Effect of pKM101 on cell killing and specificity of mutation induction by cis-diaminedichloroplatinum(II) in Escherichia coli K-12.

Cell killing and mutation induction in the lacI gene of Escherichia coli by cis-Pt(NH3)2Cl2 were studied in cells with different repair capacities, with and without pKM101. The presence of the plasmid pKM101 made repair-proficient cells more susceptible to killing by cis-Pt(NH3)2Cl2 and strongly enhanced mutation induction by that compound. Both effects were shown to be dependent upon excision repair. Characterization of the induced mutations in the lacI gene after cis-Pt(NH3)2Cl2 treatment of E. coli cells, by the LacI system, revealed that the mutagenic specificity of the Pt compound was strongly influenced by the presence of the pKM101 plasmid. With pKM101, 23% of the induced amber and ochre mutations resulted from substitutions at AT base pairs, whereas these mutations were hardly induced in cells without pKM101. These results suggest that pKM101-induced repair differs from normal SOS repair.     

Regulation of 3 beta-hydroxysteroid dehydrogenase activity by human chorionic gonadotropin, androgens, and anti-androgens in cultured testicular cells

delta 5-3 beta-Hydroxysteroid dehydrogenase is a key enzyme for testicular androgen biosynthesis and a marker for the Leydig cells. The hormonal regulation of this enzyme was studied in cultured rat testicular cells. Human chorionic gonadotropin (hCG) increased testosterone production in vitro while time course studies indicated a biphasic action of the gonadotropin on 3 beta-hydroxysteroid dehydrogenase activity. An initial stimulation (51%) of the enzyme was detected between 3 and 12 h of culture when medium testosterone was low. This is followed by an inhibition of 3 beta-hydroxysteroid dehydrogenase activity on days 2 and 3 of culture when medium testosterone was elevated. Concomitant treatment with a synthetic androgen (R1881) inhibited 3 beta-hydroxysteroid dehydrogenase activity and testosterone production in hCG-treated cultures while an anti-androgen (cyproterone acetate) increased 3 beta-hydroxysteroid dehydrogenase activity and testosterone biosynthesis. Addition of 10(-5) M spironolactone, an inhibitor of 17 alpha-hydroxylase, blocked the hCG stimulation of testosterone production but increased medium progesterone. In the absence of the secreted androgen, hCG stimulated 3 beta-hydroxysteroid dehydrogenase activity in a time- and dose-related manner. Furthermore, hCG stimulation of 3 beta-hydroxysteroid dehydrogenase activity and progesterone accumulation in spironolactone-supplemented cultures was decreased by concomitant treatment with R1881 but was not affected by cyproterone acetate. The inhibitory effect of R1881 was blocked by the anti-androgen. In the absence of hCG, treatment with testosterone, dihydrotestosterone, or R1881, but not promegestone, alone also inhibited 3 beta-hydroxysteroid dehydrogenase activity while the inhibitory effect of testosterone was blocked by cyproterone acetate. Thus, hCG stimulates 3 beta-hydroxysteroid dehydrogenase activity in cultured testicular cells. The androgenic steroidogenic end products, in turn, inhibit this enzyme. The hormonal regulation of 3 beta-hydroxysteroid dehydrogenase activity may be important in the ultrashort loop autoregulation of androgen biosynthesis.     

Synthesis of hydroxy fatty acids from 4, 7, 10, 13, 16, 19-[1-14C] docosahexaenoic acid by human platelets

Human platelets incubated in the presence of 54 microM [1-14C]22:6 produced hydroxydocosahexaenoic acid (HDHE) at about half the rate with which 12-hydroxy-5,8,10,14-eicosatetraenoic acid is produced from [1-14C]arachidonic acid. More than 90% of the radioactivity in HDHE was distributed among two major isomers, 14-HDHE and 11-HDHE. The production of HDHEs was unaffected by indomethacin but completely inhibited by 5,8,11,14-heneicosatetraynoic acid, which suggests that the hydroxy fatty acids are produced by lipoxygenase. The proportions of HDHE isomers varied with the concentration of 22:6. The ratio 14-HDHE/11-HDHE was higher at 6.8 microM 22:6 than when platelets were incubated with 54 microM 22:6. It is suggested that the amounts of these isomers produced will depend both on the availability of 22:6 as well as by competition of this acid with other acids for lipoxygenase.     

