PI (α1) polymorphism in the Japanese: Confirmation of PI*M4 and description of new PI variants

PI phenotyping by separator isoelectric focusing (SIEF) was performed on a total of 1000 unrelated Japanese individuals from two different areas of Western Japan. The PI M1M4 subtype was observed together with the six common PI M subtypes. PI*M4 was confirmed to be present but rare in the Japanese. Several new PI variants were identified by comparison runs of each variant with previously reported genetic variants. The significance of treatment of serum with dithiothreitol (DTT) followed by iodacetic acid (IAC) in determination of PI variants is also described.     

A general characteristic of tumor mitochondria: Leakage of endogenous Mg2+ on incubation with uncoupler and resultant reduction of uncoupler-stimulated ATPase activity

Previous studies showed that stimulation of mouse mitochondrial ATPase activity of tumor cells, fetal liver, and adult brain by the uncoupler 2,4-dinitrophenol was markedly suppressed during incubation of the mitochondria with the uncoupler (J.-I. Hayashi et al., 1980, Biochem. Biophys. Res. Commun.92, 261–267). The present work showed the reason for this suppression. More than half the endogenous Mg²⁺ leaked from mitochondria of all tumor cells tested, and of fetal liver and adult brain during incubation with the uncoupler, while only about 30% of the endogenous Mg²⁺ leaked from mitochondria of other normal tissues. The effect of the uncoupler on Mg²⁺ leakage from liver mitochondria changed from the fetal to the adult type within about 30 min after birth. In hypotonic medium, normal liver mitochondria also lost more than half their total Mg²⁺ and concomitantly stimulation of their ATPase activity by uncoupler was considerably reduced. Exogenously added Mg²⁺ could reverse this reduced effect of the uncoupler on ATPase activity of mitochondria from normal tissues and tumor cells. These results show that the endogenous Mg²⁺ content of mitochondria directly affects the stimulation by uncoupler of ATPase activity of mitochondria from both normal tissues and tumor cells. Thus, mitochondria of all tumor cells tested, and of fetal liver and adult brain are leaky to Mg²⁺ during incubation with uncoupler and as a result of the leakage, the stimulatory effect of the uncoupler on their ATPase activity is greatly reduced.     

A mitogen-activated protein (MAP) kinase activating factor in mammalian mitogen-stimulated cells is homologous to Xenopus M phase MAP kinase activator.

The mitogen-activated protein (MAP) kinases, a family of 40-45-kDa kinases whose activation requires both tyrosine and threonine/serine phosphorylations, are suggested to play key roles in various phosphorylation cascades. A previous study of Krebs and co-workers (Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, E. G. (1991) J. Biol. Chem. 266, 4220-4227) detected an activity in epidermal growth factor (EGF)-stimulated 3T3 cells that can stimulate inactive MAP kinases. We observed this activity in rat 3Y1 cells treated with various mitogenic factors and in PC12 cells treated with nerve growth factor (NGF). Its kinetics of activation and deactivation following EGF or NGF stimulation roughly paralleled that of MAP kinase. The MAP kinase activator required the presence of ATP and a divalent cation such as Mn2+ and Mg2+ and was inactivated by phosphatase 2A treatment in vitro. This activator has been isolated from EGF-stimulated 3Y1 cells by sequential chromatography and identified as a 45-kDa monomeric protein. It was able to activate mammalian and Xenopus MAP kinases in vitro and was very similar to Xenopus M phase MAP kinase activating factor, which was purified previously from mature oocytes (Matsuda, S., Kosako, H., Takenaka, K., Moriyama, K., Sakai, H., Akiyama, T., Gotoh, Y., and Nishida, E. (1992) EMBO J. 11, 973-982), in terms of its functional, immunological, and physicochemical properties. Thus, the same or a similar upstream activating factor may function in mitogen-induced and M phase-promoting factor-induced MAP kinase activation pathways.     

Activation of mitogen-activated protein kinase and its activator by ras in intact cells and in a cell-free system.

Mitogen-activated protein (MAP) kinase is a serine/threonine kinase whose function is thought to be essential for the transduction of mitogenic signals. MAP kinase is activated by phosphorylation induced by a variety of extracellular stimuli, and its direct upstream activator has been identified. Using amphibian and mammalian systems, we show here that ras can activate MAP kinase and its activator. Injection of v-Ha-ras p21 into Xenopus immature oocytes activated both MAP kinase and maturation-promoting factor (MPF) activities. The activation of MAP kinase preceded that of MPF, demonstrating that ras activates MAP kinase in an MPF-independent pathway. Moreover, we found that the MAP kinase activator is also activated in ras-injected oocytes. Activation of MAP kinase and its activator occurred also when the v-Ki-ras gene was conditionally induced in rat fibroblastic 3Y1 cells. Furthermore, we observed that ras activated MAP kinase and its activator in a cell-free system prepared from Xenopus oocytes. Using an antibody against the Xenopus 45-kDa MAP kinase activator, we demonstrated that the 45-kDa activator molecule was activated by ras. These findings suggest that the MAP kinase activator/MAP kinase system may be the downstream components of ras signal transduction pathways.     

