Interactions of native and modified cytochrome C with a negatively charged reverse micellar liquid interface

Native and chemically modified cytochrome C were dissolved in sodium bis(2-ethylhexyl) sulphosuccinate (AOT)-oil-buffer microemulsions. The native cytochrome C contains 19 lysine residues, these groups were modified by 1) acetic anhydride or 2) succinic anhydride. At pH 8.4 the native, acetylated and succinylated proteins carry +8, –3 and –12 elementary charges, respectively. The phase behaviour of the microemulsion systems was found to be highly dependent on the charge of the proteins. Compared to a protein free system the native protein induces a L-2 phase separation at lower temperatures. The acetylated protein has a small effect on the temperature for the phase transition, whereas in the case of succinylated cytochrome C the phase transition takes place at higher temperatures. When dissolved in AOT microemulsions, the native cytochrome C has a perturbed tertiary structure, as indicated by loss of the 695 nm absorption band, while both the modified proteins retain the same optical properties when dissolved in an AOT microemulsion as in a pure buffer solution. The pertubed structure of the native cytochrome C was further investigated by testing the stability of the reduced form of the protein dissolved in the microemulsion media. The native cytochrome is unstable at W > 10, whereas the two modified proteins were found to be stable at all W-values investigated. The average location of the three proteins was determined by pulse radiolysis. The quenching rate constant of the hydrated electron depends upon the location of the probe in the reverse micelle; the succinylated protein is localised in the aqueous core of the reverse micelles, but both the native and the acetylated forms were found to be localised close to or at the AOT interface.     

Whole inactivated SIV vaccine grown on human cells fails to protect against homologous SIV grown on simian cells

Several groups have reported protection against experimental SIV infection in macaques immunized with a whole inactivated virus vaccine. The aim of the current study was to investigate whether five macaques vaccinated with whole inactivated SIV and previously shown to be protected against challenge with two divergent strains of SIV grown on human cells could resist challenge with a subsequent homologous SIV grown on macaque cells. We show here that this same vaccine did not protect when the challenge virus was grown on primary cells of monkey origin.     

Phosphorylation of the Na,K-ATPase by Ca,phospholipid-dependent andcAMP-dependent protein kinases. Mapping of the region phosphorylated by Ca,phospholipid-dependent protein kinase

Ca,phospholipid-dependent (PKC) andcAMP-dependent (PKA) protein kinases phosphorylate the -subunit of the Na,K-ATPase from duck salt gland with the incorporation of 0.3 and 0.5 mol³²P/mol of -subunit, respectively. PKA (in contrast to PKC) phosphorylates the -subunit only in the presence of detergents. Limited tryptic digestion of the Na,K-ATPase phosphorylated by PKC demonstrates that³²P is incorporated into the N-terminal 41-kDa fragment of the -subunit. Selective chymotrypsin cleavage of phosphorylated enzyme yields a 35-kDa radioactive fragment derived from the central region of the -subunit molecule. These findings suggest that PKC phosphorylates the -subunit of the Na,K-ATPase within the region restricted by C₃ and T₁ cleavage sites.     

Production and composition of extracellular polysaccharide from cell suspension cultures of Mentha

A suspension culture of Mentha was established from callus which formed on the tips of young shoots of a Mentha hybrid (M. arvenis × M. spicata). Changes in growth parameters during a culture cycle were recorded. The general appearance of cells during division and growth, including the changes in cell form, was also represented.Suspension-cultured cells of Mentha hybrid released a large amount of extracellular polysaccharides (ECP) mainly at the logarithmic phase of the growth cycle. The ECP contained galacturonic acid as major components and arabinose, galactose, glucose, xylose, rhamnose and mannose as minor components. The ratio of the uronic acid content to total sugar content in the ECP was below 40% at day 7, but increased up to 90% at day 21. The relative contents of xylose and glucose in the ECP decreased during the culture period, while the arabinose content increased and those of rhamnose, mannose and galactose remained constant.The IR spectrum suggested that the ECP were low-methoxylated pectic polysaccharides. The presence of lignin and related compounds in the ECP was not detected. The protein content of the ECP was about 10% and the main amino acids were alanine, proline, hydroxyproline, valine, asparticacid and serine, in that order.     

Cloning,sequence analysis and expression of a cDNA encoding active phosphoenolpyruvate carboxylase of the C3 plant Solanum tuberosum

A cDNA coding for phosphoenolpyruvate carboxylase (PEPC) was isolated from a cDNA library from Solanum tuberosum and the sequence of the cDNA was determined. It was inserted into a bacterial expression vector and a PEPC^- Escherichia coli mutant could be complemented by the cDNA construct. A functional fusion protein could be synthesized in E. coli. The properties of this PEPC protein clearly resembled those of typical C3 plant enzymes.     

