Role of de novo protein synthesis in the interconversion of glucose transport systems in the yeast Pichia ohmeri

Glucose-repressed cells of the yeast Pichia ohmeri IGC 2879 transported glucose by facilitated diffusion. Derepression led to the formation of a glucose/proton symport and the simultaneous reduction of the facilitated diffusion capacity by about 70%. Cycloheximide prevented this interconversion indicating its dependence on de novo protein synthesis (proteosynthetic interconversion). In buffer with 2% glucose the glucose/proton symport suffered irreversible inactivation while the facilitated diffusion system was simultaneously restored. This reverse interconversion process did not require de novo protein synthesis as indicated by its lack of sensitivity to cycloheximide (degradative interconversion). Thus the glucose/proton symport system appeared to consist of about 70% of the facilitated diffusion proteins turned silent through association with additional protein(s) the latter being sensitive to glucose-induced repression and glucose-induced inactivation.     

Phenylglyoxal modification of the Photosystem II reaction center

Time-resolved spectroscopic techniques, including optical flash photolysis and electron spin resonance (ESR), have been used in conjunction with fluorescence-induction and dye-reduction assays to monitor electron transport in Photosystem II (PS II) subchloroplast particles incubated with the covalent modifier, phenylglyoxal. Phenylglyoxal-modified digitonin (D-10) particles from spinach are characterized by a high initial fluorescence yield (Fi) and an abolition of the variable component of fluorescence (Fv); an inhibition of PS-II-mediated reduction of dichlorophenol indophenol (DPIP) by sym-diphenylcarbazide; an abolition of flash-induced absorption transients (t1/2 greater than 2 microseconds) at 820 nm attributed to the primary electron donor, P-680+; the inhibition of photoreduction of the acceptor Qa; and the elimination of the ESR Signal 2s and Signal 2f. These observations suggest the critical participation of specific arginine residues on both the oxidizing and reducing sides of Photosystem II and also implicate phenylglyoxal as a quinone-binding site inhibitor (Golbeck, J.H. and Warden, J.T. (1984) Biochim. Biophys. Acta 767, 263-271).     

Gibberellic acid by solid state fermentation: Consistent and improved yields

Five strains each of Gibberella fujikuroi and Fusarium monoliforme were screened to select G. fujikuroi P-3, a strain capable of giving consistent production of gibberellic acid (GA(3)) by solid state fermentation (SSF). The comparative production of GA(3) by SSF and submerged fermentation (SmF) indicated better productivity with the former technique. The accumulation of GA(3) was 1.626 times higher in the case of SSF. On the basis of available carbohydrates in the media, the percent conversions were 0.096 and 0.156 in SmF and SSF, respectively. The use of coarse wheat bran of the particle size of 0.3-0.4 cm resulted in an increase of 2.5 times in the yield of GA(3). The enrichment of commercial wheat bran with soluble starch gave enhanced accumulation to an extent of 3.5 times. The relation between GA(3) production and cell growth in SSF was similar to that encountered in SmF. The consistent and improved yields to a tune of 1.22 g GA(3) per kilogram dry moldy bran (DMB) establish the potential and feasibility of SSF for the production of GA(3) by G. fujikuroi P-3. On preliminary cost analysis, a net savings of about 60% and 50% on fermentation medium cost and the expenditure on down-stream processing, respectively, as compared to the presently employed SmF technique was evident.     

Several nodulins of soybean share structural domains but differ in their subcellular locations.

Four soybean cDNA nodule-specific clones encoding nodulin-23, -26b, -27 and -44 were observed to cross-hybridize under low stringency conditions. Nucleotide sequence analysis revealed that the cDNAs contain three distinct domains: two domains with 70 to 95% homology separated by a third domain unique to each cDNA. Despite a number of nucleotide insertions and deletions, the protein sequences are conserved in the two domains which correlate with the homologous nucleotide domains. The amino terminal domain of each nodulin contains putative signal sequences for membrane translocation, although only two (nodulin-23 and -44) meet all the criteria for a functional signal. Immuno-precipitation of hybrid-release translation products of the four cDNAs revealed that nodulin-23 is associated with the peribacteroid membrane while nodulin-27 is in the cytoplasmic fraction of the nodule. These four nodulins are members of a diverse family with conserved structural features and the genes encoding them appear to have recently evolved from a common ancestor.     

Nodulin-26, a peribacteroid membrane nodulin is expressed independently of the development of the peribacteroid compartment.

The peribacteroid membrane (pbm) of root nodules is derived from the plant cell plasma membrane but contains in addition several nodule-specific host proteins (nodulins). Antibodies raised against purified pbm of soybean were used to immunoprecipitate polysomes to isolate an RNA fraction that served as a template for the synthesis of a cDNA probe for screening a nodule-specific cDNA library. Clone p1B1 was found to encode a 26.5 kDa polypeptide (nodulin-26) which is immunoprecipitable specifically with the anti-pbm serum. Nodulin-26 has features of a transmembrane protein and its structure differs from that of nodulin-24 which appears to be a surface protein of pbm. The expression of these two pbm nodulins was examined in nodules induced by Bradyrhizobium japonicum Tn5 mutants that arrest nodule development at different stages of pbm biosynthesis. Nodules that do not show release of bacteria from the infection thread express nodulin-24 at a very low level. In contrast, the expression of nodulin-26 occurs fully in nodules that form infection threads only and is not affected by the release of bacteria from the threads.     

