and 14 were inhibited by EDTA, while that of Fraction 8 was not. Fractions 8 and 13 were further purified by CM-cellulose and gel filtration and were homogeneous as judged by electrophoresis on polyacrylamide gel and cellulose acetate membrane. The molecular weights of the purified Fractions 8 and 13 were 26 000 and 22 400, respectively. Both were single peptide chains. The specific fibrinogenolytic activity of Fraction 8 was 17 mg fibrinogen/min/mg protein, while that of Fraction 13 was 100 mg fibrinogen/min/mg protein. Fraction 13 digested specifically the α(A) chain of monomeric fibrinogen to yield two cleavage products. Fraction 8 digested the β(B) chain first to yield four cleavage products. When the incubation time was prolonged, the α(A) chain was also partially digested by Fraction 8 to yield two cleavage products.     

Oxidative stress in rats fed a high-fat high-sucrose diet and preventive effect of polyphenols: Involvement of mitochondrial and NAD(P)H oxidase systems

Mitochondrial and NADPH oxidase systems and oxidative stress were investigated in 12 week high-fat high-sucrose (HFHS) diet-fed rats. A protective effect of wine polyphenol (PP) extract was also examined. In liver, maximal activities of CII and CII+III mitochondrial complexes were decreased but NADPH oxidase expression (p22^phox and p47^phox) and NADPH oxidase-dependent superoxide anion production were not modified, whereas oxidative stress (lipid and protein oxidation products and antioxidant systems) was increased with HFHS diet. In muscle, anion superoxide production was slightly increased while mitochondrial complex activities and lipid and protein oxidation products were not modified with HFHS diet. In heart, NADPH oxidase expression and superoxide anion production were increased, and maximal activity of mitochondrial respiratory chain complexes or oxidative stress parameters were not modified. Wine polyphenol extract had an inhibiting effect on liver oxidative stress and on heart NADPH oxidase expression and superoxide anion production, and on induction of hepatic steatosis with HFHS diet. Induction of mitochondrial dysfunction could be a primary event in the development of oxidative stress in liver, while in skeletal muscle and in heart the NADPH oxidase system seems to be mainly involved in oxidative stress. Wine polyphenol extract was shown to partially prevent oxidative stress in liver and heart tissues and to nearly completely prevent steatosis development in liver.     

Characterisation of secreted polysaccharides and (glyco)proteins from suspension cultures of Pyrus communis

High molecular weight material recovered from the culture filtrate of cell suspension cultured Pyrus communis was composed of 81% carbohydrate, 13% protein and 5% inorganic material. This material was separated into three fractions (one neutral (Fraction A) and two acidic (Fractions B and C)), by anion-exchange chromatography on DEAE-Sepharose CL-6B using a gradient of imidazole-HCl at pH 7.0. The monosaccharide and linkage composition of each fraction was determined after carboxyl reduction of uronic acid residues. From the combined results of the carbohydrate analyses, we conclude that the high molecular weight extracellular material consists of three major and two minor polysaccharides: a (fucogalacto)xyloglucan (36%) in the unbound neutral Fraction A; a type II arabinogalactan (as an arabinogalactan-protein, 29%) and an acidic (glucurono)arabinoxylan (2%) in Fraction B; and a galacturonan (33%) and a trace of heteromannan in Fraction C. The main amino acids in the proteins were Glx, Thr, Ser, Hyp/Pro and Gly. Further separation of Fraction B by solvent partition, SDS-PAGE and analysis by LC-MS/MS identified the major proteins as two chitanases, two thaumatin-like proteins, a beta-1,3-glucanase, an extracellular dermal glycoprotein and a pathogenesis-related protein.     

