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Biomedical subjects

N Nakajo

Publications and source records attributed to N Nakajo.

At least 19 recordsLinked to original sources
PubMed2001

Cytoplasmic occurrence of the Chk1/Cdc25 pathway and regulation of Chk1 in Xenopus oocytes.

Chk1, a nuclear DNA damage/replication G2 checkpoint kinase, phosphorylates Cdc25 and causes its nuclear exclusion in yeast and mammalian cells, thereby arresting the cell at the G2 phase until DNA repair/replication is completed. Chk1 is also involved, at least in part, in the natural G2 arrest of immature Xenopus oocytes, but it is unknown how Chk1 inhibits Cdc25 function and undergoes regulation during oocyte maturation. By using enucleated oocytes, we show here that Chk1 inhibits Cdc25 function in the cytoplasm of G2-arrested oocytes and that Cdc25 is activated exclusively in the cytoplasm of maturing oocytes. Moreover, we show that Chk1 activity is not appreciably altered during maturation, being maintained at basal levels, and that C-terminal truncation mutants of Chk1 have very high kinase activities, strong abilities to inhibit maturation, and altered subcellular localization in oocytes. These results, together with other results, suggest that the Chk1/Cdc25 pathway is involved cytoplasmically in G2 arrest of Xenopus oocytes, but moderately and independent of the G2 checkpoint, and that the C-terminal region of Chk1 negatively regulates its kinase activity and also determines its subcellular localization. Based on these results, we discuss the possibility that Chk1 (with the basal activity) may function as an ordinary regulator of Cdc25 in oocytes (and in other cell types) and that Chk1 might be hyperactivated in response to the G2 checkpoint via its dramatic conformational change.

14-3-3 Proteins↗
PubMed2000

Absence of Wee1 ensures the meiotic cell cycle in Xenopus oocytes.

Meiotic cells undergo two successive divisions without an intervening S phase. However, the mechanism of S-phase omission between the two meiotic divisions is largely unknown. Here we show that Wee1, a universal mitotic inhibitor, is absent in immature (but not mature) Xenopus oocytes, being down-regulated specifically during oogenesis; this down-regulation is most likely due to a translational repression. Even the modest ectopic expression of Wee1 in immature (meiosis I) oocytes can induce interphase nucleus reformation and DNA replication just after meiosis I. Thus, the presence of Wee1 during meiosis I converts the meiotic cell cycle into a mitotic-like cell cycle having S phase. In contrast, Myt1, a Wee1-related kinase, is present and directly involved in G(2) arrest of immature oocytes, but its ectopic expression has little effect on the meiotic cell cycle. These results strongly indicate that the absence of Wee1 in meiosis I ensures the meiotic cell cycle in Xenopus oocytes. Based on these results and the data published previously in other organisms, we suggest that absence of Wee1 may be a well-conserved mechanism for omitting interphase or S phase between the two meiotic divisions.

Animals↗
PubMed2000

No genotoxic effect of propofol in chinese hamster ovary cells: analysis by sister chromatid exchanges.

BACKGROUND: In spite of its high placental transfer, propofol is frequently used in general anesthesia and sedation during obstetric and gynecological surgery such as in vitro fertilization. This study investigated whether or not propofol has a genotoxic potential by the sister chromatid exchange assay in vitro. METHODS: Sister chromatid exchanges induced after exposure to propofol were measured in Chinese hamster ovary cells with and without metabolic activation. After propofol (0.2-20 microg ml(-1)) diluted dimethyl sulfoxide was applied for 2 h with or without S9 mix, the cells having been incubated for two metaphases (34 h) in the presence of 5'-bromo-2-deoxyuridine. N-nitrosodimethylamine and mitomycin C were used as positive controls with and without metabolic activation. The chromosomes were stained with the fluorescence plus Giemsa method, and then sister chromatid exchanges in 50 cells were counted for each concentration. RESULTS: Although increasing concentrations of propofol inhibited cell proliferation, no concentrations of propofol used in this study increased the sister chromatid exchange values, with and without metabolic activation. CONCLUSION: It was concluded that there was no indication, from the sister chromatid exchange assay in mammalian cells, of a genotoxic effect of propofol and its metabolites.

Anesthetics, Intravenous↗
PubMed1999

Two structural variants of Nek2 kinase, termed Nek2A and Nek2B, are differentially expressed in Xenopus tissues and development.

