Cell death and proliferation in Nd-YAG laser, electrocautery, and scalpel wounds on mice skin.

The purpose of this study is to compare cell death and proliferation in laser, electrocautery and scalpel wounds on the mice epidermis. Wounds were examined by transmission electron microscopy, the detection of free 3'-OH DNA ends and immunohistochemistry of proliferating cell nuclear antigen (PCNA), inducible nitric oxide synthase (iNOS), keratinocyte growth factor (KGF) and keratinocyte growth factor receptor (KGFR). Reepithelization was first observed 5 days after scalpel and laser incisions and 7 days after electrocautery incision. Ultrastructurally, keratinocytes in both electrocautery and laser wounds showed similar post-apoptotic necrotic changes. Interestingly, dividing cells were often observed 3 days after laser incision. Apoptotic index in electrocautery wounds was higher than in laser wounds, although there was no significant difference in the PCNA expression level between them. The expression of iNOS, KGF and KGFR in laser wounds was more intense than in electrocautery wounds. In scalpel wounds, keratinocytes did not show significant changes in morphology or of markers of cell death and proliferation during the observation period. Therefore, the increase in the number of dividing cells and in the expression level of iNOS, KGF and KGFR may induce earlier and thicker reepithelization in laser wounds than in electrocautery and scalpel wounds.

Extracellular pH affects platelet aggregation associated with modulation of store-operated Ca(2+) entry.

The pH dependence of store-operated Ca(2+) influx (SOCI) into human platelets, as well as its physiological consequence, aggregation, was studied. In Ca(2+)-free medium, thapsigargin (1 microM) induced a small increase in intracellular free-Ca(2+) ([Ca(2+)](i)), which was not affected by changes in extracellular pH. The addition of Ca(2+) (0.5-3 mM) after Ca(2+) store depletion caused by thapsigargin resulted in concentration-dependent increases in [Ca(2+)](i) (SOCI), which were strongly inhibited by SKF-96365 (100 microM), an inhibitor of receptor-mediated Ca(2+) entry. SOCI was inhibited by acidosis (pH 6.9) and augmented by alkalosis (pH 7.9). The addition of Ca(2+) (0.5-3 mM) to platelets, which were kept in Ca(2+)-free medium, slightly but significantly increased [Ca(2+)](i). This Ca(2+) leak entry was also decreased and increased by extracellular acidosis (pH 6.9) and alkalosis (pH 7.9), respectively, but not affected by SKF-96365. Neither thapsigargin (1 microM) stimulation in Ca(2+)-free solution nor elevation of extracellular Ca(2+) alone was sufficient to induce platelet aggregation. In contrast, the addition of Ca(2+) (1 mM) to platelets activated by thapsigargin resulted in aggregation, which was markedly inhibited by SKF-96365 (100 microM). Platelet aggregation associated with SOCI was also inhibited by extracellular acidosis (pH 6.9) and augmented by extracellular alkalosis (pH 7.9). These results suggest that acidosis-induced inhibition, as well as alkalosis-induced promotion of platelet aggregation, involve pH effects on SOCI.

[Urgent allogeneic bone marrow transplantation using a preparative regimen of cyclophosphamide anti-human thymocytes rabbit globulin in a patient with severe aplastic anemia with pneumonia].

A 16-year-old girl was diagnosed as having severe aplastic anemia (SAA) had received emergency complicated by sever pneumonia. She had an HLA-identical younger brother and been urgently transplantation with her brother's marrow following a preparative regimen of CY+rabbit antithymocyte globulin (ATG). Granulocyte transfusions carried out before and after the transplant prevented exacerbation of the pneumonia. The pneumonia was cured in association with the hematopoietic recovery after BMT. No signs or symptoms of acute or chronic graft-versus-host disease were recognized and her hematological data are normal. The rabbit ATG was thought to be effective in preventing rejection and could be used in the preparative regimen instead of total body irradiation.

[Is platelet protein S different from plasma protein S in human being?].