Transformation of 3,5-dimethoxy,4-hydroxy cinnamic acid by polyphenol oxidase from the fungus Trametes versicolor: product elucidation studies

Sinapic acid (SA), 3,5-dimethoxy,4-hydroxy cinnamic acid, was incubated with a crude polyphenol oxidase from the fungus Trametes versicolor. Some products of this transformation were isolated and their structures identified using mass spectrometry, nuclear magnetic resonance and Fourier transform infrared spectroscopy, and X-ray crystallography. It was found that the enzymatic oxidation of SA includes two distinct phases. In the initial phase SA is enzymatically transformed to r-1H-2c,6c-bis-(4'-hydroxy-3', 5'-dimethoxyphenyl)-3,7-dioxabicyclo-[3,3,0]-octane-4,8-dione, dehydrodisinapic acid dilactone. The mechanism of this reaction may involve coupling of two phenoxy radicals by the beta-beta mode and subsequent intramolecular nucleophilic attack. In the second phase dehydrodisinapic acid dilactone is transformed by polyphenol oxidase into several intermediate products, including 4-(4-(3, 5-dimethoxy-4-oxo-2,5-cyclohexadienyliden)-1, 4-dihydroxy-(E)-2-butenylidene)-2,6-dimethoxy-2, 5-cyclohexadien-1-one. The final product of the overall transformation of SA is 2,6-dimethoxy-p-benzoquinone. The obtained results were used to propose a part of the transformation pathway for the enzymatic oxidation of SA by polyphenol oxidase.     

Enhancement of antibacterial effects of epigallocatechin gallate, using ascorbic acid

Although plant polyphenols such as (−)-epigallocatechin gallate (EGCG) have antibacterial activity towards methicillin-resistant Staphylococcus aureus (MRSA), such polyphenols are unstable in solution. Because the instability of polyphenols is attributable to their oxidation, we examined the effects of antioxidants and inhibitors of polyphenol oxidation on the maintenance of polyphenol antibacterial activity. The antibacterial activity of EGCG was enhanced in the presence of ascorbic acid, and ascorbic acid was the most effective for retaining the concentration of stable EGCG. On the other hand, the antibacterial activity of EGCG was lowered in the presence of casein in spite of its suppressing effect on the EGCG decrease. The effect of EGCG on the antibiotic resistance of MRSA was also enhanced in the presence of ascorbic acid. The addition of an antioxidant may affect other pharmacological effects of polyphenols in analogous ways, although this does not mean the clinical usefulness of the addition directly.     

Protective action of fenugreek (Trigonella foenum graecum) seed polyphenols against alcohol-induced protein and lipid damage in rat liver

The study investigates the effect of fenugreek seed polyphenol extract (FPEt) on ethanol-induced damage in rat liver. Chronic ethanol administration (6 g kg(-1) day(-1) x 60 days) caused liver damage that was manifested by excessive formation of thiobarbituric-acid-reactive substances, lipid hydroperoxides, and conjugated dienes, the end products of lipid peroxidation, and significant elevation of protein carbonyl groups and diminution of sulfhydryl groups, a marker of protein oxidation. Decreased activities of enzymic and non-enzymic antioxidant levels and decreased levels of thiol groups (both non-protein and protein) were observed in ethanol-treated rats. Further, ethanol significantly increased the accumulation of 4-hydroxynonenal protein adducts, nitrated and oxidized proteins in liver which was evidenced by immunohistochemistry. Administration of FPEt to ethanol-fed rats (200 mg kg(-1) day(-1)) significantly reduced the levels of lipid peroxidation products and protein carbonyl content, increased the activities of antioxidant enzymes, and restored the levels of thiol groups. The effects of FPEt were comparable with those of a positive control, silymarin. These findings show that FPEt ameliorates the pathological liver changes induced by chronic ethanol feeding.     

Studies on the Polyuronide of Oh-gyo-tye (The Achine of Ficus awkeotsang MAKINO)

A polyuronide, main component of the water extract of achine of Ficus awkeotsang MAKINO (on-gyo-tye), was purified by ion-exchange chromatography on DEAE-cellulose. The polyuronide (Fraction IB) is homogeneous electrophoretically and consists mainly of galacturonic acid. Optical rotation of Fraction IB is and content of methoxyl group is trace. In periodate oxidation of Fraction IB, molar ratio of galacturonic acid residue and periodate consumption was 1, and formic acid formation was very small. Periodate oxidation product of Fraction IB was oxidized further with bromine and the resulted substance was hydrolyzed. In the hydrolyzate, presence of large amount of tartaric and glyoxylic acids and small amount of tartronic acid were detected by paper chromatography. Reduced viscosity of aquous solution of Fraction IB increased with decreasing of the concentration of Fraction IB solution. From these results, it was deduced that Fraction IB has a linear structure of 1→4 linkage of d-galacturonic acid, probably α-linkage.     