关于吸附精制百日咳菌苗、白喉、破伤风类毒素混合制剂配方的实验报告

本文对吸附精制百白破混合制剂的不同配方进行了实验,结果表明,新一代吸附精制百白破混合制剂最佳配方为：精制百日咳菌苗１８μｇＰＮ／ｍｌ、精制白喉类毒素为３０Ｌｆ／ｍｌ、精制破伤风类毒素为１０Ｌｆ／ｍｌ。由该配方组成的吸附精制百白破混合制剂,其中百日咳菌苗的毒性试验ＢＷＤＵ／ｍｌ、ＬＰＵ／ｍｌ、ＨＳＵ／ｍｌ三种指标均符合制检规程要求。其效力单位（ＩＵ／ｍｌ）超过规程要求一倍以上,精制白喉和破伤风类毒素的安全试验均符合规程要求,白类效力试验≥８０－１００％,破类效力试验≥０．５－４．５ＩＵ／ｍｌ。上述结果说明本文提出的配方作为新一代精制百白破混合制剂的配方是适宜和实用的。     

Directional mutation pressure,mutator mutations,and dynamics of molecular evolution

Using a general form of the directional mutation theory, this paper analyzes the effect of mutations in mutator genes on the G + C content of DNA, the frequency of substitution mutations, and evolutionary changes (cumulative mutations) under various degrees of selective constraints. Directional mutation theory predicts that when the mutational bias between A/T and G/C nucleotide pairs is equilibrated with the base composition of a neutral set of DNA nucleotides, the mutation frequency per gene will be much lower than the frequency immediately after the mutator mutation takes place. This prediction explains the wide variation of the DNA G + C content among unicellular organisms and possibly also the wide intragenomic heterogeneity of third codon positions for the genes of multicellular eukaryotes. The present analyses lead to several predictions that are not consistent with a number of the frequently held assumptions in the field of molecular evolution, including belief in a constant rate of evolution, symmetric branching of phylogenetic trees, the generality of higher mutation frequency for neutral sets of nucleotides, the notion that mutator mutations are generally deleterious because of their high mutation rates, and teleological explanations of DNA base composition. Presented at the NATO Advanced Research Workshop onGenome Organization and Evolution, Spetsai, Greece, 16–22 September 1992     

Regional Distribution and Quantitative Measurement of the Phosphoinositidase C-Linked Guanine Nucleotide Binding Proteins G1α and Gqα in Rat Brain

Abstract: Levels of the guanine nucleotide binding proteins G₁₁α and G_qα, which produce receptor regulation of phosphoinositidase C., were measured immunologically in 13 regions of rat central nervous system. This was achieved by immunoblotting membranes from these regions with antisera (CQ series) that identify these two polypeptides equally, following separation of the membranes using sodium dodecyl sulphate-polyacrylamide gel electrophoresis conditions that can resolve G_qα and G₁₁α. In all regions examined, G_qα was more highly expressed than G₁₁α. Ratios of levels of G_qα to G₁₁α varied between the regions from 5:1 to 2:1. Quantitative measurements of the levels of G_qα and G₁₁α in each region were obtained by comparison with known amounts of purified liver G_qα and G₁₁α and with E. coli expressed recombinant G_qα. Areas that expressed G_qα highly included olfactory bulb (930 ng/ mg of membrane protein), frontal cortex (700 ng/mg of membrane protein), parietal occipital cortex (670 ng/mg of membrane protein), caudate putamen (1,003 ng/mg of membrane protein), hippocampus (1,045 ng/mg of membrane protein), hypothalamus (790 ng/mg of membrane protein), and cerebellum (950 ng/mg of membrane protein). More modest levels were observed in thalamus (450 ng/mg of membrane protein), pituitary (480 ng/mg of membrane protein), optic chiasma (330 ng/mg of membrane protein), and spinal cord (350 ng/mg of membrane protein). Gna was more evenly expressed with values ranging from about 170 ng/mg of membrane protein in spinal cord and optic chiasma to close to 300 ng/mg of membrane protein in regions expressing high levels of G_qα. A third polypeptide could be identified by the CQ antisera in all brain regions. The possibility that this polypeptide is the α subunit of G₁₄ is discussed.     

