Human embryonic stem (ES) cells are predicted to be a valuable source for producing ES-derived therapeutic spare tissues to treat diseases by controlling their growth and differentiation. To understand the regulative mechanisms of their differentiation in vivo and in vitro, ES cells derived from nonhuman primates could be a powerful tool. We established four ES cell lines from cynomolgus monkey (Macaca fascicularis) blastocysts produced by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). The ES cells were characterized by the expression of specific markers such as alkaline phosphatase and stage-specific embryonic antigen-4. They were successfully maintained in an undifferentiated state and with a normal karyotype even after more than 6 months of culture. Pluripotential competence was confirmed by the formation of teratomas containing ectoderm-, mesoderm-, and endodermderivatives after subcutaneous injection into SCID mice. Differentiation to a variety of tissues was identified by immunohistochemical analyses using tissue-specific antibodies. Therefore, we established pluripotent ES cell lines derived from monkeys that are widely used as experimental animals. These lines could be a useful resource for preclinical stem cell research, including allogenic transplantation into monkey models of disease.

Ca2؉ , which enters cardiac myocytes through voltagedependent Ca 2؉ channels during excitation, is extruded from myocytes primarily by the Na ؉ /Ca 2؉ exchanger (NCX1) during relaxation. The increase in intracellular Ca 2؉ concentration in myocytes by digitalis treatment and after ischemia/reperfusion is also thought to result from the reverse mode of the Na ؉ /Ca 2؉ exchange mechanism. However, the precise roles of the NCX1 are still unclear because of the lack of its specific inhibitors. We generated Ncx1-deficient mice by gene targeting to determine the in vivo function of the exchanger. Homozygous Ncx1-deficient mice died between embryonic days 9 and 10. Their hearts did not beat, and cardiac myocytes showed apoptosis. No forward mode or reverse mode of the Na ؉ /Ca 2؉ exchange activity was detected in null mutant hearts. The Na ؉ -dependent Ca 2؉ exchange activity as well as protein content of NCX1 were decreased by ϳ50% in the heart, kidney, aorta, and smooth muscle cells of the heterozygous mice, and tension development of the aortic ring in Na ؉ -free solution was markedly impaired in heterozygous mice. These findings suggest that NCX1 is required for heartbeats and survival of cardiac myocytes in embryos and plays critical roles in Na ؉

The gene encoding a transmembrane glycoprotein LIG-1, of which the extracellular region was organized with the leucine-rich repeats and immunoglobulin-like domains, was disrupted in mice by gene targeting. LIG-1-deficient mice developed a skin change on the tail and facial area after birth. The affected skin was histologically reminiscent of the epidermis in human common skin disease 'psoriasis'. LIG-1 was expressed in basal cells of the epidermis and outer root sheath cells of hair follicles in mice. Interestingly, the LIG-1 expression was apparently down-regulated in the psoriatic lesions, suggesting that LIG-1 inversely correlates with proliferative ability of epidermal keratinocytes. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

—Complete cerebral ischemia was produced in normothermic anaesthetized cats by clamping the innominate and the left subclavian arteries combined with lowering the blood pressure. After 1 h of ischemia, ATP was no longer present in detectable amounts. Total adenine nucleotides were reduced to 34 per cent of the normal level. The breakdown of guanine nucleotides was less marked, with small amounts of GTP still being present at the end of the ischemic period. In animals with signs of functional recovery after 3–7 h of recirculation, ATP was resynthesized to 62 per cent of the control level. Total adenine nucleotides increased to 68 per cent and the adenylate energy change—[ATP + 1/2 ADP]/[AMP + ADP + ATP]—was re‐established to within 7 per cent of the pre‐ischemic value. Radiochromatography of nucleotides following intravenous injection of [14C]formate indicated a marked enhancement of postischemic purine de novo synthesis. Purine nucleosides and free bases which accumulated during ischemia, were partially re‐utilized by salvage pathways: adenosine was rephosphorylated to AMP by adenosine kinase (EC 2.7.1.20); inosine and hypoxanthine were re‐used via IMP in a reaction mediated by hypoxanthine phosphoribosyltransferase (EC 2.4.2.8).