Ammonium effects on streptonigrin biosynthesis byStreptomyces flocculus

Summary A defined medium containing glucose and ammonium as the sole carbon and nitrogen sources was developed to support growth and streptonigrin production. In this defined medium, increased initial levels of ammonium resulted in increased growth suggesting that nitrogen is the growth limiting nutrient. In some cases, increased initial ammonium levels resulted in decreased specific streptonigrin productivity, suggesting that nitrogen regulatory mechanisms may adversely affect streptonigrin biosynthesis. This suggestion that nitrogen regulation adversely affects antibiotic biosynthesis is further supported by results from two studies in which the ammonium supply to the cells was controlled. In the first study, streptonigrin productivity and final titer were enhanced by the addition of an ammonium trapping agent. In the second experiment, when ammonium chloride was fed slowly throughout the course of cultivation, the production phase was lengthened and the maximum antibiotic concentration was enhanced compared to the batch controls containing either the same initial or the same total ammonium chloride levels. Although our results indicate streptonigrin production may be subject to nitrogen regulatory mechanisms, the effect of nitrogen on streptonigrin production cannot be strictly correlated to the extracellular ammonium concentration. In fact, we observed that when ammonium was depleted from the medium, streptonigrin production ceased.     

Extracellular Striatal Dopamine and Glutamate After Decortication and Kainate Receptor Stimulation, as Measured by Microdialysis

Abstract: Disruption of corticostriatal glutamate input in the striatum decreased significantly extracellular striatal glutamate and dopamine levels. Local administration of 300 µ M concentration of excitatory receptor agonist kainic acid increased significantly extracellular striatal dopamine in intact freely moving rats. These findings support the hypothesis that glutamate exerts a tonic facilitatory effect on striatal dopamine release. The effect of kainic acid on extracellular striatal glutamate concentration in intact rats was a biphasic increase. The first glutamate increase can be explained by stimulation of presynaptic kainate receptors present on corticostriatal glutamatergic nerve terminals; the second increase is probably the result of a continuous interaction of the different striatal neurotransmitters after disturbance of their balance. Release of dopamine and glutamate was modulated differently in the intact striatum and in the striatum deprived of corticostriatal input. Dopamine release in the denervated striatum after kainate receptor stimulation was significantly lower than in intact striatum, confirming the so-called cooperativity between glutamate and kainic acid. Loss of presynaptic kainate receptors on the glutamatergic nerve terminals after decortication resulted in a loss of effect of kainic acid on glutamate release in denervated striatum. Aspartate showed no significant changes in this study.     

Assessment of the Role of the Glutathione and Pentose Phosphate Pathways in the Protection of Primary Cerebrocortical Cultures from Oxidative Stress

Abstract: Reactive oxygen species have been implicated in neuronal injury associated with various neuropathological disorders. However, little is known regarding the relationship between antioxidant enzyme capacity and resultant toxicity. The antioxidant pathways of primary cerebrocortical cultures were directly examined using a novel technique that measures pentose phosphate pathway (PPP) activity, which is enzymatically coupled to glutathione peroxidase (GPx) detoxification of hydrogen peroxide (H₂O₂). PPP activity was quantified from data obtained by gas chromatography/mass spectrometry analysis of released labeled lactate following metabolic degradation of [1,6-¹³C₂,6,6-²H₂]glucose by cerebrocortical cultures. The antioxidant capacity of these cultures was systematically evaluated using H₂O₂, and the resultant toxicity was quantified by lactate dehydrogenase release. Exposure of primary mixed and purified astrocytic cultures to H₂O₂ caused stimulation of PPP activity in a concentration-dependent fashion from 0.25 to 22.2% and from 6.9 to 66.7% of glucose metabolized to lactate through the PPP, respectively. In the mixed cultures, chelation of iron before H₂O₂ exposure was protective and resulted in a correlation between PPP saturation and toxicity. Conversely, addition of iron, inhibition of GPx, or depletion of glutathione decreased H₂O₂-induced PPP stimulation and increased toxicity. These results implicate the Fenton reaction, reflect the pivotal role of GPx in H₂O₂ detoxification, and contribute to our understanding of the etiological role of free radicals in neuropathological conditions.     

