Protein kinase C activity in renal microvillus membranes

Protein kinase C activity was found in rabbit renal microvillus membrane vesicles. C-kinase activity was assayed by examining H1 histone phosphorylation using microvillus membrane vesicles dispersed with Triton X. Calcium-activated protein kinase activity was only demonstrable in the presence of phosphatidylserine (PS). With PS (15 micrograms/ml) the Ka for activation by calcium was 1.04 microM. This was reduced to 0.38 microM by addition of diolein (3.75 micrograms/ml). These activations were dose-dependent and their combined synergistic activation could be reproduced by the combination of PS (15 micrograms/ml) and the phorbol ester, TPA (1.17 ng/ml). During microvillus membrane purification, protein kinase C activity enriched 5-fold relative to its activity in the homogenates. The activity was not due to trapped cytosol or adventitious association with microvillus membranes during homogenization. During further purification on sucrose gradients, the C-kinase activity coenriched with brush border and not with basolateral enzyme markers. We conclude that protein kinase C is a normal component of the renal microvillus membrane.     

Ginseng extract inhibits protein degradation and stimulates protein synthesis in human fibroblasts

Aqueous extracts of Panax ginseng inhibit intracellular protein degradation in confluent cultures of IMR-90 human diploid fibroblasts. The magnitude of the inhibition is similar to that observed with insulin and polypeptide growth factors. Furthermore, the inhibition of proteolysis by ginseng, like that produced by insulin and growth factors, is selective in that it applies to long-lived proteins but not to short-lived proteins. Ginseng also stimulates protein synthesis in human fibroblasts indicating that components of ginseng extract are capable of acting directly on human cells to promote protein accumulation.     

Cloning and sequencing of the beta-lactamase I gene of Bacillus cereus 5/B and its expression in Bacillus subtilis.

The beta-lactamases of Bacillus cereus have attracted interest because they are secreted efficiently, because multiple enzymes are frequently present, and because their regulation has unusual features. beta-Lactamase I of strain 5/B is produced constitutively at a high level, and the exoenzyme appears to be several thousand daltons larger than the corresponding product of strain 569/H. We have cloned the gene for 5/B beta-lactamase I in Escherichia coli and B. subtilis and have sequenced the structural portion and the regulatory regions. The 5/B enzyme is produced at a low level in E. coli RR1(pRWY200) and remains cellbound. In B. subtilis it is formed in large amounts, and over 90% of it is released into the medium. There is a large degree of homology between the promoter and leader peptide regions of the 5/B and 569/H genes; both utilize UUG as the translation initiation codon (P. S. F. Mézes, R. W. Blacher, and J. O. Lampen, (J. Biol. Chem. 260:1218-1223, 1985). Although there are significant differences in the peptide segment where processing would be expected to occur, the NH2 terminus of the major 5/B product from B. subtilis BD170(pRWY215) is His-44, which is the same as the NH2 terminus of the major 569/H product from B. subtilis BD170(pRWM5).     

Biosynthesis of riboflavin in Bacillus subtilis: origin of the four-carbon moiety.

We studied the incorporation of [1-13C]ribose and [1,3-13C2]glycerol into the riboflavin precursor 6,7-dimethyl-8-ribityllumazine, using a riboflavin-deficient mutant of Bacillus subtilis. The formation of the pyrazine ring requires the addition of a four-carbon moiety to a pyrimidine precursor. The results show that C-6 alpha, C-6, C-7, and C-7 alpha of 6,7-dimethyl-8-ribityllumazine were biosynthetically equivalent to C-1, C-2, C-3, and C-5 of a pentose phosphate. C-4 of the pentose precursor was lost through an intramolecular skeletal rearrangement. Thus, the last steps in the biosynthesis of 6,7-dimethyl-8-ribityllumazine apparently involve the same mechanism in bacteria as in fungi.     

Vesicular stomatitis virus-infected cells fuse when the intracellular pool of functional M protein is reduced in the presence of G protein.

Five highly cytolytic strains of both Indiana and New Jersey serotypes of vesicular stomatitis virus were shown to induce cell fusion in BHK-21 and R(B77) cells. Inhibition of protein synthesis after the eclipse period of viral replication is a prerequisite for vesicular stomatitis virus-induced cell fusion. Pulse-chase experiments showed that inhibition of protein synthesis would lead to a drastic reduction in the intracellular pool of M protein as compared with other proteins. A temperature-sensitive mutant defective in M protein function (G31) was the only mutant of the five complementation groups to spontaneously induce polykaryocytes at the nonpermissive temperature. Previously, G protein has been shown to play a role in vesicular stomatitis virus-induced cell fusion. These results suggest that the combination of the presence of G protein on the virus-infected cell surface and the absence of functional M protein or a reduced level of intracellular M protein promotes cell fusion. On the basis of this study, we propose that vesicular stomatitis virus infection can induce cell fusion when the functional M protein pool declines to a critical level while G protein remains on the cell surface.     

Cytoskeletal changes visualized by fluorescence microscopy during amoeba-to-flagellate and flagellate-to-amoeba transformations inPhysarum polycephalum

Summary Changes in the intracellular distribution of microtubules and microfilaments during amoeba-to-flagellate and flagellate-to-amoeba transformations inPhysarum polycephalum were examined by fluorescence microscopy using anti-tubulin antibody and NBD-phallacidin, respectively. Amoebae contained an extensive microtubular cytoskeleton, which was converted to a flagellar cone structure during transformation to flagellates in liquid medium. When flagellates reverted back to amoebae, this conical structure disintegrated prior to flagella resorption. Amoebae showed some microfilament-enriched domains along the periphery, from which numerous filamentous extrusions, probably pseudopods and filopods, emanated. Flagellates contained a ridge, a sheet-like structure, along their dorsal axis, especially in the earlier stages of flagellation. Another microfilament-enriched thick filamentous structure ran along the dorsal axis, starting from the anterior tip of the cell. This structure apparently coincided spatially with one of the bundles of microtubules. During the reversion to amoebae, other localized microfilaments were transiently observed at the posterior end. A model of cytoskeletal changes in the transformations between these two cell types was proposed.     

The fermentation process for l-phenylalanine production using an auxotrophic regulatory mutant of Escherichia coli

Summary A process for l-phenylalanine production was studied using a tyrosine auxotrophic regulatory mutant of Escherichia coli, resistant to both -2-thienyl-dl-alanine and p-fluoro-dl-phenylalanine. Fermentations were carried out in a 30-1 fermentor with intermittent feeding of glucose plus phosphate. The mutant accumulated l-phenylalanine in the fermentation broth up to 15 g/l at pH 7.0 and 33°C. Column chromatography on a strong cation exchanger was employed as the most effective step in the purification of l-phenyl-alanine from the broth. This step brought about 4-fold concentration of the product with 96% recovery.