Preparation and properties of 5′-nucleotidases from bovine milk fat globule membranes

The 5′-nucleotidase (5′-ribonucleoside phosphohydrolase, EC 3.1.3.5) from bovine milk fat globule membranes was partially purified. Two separate peaks of activity were obtained from a Sepharose column and the two fractions, designated V and VI in order of elution, were collected and characterized separately. Both V and VI exhibited pH optima between 7.0 and 7.5 for AMP, GMP and CMP in the absence of metal ions. In the presence of Mg²⁺, a second pH optimum at 10.0 was observed with both fractions. Low concentrations of MnCl₂ activated Fraction V but not Fraction VI. HgCl₂ was a potent inhibitor of both fractions. The relative rates of hydrolysis of various 5′-mononucleotides differed comparing the two fractions. Optimum temperature for P_i release was 69 °C for both fractions. Activation energies were 10 400 cal/mole and 9600 cal/mole for Fractions V and VI, respectively. For V, calculated K_m values for AMP, GMP and CMP were 0.94, 2.5 and 1.16 mM, respectively. Calculated K_m values for Fraction VI for AMP, GMP and CMP were 5.0, 3.95 and 1.73 mM, respectively. ATP was a competitive inhibitor of AMP hydrolysis by Fraction V and a noncompetitive inhibitor of AMP hydrolysis by Fraction VI. Both fractions contained chloroform-methanol-extractable phospholipid. The phospholipid distribution pattern of Fraction VI was similar to that of milk fat globule membranes. Fraction V contained only sphingomyelin and phosphatidylcholine. It is proposed that milk fat globule membranes contain two separate 5′-nucleotidases.     

Uptake and incorporation of the products of proteolysis by the rumen bacteriumBacteroides ruminicola R8/4

The polypeptides of the proteolytic rumen bacteriumBacteroides ruminicola R8/4 grown in the presence of either leaf Fraction 1 protein, bovine serum albumin, or Bactocasitone as sole nitrogen source were separated by SDS-polyacrylamide gel electrophoresis. Over 40 polypeptides were resolved; the pattern for organisms grown on Fraction 1 protein was similar but not identical to that of the serum albumin and Bactocasitone-grown bacteria. All the bacterial polypeptides were distinguishable from the polypeptides of Fraction 1 protein (and serum albumin). The stained pattern was the same for organisms sampled at intervals during the growth of a batch culture. After incubation of the growing organisms with [¹⁴C]-Fraction 1 protein, all the bacterial polypeptides were labeled. Bacteria grown in the presence of nonlabeled Fraction 1 protein and a mixture of [^14C]-labeled amino acids incorporated label into all the polypeptides; the bacteria did not grow in the absence of intact protein, and then virtually no label was incorporated from the amino acid mixture.     

Enzymatic transformation of caffeic acid with enhanced cyclooxygenase-2 inhibitory activity

Convenient enzymatic transformation of the phenylpropanoid caffeic acid (1) with polyphenol oxidase originating from pear afforded three new oxidized metabolites, caffeoxynic acid (2), caffeicinic acid (3), and isocaffeicinic acid (4), along with, 7,8-erythro-caffeicin (5) and phellinsin A (6). The structures of the three new caffeic acid oxidation products were elucidated on the basis of spectroscopic methods. The new products 2 and 3 exhibited significantly enhanced inhibitory activity against cyclooxygenase-2 (COX-2) when compared to parent caffeic acid.     

