Construction of a gamete-enriched gene pool and RNAi-mediated functional analysis in Dictyostelium discoideum

Macrocysts in Dictyostelium discoideum possess prototypic features of sexual reproduction and are useful for understanding the basic mechanisms of the reproductive process. Here, we randomly analyzed 1,071 gamete cDNAs, and then constructed a gamete-specific subtraction library, FC-IC. Nucleotide sequences of all 903 FC-IC clones were determined and clustered into 272 independent genes. Expression analysis based on real-time RT-PCR revealed 67 gamete-enriched genes, among which those involved in 'signal transduction' and 'multicellular organization' are prevalent. One of them, FC-IC0003, appeared also to be mating-type specific, and was named gmsA. RNAi-mediated silencing as well as disruption of gmsA reduced the cellular competency for sexual cell fusion, indicating the involvement of this gene in the sexual development of D. discoideum.     

Dermorphin tetrapeptide analogues with 2',6'-dimethylphenylalanine (Dmp) substituted for aromatic amino acids have high mu opioid receptor binding and biological activities

To investigate the value of the 2',6'-dimethylphenylalanine (Dmp) residue as an aromatic amino acid substitution, we prepared analogues of the mu opioid receptor-selective dermorphin tetrapeptide Tyr-D-Arg-Phe-betaAla-NH(2) (YRFB) in which Dmp or its D-isomer replaced Tyr(1) or Phe(3). Replacing Phe(3) with Dmp essentially tripled mu receptor affinity and the receptor's in vitro biological activities as determined with the guinea pig ileum (GPI) assay but did not change delta receptor affinity. Despite an inversion of the D configuration at this position, mu receptor affinity and selectivity remained comparable with those of the L-isomer. Replacing the N-terminal Tyr residue with Dmp produced a slightly improved mu receptor affinity and a potent GPI activity, even though the substituted compound lacks the side chain phenolic hydroxyl group at the N-terminal residue. Dual substitution of Dmp for Tyr(1) and Phe(3) produced significantly improved mu receptor affinity and selectivity compared with the singly substituted analogues. Subcutaneous injection of the two analogues, [Dmp(3)]YRFB and [Dmp(1)]YRFB, in mice produced potent analgesic activities that were greater than morphine in the formalin test. These lines of evidence suggest that the Dmp residue would be an effective aromatic amino acid surrogate for both Tyr and Phe in the design and development of novel opioid mimetics.     

Mechanism of persistence in HCV infection

One of the prominent features of hepatitis C virus (HCV) is persistent infection, which is assumed to be a crucial event as a result of evading host defense system. Type I interferon beta (IFN- beta) system is induced rapidly after viral infection and plays a central role in innate immunity. Upon immediate induction of type I IFN as host first defense line, interferon regulatory factor-3 (IRF-3) is phosphorylated, formed of homodimer and translocates to nucleus. IFN-beta induction due to new castle disease virus (NDV) was significantly decreasd after the expression of full HCV genome (HCR6-Rz). Similar modification was observed in cell line expressing core to the NS2 protein region (HCR6-Fse). However, this decreasing was not observed in cell line expressing NS2 to the NS5B region (HCR6-Age). IRF-3 dimer formation induced by NDV infection was also suppressed after the expression of HCR6-Rz and HCR6-Fse, but not HCR6-Age. We further analyzed using transiently expressed HCV core, E1 or E2 in HepG2 cells. The suppression of IRF-3 dimer formation was caused by HCV core protein alone. These results indicated that a new crucial biological function of HCV core protein that may be related to persistence and pathogenesis of HCV.     

