Protein Kinase C of Sympathetic Neuronal Membrane Is Activated by Phorbol Ester-Correlation Between Transmitter Release, 45Ca2+ Uptake, and the Enzyme Activity

The effects of phorbol esters [phorbol 12,13-dibutyrate (PDB), 12-O-tetradecanoylphorbol 13-acetate (TPA), and phorbol 13-acetate] were investigated on the release of [3H]norepinephrine, 45Ca2+ accumulation, and protein kinase C activity in cultured sympathetic neurons of the chick embryo. Sympathetic neurons derived from 10-day-old chick embryo were cultured in serum-free medium supplemented with insulin, transferrin, and nerve growth factor. After 3 days, neurons were loaded with [3H]-norepinephrine and the release of [3H]norepinephrine was determined before and after electrical stimulation. Stimulation at 1 Hz for 15 s increased the release of [3H]-norepinephrine over the nonstimulation period. Stimulation-evoked release gradually declined with time during subsequent stimulation periods. Incubation of neurons in Ca2+-free Krebs solution containing 1 mM EGTA completely blocked stimulation-evoked release of [3H]-norepinephrine. Stimulation-evoked release of [3H]-norepinephrine was markedly facilitated by 3 and 10 nM PDB or TPA. The spontaneous release was also enhanced by PDB and TPA. The net accumulation of 45Ca2+ during stimulation of sympathetic neurons was increased by two- to fourfold in the presence of PDB or TPA. PDB at 1-100 nM produced a concentration-dependent increase in the activation of protein kinase C. PDB at 30 nM increased the activity of protein kinase C of the particulate fraction from 0.09 to 0.58 pmol/min/mg protein. There was no significant change in protein kinase C activity of the cytosolic fraction (0.14 pmol/min/mg versus 0.13 pmol/min/mg protein). The ratio of the particulate to cytosolic protein kinase C increased from a control value of 0.62 to 4.39 after treatment with 30 nM PDB. TPA (10 and 30 nM) also increased protein kinase C activity of the particulate fraction by six- to eightfold. Phorbol 13-acetate had no effect on protein kinase C activity, [3H]norepinephrine release, and 45Ca2+ accumulation. These results provide direct evidence that activation of protein kinase C enhances Ca2+ accumulation, which in turn leads to the facilitation of transmitter release in sympathetic neurons.     

Conservation of IS66 homologue of octopine Ti plasmid DNA in Rhizobium fredii plasmid DNA

Summary DNA sequences homologous to the T-DNA region of the octopine Ti plasmid from Agrobacterium tumefaciens are found in various fast-growing Rhizobium fredii strains. The largest fragment (BamHI fragment 2) at the right-boundary region of the core T-DNA hybridizes to more than one plasmid present in R. fredii. However, one smaller fragment (EcoRI fragment 19a) adjacent to the core T-DNA shows homology only with the plasmid carrying the symbiotic nitrogen-fixation genes (pSym). Hybridization data obtained with digested R. fredii USDA193 pSym DNA suggests that the homology is mainly with two HindIII fragments, 1.7 kb and 8.8 kb in size, of the plasmid. The 1.7 kb HindIII fragment also hybridizes to two regions of the virulence plasmid of A. tumefaciens, pAL1819, a deletion plasmid derived from the octopine Ti plasmid, pTiAch5. Hybridization studies with an insertion element IS66 from A. tumefaciens indicate that the 1.7 kb HindIII fragment of R. fredii plasmid, homologous to the T-DNA and the virulence region of Ti plasmid, is itself an IS66 homologue.     

Molecular cloning and expression of a xylanase gene from Cellulomonas sp. into Escherichia coli

Summary The xylanase gene of Cellulomonas sp. NCIM 2353 was cloned in pUC 18 and selected by growth on xylan as the sole carbon source. The functional clone harboured the recombinant plasmid with an insert of 1.42 kbp, as determined by restriction mapping and Southern hydridization. The clone secreted a xylanase of 45 000 mol. wt. as determined by Western blot analysis using specific antixylanase antibodies. The DNA insert carried the full structural gene along with its promoter and possibly regulatory sequences, since xylanase activity in the clone Cs11 was inducible by xylan. Offprint requests to: D. N. Deobagkar     

Ethanol Promotes Apoptosis in Cerebellar Granule Cells by Inhibiting the Trophic Effect of NMDA

Abstract: When primary cultures of cerebellar granule neurons are grown in a physiological concentration of KCl (5 m M ) they undergo apoptosis, which can be prevented by growing the cells in the presence of N -methyl- d -aspartate (NMDA). We now show that ethanol inhibits this trophic effect of NMDA, i.e., promotes apoptosis, and also inhibits the NMDA-induced increase in intracellular Ca²⁺ concentration in cells grown in 5 m M KCl. Both effects of ethanol show a similar concentration dependence and are reversed by a high concentration of glycine, the co-agonist at the NMDA receptor. The data suggest that the effect of ethanol on apoptosis is mediated, at least in part, by inhibition of NMDA receptor function. This effect of ethanol to increase apoptosis could contribute to the previously described in vivo sensitivity of the developing cerebellum to ethanol-induced damage.     