Cell-free synthesis and immunological characterization of salivary proteins from Chironomus tentans

We have isolated the salivary proteins of the larva of the harlequin fly Chironomus tentans, and characterized its constituents by gel electrophoresis and immunological techniques. The detailed composition of saliva from individual animals is shown to be very variable, but four main protein groups can be defined. The largest, Fraction A, comprises up to five species, with molecular weights of between 820,000 and 700,000 Daltons. It includes at least two distinct antigenic species. This finding is discussed in the context of the known heterogeneity of the 75S RNA fraction which is transcribed in the Balbiani rings 1 and 2. — The other prominent protein classes in isolated saliva range in size from 230,000 down to less than 20,000 Daltons. — We have also employed antiserum against salivary proteins to investigate the products of in vitro translation of salivary gland RNA in the rabbit reticulocyte lysate system. A broad spectrum of polypeptide species is obtained which are immunologically related to salivary components, including species of over 300,000 Daltons. These latter are interpreted as unfinished Fraction A polypeptides resulting from incomplete translation of 75S RNA from BR1 and BR2. Evidence is presented to demonstrate that other salivary proteins, apart from Fraction A, are faithfully translated in the reticulocyte lysate.     

Characteristics of piraltin, a polyphenol concentrate, produced by freeze-drying of red wine

Moderate consumption of red wine is associated with a decreased risk for coronary heart disease. Apart from alcohol, an additive role for wine polyphenols has been suggested. However, the real contribution of these compounds can only be studied when available without the alcohol component. The objective of the study was to prepare a wine polyphenol concentrate not containing alcohol and to compare the quantitative and qualitative properties of this concentrate with those of the original wine from which the concentrate is made. This polyphenol concentrate, called piraltin, was made out of red wine by a freeze-drying technique. Both piraltin and the original red wine were analyzed quantitatively for the main polyphenols present: gallic acid, catechin, epicatechin and quercetin. The qualitative comparison comprised the inhibitory effect of the two products on LDL oxidation in vitro. In the process of freeze-drying recovery of the four determined flavonoids of red wine is fairly constant (average 68 +/- 7%). In a copper induced LDL oxidation assay both red wine and piraltin prolonged lag-times over 300% compared to controls without a significant difference between the two products. The freeze-dried polyphenol concentrate piraltin contains about 70% of the total polyphenol content of the original wine. This preparation technique does not cause a loss of antioxidative properties of the phenols. Piraltin creates the possibility to study the effects of wine polyphenols separately without the influence of alcohol both in vitro and in vivo.     

Pathogenicity of Vibrio penaeicida for white shrimp Litopenaeus vannamei: a cysteine protease-like exotoxin as a virulence factor

The pathogenicity of Vibrio penaeicida Strains KH-1 and AM101, their culture-free supernatant (CFS), and their protein fraction obtained by 40% of ammonium sulfate precipitation (PFs40) were assessed in experimental challenges against juvenile Litopenaeus vannamei. Live Vibrio cells, CFS, and PFs40 from the AM101 strain produced a significantly higher mortality (p < 0.05) compared to the KH-1 strain. Toxicity and median lethal doses (LD50) of Fast Protein Liquid Chromatography (FPLC) products were evaluated on L. vannamei. The first FPLC fraction sample (A) from PFs40 of the AM101 strain displayed LD50 values of 1.68 and 5.61 microg protein ind.(-1), respectively. The second FPLC process from Fraction A showed a peak (A1) also with toxic effects to shrimp. PFs40, Fraction A, and Peak A1 showed a 38.5 kDa molecular band (SDS-PAGE), with activity on a gelatin protease zymogram. The lethal effect of PFs40 and Fraction A was inhibited by Proteinase K, CuCl2, E-64, and heat (60 and 100 degrees C) treatments, but was not inhibited by EDTA-Na2, aprotinin, and soy trypsin treatments. These results and the zymogram inhibition test suggest the presence of a cysteine protease-like proteinaceous exotoxin as a dominant protease, secreted by V. penaeicida Strain AM101.     

Immunological study of lysates from the cell wall of group A Streptococcus]

The chemical composition and presence of immunogenic components in the lysates of the cell walls of group A Streptococcus, type M29, were studied. The lysates were prepared with the use of muramidase. Fc-Receptors were detected in the lysates. Within the first 30 minutes of cell wall lysis by muramidase, 4 times higher amounts of the protein reacting with fibrinogen excreted than in the subsequent 4 hours. The lysates contained immunogenic proteins. Fraction III isolated by chromatography of the 30-minute lysate on DEAE-trisacryl formed a single precipitation band with lysate antiserum. The lysate Fraction IV forming three precipitation bands contained a protein not specific of the type. The protein was identical to the protein antigen from Triton X-100 extracts of group A Streptococcus, types M1, M12 and M29. The group-specific polysaccharide was detected in the lysate Fraction I and Fraction II of the 4-hour lysate.     

