Effect of GABA A receptor activation on UT-coupled signaling pathways in rat cortical astrocytes

Cultured rat cortical astrocytes express two types of urotensin II (UII) binding sites: a high affinity site corresponding to the UT (GPR14) receptor and a low affinity site that has not been fully characterized. Activation of the high affinity site in astroglial cells stimulates polyphosphoinositide (PIP) turnover and provokes an increase in intracellular calcium concentration. We have hypothesized that the existence of distinct affinity sites for UII in rat cortical astrocytes could be accounted for by a possible cross-talk between UT and the ligand-gated ion channel GABA(A) receptor (GABA A R). Exposure of cultured astrocytes to UII provoked a bell-shaped increase in cAMP production, with an EC50 stimulating value of 0.83+/-0.04 pM, that was totally blocked in the presence of the adenylyl cyclase inhibitor SQ 22,536. In contrast, UII was found to inhibit forskolin-induced cAMP formation. In the presence of the specific PKA inhibitor H89, UII provoked a sustained stimulation of cAMP formation. Inhibition of PKA by H89 strongly reduced the stimulatory effect of UII on PIP metabolism. GABA and the GABA A R agonist isoguvacine provoked a marked inhibition of UII-induced cAMP synthesis and a significant reduction of UII-evoked PIP turnover. These data suggest that functional interaction between UT and GABA(A)R negatively regulates coupling of UT to the classical PLC/IP(3) signaling cascade as well as to the adenylyl cyclase/PKA pathway.     

Amino acid biosynthesis and metabolic flux profiling of Pichia pastoris.

Amino acid biosynthesis and central carbon metabolism of Pichia pastoris were studied using biosynthetically directed fractional (13)C labeling. Cells were grown aerobically in a chemostat culture fed at two dilution rates (0.05 h(-1), 0.16 h(-1)) with glycerol as the sole carbon source. For investigation of amino acid biosynthesis and comparison with glycerol cultivations, cells were also grown at 0.16 h(-1) on glucose. Our results show that, firstly, amino acids are synthesized as in Saccharomyces cerevisiae. Secondly, biosynthesis of mitochondrial pyruvate via the malic enzyme is not registered for any of the three cultivations. Thirdly, transfer of oxaloacetate across the mitochondrial membrane appears bidirectional, with a smaller fraction of cytosolic oxaloacetate stemming from the mitochondrial pool at the higher dilution rate of 0.16 h(-1) (for glucose or glycerol cultivation) when compared to the glycerol cultivation at 0.05 h(-1). Fourthly, the fraction of anaplerotic synthesis of oxaloacetate increases from 33% to 48% when increasing the dilution rate for glycerol supply, while 38% is detected when glucose is fed. Finally, the cultivation on glucose also allowed qualitative comparison with the flux ratio profile previously published for Pichia stipitis and S. cerevisiae grown on glucose in a chemostat culture at a dilution rate of 0.1 h(-1). This provided a first indication that regulation of central carbon metabolism in P. pastoris and S. cerevisiae might be more similar to each other than to P. stipitis.     

Translation by Escherichia coli ribosomes of alfalfa mosaic virus RNA 4 can be initiated at two sites on the monocistronic message.

Substantial evidence is provided to corroborate our previous finding that Escherichia coli ribosomes recognize two binding sites on the 5' end of alfalfa mosaic virus (AMV) RNA 4 [for a preliminary report see Castel, A., Kraal, B., Kerklaan, P. R. M., Klok, J., and Bosch, L. (1977) Proc. Natl Acad. Sci. U.S.A. 74, 5509--5513]. Translation can start at either site using AcPhe-tRNA or fMet-RNA as initiator and takes place in the same reading frame along the monocistronic mRNA. The size and composition of the isolated extra NH2-terminal fragment of the acetylphenylalanyl product were found to be in agreement with the 5' non-coding region of the messenger. Removal of the 5'-terminal cap structure of AMV RNA 4 did not influence significantly both initiation reactions. Ribosomal protein S1 was essential for binding as well as incorporation of both fMet-tRNA and AcPhe-tRNA. A similar interaction on the ribosome was found for AcPhe-tRNA directed by AMV RNA 4 as for fMet-tRNA directed by either AMV RNA 4 or MS2 RNA with respect to the influence of initiation factors. It is concluded that the heterologous plant viral messenger is reliably translated in the E. coli system and that E. coli ribosomes recognize with high specificity an extra initiation site close to the 5' extremity of the messenger. The relationship of this site to a hypothetical entry site involved in the early recognition in the initiation mechanism between ribosome and messenger is discussed.     