Nek2 kinase, a NIMA-related kinase, has been suggested to play both meiotic and mitotic roles in mammals, but its function(s) during development is poorly understood. We have isolated here cDNAs encoding a Xenopus homolog of mammalian Nek2 and have shown that Xenopus Nek2 has two structural variants, termed Nek2A and Nek2B. Nek2A, most likely a C-terminally spliced form, corresponds to the previously described human and mouse Nek2, while Nek2B is most probably a novel, C-terminally unspliced form of Nek2. As a consequence of this (probable) alternative splicing, Nek2B lacks the C-terminal 70-amino-acid sequence of Nek2A, which contains a PEST sequence (or a motif for rapid degradation). Western blot analysis reveals that Nek2A is expressed predominantly in the testis (presumably in spermatocytes) and very weakly in the stomach and, during development, only after the neurula stage. By contrast, Nek2B is expressed mainly in the ovary and in both primary and secondary oocytes and early embryos up to the neurula stage. These results suggest that Nek2A and Nek2B may play both meiotic and mitotic roles, but in a spatially and temporally complementary manner during Xenopus development, and that Nek2B, rather than Nek2A (or the conventional form of Nek2), may play an important role in early development. We discuss the possibility that a counterpart of Xenopus Nek2B might also exist and function in early mammalian development.

Alternative Splicing↗
PubMed1999

Involvement of Chk1 kinase in prophase I arrest of Xenopus oocytes.

Chk1 kinase, a DNA damage/replication G2 checkpoint kinase, has recently been shown to phosphorylate and inhibit Cdc25C, a Cdc2 Tyr-15 phosphatase, thereby directly linking the G2 checkpoint to negative regulation of Cdc2. Immature Xenopus oocytes are arrested naturally at the first meiotic prophase (prophase I) or the late G2 phase, with sustained Cdc2 Tyr-15 phosphorylation. Here we have cloned a Xenopus homolog of Chk1, determined its developmental expression, and examined its possible role in prophase I arrest of oocytes. Xenopus Chk1 protein is expressed at approximately constant levels throughout oocyte maturation and early embryogenesis. Overexpression of wild-type Chk1 in oocytes prevents the release from prophase I arrest by progesterone. Conversely, specific inhibition of endogenous Chk1 either by overexpression of a dominant-negative Chk1 mutant or by injection of a neutralizing anti-Chk1 antibody facilitates prophase I release by progesterone. Moreover, when ectopically expressed in oocytes, a Chk1-nonphosphorylatable Cdc25C mutant alone can induce prophase I release much more efficiently than wild-type Cdc25C; if endogenous Chk1 function is inhibited, however, even wild-type Cdc25C can induce the release very efficiently. These results suggest strongly that Chk1 is involved in physiological prophase I arrest of Xenopus oocytes via the direct phosphorylation and inhibition of Cdc25C. We discuss the possibility that Chk1 might function either as a G2 checkpoint kinase or as an ordinary cell cycle regulator in prophase-I-arrested oocytes.

Amino Acid Sequence↗
PubMed1999

Inhibition of styloglossus motoneurons during the palatally induced jaw-closing reflex.

The inhibition of hypoglossal motoneurons innervating the styloglossus muscle during transient jaw closing, the so-called jaw-closing reflex, was studied in cats. The application of diffuse pressure stimulation to the posterior palatal surface produced the jaw-closing reflex and inhibitory postsynaptic potentials in the styloglossus motoneurons, indicating that mechanosensory inputs from the posterior palatal mucosa sent inhibitory synaptic inputs to styloglossus motoneurons. We also demonstrated that, during the palatally induced jaw-closing reflex, the tongue extended at jaw closure and was still extended forward in the initial part of the opening phase. In all of 22 styloglossus motoneurons studied, the depression of firing was elicited after the onset of jaw closure. In 14 of 22 styloglossus motoneurons, the depression of firing was elicited in the closing phase, and in the remaining cells it was elicited in the occlusal phase. By increasing the intracellular concentration of chloride ions, the inhibitory postsynaptic potential elicited in the styloglossus motoneuron converted to a depolarizing potential. It is concluded that the inhibition of styloglossus motoneurons may be involved in the maintenance of tongue protrusions during the palatally induced jaw-closing reflex, and that inhibitory postsynaptic potentials evoked in the styloglossus motoneurons are partly due to a chloride-dependent inhibitory postsynaptic potential.

Animals↗
PubMed1998

Fabrication and characterization of multilayer supermirrors for hard X-ray optics.

Multilayer supermirrors stacked with three sets of Pt/C combinations have been fabricated on a flat float-glass and conical replica foil mirror using a magnetron DC sputtering system, and applied to X-ray optical systems in the hard X-ray region. The design of the supermirror is optimized to obtain the highest integrated reflectivity in the energy band and at the grazing angle concerned. X-ray reflectivities of 30% in the 25-35 keV band at an incidence angle of 0.3 degrees were obtained.

Journal Article↗
PubMed1998

Characterization of substrates for use in X-ray multilayer optics.

The optical performance of platinum-carbon multilayers deposited onto different substrates has been examined. Specular reflectivity and non-specular diffuse scattering were measured to study the replication of substrate roughness into the multilayer structure. Surface topography was measured before and after deposition using a scanning probe microscope and a mechanical profiler.

