Characterization of alkaline nuclease from rat liver mitochondria.

The alkaline nuclease (pH optimum 9.0) has been purified about 500-fold in 25% yield from the extract of rat liver mitochondria. The enzyme cleaves yeast RNA, poly(U), poly(U), poly(C) and denatured DNA to yield oligonucleotides with 5'-phosphoryl and 3'-hydroxyl ends. The enzyme has a molecular weight of about 60 000, a sedimentation coefficient of 4 S and an isoelectric point of 9.0. The behaviors of RNAase activity of the nuclease are identical with those of DNAase activity in column chromatography as well as in catalytic nature. The affinities of RNAase activity for substrate, Mg2+, spermidine and polyvinyl sulfate are lower than those of DNAase activity. The alkaline nuclease activity measured in the homogenate of regenerating rat liver is not significantly changed.     

Use of a feedback-insensitive α subunit of anthranilate synthase as a selectable marker for transformation of rice and potato

A selection system based on a mutant rice gene for a feedback-insensitive subunit of anthranilate synthase (OASA1D) was developed for the transformation of rice and potato. Expression of OASA1D conferred resistance to the tryptophan analog 5-methyltryptophan (5MT) in transformed cells of rice and potato. The selection system based on OASA1D and 5MT was associated with a high transformation efficiency, a short time frame for the generation of transgenic plants, simple culture procedures, and it was as effective as hygromycin B selection in rice (monocotyledon) and kanamycin selection in potato (dicotyledon). Transgenic rice and potato plants established by 5MT selection had normal morphology and accumulated tryptophan when OASA1D was expressed under the control of a constitutive promoter. These results demonstrate the efficacy of OASA1D as a selectable marker and they suggest that the 5MT selection system based on this gene will prove applicable to a wide range of plant species and culture procedures.     

Repetitive transcranial magnetic stimulation induces kf-1 expression in the rat brain

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive approach used for stimulating the brain, and has proven effective in the treatment of depression, however the mechanism of its antidepressant action is unknown. Recently, we have reported the induction of kf-1 in rat frontal cortex and hippocampus after chronic antidepressant treatment and repeated electroconvulsive treatment (ECT). In this study, we demonstrated the induction of kf-1 after rTMS in the rat frontal cortex and hippocampus, but not in hypothalamus. Our data suggest that kf-1 may be a common functional molecule that is increased after antidepressant treatment, ECT and rTMS. In conclusion, it is proposed that induction of kf-1 may be associated with the treatment induced adaptive neural plasticity in the brain, which is a long-term target for their antidepressant action.     

A novel scheme of dystrophin disruption for the progression of advanced heart failure

The precise mechanism of the progression of advanced heart failure is unknown. We assessed a new scheme in two heart failure models: (I) congenital dilated cardiomyopathy (DCM) in TO-2 strain hamsters lacking delta-sarcoglycan (SG) gene and (II) administration of a high-dose of isoproterenol, as an acute heart failure in normal rats. In TO-2 hamsters, we followed the time course of the histological, physiological and metabolic the progressions of heart failure to the end stage. Dystrophin localization detected by immunostaining age-dependently to the myoplasm and the in situ sarcolemma fragility evaluated by Evans blue entry was increased in the same cardiomyocytes. Western blotting revealed a limited cleavage of the dystrophin protein at the rod domain, strongly suggesting a contribution of endogenous protease(s). We found a remarkable up-regulation of the amount of calpain-1 and -2, and no change of their counterpart, calpastatin. After supplementing TO-2 hearts with the normal delta-SG gene in vivo, these pathological alterations and the animals' survival improved. Furthermore, dystrophin but not delta-SG was disrupted by a high dose of isoproterenol, translocated from the sarcolemma to the myoplasm and fragmented. These results of heart failure, irrespective of the hereditary or acquired origin, indicate a vicious cycle formed by the increased sarcolemma permeability, preferential activation of calpain over calpastatin, and translocation and cleavage of dystrophin would commonly lead to advanced heart failure.     

Phosphorylation of histone H2AX at M phase in human cells without DNA damage response

A variant of histone H2A, H2AX, is phosphorylated on Ser139 in response to DNA double-strand breaks (DSBs), and clusters of the phosphorylated form of H2AX (gamma-H2AX) in nuclei of DSB-induced cells show foci at breakage sites. Here, we show phosphorylation of H2AX in a cell cycle-dependent manner without any detectable DNA damage response. Western blot and immunocytochemical analyses with the anti-gamma-H2AX antibody revealed that H2AX is phosphorylated at M phase in HeLa cells. In ataxia-telangiectasia cells lacking ATM kinase activity, gamma-H2AX was scarcely detectable in the mitotic chromosomes, suggesting involvement of ATM in M-phase phosphorylation of H2AX. Single-cell gel electrophoresis assay and Western blot analysis with the anti-phospho-p53 (Ser15) antibody indicated that H2AX in human M-phase cells is phosphorylated independently of DSB and DNA damage signaling. Even in the absence of DNA damage, phosphorylation of H2AX in normal cell cycle progression may contribute to maintenance of genomic integrity.     

