Comparative pharmacology of two D1-like dopamine receptors cloned from the silkworm Bombyx mori

Dopamine (DA) is a physiologically important biogenic amine in insect peripheral and nervous tissues. We recently cloned two DA receptors (BmDopR1 and BmDopR2) from the silkworm Bombyx mori and identified them as D1-like receptors, which activate adenylate cyclase to increase intracellular cAMP levels. In this study, these two receptors were stably expressed in HEK-293 cells, and the dose-responsiveness to DA and their pharmacological properties were examined using cAMP assays. BmDopR1 showed a dose-dependent increase in cAMP levels at DA concentrations up to 10^?7 M with EC₅₀ of 3.30 nM, while BmDopR2 required 10^?6 M DA for activation. In BmDopR1-expressing cells, DA at 10^?6–10^?4 M induced 30–50% lower cAMP production than 10^?7 M DA. BmDopR2-expressing cells showed a standard sigmoidal dose–response, with maximum cAMP levels attained with 10^?5–10^?4 M DA and EC₅₀ of 1.30 μM. Both receptors had similar agonist profiles, and the typical vertebrate D1-like receptor agonist SKF-38393 was ineffective. Experiments with antagonists revealed that BmDopR1 exhibits D1-like features. However, the pharmacology of BmDopR2 was distinct from D1-like receptors; the typical vertebrate D1-like receptor antagonist SCH-23390 was less potent than the nonselective antagonist flupenthixol and the D2-like receptor antagonist chlorpromazine. The rank order of activities of several antagonists for BmDopR1 and BmDopR2 was more similar to that of Drosophila melanogaster DA receptors than Apis mellifera DA receptors. These data suggest that DA receptors could be potential targets for specific insecticides or insectistatics.     

Mechanisms of translational regulation by a human eIF5-mimic protein

The translation factor eIF5 is an important partner of eIF2, directly modulating its function in several critical steps. First, eIF5 binds eIF2/GTP/Met-tRNA(i)(Met) ternary complex (TC), promoting its recruitment to 40S ribosomal subunits. Secondly, its GTPase activating function promotes eIF2 dissociation for ribosomal subunit joining. Finally, eIF5 GDP dissociation inhibition (GDI) activity can antagonize eIF2 reactivation by competing with the eIF2 guanine exchange factor (GEF), eIF2B. The C-terminal domain (CTD) of eIF5, a W2-type HEAT domain, mediates its interaction with eIF2. Here, we characterize a related human protein containing MA3- and W2-type HEAT domains, previously termed BZW2 and renamed here as eIF5-mimic protein 1 (5MP1). Human 5MP1 interacts with eIF2 and eIF3 and inhibits general and gene-specific translation in mammalian systems. We further test whether 5MP1 is a mimic or competitor of the GEF catalytic subunit eIF2Bε or eIF5, using yeast as a model. Our results suggest that 5MP1 interacts with yeast eIF2 and promotes TC formation, but inhibits TC binding to the ribosome. Moreover, 5MP1 is not a GEF but a weak GDI for yeast eIF2. We propose that 5MP1 is a partial mimic and competitor of eIF5, interfering with the key steps by which eIF5 regulates eIF2 function.     

Roles of platelets and macrophages in the protective effects of lipopolysaccharide against concanavalin A-induced murine hepatitis

Platelets are reportedly causal in hepatitis. We previously showed that in mice, lipopolysaccharide (LPS) induces a reversible and macrophage-dependent hepatic platelet accumulation (HPA), including translocation of platelets into Disse spaces and their entry into hepatocytes. Concanavalin A (ConA), which induces hepatitis in mice via both T cells and macrophages, also induces HPA. Here, we examined the relationship between HPA and ConA-hepatitis. ConA-hepatitis and HPA were evaluated by serum transaminases, hepatic 5-hydroxytryptamine, and/or electron microscopy. Unlike LPS-induced HPA, ConA-induced HPA was only moderately dependent on phagocytic macrophages. Against expectations, platelet-depletion significantly exacerbated ConA-hepatitis, and anti-P-selectin antibody and P-selectin receptor blockade reduced both ConA-induced HPA and hepatitis. Prior induction of HPA by pretreatment with low-dose LPS powerfully reduced ConA-hepatitis. Such protection by LPS-pretreatment was not effective in mice depleted of phagocytic macrophages. In platelet-depleted mice, LPS-pretreatment severely exacerbated ConA-hepatitis. In mice depleted of both macrophages and platelets, neither ConA nor LPS-pretreatment + ConA induced hepatitis. In mice deficient in IL-1α and IL-1β (but not in TNFα), ConA-induced hepatitis was mild, and a protective effect of LPS was not detected. These results suggest that (i) there are causal and protective types of HPA, (ii) the causal type involves hepatic aggregation of platelets, which may be induced by platelet stimulants leaked from injured hepatocytes, (iii) the protective type is inducible by administration of prior low-dose LPS in a manner dependent on phagocytic (or F4/80-positive) macrophages, and (iv) IL-1 is involved in both the causal and protective types.     

