Na+/K+-ATPase Inhibition Partially Mimics the Ethanol-Induced Increase of the Golgi Cell-Dependent Component of the Tonic GABAergic Current in Rat Cerebellar Granule Cells

Cerebellar granule cells (CGNs) are one of many neurons that express phasic and tonic GABAergic conductances. Although it is well established that Golgi cells (GoCs) mediate phasic GABAergic currents in CGNs, their role in mediating tonic currents in CGNs (CGN-I_tonic) is controversial. Earlier studies suggested that GoCs mediate a component of CGN-I_tonic that is present only in preparations from immature rodents. However, more recent studies have detected a GoC-dependent component of CGN-I_tonic in preparations of mature rodents. In addition, acute exposure to ethanol was shown to potentiate the GoC component of CGN-I_tonic and to induce a parallel increase in spontaneous inhibitory postsynaptic current frequency at CGNs. Here, we tested the hypothesis that these effects of ethanol on GABAergic transmission in CGNs are mediated by inhibition of the Na⁺/K⁺-ATPase. We used whole-cell patch-clamp electrophysiology techniques in cerebellar slices of male rats (postnatal day 23–30). Under these conditions, we reliably detected a GoC-dependent component of CGN-I_tonic that could be blocked with tetrodotoxin. Further analysis revealed a positive correlation between basal sIPSC frequency and the magnitude of the GoC-dependent component of CGN-I_tonic. Inhibition of the Na⁺/K⁺-ATPase with a submaximal concentration of ouabain partially mimicked the ethanol-induced potentiation of both phasic and tonic GABAergic currents in CGNs. Modeling studies suggest that selective inhibition of the Na⁺/K⁺-ATPase in GoCs can, in part, explain these effects of ethanol. These findings establish a novel mechanism of action of ethanol on GABAergic transmission in the central nervous system.     

Separation of endocytic from exocytic coated vesicles using a novel cholinesterase mediated density shift technique

Coated vesicles isolated from rat liver perfused with diisopropylfluorophosphate (DFP) to inactivate endogenous cholinesterase contained newly synthesized secretory cholinesterase after a 30 min recovery. The cholinesterase is found in coated vesicles of presumed endocytic origin following DFP treatment and perfusion for 3 min with galactosylated cholinesterase, a ligand for the asialoglycoprotein receptor. Highly enriched populations of endocytic and exocytic coated vesicles can be separated by use of a novel cholinesterase mediated density shift technique. The two coated vesicle classes have very similar polypeptide compositions but differ significantly in the ratio of cholesterol to phospholipid.     

Lysosomal enzyme precursors in coated vesicles derived from the exocytic and endocytic pathways

The molecular forms of two lysosomal enzymes, cathepsin C and cathepsin D, have been examined in lysosomes and coated vesicles (CVs) of rat liver. In addition, the relative proportion of these lysosomal enzymes residing in functionally distinct CV subpopulations was quantitated. CVs contained newly synthesized precursor forms of the enzymes in contrast to lysosomes where only the mature forms were detected. Exocytic and endocytic CV subpopulations were prepared by two completely different protocols. One procedure, a density shift method, uses cholinesterase to alter the density of CVs derived from exocytic or endocytic pathways. The other relies on electrophoretic heterogeneity to accomplish the CV subfractionation. Subpopulations of CVs prepared by either procedure showed similar results, when examined for their relative proportion of cathepsin C and cathepsin D precursors. Within the starting CV preparation, exocytic CVs contained approximately 80-90% of the total steady-state levels of these enzymes while the level in the endocytic population was approximately 10-13%. The implications of these findings are discussed with regard to lysosome trafficking.     

Influence of harmonic radar tag attachment on nymphal Halyomorpha halys mobility,survivorship, and detectability

The brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), is a polyphagous invasive insect and currently one of the most threatening agricultural pests in the USA and globally. Nymphs are highly mobile, moving among host plants, and causing significant damage. Thus, understanding dispersal biology for all life stages is critical for the development of reliable monitoring and management programs. Here, we evaluated the influence of harmonic radar as a tool to study dispersal ecology of nymphal H. halys; we measured the impact of glues and tag attachment on survivorship and mobility in the laboratory and validated in the field that tagged and released nymphs could be tracked on baited and unbaited host and non‐host plants using harmonic radar. In the laboratory, four glues were evaluated for attaching harmonic radar tags securely to nymphs, and survivorship with attached tags was measured. There were no significant differences in survivorship or vertical and horizontal movement among nymphs with tags affixed with the glue treatments compared with the untagged control. Based on numerically greater survivorship of nymphs with tags affixed with Loctite glass glue, a field validation study of tagged nymphs released in host (apple tree) and non‐host (mowed grass) with or without H. halys pheromonal stimuli present revealed that nymphs could be successfully relocated using harmonic radar after 48 h. Among treatments, 83% of nymphs remained in baited and unbaited apple trees, 50% of nymphs remained in baited mowed grass plots, and in unbaited mowed grass plots, 17% of fifth instars, and 0% of fourth instars were retained. The absence of negative effects on mobility, survivorship, and field tracking validates that harmonic radar can be used to study dispersal ecology of nymphal H. halys.     

nanos1: a mouse nanos gene expressed in the central nervous system is dispensable for normal development

A mouse nanos (nanos1) gene was cloned and its function was examined by generating a gene-knockout mouse. The nanos1 gene encodes an RNA-binding protein, which contains a putative zinc-finger motif that exhibits similarity with other nanos-class genes in vertebrates and invertebrates. Although nanos1 is not detected in primordial germ cells, it is observed in seminiferous tubules of mature testis. Interestingly, maternally expressed nanos1 is observed in substantial amounts in oocytes, but the amount of maternal RNA is rapidly reduced after fertilization, and the transient zygotic nanos1 expression is observed in eight-cell embryos. At 12.5 days postcoitum, nanos1 is re-expressed in the central nervous system and the expression continues in the adult brain, in which the hippocampal formation is the predominant region. The nanos1 -deficient mice develop to term without any detectable abnormality and they are fertile. No significant neural defect is observed in terms of their behavior to date.     

