The presence and localization in pig and rat testes of phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK) and its substrates were investigated. PL-Ca-PK activity was found in the testis total particulate fraction and epididymal fluid, but little in the testis cytosol and matured spermatozoa obtained from the epididymis. Similarly, at least three endogenous substrates (83,000, 33,000, and 26,000) and five substrates (>100,000, 83,000,60,000, 43,000, and 19,000) for PL-Ca-PK were detected in the testis total particulate fraction and the epididymal fluid, respectively, but little or no substrates were observed in the testis cytosol and matured spermatozoa. The three substrates detected in the total particulate fraction were also observed in the testis nuclear fraction. In rat testis, PL-Ca-PK activity was detected in the total particulate fraction of germ cells. The results suggested that PL-Ca-PK system might be important in membrane- or subcellular organellarassociated functons in testis. (Endocrinology115: 2391–2399,1984)
Radiation chimeras in mice were induced by intrasplenic injection of allogeneic bone marrow cells instead of intravenous injection. Interestingly, the survival time in X-irradiated BALB/c mice inoculated intrasplenically (i.s.) with bone marrow cells from C3H/He mice was markedly prolonged as compared with that in X-irradiated BALB/c mice inoculated i.v. with bone marrow cells from C3H/He mice. However, when C57BL/6 mice were used as donors, a significant difference between i.s. injection and i.v. injection has not been found in survival time at 60 days after X irradiation. On the contrary, when bone marrow cells from BALB/c or C57BL/6 mice were injected into X-irradiated C3H/He mice, i.s. injection gave longer survival days to recipients than did i.v. injection. Based on testing their chimerism, it was suggested that lymphoid cells of donor origin were predominantly identified in almost all BALB/c or C3H/He recipients which were inoculated i.s. with bone marrow cells from C5BL/6 mice. However, somewhat incomplete chimerism was observed when the C3H/He to BALB/c donor-recipient combination was used and vice versa.
We studied the effect of thrombopoietin (TPO) on interleukin-3 (IL-3)-dependent bone marrow cell colony formation of mice to clarify the role of protein kinase C (PKC) in the signal transduction of TPO for the proliferation of primitive hematopoietic progenitors. TPO alone hardly yielded colonies. However, TPO in combination with IL-3 increased colony numbers synergistically from 2- to 4-fold, compared with those supported by IL-3 alone. Serial observation of colony development showed that TPO may hasten the appearance of colonies by shortening the dormant period (G(0)) of primitive progenitors. Immunocytochemical studies on PKC isoforms in progenitor cells stimulated with TPO have revealed that the expression pattern of PKC-epsilon is changed, but not that of PKC-alpha, -beta, -gamma, -delta, or -zeta. Selective PKC inhibitors, such as calphostin C and GF 109203X, and PKC-epsilon-specific translocation inhibitor peptide abrogated the enhancing effect of TPO on IL-3-dependent colony formation and the changes in the intracellular expression pattern of PKC-epsilon. These data taken together suggest that TPO has a direct effect on primitive progenitors and enhances IL-3-dependent colony formation, at least partly through the activation of PKC-epsilon.
Localizations of lactoferrin and lysozyme in the peripheral blood cells and bone marrow cells obtained from normal volunteers were investigated by an electron microscopic immunogold double stainings. Both lactoferrin and lysozyme were present in the same granules of neutrophils. Mast cell granules and some phagolysosomes of bone marrow reticulum cells also showed similar double stainabilities. In contrast, monocyte granules and eosinophil granules contained exclusively lysozyme. Neither lactoferrin nor lysozyme was observed in blastoid cells, megakaryocytes, platelets, erythroblasts, red blood cells, plasma cells, lymphocytes and basophils.The electron microscopic immunogold double stainings are useful for detecting a couple of antigens on a single cell.