Functional Changes in Rat Nigral GABAA Receptors Induced by Degeneration of the Striatonigral GABAergic Pathway: An Electrophysiological Study of Receptors Incorporated into Xenopus Oocytes

Abstract: Expression of rat brain γ-aminobutyric acid type A (GABA_A) receptors in Xenopus laevis oocytes can be achieved by injection of the oocytes with synaptosomes. This approach has now been applied to evaluate changes in the function of nigral GABA_A receptors after degeneration of the striatonigral GABAergic pathway induced by the unilateral infusion of kainic acid into the rat striatum. Ten days after striatal injection, synaptosomal membranes were prepared from the substantia nigra and introduced into oocytes. Nigral GABA_A receptors incorporated into the oocyte cell membrane were then characterized electrophysiologically under voltage-clamp conditions. The maximal amplitude of GABA-induced Cl^? currents in oocytes injected with synaptosomes from denervated substantia nigra was twice that observed in oocytes injected with synaptosomes from control substantia nigra. The concentration of GABA required for the half-maximal response did not differ between the two groups of oocytes. In addition, the potentiation of GABA-induced currents by the benzodiazepine diazepam (1 µM) and the steroid derivative allopregnanolone (3 µM) was increased by ～65 and 60%, respectively, in oocytes injected with synaptosomes from denervated substantia nigra compared with those injected with control synaptosomes. The concentrations of diazepam and allopregnanolone giving half-maximal responses were not affected by denervation. In contrast, the inhibitory effects of the benzodiazepine receptor inverse agonists FG 7142 (10 µM) and 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylic acid ethyl ester (1 µM) were reduced by 48 and 38%, respectively, after denervation. These results indicate that the up-regulation of nigral GABA_A receptors induced by degeneration of the striatonigral GABAergic pathway is associated with an increased efficacy of positive allosteric modulators, such as benzodiazepines and steroids, and with a reduced efficacy of negative allosteric modulators such as β-carbolines.     

A DNA element recognised by the molybdenum-responsive transcription factor ModE is conserved in Proteobacteria, green sulphur bacteria and Archaea

Background

In general, chemotaxis in Rhizobium has not been well characterized. Methyl accepting chemotaxis proteins are sensory proteins important in chemotaxis of numerous bacteria, but their involvement in Rhizobium chemotaxis is unclear and merits further investigation.

Results

A putative methyl accepting chemotaxis protein gene (mcpG) of Rhizobium leguminosarum VF39SM was isolated and characterized. The gene was found to reside on the nodulation plasmid, pRleVF39d. The predicted mcpG ORF displayed motifs common to known methyl-accepting chemotaxis proteins, such as two transmembrane domains and high homology to the conserved methylation and signaling domains of well-characterized MCPs. Phenotypic analysis of mcpG mutants using swarm plates did not identify ligands for this putative receptor. Additionally, gene knockouts of mcpG did not affect a mutant strain's ability to compete for nodulation with the wild type. Notably, mcpG was found to be plasmid-encoded in all strains of R. leguminosarum and R. etli examined, though it was found on the nodulation plasmid only in a minority of strains.

Conclusions

Based on sequence homology R. leguminosarum mcpG gene codes for a methyl accepting chemotaxis protein. The gene is plasmid localized in numerous Rhizobium spp. Although localized to the sym plasmid of VF39SM mcpG does not appear to participate in early nodulation events. A ligand for McpG remains to be found. Apparent McpG orthologs appear in a diverse range of proteobacteria. Identification and characterization of mcpG adds to the family of mcp genes already identified in this organism.     

Demonstration of elevated type I and type III procollagen mRNA levels in cutaneous wounds treated with helium-neon laser. Proposed mechanism for enhanced wound healing

To assess laser modulation of wound healing, full-thickness cutaneous wounds were produced in the backs of pigs, and subjected to treatment with helium-neon laser. For comparison, some wounds were treated with non-laser energy source (a tungsten light) or left untreated as controls. Type I and type III procollagen mRNA levels were determined in the wounds by molecular hybridization with cDNA probes. The results indicated that type I and type III mRNA levels were markedly increased at days 17 and 28 of the healing in wounds treated with He-Ne laser, when compared to control or tungsten light-treated wounds. The results suggest that helium-neon laser stimulates wound healing by enhancing procollagen gene expression. These observations may have relevance to previous clinical studies suggesting that helium-neon laser stimulates wound healing.     

Expression of a Rhizopus oryzae lipase in Pichia pastoris under control of the nitrogen source-regulated formaldehyde dehydrogenase promoter

A Rhizopus oryzae lipase gene has been expressed in Pichia pastoris as a reporter using the formaldehyde dehydrogenase 1 promoter (PFLD1) of this organism, which has been reported to be strongly and independently induced by either methanol as sole carbon source or methylamine as sole nitrogen source. Levels of lipase expressed and secreted under the control of the PFLD1 at different induction conditions have been compared to those obtained with the commonly used alcohol oxidase 1 promoter (PAOX1) in small (shake flask) and 1l bioreactor batch cultures. PFLD1-controlled heterologous gene expression was strongly repressed by excess of either glycerol or glucose-but not sorbitol-during growth using methylamine both as sole nitrogen source and inducing substrate. Co-induction of PFLD1 with methanol and methylamine resulted in a synergistic effect on extracellular lipase expression levels. In all tested conditions, the substitution of ammonium for methylamine as carbon source provoked a clear decrease in the specific growth rate and yield of biomass per gram of carbon source. Overall, this study demonstrates that the PFLD1 promoter is at least as efficient as the PAOX1 for extracellular expression of heterologous proteins in P. pastoris bioreactor cultures and provides a first basis for the further design of methanol-free high cell density fed-batch cultivation strategies for controlled overproduction of foreign proteins in P. pastoris.