UV irradiation inhibits initiation of DNA replication from oriC in Escherichia coli

Summary Irradiation of Escherichia coli with UV light causes a transient inhibition of DNA replication. This effect is generally thought to be accounted for by blockage of the elongation of DNA replication by UV-induced lesions in the DNA (a cis effect). However, by introducing an unirradiated E. coli origin (oriC)-dependent replicon into UV-irradiated cells, we have been able to show that the environment of a UV-irradiated cell inhibits initiation of replication from oriC on a dimer-free replicon. We therefore conclude that UV-irradiation of E. coli leads to a trans-acting inhibition of initiation of replication. The inhibition is transient and does not appear to be an SOS function.     

Influence of Co2+, Ni2+ and Fe2+ on the production of tetrapyrroles by Methanosarcina barkeri

Summary The selective formation of three tetrapyrroles, Co-containing corrinoids, Ni-containing factor F₄₃₀ and Fe-containing cytochromes (haems) by Methanosarcina barkeri Fusaro (DSM 804) was achieved as a function of the concentrations of Co²⁺, Ni²⁺ and Fe²⁺ in a methanol minimmum medium. It was found that about 70% of the total tetrapyrroles synthesized was excreted into the culture supernatant. Hence, the continuous production of tetrapyrroles in a fixed-bed reactor (supporter: porous diatomaceous clay) was carried out at a dilution rate of 10 day^-1 (850 ml medium/85 ml column/day). The effluent discharged from the reactor contained the excreted tetrapyrroles, the concentrations of which were dependent upon the Co²⁺, Ni²⁺ and Fe²⁺ concentrations in the feed medium. The maximum productivities from the reactor (1 l basis) were 52 M corrinoids/day, 24 M F₄₃₀/day and 8 M haems/day, respectively.     

The effect of hyaluronate and its oligosaccharides on endothelial cell proliferation and monolayer integrity

Hyaluronidase treatment of hyaluronic acid produced a series of oligosaccharides. Those between 3 and 16 disaccharides in length stimulated angiogenesis in vivo and the proliferation of tissue cultured endothelial cells in vitro. This effect appears to be cell type specific, as no stimulation of fibroblasts or smooth muscle cells was observed. Endothelial cells were found to endocytose both high- and low-molecular-mass hyaluronate, which might be receptor mediated. Fibroblasts and smooth muscle cells, cultured under the same conditions, showed negligible uptake of hyaluronate. Thus, the cell-specific effects may be due to the differences in internalization of hyaluronate. High-molecular-weight hyaluronate both inhibited endothelial cell proliferation and disrupted newly formed monolayers. These data are consistent with the ability of hyaluronate to inhibit new blood vessel formation in vivo and also suggest that hyaluronate metabolism plays a pivotal role in the regulation of angiogenesis.     

Characterization and differential expression of human vascular smooth muscle myosin light chain 2 isoform in nonmuscle cells

The 20-kDa regulatory myosin light chain (MLC), also known as MLC-2, plays an important role in the regulation of both smooth muscle and nonmuscle cell contractile activity. Phosphorylation of MLC-2 by the enzyme MLC kinase increases the actin-activated myosin ATPase activity and thereby regulates the contractile activity. We have isolated and characterized an MLC-2 cDNA corresponding to the human vascular smooth muscle MLC-2 isoform from a cDNA library derived from umbilical artery RNA. The translation of the in vitro synthesized mRNA, corresponding to the cDNA insert, in a rabbit reticulocyte lysate results in the synthesis of a 20,000-dalton protein that is immunoreactive with antibodies raised against purified chicken gizzard MLC-2. The derived amino acid sequence of the putative human smooth muscle MLC-2 shows only three amino acid differences when compared to chicken gizzard MLC-2. However, comparison with the human cardiac isoform reveals only 48% homology. Blot hybridizations and S1 nuclease analysis indicate that the human smooth muscle MLC-2 isoform is expressed restrictively in smooth muscle tissues such as colon and uterus and in some, but not all, nonmuscle cell lines. Previously reported MLC-2 cDNA from rat aortic smooth muscle cells in culture is ubiquitously expressed in all muscle and nonmuscle cells, and it was suggested that both smooth muscle and nonmuscle MLC-2 proteins are identical and are probably encoded by the same gene. In contrast, the human smooth muscle MLC-2 cDNA that we have characterized from an intact smooth muscle tissue is not expressed in skeletal and cardiac muscles and also in a number of nonmuscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)