Release of Ca2+ from a non-mitochondrial store site in peritoneal macrophages treated with saponin by inositol 1,4,5-trisphosphate.

The effects of inositol 1,4,5-trisphosphate, prepared from human erythrocyte ghosts, on Ca2+ release from intracellular store sites were studied in saponin-treated guinea pig peritoneal macrophages. Micromolar concentrations of inositol 1,4,5-trisphosphate released Ca2+ within 1 min from store sites which had accumulated Ca2+ in the presence of 10 mM-NaN3. In the presence of 10 mM-NaN3, the Ca2+ accumulated in the presence of oxalate was seen in the endoplasmic reticulum of saponin-treated macrophages by electron microscopy, indicating that the site of Ca2+ released by inositol 1,4,5-trisphosphate may be endoplasmic reticulum-like membranes. When the concentrations of free Ca2+ were over 3.5 X 10(-6) M, the release of Ca2+ by this agent was inhibited. This inhibition may be due to either the higher concentration of extra-vesicular free Ca2+ or the larger accumulation of Ca2+ into the store site or perhaps both effects. MgCl2 also had an inhibitory effect on the Ca2+ release. Inositol 1,4,5-trisphosphate also released Ca2+ from cardiac sarcoplasmic reticulum, but not from erythrocyte inside-out vesicles.     

Isolation and primary culture of adult human hepatocytes

Summary Biopsy tissue of adult human liver was gently dissociated with collagenase followed by Dispase. By repeated low g centrifugation, a large number of almost pure, viable hepatocytes was obtained. This is the first report of a successful procedure for obtaining adult human hepatocytes for study in tissue culture. The isolated cells have the typical morphology of liver parenchyma, and these characteristics persist throughout the period of culturing. Evidence of their function is indicated by albumin synthesis. This procedure is now being used to study human hepatocyte functions in vitro and the effects of a variety of agents including carcinogens and viruses.     

Thermal analysis of the spores of Bacillus cereus with special reference to heat activation.

The heat activation of bacterial spores was studied by means of differential thermal analysis in the temperature range 30-110 degrees C using the spores of Bacillus cereus. The thermogram showed three endothermic peaks at 56, 95, and 103 degrees C with one exothermic peak at 105 degrees C during the heating process. The spore coat separated from the native spores also showed a peak at 56 degrees C on its heating thermogram. The peak at 56 degrees C was reversible for both native spores and the spore coat. It was suggested that this peak at 56 degrees C might be related to the heat-activation process that takes place in the spore-coat region. It seems that the peak is due to the denaturation or the structural change of the spore-coat protein that might facilitate either the permeation of germination stimulators or the release of some germination inhibitor into or out of the spores.     

Computer Simulation of Fermentation Systems

Results of batch fermentation of gluconic acid by Pseudomonas ovalis were graphically analyzed to obtain a kinetic model to represent the data. Since gluconic acid was produced by the hydrolysis of a lactone intermediate, the model was necessarily represented by a set of kinetic equations. A computer simulation technique involving the use of the MIDAS program was developed to solve the system of nonlinear equations and to check the appropriateness of the model. Since the maximal specific growth rate and the rate constant for the production of the lactone intermediate varied with time, function generators were used to simulate these system parameters. The merit of using the MIDAS program was considered in relation to analysis and model testing in microbiological processes of similar types.     

Biologically active oligodeoxyribonucleotides-IX. Synthesis and anti-HIV-1 activity of hexadeoxyribonucleotides, TGGGAG, bearing 3′- and 5′-end-modification

We have determined that hexadeoxyribonucleotides (5′TGGGAG3′), with modified aromatic groups such as a trityl group at the 5′-end, have anti-HIV-1 activity in vitro. The 6-mer bearing a 3,4-dibenzyloxybenzyl (3,4-DBB) group at the 5′-end had the most potent activity and the least cytotoxicity. When the 3′-end of the 5′-(3,4-DBB)-modified 6-mer was substituted with a 2-hydroxyethylphosphate, a 2-hydroxyethylthiophosphate, or a methylphosphate group at the 3′-end, anti-HIV-1 activity increased. Moreover, among various 3′- and 5′-end-modified 6-mers that were tested, the 6-mer (R-95288) bearing a 3,4-DBB group at the 5′-end and a 2-hydroxyethylphosphate group at the 3′-end was the most stable, when incubated with mouse, rat, or human plasma. Therefore, R-95288 was chosen as the best candidate for possible use in therapy on the basis of its anti-HIV-1 activity.     