A comparison of the polypeptide isoelectric points and antigenic determinant sites of the large subunit of fraction 1 protein from Lycopersicon esculentum, Nicotiana tabacum and Petunia hybrida.

The large subunit of Fraction 1 protein from Lycopersicon esculentum, Nicotiana tabacum and Petunia hybrida has been examined by isoelectric focusing of the S-carboxymethylated polypeptides, and by double immunodiffusion with antiserum raised against Fraction 1 protein. The immunological results reveal heterogeneity in the large subunit primary structure not identified by isoelectric focusing. A variable phylogeny can be generated depending on whether serological or electrofocusing criteria are used.     

The Effects of Nitrate Supply and Grain Reserves on Fraction I Protein Level in the First Leaf of Barley

The timing and rate of nitrate application to barley seedlingsgrown under control and shade conditions can appreciably affectthe maximum amount of Fraction I protein attained in the firstleaf lamina. In unshaded seedlings early application resultsin a higher maximum amount of Fraction I protein per lamina,but not per unit lamina fresh weight. Late application of nitratehowever delays the age at which Fraction I protein reaches amaximum both in absolute terms and as a proportion of totalsoluble protein. For both control and shade-grown material earlyand higher rates of nitrate supply increase the maximum amountof soluble protein in the leaf but not the proportion representedby Fraction I protein. Lower rates of nitrate application havemuch less effect on first-leaf protein synthesis when applicationis given late. This is thought to be due to competition fromthe rapidly developing second leaf. Studies on the soluble protein content of shaded first-leaflaminae have shown that although grain size affects the maximumamount of Fraction I protein attained it does not alter theage at which this is attained; nor is the proportion of thetotal soluble protein accounted for by Fraction I protein affectedby grain size or grain nitrogen content. A model is proposed to explain the contribution made by grainreserves and exogenous carbon and nitrogen supply to the developmentof the soluble protein content of the first leaf.     

Synthesis of low-phenylalanine polypeptides

Low-phenylalanine-peptides for dietotherapy of phenylketonuria (PKU) were prepared from soybean protein isolate. Soluble fraction of soybean protein isolate was hydrolysed by alpha-chymotrypsin then followed by carboxypeptidase-A. Molecular weight distribution and amino acid analysis were made on the resultant polypeptides. The chymotrypsin hydrolysate was divided into two fractions, Fraction I (molecular weight greater than 2500) and Fraction II (molecular weight between 1000 and 2500). The phenylalanine content of Fraction I (3.1%) was lower than that of Fraction II (5%), indicating the nonuniform distribution of phenylalanine in soy bean protein. Carboxypeptidase hydrolysis of Fraction I further reduced the phenylalanine concentration to 2.3%, approximately half of the original concentration in soybean protein isolate.     

Pre-fractionation of archival frozen tumours for proteomics applications

We report a protocol for pre-fractionation of proteins from frozen tumours. Using this protocol two separate protein fractions are extracted: Fraction 1 is enriched for cytosolic and Fraction 2 for nuclear/nuclear membrane proteins. The accuracy and reproducibility of the protocol were demonstrated by Western blot analyses and SELDI-TOF-MS profilings. The resulting protein profiles were similar within the respective groups of Fractions 1 and 2 samples, without differences between freshly prepared tumours or those obtained as frozen tissue from biobanks.     

A simple electrophoretic procedure for the determination of the polypeptide composition of the subunits of Fraction 1 protein

A rapid and convenient method is described for resolving the polypeptide composition of Fraction 1 protein. Using crude leaf extracts of a number of Lycopersicon species, Fraction 1 protein was first separated by polyacrylamide gel electrophoresis and the gel slices containing the protein were isoelectrofocused in the presence of 8 m urea. Isoelectric focusing was also applied directly on subunits in gel slices obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polypeptide composition produced is in agreement with previous determinations obtained by more elaborated techniques.