Cerebral Metabolic Compartmentation as Revealed by Nuclear Magnetic Resonance Analysis of D-[1-13C]Glucose Metabolism

Abstract: Nuclear magnetic resonance (NMR) was used to study the metabolic pathways involved in the conversion of glucose to glutamate, γ-aminobutyrate (GABA), glutamine, and aspartate. d -[1^-13C]Glucose was administered to rats intraperitoneally, and 6, 15, 30, or 45 min later the rats were killed and extracts from the forebrain were prepared for ¹³C-NMR analysis and amino acid analysis. The absolute amount of ¹³C present within each carbon-atom pool was determined for C-2, C-3, and C-4 of glutamate, glutamine, and GABA, for C-2 and C-3 of aspartate, and for C-3 of lactate. The natural abundance ¹³C present in extracts from control rats was also determined for each of these compounds and for N-acetylaspartate and taurine. The pattern of labeling within glutamate and GABA indicates that these amino acids were synthesized primarily within compartments in which glucose was metabolized to pyruvate, followed by decarboxylation to acetyl-CoA for entry into the tricarboxylic acid cycle. In contrast, the labeling pattern for glutamine and aspartate indicates that appreciable amounts of these amino acids were synthesized within a compartment in which glucose was metabolized to pyruvate, followed by carboxylation to oxaloacetate. These results are consistent with the concept that pyruvate carboxylase and glutamine synthetase are glia-specific enzymes, and that this partially accounts for the unusual metabolic compartmentation in CNS tissues. The results of our study also support the concept that there are several pools of glutamate, with different metabolic turnover rates. Our results also are consistent with the concept that glutamine and/or a tricarboxylic acid cycle intermediate is supplied by astrocytes to neurons for replenishing the neurotransmitter pool of GABA. However, a similar role for astrocytes in replenishing the transmitter pool of glutamate was not substantiated, possibly due to difficulties in quantitating satellite peaks arising from ¹³C-¹³C coupling.     

Prolactin-Stimulated Mitogenesis of Cultured Astrocytes Is Mediated by a Protein Kinase C-Dependent Mechanism

Abstract: Prolactin (PRL) has been reported to activate cellular proliferation in nonreproductive tissue, such as liver, spleen, and thymus. Recently, we have extended the possible role of PRL as a mammalian mitogen by demonstrating a mitogenic effect of PRL in cultured astrocytes. Although the cellular mechanisms by which PRL regulates cell growth are not fully understood, protein kinase C (PKC) has been implicated as one of the transmembrane signaling systems involved in the regulation of PRL-induced cell proliferation in Nb2 lymphoma cells and liver. In the present studies, we examined the possible role of PKC in PRL-induced proliferation of cultured astrocytes. Incubation of cultured astrocytes with 1 nM PRL resulted in a rapid translocation of PKC from the cytosol to the membrane, with maximal PKC activity in the membrane occurring 30 min after exposure to PRL. Translocation of PKC activity occurred over a physiological range of PRL, with maximal PKC activation occurring at 1 nM. At concentrations greater than 10 nM PRL, there was a decrease in the amount of PKC activity associated with the membrane fraction compared with that of cells stimulated with 1 nM PRL. Incubation of astrocytes with PRL in the presence of the PKC inhibitors staurosporine, 1-(-5-isoquinolinesulfonyl)-2-methylpiperazine, or polymyxin B blocked the PRL-induced increase in cell number with IC₅₀ values of approximately 2 nM, 10 μM, and 6 μM, respectively. PKC is the only known cellular receptor for 12-O-tetradecanoylphorbol 13-acetate (TPA), which stimulates the translocation of PKC from the cytosol to the membrane. Incubation of astrocytes with 20 nM TPA resulted in an increase in the expression of proliferating cell nuclear antigen and cell number, whereas 4α-phorbol 12,13-didecanoate, an inactive phorbol ester, was ineffective. To examine further the effect of TPA and PRL on cellular proliferation, cultured astrocytes were incubated with increasing concentrations of TPA in the presence or absence of a minimal effective dose of PRL (100 pM). In the absence of PRL, incubation with TPA resulted in an inverted U-shaped dose-response curve, with 100 nM TPA resulting in a maximal increase in cell number. In the presence of 100 pM PRL, the TPA dose-response curve was shifted to the left, with maximal activity occurring with 10 nM TPA. Chronic stimulation of astrocytes with 500 nM TPA depleted the cells of PKC and blocked the PRL-induced increase in cell number. Finally, TPA treatment decreased cell-surface binding of ¹²⁵I-PRL. These data indicate that the PKC is involved in the mitogenic effect of PRL in cultured astrocytes.