Distinct functions of down-regulation-sensitive and -resistant types of protein kinase C in rabbit aortic smooth muscle cells

In quiescent cultures of rabbit aortic smooth muscle cells, 12-O-tetradecanoylphorbol-13-acetate (TPA) induced DNA synthesis to some extent in the presence of rabbit plasma-derived serum but inhibited the rabbit whole blood serum (WBS)-induced DNA synthesis and increase in cytoplasmic free Ca2+ concentration Ca2+]i). Prolonged treatment of the cells with phorbol-12,13-dibutyrate (PDBu) caused the partial down-regulation of protein kinase C to a level of 25-35% of that in control cells. In these PDBu-pretreated cells, TPA neither induced DNA synthesis nor inhibited the WBS-induced DNA synthesis, but still inhibited the WBS-induced increase in [Ca2+]i. These results suggest that there are down-regulation-sensitive and -resistant types of protein kinase C in rabbit aortic smooth muscle cells; that the down-regulation-sensitive type has the proliferative and antiproliferative actions whereas the down-regulation-resistant type lacks them; and that the down-regulation-resistant type has the activity to inhibit the WBS-induced increase in [Ca2+]i.     

A putative GDP–GTP exchange factor is required for development of the excretory cell in Caenorhabditis elegans

The Caenorhabditis elegans excretory cell extends tubular processes, called canals, along the basolateral surface of the epidermis. Mutations in the exc-5 gene cause tubulocystic defects in this canal. Ultrastructural analysis suggests that exc-5 is required for the proper placement of cytoskeletal elements at the apical epithelial surface. exc-5 encodes a protein homologous to guanine nucleotide exchange factors and contains motif architecture similar to that of FGD1, which is responsible for faciogenital dysplasia. exc-5 interacts genetically with mig-2, which encodes Rho GTPase. These results suggest that EXC-5 controls the structural organization of the excretory canal by regulating Rho family GTPase activities.     

Sequential polymerization of flagellin A and flagellin B into Caulobacter flagella

The mode of polymerization of two species of flagellins, flagellin A and flagellin B, in polar flagella of Caulobacter crescentus was examined. By immunological staining we found that 1 to 1.2 μm of the portion of the flagellar filament proximal to the cell was composed of flagellin B, whereas about 5 μm of the distal portion was composed of flagellin A. This result, together with the previous observation that a flagellin B-less mutant cannot form normal flagella but instead forms stubs in spite of their high level of flagellin A synthesis, indicates that flagellin B is very important for the formation of complete flagella and/or for the initiation of filament formation from the hook.     

RNA-Induced Interference with Animal Virus Infection

Some RNAs, including both single- and double-stranded RNAs, when incubated with chick embryo cell culture induce cellular resistance against viruses. Evidence was now obtained indicating that the induction of cellular resistance by RNA depends on the cellular metabolic activity, especially on the synthesis of cellular RNA and protein. Thus, inhibitors of RNA and protein synthesis, actinomycin D and cycloheximide, were found to inhibit the development of an antiviral state when added before, or during the relatively early period of, incubation of the cells with RNA. In the course of induction of cellular resistance, three stages may be distinguished, the priming stage, the developing stage, and the established resistant stage.     

A new species of Trichogramma (Hymenoptera: Trichogrammatidae) parasitic on eggs of the alderfly Sialis melania (Neuroptera: Sialidae) from Japan,with comments on its phylogeny and male wing polymorphism