Efflux of sphingomyelin, cholesterol, and phosphatidylcholine by ABCG1

Cholesterol and phospholipids are essential to the body, but an excess of cholesterol or lipids is toxic and a risk factor for arteriosclerosis. ABCG1, one of the half-type ABC proteins, is thought to be involved in cholesterol homeostasis. To explore the role of ABCG1 in cholesterol homeostasis, we examined its subcellular localization and function. ABCG1 and ABCG1-K120M, a WalkerA lysine mutant, were localized to the plasma membrane in HEK293 cells stably expressing ABCG1 and formed a homodimer. A stable transformant expressing ABCG1 exhibited efflux of cholesterol and choline phospholipids in the presence of BSA, and the cholesterol efflux was enhanced by the presence of HDL, whereas cells expressing ABCG1-K120M did not, suggesting that ATP binding and/or hydrolysis is required for the efflux. Mass and TLC analyses revealed that ABCG1 and ABCA1 secrete several species of sphingomyelin (SM) and phosphatidylcholine (PC), and SMs were preferentially secreted by ABCG1, whereas PCs were preferentially secreted by ABCA1. These results suggest that ABCA1 and ABCG1 mediate the lipid efflux in different mechanisms, in which different species of phospholipids are secreted, and function coordinately in the removal of cholesterol and phospholipids from peripheral cells.     

The protozoan inositol phosphorylceramide synthase: a novel drug target that defines a new class of sphingolipid synthase

Sphingolipids are ubiquitous and essential components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is conserved up to the formation of sphinganine. However, a divergence is apparent in the synthesis of complex sphingolipids. In animal cells, ceramide is a substrate for sphingomyelin (SM) production via the enzyme SM synthase. In contrast, fungi utilize phytoceramide in the synthesis of inositol phosphorylceramide (IPC) catalyzed by IPC synthase. Because of the absence of a mammalian equivalent, this essential enzyme represents an attractive target for anti-fungal compounds. In common with the fungi, the kinetoplastid protozoa (and higher plants) synthesize IPC rather than SM. However, orthologues of the gene believed to encode the fungal IPC synthase (AUR1) are not readily identified in the complete genome data bases of these species. By utilizing bioinformatic and functional genetic approaches, we have isolated a functional orthologue of AUR1 in the kinetoplastids, causative agents of a range of important human diseases. Expression of this gene in a mammalian cell line led to the synthesis of an IPC-like species, strongly indicating that IPC synthase activity is reconstituted. Furthermore, the gene product can be specifically inhibited by an anti-fungal-targeting IPC synthase. We propose that the kinetoplastid AUR1 functional orthologue encodes an enzyme that defines a new class of protozoan sphingolipid synthase. The identification and characterization of the protozoan IPC synthase, an enzyme with no mammalian equivalent, will raise the possibility of developing anti-protozoal drugs with minimal toxic side affects.     

Rad51 suppresses gross chromosomal rearrangement at centromere in Schizosaccharomyces pombe

Centromere that plays a pivotal role in chromosome segregation is composed of repetitive elements in many eukaryotes. Although chromosomal regions containing repeats are the hotspots of rearrangements, little is known about the stability of centromere repeats. Here, by using a minichromosome that has a complete set of centromere sequences, we have developed a fission yeast system to detect gross chromosomal rearrangements (GCRs) that occur spontaneously. Southern and comprehensive genome hybridization analyses of rearranged chromosomes show two types of GCRs: translocation between homologous chromosomes and formation of isochromosomes in which a chromosome arm is replaced by a copy of the other. Remarkably, all the examined isochromosomes contain the breakpoint in centromere repeats, showing that isochromosomes are produced by centromere rearrangement. Mutations in the Rad3 checkpoint kinase increase both types of GCRs. In contrast, the deletion of Rad51 recombinase preferentially elevates isochromosome formation. Chromatin immunoprecipitation analysis shows that Rad51 localizes at centromere around S phase. These data suggest that Rad51 suppresses rearrangements of centromere repeats that result in isochromosome formation.     

Molecular Characterization of a Deep-Sea Methanotrophic Mussel Symbiont that Carries a RuBisCO Gene

In our previous investigation on the genes of 1,5-ribulose bisphosphate carboxylase/oxygenase (RuBisCO; EC 4.1.1.39) in deep-sea chemoautotrophic and methanotrophic endosymbioses, the gene encoding the large subunit of RuBisCO form I (cbbL) had been detected in the gill of a mussel belonging to the genus Bathymodiolus from a western Pacific back-arc hydrothermal vent. This study further examined the symbiont source of the RuBisCO cbbL gene along with the genes of 16S ribosomal RNA (16S rDNA) and particulate methane monooxygenase (EC 1.14.13.25; pmoA) and probed for the presence of the ATP sulfurylase gene (EC 2.7.7.4; sopT). The 16S rDNA sequence analysis indicated that the mussel harbors a monospecific methanotrophic Gammaproteobacterium. This was confirmed by amplification and sequencing of the methanotrophic pmoA, while thiotrophic sopT was not amplified from the same symbiotic genome DNA. Fluorescence in situ hybridization demonstrated simultaneous occurrence of the symbiont-specific 16S rDNA, cbbL and pmoA, but not sopT, in the mussel gill. This is the first molecular and visual evidence for a methanotrophic bacterial endosymbiont that bears the RuBisCO cbbL gene relevant to autotrophic CO₂ fixation.