Modulation of Second Messengers in the Nervous System of Larval Manduca sexta by Muscarinic Receptors

Abstract: Measurements were made of the effects of muscarinic agents on endogenous levels of cyclic AMP and cyclic GMP, and the turnover of radiolabeled inositol phosphates in the abdominal nervous system of larval Manduca sexta . Cyclic AMP levels were increased by treatment with 3-isobutyl-1-methylxanthine or tetrodotoxin, but the muscarinic agonist oxotremorine-M and the muscarinic antagonist scopolamine had no consistent effects. In contrast, cyclic GMP levels were significantly increased by oxotremorine-M and by oxotremorine-M in the presence of 3-isobutyl-1-methylxanthine and tetrodotoxin but not in the presence of scopolamine. Using lithium to inhibit the recycling of inositol phospholipid metabolites in isolated nerve cords, we detected a small but consistent increase in inositol phosphate production by oxotremorine-M. The primary inositol metabolite generated during a 5-min exposure to oxotremorine-M co-eluted from ion-exchange columns with inositol-1-monophosphate, although other more polar metabolites were also detected. This agonist-evoked increase in inositol phosphate production was unaffected by tetrodotoxin but inhibited by scopolamine, suggesting that it is directly mediated by muscarinic receptors. Further evidence for coupling between muscarinic receptors and inositol metabolism was obtained using a cell-free preparation of nerve cord membranes labeled with [³H]inositol. Incubation with oxotremorine-M evoked a significant increase in labeled inositol bisphosphate, consistent with muscarinic receptors coupling to phosphatidylinositol metabolism. The accumulation of inositol bisphosphate in cell-free preparations suggests that the normal breakdown to inositol monophosphate requires cytosolic components. Together, these results indicate that muscarinic acetylcholine receptors in Manduca couple predominantly to the inositol phospholipid signaling system, although some receptors may modulate cyclic GMP.     

An endophytic <Emphasis Type="Italic">Neurospora</Emphasis> sp. from <Emphasis Type="Italic">Nothapodytes foetida</Emphasis> producing camptothecin

The medicinal plant, Nothapodytes foetida, contains a number of important alkaloids like camptothecin (an anticancer drug molecule), but its concentration is less to meet the existing demand of this important molecule, in an effort for accessible availability of camptothecin. An endophyte (designated ZP5SE) was isolated from the seed of Nothapodytes foetida and was examined as a potential source of anticancer drug lead compound, i.e., camptothecin, when grown in Sabouraud liquid culture media under shake flask conditions. The presence of an anticancer compound (camptothecin) in this fungus was confirmed by chromatographic and spectroscopic methods in comparison with authentic camptothecin. Isolated endophyte (Neurospora crassa) producing camptothecin may become an easily accessible source for the production of a precursor anticancer drug molecule in the future at a large scale. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2008, Vol. 44, No. 2, pp. 225–231. The text was submitted by the authors in English.     

Molecular cloning and characterization of amylase from soil metagenomic library derived from Northwestern Himalayas

The increasing demand for novel biocatalysts stimulates exploration of resources from soil. Metagenomics, a culture independent approach, represent a sheer unlimited resource for discovery of novel biocatalysts from uncultured microorganisms. In this study, a soil-derived metagenomic library containing 90,700 recombinants was constructed and screened for lipase, cellulase, protease and amylase activity. A gene (pAMY) of 909 bp encoding for amylase was found after the screening of 35,000 Escherichia coli clones. Amino acid sequence comparison and phylogenetic analysis indicated that pAMY was closely related to uncultured bacteria. The molecular mass of pAMY was estimated about 38 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Amylase activity was determined using soluble starch, amylose, glycogen and maltose as substrates. The maximal activity (2.46 U/mg) was observed at 40 °C under nearly neutral pH conditions with amylose; whereas it retains 90% of its activity at low temperature with all the substrates used in this study. The ability of pAMY to work at low temperature is unique for amylases reported so far from microbes of cultured and uncultured division.     