Microtubule-associated-protein (MAP) kinase activated by nerve growth factor and epidermal growth factor in PC12 cells. Identity with the mitogen-activated MAP kinase of fibroblastic cells

Treatment of PC12 cells with either nerve growth factor (NGF), a differentiating factor, or epidermal growth factor (EGF), a mitogen, resulted in 7-15-fold activation of a protein kinase activity in cell extracts that phosphorylated microtubule-associated protein (MAP) 2 on serine and threonine residues in vitro. Both the NGF-activated kinase and the EGF-activated kinase could be partially purified by sequential chromatography on DEAE-cellulose, phenyl-Sepharose and hydroxylapatite, and were identical with each other in their chromatographic behavior, apparent molecular mass (approximately 40 kDa) on gel filtration, substrate specificity, and phosphopeptide-mapping pattern of MAP2 phosphorylated by each kinase. Moreover, both kinases were found to be indistinguishable from a mitogen-activated MAP kinase previously described in growth-factor-stimulated or phorbol-ester-stimulated fibroblastic cells, based on the same criteria. Kinase assays in gels after SDS/polyacrylamide gel electrophoresis revealed further that the NGF- or EGF-activated MAP kinase in PC12 cells, as well as the EGF-activated MAP kinase in fibroblastic 3Y1 cells resided in two closely spaced polypeptides with an apparent molecular mass of approximately 40 kDa. In addition, these MAP kinases were inactivated by either acid phosphatase treatment or protein phosphatase 2A treatment. These results indicate that MAP kinase may be activated through phosphorylation by a differentiating factor as well as by a mitogen. MAP kinase activation by EGF was protein kinase C independent; it reached an almost maximal level 1 min after EGF treatment and subsided rapidly within 30-60 min. On the other hand, NGF-induced activation of MAP kinase was partly protein kinase C dependent and continued for at least 2-3 h.     

Primary structure of two linker chains of the extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus

Two types of linker subunits (linkers 1 and 2) of the extracellular hemoglobin of Tylorrhynchus heterochaetus have been isolated as disulfide-linked homodimers by C18 reverse-phase chromatography. These subunits constituted 6 and 13%, respectively, of total protein area on the chromatogram. The complete amino acid sequences of linkers 1 and 2 were determined by automated Edman sequencing of the peptides derived by digestions with lysyl endopeptidase, trypsin, chymotrypsin, Staphylococcus aureus V8 protease, pepsin, and endoproteinase Asp-N. The linker 1 consisted of 253 amino acid residues (the calculated molecular mass, 28,200 Da), while the linker 2 consisted of 236 residues (26,316 Da). The two chains showed 27% sequence identity. The amino acid sequences of Tylorrhynchus linkers 1 and 2 also showed 23-27% homology with the recently determined sequence of a linker chain of Lamellibrachia hemoglobin (Suzuki, T., Takagi, T., and Ohta, S. (1990) J. Biol. Chem. 265, 1551-1555). In the three linker chains, half-cystine residues were highly conserved; 8 out of 13 residues are identical, suggesting that such residues would contribute to the formation of intrachain disulfide bonds essential for the protein folding of the linker polypeptides. Based on the exact molecular masses of the linker and the heme-containing subunits, the molar ratios estimated for the subunits and the minimum molecular weights per 1 mol of heme, a model is proposed for the subunit structure of the Tylorrhynchus hemoglobin, consisting of 216 polypeptide chains, 192 heme-containing chains, and 24 linker chains.     

Origins of laboratory mice deduced from restriction patterns of mitochondrial DNA

To determine the origins of laboratory mice, the restriction patterns of mitochondrial DNAs (mtDNAs) from various strains were compared with those of relevant subspecies and/or races of Mus musculus. In most strains and substrains of laboratory mice examined (50/55), the cleavage patterns were identical to those of the European subspecies M. m. domesticus. Those that varied include two sublines of NZB, the strain NZC, and the Japanese strain RR. The NZB and NZC patterns were identical to that of the European subspecies M. m. brevirostris, which itself has restriction patterns similar to M. m. domesticus. On the other hand, the RR pattern was identical to M. m. molossinus-like mice trapped in Western China and slightly different from Japanese M. m. molossinus. These findings suggest that the strains NZB and NZC stemmed from a European founder stock which differed from the ancestral stocks of other laboratory strains and that the ancestral mice of the RR strain had been transported from China to Japan. Therefore, most laboratory strains of mice are derived from the European subspecies M. m. domesticus while M. m. brevirostris and M. m. molossinus have made minor contributions. M. m. musculus does not appear to have made any contribution.     

Cytochemical evidence of the origin of the dense tubular system in the mouse platelet

Summary Glucose-6-phosphatase (G6Pase) was used as a marker enzyme for the endoplasmic reticulum in mouse megakaryocytes and platelets. G6Pase activity was localized in the dense tubular system of the platelets. Enzyme activity was also observed in the nuclear envelope, and in the rough endoplasmic reticulum of the megakaryocytes. However, the Golgi apparatus of the megakaryocyte was never involved. The present study has added new cytochemical evidence for the hypothesis that the dense tubular system of the platelet originates from the endoplasmic reticulum of the megakaryocyte.