Protein-free models for the proton-coupled reduction of haemoproteins

Reduction of the bis-pilocarpate-haemin complex at pH greater than or equal to 10 involves the simultaneous uptake of an electron by the Fe(III) ion and a proton by the pendant alkoxide group of an axial ligand. This provides a protein-free model for reactions such as the proton-coupled reduction of cytochromes which involve cooperative Coulombic interaction between two non-bonded sites.     

Critical importance of microsome concentration in mutagenesis assay with V79 Chinese hamster cells.

For optimum mutagensis in V79 Chinese hamster cells, the amount of liver postmitochondrial fraction in the assay was found to be of critical importance, depending on the chemicals being tested. Benzo[a]pyrene (BP) required lower (1-5%) concentrations of the liver 15 000 X g supernatant (S15) from methylcholanthrene pretreated rats for a maximum induction of cytotoxicity and mutagenicity, as determined by 8-azaguanine- and ouabain-resistance. A sharp peak of mutagenicity and cytotoxicity was induced by 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-diol BP) at a concentration of 1% of the S15 fraction. Little or no response was induced by these compounds with the S15 concentrations of more than 10%. Similarly, aflatoxin B1 induced a sharp peak of mutagenicity and cytotoxicity at a concentration of 2% of the liver S15 fraction from Aroclor-pretreated rats. Under the same condition, non-carcinogenic aflatoxin G2 did not induce cytotoxicity and mutagenicity. Analysis of BP metabolites by high-pressure liquid chromatography indicates that with the 30% S15 fraction, more than 80% of BP was metabolized during the first 15 min, while with the 2% S15 fraction, 7,8-diol BP increased continuously throughout the 120-min incubation period, suggesting a strong metabolic competition to rapidly remove BP and 7,8-diol BP with a high concentration of the S15. In contrast with these compounds, N-nitrosodimethylamine induced mutagenicity and cytotoxicity which increased linearly in proportion to the increasing amount of the S15 fraction from phenobarbitone- and Aroclor-pretreated rats. Various nitrosamines with different lipophilicity were examined at a high (30%) and low (2%) concentration of the S15 fraction from Aroclor-pretreated rats, in which ratios of mutation frequencies at 30% and 2% correlated inversely with lipophilicity of the compound. This result suggests that the lipid solubility of test compounds may be one factor which determines the concentration of post-mitochondrial supernatant for optimum mutagenesis.     

Rat and human embryo and post-natal sera contain a potent endogenous competitor of estrogen-rat alpha-fetoprotein interactions

A highly active inhibitor of the binding of estrone and estradiol-17β to rat alpha-fetoprotein is demonstrated for the first time in embryo, immature and adult rat sera as well as in fetal and adult human sera. The competitive character and the narrow specificity of this inhibition effect is shown. The major compound responsible for this activity is isolated by successive column Sephadex LH₂₀ and thin layer chromatography : it is characterized as a nonpolar, nonphenolic, dialysable and thermostable substance, unreactive towards anti-estrone and anti-estradiol-17β anti-bodies. The possible biological role of an endogenous non-estrogen ligand of rodent fetoproteins is discussed.     

Grain size,sucrose level and starch accumulation in developing rice grain

Five rices (Oryza sativa L.) differing in final grain size were studied at the midmilky stage to determine if any factor could be identified which might limit rate of starch accumulation. Only UDP glucose pyrophosphorylase activity increased with increasing grain size. Detached rice panicles incubated in liquid medium containing 1% sucrose and 0.1% glutamine, in addition to minerals and vitamins, produced grains similar to those on intact plants. Sucrose level (0–1.5%) in the medium determined the extent of dry matter and starch accumulation and influenced physiological development of the ripening grains. Chemical and enzymic composition of the grain were similar to previously reported levels in grains of intact panicles analysed at regular intervals after anthesis. Addition of 3-P glycerate or K⁺ to the medium did not improve dry matter accumulation in the developing grain.     

Influence of dicarboxylic phosphatidylcholines on phosphatidylcholine liposomes as revealed by gel chromatography and electron microscopy