Molecular phylogeny in Indian Tinospora species by DNA based molecular markers

The phylogenetic relationships among the three species of Tinospora found in India are poorly understood. Morphology does not fully help to resolve the phylogeny and therefore a fast approach using molecular analysis was explored. Two molecular approaches viz Random Amplified Polymorphic DNA (RAPD) assay and restriction digestion of ITS1-5.8S-ITS2 rDNA (PCR-RFLP) were used to evaluate the genetic similarities between 40 different accessions belonging to three species. Of the 38 random primers used only six generated the polymorphism, while as three out of 11 restriction enzymes used gave polymorphic restriction patterns. The average proportion of polymorphic markers across primers was 95%, however restriction endonucleases showed 92% polymorphism. RAPD alone was found suitable for the species diversions. In contrast PCR- RFLP showed bias in detecting exact species variation. The correlation between the two markers was performed by Jaccard's coefficient of similarity. A significant (r= 0.574) but not very high correlation was obtained. Further to authenticate the results obtained by two markers, sequence analysis of ITS region of ribosomal DNA (ITS1 and ITS2, including 5.8S rDNA) was performed. Three independent clones of each species T. cordifolia, T. malabarica and T. crispa were sequenced. Phylogenetic relationship inferred from ITS sequences is in agreement with RAPD data.     

Emergence of sperm from female storage sites has egg-influenced and egg-independent phases in Drosophila melanogaster

The coordinated introduction of sperm and eggs is a prerequisite of high fertilization efficiency. In Drosophila melanogaster, as in most internally fertilizing animals, mated females store sperm prior to fertilization. Yet the regulation of sperm exit from these storage sites is poorly understood. To test one likely factor that could coordinate gamete availability, we quantified sperm exit from storage in three types of female: genetically matched females that were normal or eggless, and an additional wild-type control. Long-term depletion of sperm stores in normal females and eggless females occurs at similar rates. However, soon after mating, egg presence appears to accelerate the transition from one storage stage to the next. Since male ejaculate components and female factors contribute to sperm depletion, opportunities exist for both cooperation and conflict between the sexes in sperm storage dynamics.     

Silencing of MBD1 and MeCP2 in prostate-cancer-derived PC3 cells produces differential gene expression profiles and cellular phenotypes

Alterations in genomic CpG methylation patterns have been found to be associated with cell transformation and neoplasia. Although it is recognized that methylation of CpG residues negatively regulates gene expression, how the various MBPs (methyl-binding proteins) contribute to this process remains elusive. To determine whether the two well characterized proteins MeCP2 (methyl-CpG-binding protein 2) and MBD1 (methyl-CpG-binding domain 1) have distinct or redundant functions, we employed RNAi (RNA interference) to silence their expression in the prostate cancer-derived PC3 cell line, and subsequently compared cell growth, invasion and migration properties of these cell lines in addition to their respective mRNA-expression profiles. Cells devoid of MeCP2 proliferated more poorly compared with MBD1-deficient cells and the parental PC3 cells. Enhanced apoptosis was observed in MeCP2-deficient cells, whereas apoptosis in parental and MBD1-deficient cells appeared to be equivalent. Boyden chamber invasion and wound-healing migration assays showed that MBD1-silenced cells were both more invasive and migratory compared with MeCP2-silenced cells. Finally, gene chip microarray analyses showed striking differences in the mRNA-expression profiles obtained from MeCP2- and MBD1-depleted cells relative to each other as well as when compared with control cells. The results of the present study suggest that MeCP2 and MBD1 silencing appear to affect cellular processes independently in vivo and that discrete sets of genes involved in cellular proliferation, apoptosis, invasion and migration are targeted by each protein.     

Addition of exogenous carbon and nitrogen sources to aphid exuviae modulates synthesis of proteases and chitinase by germinating conidia of Beauveria bassiana

Secretion of catabolic extracellular enzymes (ECE) is the hallmark of the infection of insects through the cuticle by entomopathogenic fungi (EPF). In this paper, we show that germinating conidia of Beauveria bassiana (Bb) regulate the synthesis of ECE through a multiple control mode during the initial stages of germination. We tested Bb conidial growth on aphid exuviae with or without supplementation of additional carbon and/or nitrogen (C/N) compounds. To understand the interrelation between conidial germination during growth, the synthesis of ECE activity, free amino nitrogen (FAN), glucose and fungal dry weight biomass were measured. Immediately (0.25 h) upon incubation of conidia, activity of subtilisin-like Pr1 and trypsin-like Pr2 enzymes and chitinase (NAGase) was observed in the culture filtrates. At 0.25 h, addition of exogenous C-source resulted in higher activities of Pr1 and Pr2, respectively. Conversely at 0.25 h, addition of N-sources repressed the synthesis of Pr2, but that of Pr1. C/N repression was observed only for exponentially growing mycelia. NAGase activity remained at basal level and unaffected by added C/N. We conclude that C/N repression occurs only when it is necessary for the Bb infective structures to establish a nutritional relationship with the host structures.