Stimulation of extracellular protein production in Bacillus brevis 47

Summary Bacillus brevis 47 was cultivated in 2-1 fermentors to study the effect of medium supplementation on extracellular protein production. Additional polypeptone, when supplied initially or at 12 h (late exponential phase), had little stimulatory effect on extracellular protein levels, which reached 6–7 g/l after 48h. A large increase in protein production was observed, however, when polypeptone was added at 21 h (stationary phase). This addition resulted in the accumulation in the medium of 14 g/l protein after 48 h, and a total of 16 g/l when cell-bound protein was included. In all cases, glucose was consumed only very slowly.     

Synergistic release of arachidonic acid from platelets by activators of protein kinase C and Ca2+ ionophores. Evidence for the role of protein phosphorylation in the activation of phospholipase A2 and independence from the Na+/H+ exchanger

The protein kinase C activators phorbol myristate acetate (PMA), mezerein, oleoylacetylglycerol, and (-)-indolactam V, although without direct effect on arachidonic acid release, greatly enhance the release of platelet arachidonic acid caused by the Ca2+ ionophores A23187 and ionomycin. In contrast, 4 alpha-phorbol 12,13-didecanoate and (+)-indolactam V, which lack the ability to activate kinase C, do not potentiate arachidonate release. Release of arachidonic acid occurs without activation of phospholipase C and is therefore mediated by phospholipase A2. Synergism between PMA and A23187 is not affected by inactivation of the Na+/H+ exchanger with dimethylamiloride. The time course and dose-response for the effect of PMA at 23 degrees C closely correlate with the phosphorylation of a set of relatively "slowly" phosphorylated proteins (P20, P35, P41, P60), but not the rapidly phosphorylated P47 protein. P20 is myosin light chain, and P41 is probably Gi alpha, but the other proteins have not been positively identified. Depletion of metabolic ATP stores by antimycin A plus 2-deoxyglucose abolishes both protein phorphorylation and the potentiation of arachidonate release by PMA, but does not prevent fatty acid release by the ionophores. Similarly, the kinase C inhibitors H-7 and staurosporine produce, respectively, partial and complete inhibition of PMA-potentiated arachidonic acid release and protein phosphorylation, without affecting the direct response to ionophores. These results indicate that protein phosphorylation, mediated by kinase C, promotes the phospholipase A2 dependent release of arachidonic acid in platelets when intracellular Ca2+ is elevated by Ca2+ ionophores.     

Distribution of lipid-binding regions in human apolipoprotein B-100

The distribution of lipid-binding regions of human apolipoprotein B-100 has been investigated by recombining proteolytic fragments of B-100 with lipids and characterizing the lipid-bound fragments by peptide mapping, amino acid sequencing, and immunoblotting. Fragments of B-100 were generated by digestion of low-density lipoproteins (LDL) in the presence of sodium decyl sulfate with either Staphylococcus aureus V8 protease, pancreatic elastase, or chymotrypsin. Particles with electron microscopic appearance of native lipoproteins formed spontaneously when detergent was removed by dialysis from enzyme digests containing fragments of B-100 and endogenous lipids, or from incubation mixtures of delipidated B-100 fragments mixed with microemulsions of exogenous lipids (cholesteryl oleate and egg phosphatidylcholine). Fractionation of the recombinant particles by isopycnic or density gradient ultracentrifugation yielded complexes similar to native LDL with respect to shape, diameter, electrophoretic mobility, and surface and core compositions. Circular dichroic spectra of these particles showed helicity similar to LDL but a somewhat decreased content of beta-structure. Most of the fragments of B-100 were capable of binding to lipids; 12 were identified by direct sequence analysis and 14 by reaction with antisera against specific sequences within B-100. Our results indicate that lipid-binding regions of B-100 are widely distributed within the protein molecule and that proteolytic fragments derived from B-100 can reassociate in vitro with lipids to form LDL-like particles.     

A 40 kilodalton rat liver nuclear protein binds specifically to apolipoprotein B mRNA around the RNA editing site.

