Antitumor Protein-containing Polysaccharides from a Chinese Mushroom Fengweigu or Houbitake,Pleurotus sajor-caju (Fr.) Sings

Protein-containing polysaccharides extracted from fruiting bodies of a Chinese fungus named Feng Wei Gu, were fractionated and purified, and their antitumor activities were tested, out of which the following active fractions were obtained.

FI_o-a: A protein-containing xyloglucan, MW 280,000, polysaccharide: protein=76: 24 (w/w), polysaccharide consisting of Man: Gal: Xyl: Glc = 2: 12: 42: 42 (molar ratio). + 25.3°.

FA-2: A protein-containing mannogalactan, MW 120,000, polysaccharide: protein = 76 : 16 (w/w), consisting of Xyl : Man: Gal = 9 : 35 : 56 (molar ratio), + 98.5°.

FII-1: A Protein-containing xylan (62: 21 w/w). MW 200,000, +8.7°.

FIII-1a: A protein-containing glucoxylan (15: 71 w/w), +30.7°, MW 90,000, consisting of Glc : Xyl = 40 : 44 (molar ratio).

FIII-2a: A protein-containing xyloglucan, MW 70,000, polysaccharide: protein = 69: 3 (w /w), polysaccharide consisting of Xyl: Glc = 36 : 62 (molar ratio). + 38.6°.     

Antitumor Active Fucoidan from the Brown Seaweed,Umitoranoo (Sargassum thunbergii)

Neutral and acidic polysaccharides and their protein complexes were fractionated and purified from the brown seaweed umitoranoo (Sargassum thunbergii) by fractional extraction, iron-exchange chromatography, and gel filtration. Thirty-one polysaccharide fractions were obtained and tested for antitumor activity in mice with Ehrlich carcinoma transplanted i.p. Two of the fractions, GIV-A ( – 127° and mol. wt., 19,000) and GIV-B ( – 110° and mol. wt., 13,500) had such activity. On the basis of chemical and spectral analyses, these compounds were found to be a fucoidan or L-fucan containing approx. 30% sulfate ester groups per fucose residue, about 10% uronic acid, and less than 2% protein.     

Cytotoxicity of 7-Ketocholesterol toward Cultured Rat Hepatocytes and the Effect of Vitamin E

The effects of 7-ketocholesterol on rat hepatocytes prepared by collagenase perfusion were examined. The viability of cells incubated with 100 μm 7-ketocholesterol was significantly lower than those with cholesterol, although the LDH activity in the cultured medium remained unchanged during the incubation. Hepatocytes treated with 7-ketocholesterol produced large amounts of ·NO and in the early stage of incubation. Treatment of the hepatocytes with Carboxy-PTIO, which selectively scavenged ·NO, or with l-NMMA, an inhibitor of ·NO synthase, increased the cell viability. The addition of 7-ketocholesterol to the culture medium tended to increase the ratio of total sterol to phospholipid of the hepatocytes in a time-dependent manner without changing the content of phospholipid. No lipid peroxidation or oxidation of the cellular SH groups, protein SH and glutathione, was apparent. Vitamin E added 1 h before the addition of 7-ketocholesterol prevented the hepatocytes from cell death by suppressing the incorporation of 7-ketocholesterol into the hepatocytes and by scavenging .     

Quantitative Determination of 14C-Ethylene Produced by Plant Tissues

Pseudomonas testosteroni ATCC 11996 was found to produce a novel bile acid sulfate sulfatase that hydrolyzes the sulfate ester bond in lithocholic acid sulfate (LCA-S). The enzyme synthesis was induced by several kinds of bile acids including LCA-S. Mn²⁺ functioned as an essential component for the enzyme synthesis and suppressed it. This sulfatase hydrolyzes LCA-S to isolithocholic acid and sulfuric acid with inversion of α- to β-configuration of the hydroxyl group at the third position of lithocholic acid.     