In order to study the physiological role played by cyclic nucleotide-independent phosvitin kinase in pig testis, effects of heparin (an inhibitor of RNA synthesis) and polyamines (stimulators of the synthesis) on the enzyme, distribution in nuclei of the enzyme, and identification of endogenous substrates for the enzyme were examined. The enzyme was shown to be inhibited by heparin, nd the inhibition was removed by the addition of polyamines, when phosvitin (the best exogenous substrate for the enzyme) was used as substrate. The inhibition by heparin and inhibition removal by polyamines was dependent on Mg2+ concentration. The enzyme was found to be present in pig testis nuclei as well as in the cytosol. The enzyme phosphorylated chromatin-associated non-histone proteins in the testis nuclei. Of the non-histone proteins, high mobility group (HMG) 14 and 17 were identified as substrates for the enzyme. Phosphorylation of HMG 14 and 17 was also inhibited by heparin and inhibition of HMG 14 phosphorylation was removed by the addition of polyamines, although the effect was less dependent on Mg2+ concentration. These results suggested that phosvitin kinase was involved in the regulation of RNA synthesis int he testis.
We investigated whether antisense oligodeoxynucleotides complementary to bcr/abl mRNA or protein kinase antagonists display antitumor activity on Ph -positive leukemia cell lines. bcr/abl antisense oligomers showed inhibitory effects on the in vitro growth of Ph1-positive leukemia cell lines in liquid culture, and further displayed an inhibitory effect on transformed murine hematopoietic cells using transfection with a retroviral vector expressing p210bcr/abl oncoprotein. However, in vitro treatment with a bcr/abl antisense oligomer did not completely abolish the expression of bcr/abl mRNA and did not display the desired “killing effect” on Ph1-positive leukemia cells. On the other hand, investigation of the effect on Ph1-positive leukemia cells by various types of protein kinase antagonists revealed that herbimycin A, a protein tyrosine kinase antagonist, displays preferential and remarkable suppression of the growth of Ph1-positive leukemia cells and P210bcr/abl associated transformed cells by virtue of suppressing bcr/abl protein tyrosine kinase activity. These results may provide important future insights in developing a new category of antitumor therapy by targeting oncogene products.
Ultrastructural localization of lactoferrin in human blood cells which were obtained from 11 normal volunteers was studied by an immunogold staining method.Lactoferrin was detected in neutrophils, mast cells and reticulum cells. On the other hand, no lactoferrin was detected in monocytes, eosinophils, basophils, megakaryocytes, platelets, lymphocytes, plasma cells or erythroid cells. Generally, lactoferrin was contained in cytoplasmic granules in these lactoferrin-positive cells. No lactoferrin was detected in the nucleus, perinuclear space, rough endoplasmic reticulum, Golgi apparatus or mitochondria. In neutrophils, lactoferrin was detected in myelocytes, metamyelocytes, band cells and polymorphonuclear leucocytes. Lactoferrin was contained preferently in secondary granules and scarcely in primary granules. In mast cells, the granules were remarkably positive for lactoferrin. In reticulum cells, lactoferrin was occasionally contained in phagolysosomes.
Some experiments were studied in order to clarify whether abnormal coagulation in liver cirrhosis and hepatoma may be due to abnormal fibrinogen or not. Abnormal fibrinogen might be suggested by prolonged and decreased fibrin monomer polymerization with thrombin or reptilase.The purified fibrinogen revealed same abnormal patterns which look like in plasma.It was suggested that these defects in fibrinogen were associated with delayed polymerization of the fibrin monomer.However, immunoelectrophoresic study of the purified fibrinogen showed no abnormality and S. D. S-polyacrylamide gel electrophoresis of them showed normal mobility and amount of Aα, Bβ and γ-chains.Sialic acid content of them were examined in order to detect its character.It became clear that its content were higher in the patients fibrinogen than in normal controls.From these results, it may be supposed that there is functionally the abnormal fibrinogen in some patients with liver disease and the high sialic acid content might play an important role in the abnormal coagulation.
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