Genomic organization of mouse Dishevelled genes

We report the isolation and characterization of two new genomic loci corresponding to the mouse Dishevelled (Dvl) genes Dvl2 and Dvl3. The Dvl genes are homologs of the Drosophila dsh segment polarity gene, and are involved in the Wnt/wingless signal transduction pathway. Dvl2 and Dvl3 genomic clones were isolated from a mouse 129 strain λFIXII genomic library and have identical exon/intron organization to Dvll. All three Dishevelled genes span 15 exons and 14 introns and have a number of conserved splice junction sites.     

Cloning and expression of transaldolase from potato

We have isolated a cDNA encoding transaldolase, an enzyme of the pentose-phosphate pathway, from potato (Solanum tuberosum). The 1.5 kb cDNA encodes a protein of 438 amino acid residues with a molecular mass of 47.8 kDa. When the potato cDNA was expressed in Escherichia coli a 45 kDa protein with transaldolase activity was produced. The first 62 amino acids of the deduced amino acid sequence represent an apparent plastid transit sequence. While the potato transaldolase has considerable similarity to the enzyme from cyanobacteria and Mycobacterium leprae, similarity to the conserved transaldolase enzymes from humans, E. coli and Saccharomyces cerevisiae is more limited. Northern analysis indicated that the transaldolase mRNA accumulated in tubers in response to wounding. Probing the RNA from various potato tissues indicated that the transaldolase mRNA accumulation to higher levels in the stem of mature potato plants than in either leaves or tubers. These data are consistent with a role for this enzyme in lignin biosynthesis.     

The translational stop signal: Codon with a context, or extended factor recognition element?

Wide ranging studies of the readthrough of translational stop codons within the last 25 years have suggested that the stop codon might be only part of the molecular signature for recognition of the termination signal. Such studies do not distinguish between effects on suppression and effects on termination, and so we have used a number of different approaches to deduce whether the stop signal is a codon with a context or an extended factor recognition element. A data base of natural termination sites from a wide range of organisms (148 organisms, 40000 sequences) shows a very marked bias in the bases surrounding the stop codon in the genes for all organisms examined, with the most dramatic bias in the base following the codon (+4). The nature of this base determines the efficiency of the stop signal in vivo, and in Escherichia coli this is reinforced by overexpressing the stimulatory factor, release factor-3. Strong signals, defined by their high relative rates of selecting the decoding release factors, are enhanced whereas weak signals respond relatively poorly. Site-directed cross-linking from the +1, and bases up to +6 but not beyond make close contact with the bacterial release factor-2. The translational stop signal is deduced to be an extended factor recognition sequence with a core element, rather than simply a factor recognition triplet codon influenced by context.     

Octopamine mediates rapid stimulation of protein kinase A in the antennal lobe of honeybees

In the honeybee octopamine mediates mechanisms of arousal that interfere with the appetitive proboscis extension response to food-indicating chemosensory stimuli. This study demonstrates that injections of octopamine or cyclic adenosine monophosphate (cAMP) into the primary chemosensory neuropil of the honeybee, the antennal lobe, evokes a rapid and transient activation of cAMP-dependent protein kinase (PKA). Other monoamines detectable in the antennal lobe, dopamine and serotonin, do not affect the level of PKA activity. Stimulation of the bees' antenna with the appetitive stimulus water or sucrose solution in vivo also causes a short-term activation of PKA in the antennal lobe. The increased PKA activity can be detected immediately (0.5 s) after stimulation but reverts to the basal level within 3 s. This effect can be abolished by monoamine depletion with reserpine. Since octopamine is the only monoamine that stimulates PKA, it appears to mediate the PKA activation after sucrose stimulus and may contribute to the processing of this chemosensory input. © 1995 John Wiley & Sons, Inc.