A new species of Trichogramma that parasitizes Sialis melania eggs is described as Trichogramma tajimaense Yashiro, Hirose and Honda, sp. nov. from Japan. Its phylogenetic position is based on a DNA‐based analysis, and data regarding its male wing polymorphism are also presented. The view that T. tajimaense is closely related to T. semblidis, another parasitoid of Sialis eggs, is supported by the results of a phylogenetic analysis, as well as by the biological and morphological similarities between both species. Trichogramma tajimaense is also similar in male wing polymorphism to T. kurosuae, a gregarious egg parasitoid of the lepidopteran Ivela auripes, as both Trichogramma species exhibit male wing trimorphism (fully alate, brachypterous and apterous forms) in contrast to the male wing dimorphism (fully alate and apterous forms) of T. semblidis. However, no phylogenetic analysis reveals a close relationship between T. tajimaense and T. kurosuae, and a difference exists between these two species in the mean percentage of flightless (brachypterous and apterous) males that emerge from a host egg mass; 96% of T. tajimaense males are incapable of flight, whereas about 50% of T. kurosuae males are flightless. Because all or almost all males of T. semblidis parasitizing Sialis eggs are apterous, T. tajimaense is more similar to T. semblidis than to T. kurosuae in the proportion of flightless males. In addition, male wing polymorphisms of Trichogramma in relation to mating systems could also show a similarity between T. tajimaense and T. semblidis when considering both species as quasi‐gregarious parasitoids of Sialis eggs.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A new haptenic compound, a muramyl dipeptide (MDP) derivative (designated as L4-MDP-ONB) cross-reactive with Bacillus Calmette Guerin (BCG) was synthesized. The cross-reactivity of L4-MDP hapten to BCG was demonstrated from the following evidence; (a) lymph node cells from BCG-primed C3H/HeN mice exhibited appreciable L4-MDP-specific proliferative responses to the in vitro stimulation of L4-MDP-modified syngeneic cells (L4-MDP-self); (b) inoculation of L4-MDP-self into footpads of BCG-primed C3H/HeN mice elicited ample delayed type-hypersensitivity (DTH) responses in vivo as measured by footpad swelling; and (c) BCG-primed mice contained L4-MDP-reactive helper T cell activity which functions to augment the generation of effector T cell responses to cell surface antigens. This crossreactivity between L4-MDP hapten and BCG as measured by the helper T cell activity was applied to enhanced induction of tumor-specific immunity. When BCG-primed C3H/HeN mice were immunized with L4-MDP-modified syngeneic X5563 tumor cells, these mice could generate augmented tumor-specific in vivo protective (tumor neutralizing) immunity as well as in vitro cytotoxic T cell responses. These results indicate the effectiveness of L4-MDP hapten in augmenting tumor-specific immunity. The present approach is discussed in the context of potential advantages of this new hapten for its future application to clinical tumor systems.     

Endothelin Evokes Efflux of Glutamate in Cultures of Rat Astrocytes

Abstract: Excessive release of glutamate, from glial cells as well as neurons, is thought to be a major cause of neuronal death in ischemia. To investigate glutamate release from glial cells, we measured glutamate efflux from cultures of rat astrocytes preloaded with l -[³H]-glutamate. Glutamate efflux was induced by either 60 m M KCl or Na⁺-free medium, suggesting that the efflux is due to the reversed operation of a Na⁺- and K⁺-coupled glutamate uptake machinery. While investigating various neuropeptides and neurotransmitters, we found that endothelin (ET) specifically induced efflux of glutamate. Northern blot analysis and binding study showed that the ET type B receptor (ET_B-R) subtype was expressed two to three times more densely than the ET type A receptor (ET_A-R) in astrocytes. The ET_B-R antagonist IRL 2500 partially inhibited efflux of glutamate induced by 1 n M ET-1 in a concentration-dependent manner, causing a maximal inhibition of 60% at 1 µ M . However, the ET_A-R antagonist BQ-123 did not cause significant inhibition even at 10 µ M . Combination of both antagonists completely inhibited the ET-1-induced efflux. These results indicate that both receptor subtypes are involved in efflux of glutamate with a major contribution from the ET_B-R. Our findings suggest that ET, which is known to be released in ischemia, may exacerbate neurodegeneration by stimulating efflux of glutamate.     