Purification and Characterization of a Cysteine Protease from Corms of Freesia,Freesia reflacta

A protease (freesia protease B) has been purified to electrophoretic homogeneity from corms of freesia, Freesia reflacta by five steps of chromatography. Its M_r was estimated to be about 26,000 by SDS–PAGE. The optimum pH of the enzyme was 6.0–7.0 at 30°C using casein as a substrate. The enzyme was strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethanesulphonylfluoride and EDTA. These results indicate that freesia protease B is a cysteine protease. Nine sites of oxidized insulin B-chain were cleaved by freesia protease B in 24 h of hydrolysis. The four cleavage sites among them resembled those of papain. From the digestion of five peptidyl substrates the specificity of freesia protease B was found to be approximately broad, but the preferential cleavage sites were negatively charged residues at positions. Freesia protease B preferred also the large hydrophobic amino acid residues at the P₂ position, in a similar manner to papain. The amino terminal sequence of freesia protease B was identical with those of papain in regard to the conservative residues of cysteine protease.     

Determination of CGTase from Bacillus ohbensis and Its Optimum pH Using HPLC

Glucose is widely known to be required during superoxide generation in phagocytic cells. However, when an specific chemiluminescence probe with the Cypridina luciferin analog 2-methyl-6-(p-methoxyphenyl)-3, 7 -dihydroimidazo[ 1,2-a]pyrazin-3-one (MCLA) was used, about 60% of the chemiluminescence remained in stimulated macrophages in the presence of the glycolytic inhibitor 2-deoxyglucose. -nonspecific luminol-dependent chemiluminescence disappeared when the same drug was added. These results clearly demonstrate that the generation of by macrophages is not completely glucose-dependent, and strongly suggest that macrophages have both glucoseindependent NADPH-supplying pathway(s) and glucose dependent pathway(s) which generate reactive oxygen species other than .     

Molecular Characteristics of Water-soluble Polysaccharide Extracted from Jelly Fig (Ficus awkeotsang Makino) Seeds

The molecular characteristics of polysaccharide obtained by extracting with water from jelly fig (Ficus awkeotsang Makino) seeds were elucidated by measuring the weight-average molecular weight (M_w) and radius of gyration (R_G), indicating the molecular expanse, by the light-scattering method.

The M_w and R_G values of the water-soluble jelly fig polysaccharide were 84–130 × 10⁴ and 240–450 mm, respectively, these values being larger than those of commercial LM pectin and HM pectin. In addition, the M_w and R_G values of the water-soluble jelly fig polysaccharide initially increased after being extracted, indicating the highest values 5 hours after the extraction, and thereafter decreased. These changes in the time-course of the molecule reflected well the changes with time in the mechanical characteristics and network structure of the water-soluble jelly fig polysaccharide gel. Exponent α in the expression was found to be 0.37. From these results, the conformation of the water-soluble jelly fig polysaccharide molecule after association by the contained inorganic elements proved to be of globular form rather than a random coil shape as a result of contraction of the molecule.     

Lipolysis by Intracellular Lipase of Streptococcus lactis against Its Neutral Lipids Obtained by Growth at Low Temperature

Lipolytic activities of intracellular lipase obtained from Streptococcus lactis 527 cells grown at 30°C were determined using bacterial neutral lipids extracted from cells grown at 10 and 30°C. The amounts of free fatty acids liberated from lipids by lipase were in the order: 30°C neutral lipid > 10°C neutral lipid > triolein > intracellular membrane fraction. Glycerides hydrolyzed partially by lipase were detected on thin-layer plates and were composed of 1,3- and 1,2-diglycerides, fatty acids and unhydrolyzed triglycerides. Fatty acids liberated from neutral lipids by lipase were determined by gas chromatography. It was found that the major acid was cy-C₁₀ and the minor among the acids liberated from 10°C neutral lipid, whereas the major acid was and the minors and cy-C₁₀ from 30°C lipid.     

The Chemistry of Ilexol. IV.

It has now been shown that Acetate A previously obtained by isomerizing ilexol acetate is nothing other than urs-13 (18)-en-3β-yl acetate prepared from α-amyrin, furnishing conclusive evidence for its conversion into one of the compounds of the ursane series.

Isoilexol, oxidized with chromium trioxide at room temperature, afforded a ketone named isoilexone, C₃₀H₄₈O, m.p. 194–195°, +75.90° (c, 0.527), and oxidized with the same oxidant at 70–80°, another ketone named isoilexenedione, C₃₀H₄₆O₂, m.p. 221-223°, +16.28° (c, 0.307).