Leishmania mexicana amazonensis: Surface charge of amastigote and promastigote forms

The surface charge of Leishmania mexicana amazonensis was evaluated by means of the binding of colloidal iron hydroxyde particles at pH 1.8 and cationized ferritin particles at pH 7.2 to the cell surface, visualizated by electron microscopy and by direct measurements of the electrophoretic mobility of cells suspended in solutions of different pH. The following forms of the parasite were analysed: amastigotes (surrounded or not by the membrane of the endocytic vacuole, isolated from lesions), transitional forms, and infective (5 passages) and noninfective (176 passages) promastigotes. The results obtained indicate that the surface of L. m. amazonensis contains both negatively and positively charged dissociating groups and that changes occur in the surface charge during amastigote-promastigote transformation. Treatment of the parasite with neuraminidase significantly reduced the electrophoretic mobility of the cells. Neuraminidase-treated cells recovered their normal electrophoretic mobility when incubated for 8 hr in fresh culture medium by a process that is inhibited by puromycin.     

Chromosomal location of the active NORs in theSteropleurus martorelli complex

The chromosomal location of the active NORs has been analyzed by a silver impregnation procedure in theSteropleurus martorelli complex. A primary NOR, which is always present at the first meiotic prophase, has been found in each of the four described races. In addition to this, all races possess one or two secondary NORs which are less active than the former and can be occasionally shown. Usually only one of the two homologous chromosomes has been found to be involved with nucleolus organisation.These results are discussed in relation to hypotheses on the chromosome differentiation of this species complex.     

Residual beta-cell function in type II diabetes and evaluation of the hepatic insulin extraction

Insulin and C-peptide (free insulin and C-peptide in insulin-treated patients) were measured after glucose stimulation in nine Type II diabetics on chlorpropamide, eleven insulin-treated maturity-onset diabetics and in 8 normal controls. Dissociation between C-peptide and insulin response to glucose was observed in several diabetics. The relation between incremental molar areas under C-peptide and insulin curves, after glucose challenge (delta CPR - delta IRI/delta CPR) were used to evaluate the hepatic insulin extraction in all but the insulin-treated diabetics. The lower insulin requirements and better control of the short-duration insulin-treated maturity-onset diabetics in relation to the long-term ones could not be explained either by the residual insulin secretion or by the level of "insulin antibodies". The chlorpropamide-responsive patients presented higher insulin levels after the glucose challenge and a lower hepatic insulin extraction than the non-responsive ones.     

Comparative studies of seed proteins of the genusArtocarpus with respect to lectins

Proteins from two species of the genusArtocarpus (A. integrifolia L. andA. incisa L.) were compared by ammonium sulphate fractionation, molecular sieve chromatography and SDS-polyacrylamide gel electrophoresis, with special attention to the lectins. The protein content and hemagglutinating activity were markedly different in the two seeds. The protein pattern obtained by both molecular sieve chromatography and SDS-polyacrylamide gel electrophoresis were quite different. The only similarities found were the elution volume of the lectins in the Sephadex G-100 column and the lectin bands (11 500 and 15 000 daltons) in SDS-polyacrylamide gel electrophoresis.     

In vitro cleavage specificity of the adenovirus type 2 proteinase

Two in vitro proteinase assay systems were developed and used to study the peptide bond specificity and substrate specificity of the adenovirus endoproteinase. Five adenovirus precursor proteins (PVI, PVII, PVIII, 87K, 11K), all found in the virion of the ts1 mutant grown at the nonpermissive temperature, were digested by the proteinase. All, except 11K, were cleaved to their mature counterparts. Some of the proteins, particularly the 87K terminal protein, were processed via cleavage intermediates similar to those found in vivo. The data suggest that the proteinase specifically hydrolyses Gly-Ala bonds. The high specificity for the natural substrates and the failure to cleave foreign proteins suggest that cleavage activity is determined not only by primary sequence but also by other physical features of the substrate. Enzyme activity was inhibited by diisopropylfluorophosphate, showing that it is a serine proteinase.     

Simple computer method for evaluation of lateral diffusion coefficients from fluorescence photobleaching recovery kinetics.

A method is presented for the analysis of fluorescence photobleaching recovery curves. Based on the simplified kinetic expression of Yguerabide, J., J.A. Schmidt, and E.E. Yguerabide (1982, Biophys. J., 40:69-75), a linearization procedure is described that permits unequivocal determination of all diffusion parameters. The presence of additional membrane flow or multiple diffusion coefficients can easily be detected by this method, and simple corrections for the presence of these alternative recovery processes can be made by the use of a regular mini-computer. The validity of the method is tested on simulated recovery curves, varying the contribution of flow, multiple diffusion coefficients, and statistical noise due to counting error.