Apolipoprotein (apo) B-48 mRNA is the product of RNA editing which consists of a C----U conversion changing a CAA codon encoding Gln-2153 in apoB-100 mRNA to a UAA stop codon in apoB-48 mRNA. In the adult rat, RNA editing occurs both in the small intestine and the liver. We have studied the ability of rat liver nuclear extracts to bind to synthetic apoB mRNA segments spanning the editing site. Using an RNA gel mobility shift assay, we found the sequence-specific binding of a protein(s) to a 65-nucleotide apoB-100 mRNA. UV crosslinking followed by T1 ribonuclease digestion and SDS-polyacrylamide gel electrophoresis demonstrated the formation of a 40 kDa protein-RNA complex when 32P-labeled apoB-100 mRNA was incubated with a rat liver nuclear extract but not with HeLa nuclear extract. Binding was specific for the sense strand of apoB mRNA, and was not demonstrated with single-stranded apoB DNA, or antisense apoB RNA. The complex also failed to form if SDS was present during the UV light exposure. Binding experiments using synthetic apoB mRNAs indicate that the 40 kDa protein would also bind to apoB-48 mRNA but not apoA-I, apoA-IV, apoC-II or apoE mRNA. Experiments using deletion mutants of apoB-100 mRNA indicate efficient binding of wildtype 65-nucleotide (W65), 40-nucleotide (W40) and 26-nucleotide (W26) apoB-100 mRNA segments, but not 10-nucleotide (or smaller) segments of apoB-100 mRNA to the 40 kDa protein. In contrast, two other regions of apoB-100 mRNA, B-5' (bases 1128-3003) and B-3' (bases 11310-11390), failed to bind to the protein. The 40 kDa sequence-specific binding protein in rat liver nuclear extract may play a role in apoB-100 mRNA editing.     

5,6,7,8-Tetrahydromethanopterin-dependent enzymes involved in methanogenesis

Abstract In the process of methanogenesis, 5,6,7,8-tetrahydromethanopterin (H₄MPT) is the carrier of the C₁ unit at the formyl through methyl state of reduction. By the transfer of a formyl group from formylmethanofuran, 5-formyl- and 10-formyl-H₄MPT are formed in hydrogenotrophic and methylotrophic organisms, respectively. Cyclohydrolysis of the 5- and 10-formyl derivatives then yields 5,10-methenyl-H₄MPT, which is reduced in two subsequent coenzyme F₄₂₀-dependent reactions to 5-methyl-H₄MPT. Following the transfer of the methyl group to coenzyme M, the substrate of the terminal step in methanogenesis, methylcoenzyme M, is produced. In this paper properties of the enzymes catalyzing the individual H₄MPT-dependent reactions are discussed.     

Transformation with a mutant Arabidopsis acetolactate synthase gene renders tobacco resistant to sulfonylurea herbicides

Summary A gene encoding acetolactate synthase was cloned from a chlorsulfuron-resistant mutant of Arabidopsis. The DNA sequence of the mutant gene differed from that of the wild type by a single base pair substitution. When introduced into tobacco by Ti plasmid-mediated transformation the gene conferred a high level of herbicide resistance. These results suggest that the cloned gene may confer agronomically useful levels of herbicide resistnace in other crop species, and that it may be useful as a selectable marker for plant transformation experiments.     

Correction of chlorophyll-defective male-sterile winter oilseed rape (Brassica napus) through organelle exchange: Characterization of the chlorophyll deficiency

Jarl, C. I., Ljungberg, U. K. and Bornman, C. H. 1988. Correction of chlorophyll-defective male-sterile winter oilseed rape ( Brassica napus ) through organelle exchange: Characterization of the chlorophyll deficiency. - Physiol. Plant. 72: 505–510.
As is known, the introduction of male-sterile Raphanus sativus L. cytoplasm into Brassica napus L. results in male-sterile oilseed rape plants, which display a temperature-related chlorophyll defect. The influences of temperature and irradiance on this defect were investigated. Compared to a line of normal (green phenotype) male-fertile oilseed rape, the male-sterile line had reduced chlorophyll content, fewer chloroplasts per cell, an altered ultrastructure of the chloroplasts and reduced activities of both photosystems, although the relative amounts of the photosystems and the chlorophyll a/b ratio were similar. The lower activity of the photosystems is explained by a decreased functional antennae size and a reduced efficiency in the interactions between the nuclear-encoded light-harvesting proteins and the reaction centres coded for by the plastome. Some thylakoid polypeptides differed in proportion between the male-fertile line with green phenotype and the male-sterile line with chlorotic phenotype. Characters, in which the two lines exhibited differences, are ascribed to difficulties in molecular communication between the oilseed rape nucleus and the radish cytoplasm, which are combined in the deficient male-sterile line.     