Biological function of the dTDP-rhamnose synthesis pathway in Streptococcus mutans.

We have cloned a new gene locus that comprises three genes concerned with the biosynthesis of the serotype c-specific polysaccharide antigen in Streptococcus mutans. The genes encode proteins exhibiting significant homology to the rfbA, rfbB, and rfbD gene products that are involved in the anabolism of dTDP-L-rhamnose from D-glucose-1-phosphate. This anabolism pathway pertains to biosynthesis of the O antigen of lipopolysaccharide in gram-negative bacteria. The cell extract of Escherichia coli expressing each of the cloned genes of S. mutans exhibited enzymatic activity corresponding to the homologous counterpart of the rfb gene products. Rhamnose was not detected in the cell wall preparation purified from the mutant in which each of the three cloned genes was insertionally inactivated. Rabbit antiserum against S. mutans serotype c-specific antigen did not react with the autoclaved extracts from these mutants. These results indicate that the gene products identified in the present study are involved in the dTDP-L-rhamnose synthesis pathway and that the pathway relates to the biosynthesis of the serotype-specific polysaccharide antigen of S. mutans. Southern hybridization analysis revealed that genes homologous to the cloned genes involved in the dTDP-L-rhamnose synthesis pathway were widely distributed in a variety of streptococci. This is the first report of the biological function of the dTDP-rhamnose pathway in streptococci.     

Liquid chromatographic-atmospheric pressure chemical ionization mass spectrometric determination of anandamide and its analogs in rat brain and peripheral tissues

A simple and selective method for the determination of anandamide (arachidonoylethanolamide), an endogenous cannabinoid receptor ligand, and its analogs with liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS) was developed. The calibration curve for standard anandamide was linear over the range 625 fmol-125 pmol per injection (r=0.999) with a precision of 1.0% (C.V.) at 25 pmol. The detection limit attained was 200 fmol per injection at a signal-to-noise ration of 2. Anandamide and its analogs were extracted from rat brain and peripheral tissues according to the method of Folch, and the recovery of anandamide from rat brain homogenates was 67.0–72.6%. The method was applied to their determination in rat brain and peripheral tissues.     

Different spatial distribution of mRNAs for activin receptors (type IIA and IIB) and follistatin in developing embryos of Xenopus laevis

Spatial distribution of mRNAs for activin receptors and follistatin was studied by Northern blot hybridization using RNAs from different parts of dissected Xenopus embryos. mRNAs of two activin receptors (type IIA and IIB) occurred uniformly in pre-gastrular embryos, but occurred in larger amounts in ectoderm (in gastrulae), neural plate (in neurulae) and anterior (head) regions (in tailbud embryos) than in other embryonic regions. By contrast, follistatin mRNA appeared almost exclusively in the dorsal mesoderm including invaginating organizer region at the gastrula stage, in notochord and in dorsal ectoderm at the neurula stage, then in anterior part at the tailbud stage. The localized patterns of the distribution of these mRNAs may be due to the regionally different zygotic expression of genes in embryos at later stages. From the relatively widespread pattern of distribution of their mRNAs, we assume that both type IIA and type IIB activin receptors have broad functions in ectodermal and neural differentiation. On the other hand, follistatin mRNA showed quite a restricted pattern of expression, and therefore, we assume that follistatin may have functions more specifically related to the sites of expression of its mRNA. Thus, follistatin may be involved in the differentiation of notochord itself and/or directly be responsible for organizer functions such as neural induction and subsequent differentiation of induced neural tissues at the gastrula and later stages.