Beta-amyloid from Alzheimer disease brains inhibits sprouting and survival of sympathetic neurons

The significance of the amyloid plaque core proteins (APCP) in Alzheimer's disease (AD) and its consequences for neuronal survival have been controversial. To address this problem we purified the APCP and beta A obtained from brains with AD, and assessed their biological effects in tissue culture. APCP and beta A caused severe toxicity to chick and rat sympathetic and sensory neurons whose survival is dependent upon NGF. This toxicity was dose dependent and reversible at low doses. APCP and beta A prevented sprouting of neurites in freshly plated neurons. In established cultures addition of these molecules caused vacuolation and fragmentation of neurites and disintegration of neuronal soma. We suggest that the deposition of APCP in AD may be partly responsible for the destruction of the neuritic arbor, thereby contributing to the formation of the neuritic plaque and to neuronal death.     

Stimulated rise in neuronal calcium is faster and greater in the nucleus than the cytosol

Calcium is an important regulator of a variety of neuronal activities including gene expression. However, it is not clear how Ca2+ influx affects intracellular Ca2+ concentration [( Ca2+]i) in the nucleus. We have taken advantage of laser photometry, the Ca2(+)-sensitive dye Indo-1 that allows ratio imaging, and confocal microscopy to eliminate the influences of unequal cell geometry and dye distribution. We show that Ca2+ influx into sympathetic neurons causes a significantly greater and faster increase in [Ca2+]i in the nucleus than in the cytosol. The differential increase in nuclear [Ca2+]i was apparent when Ca2+ entered from the extracellular medium during K+ depolarization, ionomycin or acetylcholine treatment, and brief periods of electrical stimulation. When intracellular Ca2+ was mobilized by caffeine the rise in nuclear [Ca2+]i was again greater than in any other region of the neuron. The increased nuclear Ca2+ levels were uniform throughout the nucleus and not associated with the nuclear envelope. The differential rise in nuclear Ca2+ was eliminated by acridine orange binding to nucleic acids. Nonexcitable cells (astrocytes, oligodendrocytes, and fibroblasts) did not show differential distribution of Ca2+ after ionomycin treatment. These results support the idea that activity-dependent gene regulation in sympathetic neurons may be mediated by changes in Ca2+ concentration at the level of the chromatin material.     

Isolation and characterization of halotolerant Streptomyces radiopugnans from Antarctica soil

An actinomycete wild strain PM0626271 (= MTCC 5447), producing novel antibacterial compounds, was isolated from soil collected from Antarctica. The taxonomic status of the isolate was established by polyphasic approach. Scanning electron microscopy observations and the presence of LL‐Diaminopimelic acid in the cell wall hydrolysate confirmed the genus Streptomyces. Analysis of 16S rRNA gene sequence showed highest sequence similarity to Streptomyces radiopugnans (99%). The phylogenetic tree constructed using near complete 16S rRNA gene sequences of the isolate and closely related strains revealed that although the isolate fell within the S. radiopugnans gene subclade, it was allocated a different branch in the phylogenetic tree, separating it from the majority of the radiopugnans strains. Similar to type strain, S. radiopugnans R97^T, the Antarctica isolate displayed thermo tolerance as well as resistance to ⁶⁰Co gamma radiation, up to the dose of 15 kGy. However, media and salt tolerance studies revealed that, unlike the type strain, this isolate needed higher salinity for its growth. This is the first report of S. radiopugnans isolated from the Antarctica region. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Streptomyces radiopugnans MTCC 5447 is JQ723477 .

Significance and Impact of the Study

The study presents the first report of isolation of Streptomyces radiopugnans from Antarctica. To date, there is only one publication regarding S. radiopugnans R97^T isolated from radiation‐polluted soil. Like the type strain, Antarctica isolate was thermotolerant and radiotolerant, but in addition, it required salts for growth and did not degrade phenol. We envisaged that metabolic pattern of the same species varies based on acclimatization in its native ecological habitat. Additionally, Antarctica isolate had produced novel antibacterial compounds (patent‐US2012/0156295). The study highlighted that least explored extreme regions like Antarctica are rich resources of novel microbial strains producing novel bioactive compounds.     

Catechin induced modulation in the activities of thyroid hormone synthesizing enzymes leading to hypothyroidism

Catechins, the flavonoids found in abundance in green tea, have many beneficial health effects such as antioxidative, anticarcinogenic, anti-inflammatory, antiallergic, and hypotensive properties. However, flavonoids have antithyroid/goitrogenic effect, although less information is available about the effect of pure catechin on thyroid physiology. The present investigation has been undertaken to explore the effect of catechin administration on thyroid physiology in rat model. For the in vivo experiment catechin was injected intraperitoneally (i.p.) at doses of 10, 20 and 30 mg/kg body to male albino rats for 15 and 30 days, respectively, and thyroid activities were evaluated with respect to determination of serum levels of thyroid hormones, thyroid peroxidase, 5′-deiodinase I (5′-DI), and Na⁺, K⁺-ATPase activities that are involved in the synthesis of thyroid hormone. Catechin decreased the activities of thyroid peroxidase and thyroidal 5′-deiodinase I, while Na⁺, K⁺-ATPase activity significantly increased in dose-dependent manner; substantial decrease in serum T3 and T4 levels coupled with significant elevation of serum TSH were also noted. Histological examinations of the thyroid gland revealed marked hypertrophy and/or hyperplasia of the thyroid follicles with depleted colloid content. In in vitro study, short-term exposure of rat thyroid tissue to catechin at the concentrations of 0.10, 0.20, and 0.30 mg/ml leads to decrease in the activities of thyroid peroxidase and 5′-deiodinase I, while the activity of thyroidal Na⁺, K⁺-ATPase remains unaltered even at high concentration of catechin treatment. The present study reinforces the concept that catechin, tea flavonoids possess potent antithyroid activity as evidenced from in vivo and in vitro studies.     

The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

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