The effect of dicarboxylic phosphatidylcholines (glutarylphosphatidylcholine) on the structural changes of phosphatidylcholine liposomes is examined by using multilamellar liposomes prepared with egg phosphatidylcholine or dipalmitoylphosphatidylcholine and by varying the surface charge by addition of dicetyl phosphate. Investigations are performed by gel chromatography and electron microscopy. Glutarylphosphatidylcholine is in micellar form (rod-like micelles or globular micelles). The structures obtained depend on the fatty acid saturation of liposomes and on the charge of liposome (addition or not of dicetyl phosphate). With egg phosphatidylcholine/glutarylphosphatidylcholine dispersions, an aspect more similar to myelinic figures than liposomes is observed, while in the presence of dicetyl phosphate, liposomes similar to control egg phosphatidylcholine liposomes are obtained. Gel chromatography on Sepharose 4B and turbidity measurements prove that dicetyl phosphate increases the stability of egg phosphatidylcholine/glutarylphosphatidylcholine mixtures. On the other hand, in dipalmitoylphosphatidylcholine/glutarylphosphatidylcholine dispersions, incorporation of dicetyl phosphate destabilizes bilayer structure and the formation of mixed micelles occurs. Viscosity measurement shows, in the presence of dicetyl phosphate, an increased fluidity for dipalmitoylphosphatidylcholine/glutarylphosphatidylcholine dispersions, in agreement with the micellar organization. These data confirm that the disorganization of liposomal membranes by dicarboxylic phosphatidylcholine depends on the fatty acid composition of phosphatidylcholine and on the presence of dicetyl phosphate.     

Chromatographic resolution of amino acid adducts of aliphatic halides

Numerous xenobiotics are known to be bioactivated and to covalently bind to proteins, but the resulting amino acid adducts (AAAs) are unknown. In this study the AAAs of twelve ¹⁴C-labeled aliphatic halides were examined after formation in an in vitro microsomal system. After exhaustive solvent extraction of the precipitated microsomal protein, the AAAs were isolated by Pronase digestion, followed by filtration through a 500 mol. wt. exclusion membrane. The liberated AAAs were applied to a constant flow DC-4A cation exchange column, resolved by stepwise buffer elution, collected and counted for radioactivity. Column recovery for applied radioactivity was 100 ± 4%. Generally, 1–4 different AAAs (defined by eluting radioactivity) were resolved, with each organohalogen displaying a characteristic elution profile. Methyl iodide, trichloroethylene and 1,2-dichloroethylene had a single major AAA while bromotrichloromethane, 1,2-dibromoethane, 1,1,1-trichloroethane, 1,2-dichloroethane, 1,1,2-trichloroethane, 2-bromo-2-chloro-1,1,1-trifluoroethane, chloroform and carbon tetrachloride had up to 4 AAAs or more, indicating combinations of binding site(s) and reactive intermediate(s). The single AAA formed following incubation of methyl iodide with the microsomes was identified as S-methylcysteine. Thus, this method appears capable of resolving binding sites and is the initial isolation step for identifying specific adducts to proteins.     

Ligand-exchange chromatographic resolution ofDl-amino acids in the presence of nucleic acids

Summary Nine representativeDl-amino acids were resolved using ligand-exchange chromatography whose mobile phase containe Cu (II) and ribonucleic acids. It was also found that deoxyribonucleic acids could be substituted for RNAs. In the presence of 5-GMP, 4XMP, (a mixture of 5-GMP,-AMP,-CMP, and-UMP), and oligomer of RNA,L-amino acids, which seemed to give more stable complexes, were eluted more slowly than the opposite isomers.     

肾综合征出血热病毒糖蛋白的提纯和鉴定

以感染肾综合征出血热病毒(HFRSV)的Vero E6细胞为材料,用免疫亲和层析结合制备聚丙烯酰胺凝胶电泳(PAGE)从感染细胞中提纯了HFRSV两种糖蛋白。先用免疫亲和层析从感染细胞的粗制抗原中获得含有四种蛋白的混合液,用[~3H]-氨基葡萄糖在感染细胞中标记病毒糖蛋白,观察到[~3H]-氨基葡萄糖只结合入78K和57K的病毒蛋白。再用制备SDS-PAGE从HFRSV混合液中提纯78K和57K两种蛋白。实验证明这两种糖蛋白均具中和抗原决定簇,57K的糖蛋白尚具血凝活性,初步鉴定表明这两种糖蛋白相当于文献报道的HFRSV G_1和G_2。     

A novel magnetic silica support for use in chromatographic and enzymatic bioprocessing

A limited number of support matrices have so far been developed for use in magnetically stabilized fluidized bed (MSFB) applications. We have developed a versatile magnetic silica support which can be derivatized readily for both adsorption chromatography and enzyme immobilization by well-known techniques. A magnetic pellicular bead is prepared by electrostatically depositing alternating layers of colloidal silica and cationic polymer onto macroscopic nickel core particles. The polymer is then burned out and the silica partially sintered to yield a porous shell with 5-80 m(2)/g of surface area. This magnetic composite was tested as a support for immobilizing invertase. Sucrose was continuously converted to its component monosaccharides with nearly constant activity over the first 8 days and retention of 50% of initial activity after 25 days.