Commitment of fetal male germ cells to spermatogonial stem cells during mouse embryonic development

The continuous production of mammalian sperm is maintained by the proliferation and differentiation of spermatogonial stem cells that originate from primordial germ cells (PGCs) in the early embryo. Although spermatogonial stem cells arise from PGCs, it is not clear whether fetal male germ cells function as spermatogonial stem cells able to produce functional sperm. In the present study, we examined the timing and mechanisms of the commitment of fetal germ cells to differentiate into spermatogonial stem cells by transplantation techniques. Transplantation of fetal germ cells into the seminiferous tubules of adult testis showed that donor germ cells, at 14.5 days postcoitum (dpc), were able to initiate spermatogenesis in the adult recipient seminiferous tubules, whereas no germ cell differentiation was observed in the transplantation of 12.5-dpc germ cells. These results indicate that the commitment of fetal germ cells to differentiate into spermatogonial stem cells initiates between embryonic days 12.5 and 14.5. Furthermore, the results suggest the importance of the interaction between germ cells and somatic cells in the determination of fetal germ cell differentiation into spermatogonial stem cells, as normal spermatogenesis was observed when a 12.5-dpc whole gonad was transplanted into adult recipient testis. In addition, sperm obtained from the 12.5- dpc male gonadal explant had the ability to develop normally if injected into the cytoplasm of oocytes, indicating that normal development of fetal germ cells in fetal gonadal explant occurred in the adult testicular environment.     

Skewed X-inactivation in cloned mice

In female mammals, dosage compensation for X-linked genes is accomplished by inactivation of one of two X chromosomes. The X-inactivation ratio (a percentage of the cells with inactivated maternal X chromosomes in the whole cells) is skewed as a consequence of various genetic mutations, and has been observed in a number of X-linked disorders. We previously reported that phenotypically normal full-term cloned mouse fetuses had loci with inappropriate DNA methylation. Thus, cloned mice are excellent models to study abnormal epigenetic events in mammalian development. In the present study, we analyzed X-inactivation ratios in adult female cloned mice (B6C3F1). Kidneys of eight naturally produced controls and 11 cloned mice were analyzed. Although variations in X-inactivation ratio among the mice were observed in both groups, the distributions were significantly different (Ansary-Bradley test, P<0.01). In particular, 2 of 11 cloned mice showed skewed X-inactivation ratios (19.2% and 86.8%). Similarly, in intestine, 1 of 10 cloned mice had a skewed ratio (75.7%). Skewed X-inactivation was observed to various degrees in different tissues of different individuals, suggesting that skewed X-inactivation in cloned mice is the result of secondary cell selection in combination with stochastic distortion of primary choice. The present study is the first demonstration that skewed X-inactivation occurs in cloned animals. This finding is important for understanding both nuclear transfer technology and etiology of X-linked disorders.     

Transformation of lactulose trihydrate into anhydrous lactulose by fluidized bed drying and its characterization

Stable anhydrous lactulose was produced from lactulose trihydrate by stepwise heating on a fluidized bed. The processes were performed on stable powder forms. The anhydrous lactulose was characterized by an opaque white appearance, a coarse surface structure with random cracks and indentations, a high degree of crystallization, stability under humid conditions, and by X-ray powder diffraction, differential thermal analysis, and differential thermogravimetry. Those characteristics were different from those of the original trihydrate, which was transparent, had a smooth surface and a higher degree of crystallization, was stable under humid conditions and had different X-ray powder diffraction, differential thermal analysis, and thermogravimetric characteristics. The transformation was enhanced when the inlet temperature was 45-55 degrees C or when the temperature of the fluidized bed was over 40 degrees C. At these cutoff temperatures, both crystalline forms were observed.     

Quantitative analysis of estrogen receptor proteins in rat ovary

The mRNAs of estrogen receptor beta (ERbeta), and its splice variant, ERbeta2, are abundant in granulosa cells in the ovary. With the use of antibodies, ERbeta protein has also been shown to be abundantly expressed, but to date no ERbeta2 protein has been demonstrated in the ovary. ERbeta2 has a peptide, 18 amino acids in length, inserted into its ligand-binding domain, resulting in a reported 35-fold reduction in its affinity for estrogen (E2). ERalpha, ERbeta1 and ERbeta2 were quantified by Western blotting and by RT-PCR and their cellular localization in the ovary was examined by immunohistochemistry. In 3- and 5-week-old virgin, pregnant, lactating and post-lactating rats, the level of ERalpha protein ranged between 1.6 and 3.8 fmol/microg total protein. That of ERbeta was 8.8-11.2 and of ERbeta2, in the same samples, 4.1-5.9 fmol/microg total protein. ERbeta2 and ERbeta1 proteins were, therefore, present in approximately equal amounts in the ovary throughout the various reproductive stages. The major ERbeta proteins in rat ovary, detected by their molecular weights on Western blots, were ERbeta1-530 and ERbeta2-548 (530+18 amino acids (aa)). Immunohistochemical staining revealed that ERbeta and ERbeta2 were expressed predominantly in granulosa cells of growing follicles, while ERalpha was found only in theca cells. In some theca cells, both ERalpha, ERbeta2 were expressed. The data suggests that in theca cells, where it is co expressed with ERalpha, ERbeta2 could function as a repressor of ERalpha. However, in granulosa cells where no ERalpha is detectable, and where E2 levels are high, ERbeta2, with its low affinity for E2, could be an important sensor through which E2 exerts regulatory control.