Induction of hepatic injury by hepatitis C virus-specific CD8+ murine cytotoxic T lymphocytes in transgenic mice expressing the viral structural genes

In the present study, we generated killer cells specific for hepatitis C virus (HCV) structural protein by re-stimulation of immune spleen cells from H-2(d) haplotype transgenic (Tg) mice, expressing the core, E1, E2, and NS2 genes of HCV regulated by the Cre/loxP switching system. The generated killer cells were conventional CD8(+)L(d) class-I MHC molecule-restricted cytotoxic T lymphocytes (CTLs) and specific for the HCV E1 structural protein. Because the CTLs could also kill hepatocytes from the Tg mice expressing HCV structural proteins in vitro, we attempted to transfer those CTLs intravenously into interferon regulatory factor-1 (IRF-1) negative, CD8-deficient Tg mice representing the HCV structural genes on hepatocytes to examine whether the inoculated CD8(+) CTLs can eliminate hepatocytes expressing the HCV genes in vivo. We observed an elevation of serum ALT level as well as damage of the liver tissue histologically. To our knowledge, this is the first demonstration to show that HCV-specific CD8(+) CTLs specifically attack hepatocytes expressing the HCV structural proteins both in vitro and in vivo.     

DNA probes on beads arrayed in a capillary, 'Bead-array', exhibited high hybridization performance

A DNA analysis platform called 'Bead-array' is presented and its features when used in hybridization detection are shown. In 'Bead-array', beads of 100- micro m diameter are lined in a determined order in a capillary. Each bead is conjugated with DNA probes, and can be identified by its order in the capillary. This probe array is easily produced by just arraying beads conjugated with probes into the capillary in a fixed order. The hybridization is also easily completed by introducing samples (1-300 micro l) into the capillary with reciprocal flow. For hybridization detection, as little as 1 amol of fluorescent-labeled oligo DNA was detected. The hybridization reaction was completed in 1 min irrespective of the amount of target DNA. When the number of target molecules was smaller than that of probe molecules on the bead, 10 fmol, almost all targets were captured on the bead. 'Bead-array' enables reliable and reproducible measurement of the target quantity. This rapid and sensitive platform seems very promising for various genetic testing tasks.     

Effect of 10-aza-substitution on benzo[a]pyrene mutagenicity in vivo and in vitro

Benzo[a]pyrene (BaP), an environmental carcinogen, shows genotoxicity after metabolic transformation into the bay-region diol epoxide, BaP-7,8-diol 9,10-epoxide. 10-Azabenzo[a]pyrene (10-azaBaP), in which a ring nitrogen is located in the bay-region, is also a carcinogen and shows mutagenicity in the Ames test in the presence of the rat liver microsomal enzymes. In order to evaluate the effect of aza-substitution on in vivo genotoxicity, BaP and 10-azaBaP were assayed for their in vivo mutagenicity using the lacZ-transgenic mouse (MutaMouse). BaP was potently mutagenic in all of the organs examined (liver, lung, kidney, spleen, forestomach, stomach, colon, and bone marrow), as described in our previous report, whereas, 10-azaBaP was slightly mutagenic only in the liver and colon. The in vitro mutagenicities of BaP and 10-azaBaP were evaluated by the Ames test using liver homogenates prepared from several sources, i.e. CYP1A-inducer-treated rats, CYP1A-inducer-treated and non-treated mice, and humans. BaP showed greater mutagenicities than 10-azaBaP in the presence of a liver homogenate prepared from CYP1A-inducer-treated rodents. However, 10-azaBaP showed mutagenicities similar to or more potent than BaP in the presence of a liver homogenate or S9 from non-treated mice and humans. These results indicate that 10-aza-substitution markedly modifies the nature of mutagenicity of benzo[a]pyrene in both in vivo and in vitro mutagenesis assays.     

Caenorhabditis elegans reticulon interacts with RME-1 during embryogenesis

Reticulon (RTN) family proteins are localized in the endoplasmic reticulum (ER). At least four different RTN genes have been identified in mammals, but in most cases, the functions of the encoded proteins except mammalian RTN4-A and RTN4-B are unknown. Each RTN gene produces 1-3 proteins by different promoters and alternative splicing. In Caenorhabditis elegans, there is a single gene (rtn gene) encoding three reticulon proteins, nRTN-A, B, and C. mRNA of nRTN-C is expressed in germ cells and embryos. However, nRTN-C protein is only expressed during embryogenesis and rapidly disappears after hatch. By yeast two-hybrid screening, two clones encoding the same C-terminal region of RME-1, a protein functioning in the endocytic recycling, were isolated. These findings suggest that nRTN-C functions in the endocytic pathway during embryogenesis.