Platelet suspension was obtained from normal platelet rich plasma (PRP) by the gel filtration method using Sepharose 2B and platelet pellet was prepared from the platelet suspension after centrifugation. A platelet preparation was prepared from the solubilized platelet pellet. Purified protein S was obtained from plasma and platelet preparation using immuno-affinity column chromatography. Total antigen of protein S was measured by enzyme immunoassay and activity of protein S was measured as a cofactor of activated protein C. The ratio of purified protein S activity to total antigen in normal plasma and platelet was 2.3 and 2.1, respectively. According to immunoblot and crossed immunoelectrophoresis studies, the molecular weight of platelet protein S was different from that of plasma protein S. Localization of protein S in platelet alpha-granules was demonstrated by immunoelectronmicroscopy.

Ornithine decarboxylase antizyme in kidneys of male and female mice.

Antizyme, a protein inhibitor of ornithine decarboxylase (ODC), was shown to be induced in mouse kidney by repeated injection of putrescine. Antizyme was also present as a complex with ODC in the kidney of untreated mouse. The amount of the renal ODC-antizyme complex was 3-fold higher in male mice than in female mice. On the contrary, the proportion of ODC present as a complex with antizyme was 24-fold higher in females than in males, and the decay of renal ODC activity after cycloheximide treatment was about 5-fold more rapid in females than in males. Administration of testosterone to female mice, a procedure known to prolong the half-life of renal ODC, increased both ODC activity and the content of ODC-antizyme complex, but decreased the antizyme/ODC ratio in the kidney. These results are consistent with the previous observation in HTC cells that the decay rate of ODC activity in the presence of cycloheximide correlated well with the proportion of ODC present as a complex with antizyme, suggesting the ubiquitous role of antizyme in ODC degradation.

Identical catalytic-centre activity for mouse kidney and rat liver ornithine decarboxylases as determined with antizyme and affinity labelling.

Since the catalytic-centre activity of mouse kidney ornithine decarboxylase (ODC) has been assumed to be twice as high as that of rat liver ODC, we compared relative catalytic-centre activity of the two enzymes by titration with antizyme, which inhibits ODC by stoichiometric binding. In either a crude or a purified state, both enzymes were inhibited by rat liver antizyme to the same extent, indicating that they have nearly identical catalytic-centre activities. This conclusion was supported by comparison of affinity labelling of the enzymes with alpha-difluoromethyl[14C]ornithine.

Existence of antizyme and ornithine decarboxylase-antizyme complex in RK13 kidney cells.

It has been reported that 'antizyme', a protein inhibitor of ornithine decarboxylase (ODC) induced by its product, is not found in rat or mouse kidney. We determined whether antizyme was present in rabbit kidney cells (RK13) in culture. Antizyme could be induced in these cells by putrescine treatment, a substantial portion being in the particulate fraction in contrast with hepatic antizyme. Furthermore, ODC-antizyme complex was present even in untreated cells. Pretreatment of cells with putrescine increased the relative amount of ODC-antizyme complex and accelerated decay of ODC. These results support the ubiquitous existence of antizyme and its role in ODC degradation.

2',3'-cyclic nucleotide 3'-phosphohydrolase activity in rat visual pathways with experimental allergic encephalomyelitis.

The enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) is one of the important markers for myelin synthesis or demyelination. We measured the CNPase activities in the visual pathway tissues of Lewis strain rats with experimental allergic encephalomyelitis (EAE) induced by inoculation of myelin basic protein from the brain. The enzyme activities in the retina, optic nerve, optic chiasma and lateral geniculate body were reduced to about 50-80% of that of controls at 15 days after myelin basic protein inoculation when symptoms of EAE were most severe. However, the activities recovered at 19 days except in the retina when the symptoms of EAE disappeared. Furthermore, the activities of the optic nerve and chiasma increased to a level higher than that of the controls at this time. By histopathological study, infiltration of inflammatory cells and focal demyelination were found in the optic nerve and lumbar spinal cord at 15 days after myelin basic protein inoculation. It was considered that the reduction of CNPase activity and its recovery and increase reflect demyelination and activation of oligodendroglia for remyelination, respectively.