In Vitro Peptidoglycan Synthesis Which is Very Sensitive to Cephalexin and Penicillin G in Escherichia coli: Presumable Septum-forming Reaction Sequence

Hydrangeae Dulcis Folium, the fermented and dried leaves of Hydrangea macrophylla SER. var. thunbergii MAKINO, suppressed D-galactosamine-induced liver injury by 85.2% when added to the diet at 1% and fed to rats for fifteen days. The hepatoprotective effect is more potent than that of a milk thistle extract and turmeric powder. Some fractionated extracts showed hepatoprotective activity in the D-galactosamine-induced in vitro liver injury model.     

Understanding the X chromosome inactivation cycle in mice: A comprehensive view provided by nuclear transfer

During mouse development, imprinted X chromosome inactivation (XCI) is observed in preimplantation embryos and is inherited to the placental lineage, whereas random XCI is initiated in the embryonic proper. Xist RNA, which triggers XCI, is expressed ectopically in cloned embryos produced by somatic cell nuclear transfer (SCNT). To understand these mechanisms, we undertook a large-scale nuclear transfer study using different donor cells throughout the life cycle. The Xist expression patterns in the reconstructed embryos suggested that the nature of imprinted XCI is the maternal Xist-repressing imprint established at the last stage of oogenesis. Contrary to the prevailing model, this maternal imprint is erased in both the embryonic and extraembryonic lineages. The lack of the Xist-repressing imprint in the postimplantation somatic cells clearly explains how the SCNT embryos undergo ectopic Xist expression. Our data provide a comprehensive view of the XCI cycle in mice, which is essential information for future investigations of XCI mechanisms.     

Cognitive Function Related to the Sirh11/Zcchc16 Gene Acquired from an LTR Retrotransposon in Eutherians

Gene targeting of mouse S ushi- i chi-related r etrotransposon h omologue 11 / Z inc finger CCHC domain-containing 16 (Sirh11/Zcchc16) causes abnormal behaviors related to cognition, including attention, impulsivity and working memory. Sirh11/Zcchc16 encodes a CCHC type of zinc-finger protein that exhibits high homology to an LTR retrotransposon Gag protein. Upon microdialysis analysis of the prefrontal cortex region, the recovery rate of noradrenaline (NA) was reduced compared with dopamine (DA) after perfusion of high potassium-containing artificial cerebrospinal fluid in knockout (KO) mice. These data indicate that Sirh11/Zcchc16 is involved in cognitive function in the brain, possibly via the noradrenergic system, in the contemporary mouse developmental systems. Interestingly, it is highly conserved in three out of the four major groups of the eutherians, euarchontoglires, laurasiatheria and afrotheria, but is heavily mutated in xenarthran species such as the sloth and armadillo, suggesting that it has contributed to brain evolution in the three major eutherian lineages, including humans and mice. Sirh11/Zcchc16 is the first SIRH gene to be involved in brain function, instead of just the placenta, as seen in the case of Peg10, Peg11/Rtl1 and Sirh7/Ldoc1.     

Budding yeast mms4 is epistatic with rad52 and the function of Mms4 can be replaced by a bacterial Holliday junction resolvase

MMS4 of Saccharomyces cerevisiae was originally identified as the gene responsible for one of the collection of methyl methanesulfonate (MMS)-sensitive mutants, mms4. Recently it was identified as a synthetic lethal gene with an SGS1 mutation. Epistatic analyses revealed that MMS4 is involved in a pathway leading to homologous recombination requiring Rad52 or in the recombination itself, in which SGS1 is also involved. MMS sensitivity of mms4 but not sgs1, was suppressed by introducing a bacterial Holliday junction (HJ) resolvase, RusA. The frequencies of spontaneously occurring unequal sister chromatid recombination (SCR) and loss of marker in the rDNA in haploid mms4 cells and interchromosomal recombination between heteroalleles in diploid mms4 cells were essentially the same as those of wild-type cells. Although UV- and MMS-induced interchromosomal recombination was defective in sgs1 diploid cells, hyper-induction of interchromosomal recombination was observed in diploid mms4 cells, indicating that the function of Mms4 is dispensable for this type of recombination.     

In vitro and in vivo gene transfer by an optimized alpha-cyclodextrin conjugate with polyamidoamine dendrimer

The purpose of the present study is to optimize the structure of the polyamidoamine starburst dendrimer (dendrimer) conjugate with alpha-cyclodextrin (alpha-CDE conjugate) as a nonviral vector. alpha-CDE conjugates of dendrimer (generation 3, G3) with various average degrees of substitution (DS) of alpha-CyD of 1.1, 2.4, and 5.4 were prepared. alpha-CDE conjugates formed the complexes with pDNA, resulting in a change of the particle sizes of pDNA complexes, but the distinction of physicochemical properties among their vector/pDNA complexes was only very slight. The membrane-disruptive ability of alpha-CDE conjugates on liposomes encapsulating calcein and their cytotoxicity to NIH3T3 and HepG2 increased with an increase in the DS value of alpha-CyD. In vitro gene transfer activity of alpha-CDE conjugates in both NIH3T3 and HepG2 cells augmented as the charge ratio (vector/pDNA) increased, and the activity of alpha-CDE conjugate (DS 2.4) was the highest at higher charge ratios among dendrimer (G3), the three alpha-CDE conjugates, and TransFast. After intravenous administration of pDNA complexes in mice, alpha-CDE conjugate (DS 2.4) delivered pDNA more efficiently in spleen, liver, and kidney, compared with dendrimer and other alpha-CDE conjugates (DS 1.1 and 5.4). The potential use of alpha-CDE conjugate (G3, DS 2.4) could be expected as a nonviral vector in vitro and in vivo, and these data may be useful for design of alpha-CyD conjugates with other nonviral vectors.     