Bacillus cereus from blood cultures: virulence genes, antimicrobial susceptibility and risk factors for blood stream infection

We characterized the profiles of virulence genes and antimicrobial susceptibility of Bacillus cereus isolates from blood cultures as well as the risk factors for blood stream infections (BSIs). The diversity of virulence gene patterns was found to be wide among 15 B. cereus isolates from BSIs and also among 11 isolates from contaminated blood cultures. The MicroScan broth microdilution method yielded results corresponding with those of the agar dilution (reference) method for levofloxacin, linezolid, and vancomycin, while the Etest results were consistent with the reference results for clindamycin, gentamicin, imipenem, levofloxacin, and linezolid. Compared with the reference values, however, some isolates showed marked differences of the minimum inhibitory concentrations (MICs) for ampicillin and clindamycin when determined using the MicroScan method, or the MICs for ampicillin, meropenem, and vancomycin when determined using the Etest method. Significantly more patients were treated with antimicrobials for more than 3 days during the 3-month period before isolation in the BSI group. Prior antimicrobial therapy may be a risk factor for BSIs due to B. cereus.     

Single-embryo metabolomics and systematic prediction of developmental stage in zebrafish

Metabolites, the end products of gene expression in living organisms, are tightly correlated with an organism's development and growth. Thus, metabolic profiling is a potentially important tool for understanding the events that have occurred in cells, tissues, and individual organisms. Here, we present a method for predicting the developmental stage of zebrafish embryos using novel metabolomic non-target fingerprints of "single-embryos". With this method, we observed the rate of development at different temperatures. Our results suggest that this method allows us to analyse the condition, or distinguish the genotype, of single-embryos before expression of their ultimate phenotype.     

Polymerization of amino acid derivatives by polymer catalysts. II. Polymerization by copolymer catalysts containing a tertiary amine as a component

The polymerizations of D ,-L β-phenylalanine NCA, p–nitro-D ,L -β-phenylalanine NCA, and o,p-dinitro-D ,L -β-phenylalanine NCA were investigated, homopolymers and copolymers of N-vinyl-2-ethylimidazole or 2-Vinylpyridine being used as catalysts. When N-vinylpyrrolidone and N,N-diethylacrylamide, which are capable of forming hydrogen bonds with the NCA's, were used as comonomers with N-vinyl-2-ethylimidazole, the copolymer catalysts were found to bring about a faster polymerization than poly-N-vinyl-2-ethylimidazole. However, when styrene, which has no particular interaction with the NCA's, was used as a comonomer with 2-vinylpyridine, the copolymer catalyst was found to give a slower polymerization than poly-2-vinylpyridine. Electronic spectroscopy showed that the charge-transfer complex between copolymer catalysts and the NCA's plays an important role in the polymerization. The experimental results are discussed in terms of the effectiveness of the copolymer catalysts for forming hydrogen bonds or charge-transfer complexes with the NCA's.     

Polymerization of optical isomers of phenylalanine N-carboxyanhydride by various secondary amines

In the polymerization of phenylalanine NCA initiated by some secondary amines, the two enatimorphs of phenylalanine NCA were polymerized with the same rate, which was almost twice as high, as that found for the racemic mixture. This stereoselectivity was observed only when the polymerization was initiated by secondary amines which are sterically crowded and reluctant to undergo a nucleophilic addition to NCA. Poly(DL -phenylalanine) produced in the stereoselective polymerization had a higher molecular weight than that produced in nonstereoselective polymerization. These findings point to the possibility that the stereoselectivity arises only in those polymerizations which are propagated by the activated monomers and not in the propagation involving the terminal amine of the growing polymer. A possible mechanism for the stereoselective polymerization is proposed and examined.