Huang-Milon reduction of these ketones afforded one and the same deoxy compound named isoilexene, C₃₀H₅₀, m.p. 183–185°, +48.34° (c, 0.290), which might well be assumed to constitute a pair of optical antipodes with olean-13 (18)-ene, C₃₀H₅₀, m.p. 184-185°, ?48.50° (c, 0.545).     

Changes in Amino Acid Composition during Germination of Soybean

Malonogalactan, a malonylated polysaccharide (—74° (c=1.6, H₂O)) produced by Penicillium citrinum, consisted of d-galactose and malonic acid in the approximate molar ratio of 3:1. Molecular weight of the demalonylated galactan (-99° (c=4.6, H₂O)) was about 40,000. From the data regarding optical rotation, nuclear magnetic resonance spectrum, infrared spectrum, glycosidase susceptibility, periodate oxidation, Smith degradation, methylation and acid hydrolysis, the possible structure of the Penicillium malonogalactan is deduced as follows: A galactan, 1,5-β-galactofuranoside polymer esterified with malonic acid at the position of 2 or 3.     

Substrate Specificity of α-Glucosidase II in Rice Seed

The substrate specificity of rice α-glucosidase II was studied. The enzyme was active especially on nigerose, phenyl-α-maltoside and maltooligosaccharides. The actions on isomaltose and phenyl-α-glucoside were weak, and on sucrose and methyl-α-glucoside, negligible. The α-glucans, such as soluble starch, amylopectin, β-limit dextrin, glycogen and amylose, were also hydrolyzed.

The ratio of the maximum velocities for hydrolyses of maltose (G₂), nigerose (N), kojibiose (K), isomaltose (I), phenyl-α-maltoside (?M) and soluble starch (SS) was estimated to be 100: 94.4: 14.2: 7.1: 89.5: 103.1 in this order, and that for hydrolyses of malto-triose (G₃), -tetraose (G₄), -pentaose (G₅), -hexaose (G₆), -heptaose (G₇), -octaose (G₈), and amyloses ( and ), 113: 113: 113: 106: 113: 100: 106: 106. The Km values for N, K, I, ?M and SS were 2.4 mm, 0.58 mm, 20 mm, 1.6 mm and 5.0 mg/ml, respectively; those for G₂, G₃, G₄, G₅, G₆, G₇, G₈, and , 2.4 mm, 2.2 mm, 2.1 mm, 1.5 mm, 1.0 mm, 1.1 mm, 0.95 mm, 1.5 mm and 1.1 mm.

Rice α-glucosidase II is considered an enzyme with a preferential activity on maltooligosaccharides.     

Hydrolysis Rate of Resistant Pentosan of White Birch Wood in Sulfuric Acid of Medium Strength

The purpose of this study is to find optimal conditions for pre-hydrolysis in the new wood saccharification process with strong sulfuric acid. In the experiment, the hydrolysis rate of resistant fraction of pentosan of white birch (Shirakamba, Betula platyphylla Sukatchev var. japonica Hara) wood and the decomposition rate of xylose are measured in acid concentrations ranging from 30 to 60% at temperatures ranging from 30 to 90°C. The hydrolysis of resistant pentosan of white birch and the decomposition of xylose are the first-order reactions. The first-order reaction constant of hydrolysis of resistant pentosan, k_B min^-1, is expressed by the following empirical equations as the function of percentage concentration of sulfuric acid, C, and reaction temperature described by absolute temperature, T°K, ranging from 40 to 80°C:

where sulfuric acid concentrations range from 30 to 50%;

where sulfuric acid concentration is 60%.

The first-order reaction constant of decomposition of xylose, k₂ min^-1, is expressed by the following empirical equation as the function of sulfuric acid strength described by acidity function, H₀, and reaction temperature described by absolute temperature, T°K, in sulfuric acid concentrations ranging from 30 to 60% at temperatures within the range of 40 to 100°C.

where C is sulfuric acid strength described by acidity function, H₀.     

Radiation Chemistry of Foods

When 10^?3m cysteine solution was irradiated in the presence of glucose at the concentration of ten-fold of cysteine, the G-values of products produced from cysteine were similar to those from 10^?3m cysteine solution. On the other hand, the yield of carbonyl compound from glucose was suppressed completely by interaction between cysteine and radicals which are secondarily produced from glucose.