Biotic and Abiotic Factors Controlling Respiration Rates of Above- and Belowground Woody Debris of Fagus crenata and Quercus crispula in Japan

As a large, long-term pool and source of carbon and nutrients, woody litter is an important component of forest ecosystems. The objective of this study was to estimate the effect of the factors that regulate the rate of decomposition of coarse and fine woody debris (CFWD) of dominant tree species in a cool-temperate forest in Japan. Respiration rates of dead stems, branches, and coarse and fine roots of Fagus crenata and Quercus crispula felled 4 years prior obtained in situ ranged from 20.9 to 500.1 mg CO₂ [kg dry wood]^–1 h^–1 in a one-time measurement in summer. Respiration rate had a significant negative relationship with diameter; in particular, that of a sample of Q. crispula with a diameter of >15 cm and substantial heartwood was low. It also had a significant positive relationship with moisture content. The explanatory variables diameter, [N], wood density, and moisture content were interrelated. The most parsimonious path model showed 14 significant correlations among 8 factors and respiration. Diameter and [C] had large negative direct effects on CFWD respiration rate, and moisture content and species had medium positive direct effects. [N] and temperature did not have direct or indirect effects, and position and wood density had indirect effects. The model revealed some interrelationships between controlling factors. We discussed the influence of the direct effects of explanatory variables and the influence especially of species and position. We speculate that the small R ² value of the most parsimonious model was probably due to the omission of microbial biomass and activity. These direct and indirect effects and interrelationships between explanatory variables could be used to develop a process-based CFWD decomposition model.     

The geranylgeranyl moiety but not the methyl moiety of the smg-25A/rab3A protein is essential for the interactions with membrane and its inhibitory GDP/GTP exchange protein.

The smg-25A/rab3A protein (smg p25A), a member of the small GTP-binding protein superfamily, has a C-terminal structure of Cys-Ala-Cys which is post-translationally processed: both cysteine residues are geranylgeranylated followed by the carboxyl methylation of the C-terminal cysteine residue. We reported previously that this posttranslational processing is essential for the interactions of smg p25A with membrane and its inhibitory GDP/GTP exchange protein, named smg p25A GDP dissociation inhibitor (GDI). In this study, we examined which posttranslational modification of smg p25A is necessary for these interactions. The smg p25A which was not posttranslationally processed was produced in Escherichia coli and purified. This protein was then geranylgeranylated at both of the 2 cysteine residues by use of a bovine brain geranylgeranyltransferase in a cell-free system (recombinant smg p25A-GG). By use of this recombinant smg p25A-GG, its membrane-binding activity and its sensitivity to smg p25A GDI were compared with those of the fully posttranslationally processed form of bovine brain smg p25A (smg p25A-GG-Me) and the posttranslationally unprocessed form of bacterial smg p25A (recombinant smg p25A). The membrane-binding activity and sensitivity to smg p25A GDI were similar between the recombinant smg p25A-GG and smg p25A-GG-Me, although recombinant smg p25A lacked both activities. These results indicate that the geranylgeranyl moiety of smg p25A is essential and sufficient for its interactions with membrane and smg p25A GDI and that the methyl moiety is not essential for these interactions.     

Physiological and Genomic Features of a Novel Sulfur-Oxidizing Gammaproteobacterium Belonging to a Previously Uncultivated Symbiotic Lineage Isolated from a Hydrothermal Vent

Strain Hiromi 1, a sulfur-oxidizing gammaproteobacterium was isolated from a hydrothermal vent chimney in the Okinawa Trough and represents a novel genus that may include a phylogenetic group found as endosymbionts of deep-sea gastropods. The SSU rRNA gene sequence similarity between strain Hiromi 1 and the gastropod endosymbionts was approximately 97%. The strain was shown to grow both chemolithoautotrophically and chemolithoheterotrophically with an energy metabolism of sulfur oxidation and O₂ or nitrate reduction. Under chemolithoheterotrophic growth conditions, the strain utilized organic acids and proteinaceous compounds as the carbon and/or nitrogen sources but not the energy source. Various sugars did not support growth as a sole carbon source. The observation of chemolithoheterotrophy in this strain is in line with metagenomic analyses of endosymbionts suggesting the occurrence of chemolithoheterotrophy in gammaproteobacterial symbionts. Chemolithoheterotrophy and the presence of homologous genes for virulence- and quorum sensing-related functions suggest that the sulfur-oxidizing chomolithotrophic microbes seek animal bodies and microbial biofilm formation to obtain supplemental organic carbons in hydrothermal ecosystems.