The novel imprinted carboxypeptidase A4 gene ( CPA4) in the 7q32 imprinting domain

By a search for novel human imprinted genes in the vicinity of the imprinted gene MEST, at chromosome 7q32, we identified the carboxypeptidase A4 gene ( CPA4) in a gene cluster of the carboxypeptidase family, 200 kb centromeric to MEST. Because CPA4 was originally identified as a protein induced in a prostate cancer cell line (PC-3) by histone deacetylase inhibitors, and was located at the putative prostate cancer-aggressiveness locus at 7q32, we investigated its imprinting status in fetal tissues and in adult benign hypertrophic prostate (BPH). RT-PCR using four intragenic polymorphisms as markers showed that CPA4 was expressed preferentially from the maternal allele in the fetal heart, lung, liver, intestine, kidney, adrenal gland, and spleen, but not in the fetal brain. It was also preferentially expressed in the BPH. These findings support that CPA4 is imprinted and may become a strong candidate gene for prostate cancer-aggressiveness. As a Silver-Russell syndrome (SRS) locus has been proposed to be located to a region near MEST and to be involved in imprinting, CPA4 would have been a candidate gene for SRS. However, analysis of ten SRS patients revealed no mutations in CPA4.     

Imprinting regulation of the murine Meg1/Grb10 and human GRB10 genes; roles of brain-specific promoters and mouse-specific CTCF-binding sites

The imprinted mouse gene Meg1/Grb10 is expres sed from maternal alleles in almost all tissues and organs, except in the brain, where it is expressed biallelically, and the paternal allele is expressed preferentially in adulthood. In contrast, the human GRB10 gene shows equal biallelic expression in almost all tissues and organs, while it is almost always expressed paternally in the fetal brain. To elucidate the molecular mechanisms of the complex imprinting patterns among the different tissues and organs of humans and mice, we analyzed in detail both the genomic structures and tissue-specific expression profiles of these species. Experiments using 5′-RACE and RT–PCR demonstrated the existence in both humans and mice of novel brain- specific promoters, in which only the paternal allele was active. The promoters were located in the primary differentially methylated regions. Interest ingly, CTCF-binding sites were found only in the mouse promoter region where CTCF showed DNA methylation-sensitive binding activity. Thus, the insulator function of CTCF might cause reciprocal maternal expression of the Meg1/Grb10 gene from another upstream promoter in the mouse, whereas the human upstream promoter is active in both parental alleles due to the lack of the corresponding insulator sequence in this region.     

Generation of pluripotent stem cells from neonatal mouse testis

Although germline cells can form multipotential embryonic stem (ES)/embryonic germ (EG) cells, these cells can be derived only from embryonic tissues, and such multipotent cells have not been available from neonatal gonads. Here we report the successful establishment of ES-like cells from neonatal mouse testis. These ES-like cells were phenotypically similar to ES/EG cells except in their genomic imprinting pattern. They differentiated into various types of somatic cells in vitro under conditions used to induce the differentiation of ES cells and produced teratomas after inoculation into mice. Furthermore, these ES-like cells formed germline chimeras when injected into blastocysts. Thus, the capacity to form multipotent cells persists in neonatal testis. The ability to derive multipotential stem cells from the neonatal testis has important implications for germ cell biology and opens the possibility of using these cells for biotechnology and medicine.     

The novel dominant mutation Dspd leads to a severe spermiogenesis defect in mice

Spermiogenesis is a complex process that is regulated by a plethora of genes and interactions between germ and somatic cells. Here we report a novel mutant mouse strain that carries a transgene insertional/translocational mutation and exhibits dominant male sterility. We named the mutation dominant spermiogenesis defect (Dspd). In the testes of Dspd mutant mice, spermatids detached from the seminiferous epithelium at different steps of the differentiation process before the completion of spermiogenesis. Microinsemination using spermatids collected from the mutant testes resulted in the birth of normal offspring. These observations indicate that the major cause of Dspd infertility is (are) a defect(s) in the Sertoli cell-spermatid interaction or communication in the seminiferous tubules. Fluorescent in situ hybridization (FISH) analysis revealed a translocation between chromosomes 7F and 14C at the transgene insertion site. The deletion of a genomic region of chromosome 7F greater than 1 megabase and containing at least six genes (Cttn, Fadd, Fgf3, Fgf4, Fgf15, and Ccnd1) was associated with the translocation. Cttn encodes the actin-binding protein cortactin. Immunohistochemical analysis revealed localization of cortactin beside elongated spermatids in wild-type testes; abnormality of cortactin localization was found in mutant testes. These data suggest an important role of cortactin in Sertoli cell-spermatid interactions and in the Dspd phenotype.