The roles of phospholipase C activation and alternative ADAR1 and ADAR2 pre-mRNA splicing in modulating serotonin 2C-receptor editing in vivo

The serotonin 2C receptor (5-HT2CR), a Gq-protein-coupled neurotransmitter receptor, exists in multiple isoforms that result from RNA editing of five exonic adenosines that are converted to inosines. In the adult brain, editing of 5-HT2C pre-mRNA exhibits remarkable plasticity in response to environmental and neurochemical stimuli. Here, we investigated two potential mechanisms underlying these plastic changes in adult 5-HT2CR editing phenotypes in vivo: activation of phospholipase C (PLC) and alternative splicing of pre-mRNA encoding the editing enzymes ADAR1 and ADAR2. Studies on two inbred strains of mice (C57Bl/6 and Balb/c) revealed that sustained stimulation of PLC—a downstream effector of activated Gαq protein—increased editing of forebrain neocortical 5-HT2C pre-mRNA at two sites known to be targeted by ADAR2. Moreover, changes in relative expression of the alternatively spliced “a” and “b” mRNA isoforms of ADAR1 and ADAR2 also correlate with changes in 5-HT2CR editing. The site-specific changes in 5-HT2CR editing detected in mice with different “a” over “b” ADAR mRNA isoform ratios only partially overlap with those evoked by sustained PLC activation and are best explained by the increased editing efficiency of ADAR1. Thus, activation of PLC and alternative splicing of ADAR pre-mRNA have both overlapping and specific roles in modulating 5-HT2CR editing phenotypes.     

Limited digestion of RNA polymerase from Escherichia coli by trypsin (effect of rifamycin and DNA on the integrity of sigma subunit)

A modified polyacrylamide gel electrophoresis technique is used to separate the polypeptides after digestion of $\text{E. coli}$ RNA polymerase with various concentration of trypsin. The subunits β and β′ and two large breakdown products of molecular weight of 147,000 and 141,000 are distinctly separated. At a very low level of trypsin σ and α are not cleaved while two major breakdown products of molecular weights of 110,000 and 43,000 appear from the larger subunits. At a still higher level of trypsin σ is converted to a polypeptide of molecular weight of 86,000 and other small fragments. DNA protects, to some extent, the σ and this polypeptide and also β and the two large breakdown products from trypsin digestion. It is also observed that rifamycin, an inhibitor of RNA synthesis, enhances the tryptic digestion of σ, only in the absence of MgCl₂.     

Interspecific transformation and DNA characteristics in Allomyces

Summary Heterologous deoxyribonucleic acid treatment in Allomyces has been shown to transfer epigynous versus hypogynous character in the recipient species.A certain proportion of inverted sexual arrangements have been consistently detected in the acceptors.The uptake of native DNA was demonstrated using labelled ³²P-nucleic acid. The uptake was found to be higher in homologous (controls) than heterologous receptors.Chromatographic fractionation of the total DNA reveals 3 types differing in their Tm values and therefore GC ratios; these appear to be localized in nuclei, mitochondria and residual cytoplasm.     

Characterization of Neurospora crassa ��-Actinin

α-Actinin, an actin-binding protein of the spectrin superfamily, is present in most eukaryotes except plants. It is composed of three domains: N-terminal CH-domains, C-terminal calcium-binding domain (with EF-hand motifs), and a central rod domain. We have cloned and expressed Neurospora crassa α-actinin as GST and GFP fusion proteins for biochemical characterization and in vivo localization, respectively. The intracellular localization pattern of α-actinin suggests that this protein is intimately associated with actin filaments and plays an important role in the processes of germination, hyphal elongation, septum formation, and conidiation. These functions were confirmed by the experiments on the effect of α-actinin gene deletion in N. crassa.     

Suitability of five species of stored-product insects as hosts for development and reproduction of the parasitoid Anisopteromalus calandrae (Hymenoptera: Pteromalidae)

We investigated the ability of two populations of Anisopteromalus calandrae (Hymenoptera: Pteromalidae), to parasitize and develop on late instars of five different stored-product insects that typically complete their development inside seeds of grain or legume species or other dry commodity. The host species were the cigarette beetle, Lasioderma serricorne (F.); cowpea weevil, Callosobruchus maculatus (F.); rice weevil, Sitophilus oryzae (L.); lesser grain borer, Rhyzopertha dominica (F.); and Angoumois grain moth, Sitotroga cerealella (Olivier). Experiments were conducted in the laboratory in a no-choice design by using petri dishes (15 by 100 mm) as experimental arenas with 20 host larvae. A. calandrae females from populations originating in Georgia (GA) and Oklahoma (OK) were introduced singly into experimental arenas and allowed to sting and oviposit for 24 h. Parasitism by the OK population was greater than that for the GA population across all hosts. However, no or very low parasitism was found on Angoumois grain moth for either population in this experiment. The highest number of parasitoid progeny was recorded on cowpea weevil (15.9) followed by rice weevil (11.5) and cigarette beetle (10.8) for the OK population. A similar trend was observed in the GA population. The highest proportion of female progeny was produced on cowpea weevil (73.0%) by the OK population. Conversely, a higher proportion of female progeny was produced on rice weevil (64.6%) by the GA population than produced by the OK population. Parasitoid adults were significantly larger and heavier when they developed on cowpea weevil irrespective of parasitoid population. The possible application of these results for biological control of stored-product insects is discussed.     

Plasticity of 5-HT 1A receptor-mediated signaling during early postnatal brain development

The presence of serotonin 1A receptor (5-HT(1A)-R) in the hippocampus, amygdala, and most regions of the frontal cortex is essential between postnatal day-5-21 (P5-21) for the expression of normal anxiety levels in adult mice. Thus, the 5-HT(1A)-R plays a crucial role in this time window of brain development. We show that the 5-HT(1A)-R-mediated stimulation of extracellular signal-regulated kinases 1 and 2 (Erk1/2) in the hippocampus undergoes a transition between P6 and P15. At P6, a protein kinase C (PKC) isozyme is required for the 5-HT(1A)-R -->Erk1/2 cascade, which causes increased cell division in the dentate gyrus. By contrast, at P15, PKC alpha participates downstream of Erk1/2 to augment synaptic transmission through the Schaffer Collateral pathway but does not cause increased cell division. Our data demonstrate that the 5-HT(1A)-R -->Erk1/2 cascade uses PKC isozymes differentially, first boosting the cell division to form new hippocampal neurons at P6 and then undergoing a plastic change in mechanism to strengthen synaptic connections in the hippocampus at P15.     

Developmentally regulated proteases in Allomyces arbuscula

A calcium-requiring neutral protease has been detected in the vegetative mycelia of Allomyces arbuscula. The half maximum activation of the enzyme required 0.7 mM and 2.8 mM Ca²⁺ in the crude and partially-purified preparation, respectively. Coinciding with differentiation of zoosporangia, there is a massive induction of another neutral protease which does not require Ca²⁺ for its activity and is of the serine type.