Constitutive and transport-related endocytotic pathways in turtle bladder epithelium

Summary Proton secretion in the urinary bladder of the fresh-water turtle is mediated by a proton pump located in the apical membrane of a population of cells characteristically rich in carbonic anhydrase. Earlier studies have demonstrated that these cells exhibit apical-membrane endocytotic and exocytotic processes which are thought to be involved in the regulation of the rate of proton transport via alterations in the number of pumps within the apical membrane. In this study, we sought to characterize these processes using two different methods. Analysis of transepithelial impedance yielded estimates of membrane capacitance which could be related to membrane area, thereby allowing one to monitor net changes in apical-membrane area resulting from changes in the net rates of endo-and exocytosis. Uptake of the fluid-phase marker FITC-dextran provided a measure of net extracellular volume uptake which was related to net rates of endocytosis. Our major conclusions are summarized as follows. The bladder cells exhibit a high baseline rate of endocytosis which appears to be a constitutive process similar to pinocytosis. This process is completely inhibited when ambient temperature is reduced to 15°C. In addition, serosal application of 0.5mm acetazolamide causes a transient increase in the rate of endocytosis, concomitant with a decrease in the rate of transport. Reduction of ambient temperature to 15°C reduces the rate of acetazolamide-induced endocytosis, but does not abolish it. Addition of 1mm serosal azide not only prevents the acetazolamide-induced increase in endocytosis, but also prevents the decrease in transport caused by acetazolamide. Azide has no effect on the baseline rate of endocytosis, nor does it prevent inhibition of carbonic anhydrase by acetazolamide. The specificity of azide, coupled with the different temperature sensitivities, demonstrate that the constitutive and transport-dependent endocytotic pathways are distinct processes. The observation that azide prevents both the acetazolamide-induced increase in endocytosis and the decrease in transport strongly supports the notion that endocytosis of proton-pump-containing membrane is requisite for the inhibition of transport by acetazolamide. Finally, the results also demonstrate that acetazolamide does not inhibit proton secretion simply by inhibiting carbonic anhydrase.     

MPTP in mice: treatment, distribution and possible source of contamination

Mice were treated daily with [3H]MPTP (30 mg/kg, 1 uCi, s.c.) for 1, 3, and 10 days to determine the fate and localization of tritiated compounds. An untreated mouse was housed either in the same cage ("cage-mate control") or in an adjacent cage separated by mesh-wire ("near-neighbor control"). The radioactivity measured in blood, brain, liver, and remaining body of [3H]MPTP-treated mice was dependent on the total dose of the drug the animals received and did not vary with the type of tissue analyzed. Significant amounts of radioactivity were found in the tissues of the "cage-mate control" mice, but not of the "near-neighbor control" mice. The route of transmission appears to be through the urine, as the urine of [3H]MPTP-treated mice was highly radioactive after the drug injection. Only traces of radioactivity were found in their feces and there was no increase in the background radiation in the environment of the cages, indicating that the tritiated compounds were not exhaled. Proper disposal of urinary products of MPTP-treated animals is therefore necessary to reduce the risk of possible drug contamination in humans.     

The taxonomic status of "Halobacterium marismortui" from the Dead Sea: a comparison with Halobacterium vallismortis

A Halobacterium strain, isolated by Ginzburg et al. from the Dead Sea in the late 1960's, often referred to as "Halobacterium marismortui" or "Halobacterium of the Dead Sea" (deposited in the American Type Culture Collection as ATCC 43049) was compared with Halobacterium (Haloarcula) vallismortis ATCC 29715. The strains appeared to be very closely related, as shown by the near identity of their 5S and 16S ribosomal RNA's, and a large number of other common properties. Distinct differences exist, however, in cell morphology, and in their potency to utilize different sugars and other compounds.