Methionine could not suppress the yield of carbonyl compound from glucose, and, G-values of products from methionine varied in comparison with those from solution containing methionine only.

From the results using scavenger, it was concluded that oxidation to methionine sulfoxide and cleavage to α-aminobutyric acid was caused by OH and attack, respectively.     

New Strains of Botryodiplodin-producing Fungi

The light-emitting species of chemiluminescence produced in rat liver homogenate on adding autoxidized linseed oil (AOLO) were investigated. The chemiluminescent intensity of liver homogenate was strongly enhanced by the addition of AOLO and showed a proportional relationship to the amount of AOLO. The chemiluminescence was reduced with singlet oxygen (¹O₂) quenchers and free radical scavengers. Among them, β-carotene showed the most effective quenching. The emission spectrum had broad bands in the visible region with eminent chemiluminescent lines at 520, 575 and 640 nm due to the simultaneous transition, . An additional weak line was found at 480 nm corresponding to . In the presence of β-carotene, lines corresponding to the simultaneous transition of ¹O₂ disappeared. These results indicate that the liver homogenate with AOLO generated singlet molecular oxygen as one of the major light-emitters of the chemiluminescence. A possible mechanism for the generation of ¹O₂ is by decomposition of peroxy radicals derived from AOLO in the liver homogenate.     

Red Algae (Eucheuma muricatum) Mucilage- Complex Formation and Mechanism

Eucheuma muricatum mucilage which was extracted and purified after irradiation of the seaweed with γ-ray of ⁶⁰Co formed a complex with , and exhibited a new absorption band at 555 nm. The absorbancy observed at that time depended on the concentration of urea and on the temperature. The curves representing relations between absorbancy at 555 nm and the above factors have two inflection points. The fact that their inflection points shift toward the lower temperature side with the increase in urea concentration suggests that the coloring phenomenon may relate closely to the transition of the mucilage. It was also found that the absorbancy at 555 nm depended on the content of pyruvic acid residue in the same mucilages, the absorbancy decreased with the increase pyruvic acid residues, and that the steric hindrance caused by a sugar residue of large demension affected the stable from containing viscous polysaccharide.     

Studies on Bacterial Protease

A crystalline alkaline protease was prepared from B. amylosacchariticus, which was isolated as a strain of saccharogenic α-amylase-producing Bacillus subtilis. The enzyme was most active at pH values between 10.3 and 10.7 towards casein and was stable at pH values from 6 to 11 on twenty hour incubation at 30°C. Calcium ions were effective to stabilize the enzyme especially at higher temperatures. The enzyme was markedly inactivated by DFP as well as protease inhibitor from potato and slightly by surface active agents, but not affected by sulfhydryl reagents and divalent metal ions except Hg⁺⁺ .Hemoglobin was the best substrate for the enzyme and more than 20% of the peptide bonds were hydrolyzed. Of numerous synthetic peptides tested, only the two compounds, and , were found to be hydrolyzed. A cyclic peptide, gramicidin S, was split by the enzyme only at the peptide bond of -l-valyl-l-ornithyl-. Methyl n-butyrate and tributyrin were also good substrates for the alkaline protease obtained here.     

Aromatic Amino Acid Residues in Japanese-radish Peroxidase a and Apoenzyme†

The nature of aromatic amino acid residues in Japanese-radish peroxidase a and the apoprotein was investigated by means of spectrophotometry and fluorospectrophotometry. The tyrosine residues in the holoenzyme were masked in the alkali-titration, giving an abnormally high value of 12.6, while they were exposed in the apoenzyme, exhibiting a value of 10.8. The difference spectra in the ultraviolet region between the holo-and apo-enzyme showed characteristic bands of tryptophan and phenylalanine as well as tyrosine. The perturbation of the aromatic amino acid residues by 50% ethyleneglycol was observed in the apoenzyme but not in the holoenzyme. The fluorophotometric experiments also revealed that the aromatic amino acid residues were in different environments in the holo- and apoenzyme. The difference between the conformation of peroxidase and that of the apoprotein was discussed.