Journal Article↗
PubMed1997

Meiotic cell cycle in Xenopus oocytes is independent of cdk2 kinase.

In vertebrates, M phase-promoting factor (MPF), a universal G2/M regulator in eukaryotic cells, drives meiotic maturation of oocytes, while cytostatic factor (CSF) arrests mature oocytes at metaphase II until fertilization. Cdk2 kinase, a G1/S regulator in higher eukaryotic cells, is activated during meiotic maturation of Xenopus oocytes and, like Mos (an essential component of CSF), is proposed to be involved in metaphase II arrest in mature oocytes. In addition, cdk2 kinase has been shown recently to be essential for MPF activation in Xenopus embryonic mitosis. Here we report injection of Xenopus oocytes with the cdk2 kinase inhibitor p21Cip in order to (re)evaluate the role of cdk2 kinase in oocyte meiosis. Immature oocytes injected with p21Cip can enter both meiosis I and meiosis II normally, as evidenced by the typical fluctuations in MPF activity. Moreover, mature oocytes injected with p21Cip are retained normally in metaphase II for a prolonged period, whereas those injected with neutralizing anti-Mos antibody are released readily from metaphase II arrest. These results argue strongly against a role for cdk2 kinase in MPF activation and its proposed role in metaphase II arrest, in Xenopus oocyte meiosis. We discuss the possibility that cdk2 kinase stored in oocytes may function, as a maternal protein, solely for early embryonic cell cycles.

Amino Acid Sequence↗
PubMed1997

Excitation of hypoglossal motoneurons responsible for tongue protrusions is associated with palatally induced jaw-closing reflex.

The excitation of hypoglossal motoneurons innervating the genioglossus and geniohyoid muscles during transient jaw closing, the so-called jaw-closing reflex, was studied in cats. The application of diffuse pressure stimulation to the posterior palatal surface produced the jaw-closing reflex, and it was found that mechanosensory inputs from the posterior palatal mucosa sent excitatory synaptic inputs to both genioglossus and geniohyoid motoneurons. We demonstrated that, during the palatally induced jaw-closing reflex, the tongue extended at jaw closure and was still extended forward in the initial part of the opening phase. In five of 27 genioglossus motoneurons and nine of 23 geniohyoid motoneurons, the onset of burst was elicited before the onset of jaw closure. The remaining cells produced the onset of burst in the closing phase and in the initial part of the occlusal phase. However, the onset of excitatory postsynaptic potentials was 75-180 ms (n=20), earlier than that of jaw closure. During the jaw-closing reflex, the genioglossus and geniohyoid motoneurons were excited during the same phase of jaw movements and there was no difference in the onset of firing between the genioglossus and geniohyoid motoneurons. It is concluded that the excitation of the genioglossus and geniohyoid motoneurons may be associated with tongue protrusions during the palatally induced jaw-closing reflex.

Animals↗
PubMed1995

Occurrence of a novel fucose-containing pentaglycosylceramide with blood-group-B active determinant in Xenopus blastula cells: its possible involvement in cell-cell adhesion.

For understanding of the biological function of glycoconjugates during embryogenesis and morphogenesis, Xenopus laevis is considered a very useful animal model. We have found that blood-group-active molecules characteristically were distributed in the cell-cell contact region of Xenopus blastula cells. The chemical nature of blood-group-active glycoconjugates, including glycosphingolipids, is little known. T.l.c.-immunostaining using anti-blood-group-antigen antibodies showed that many species of blood group-B-active glycosphingolipids existed in the neutral glycosphingolipid fraction extracted from Xenopus laevis eggs. Among the B-active glycosphingolipids detected, two major components with the fastest mobility on a t.l.c. plate, tentatively termed XN-1 and XN-2, were isolated, and their chemical structures were characterized by gas chromatography-mass spectrometry, immunological anlaysis, fast-atom-bombardment mass spectrometry and 1H-n.m.r. spectroscopy. Both XN-1 and XN-2 had an identical pentaoligosaccharide structure, but differed in their ceramide moiety. The chemical structure is: [table: see text]. This is a novel type of pentaglycosylceramide with blood-group B activity, in that it lacks N-acetylhexosamine in its core carbohydrate structure. In this paper, a possible involvement of the blood-group antigen in the cell-adhesion process of Xenopus embryonic cells is discussed.

ABO Blood-Group System↗
PubMed1994

Suppression of DNA replication via Mos function during meiotic divisions in Xenopus oocytes.

Meiosis is characterized by the absence of DNA replication between the two successive divisions. In Xenopus eggs, the ability to replicate DNA develops during meiotic maturation, but is normally suppressed until fertilization. Here we show that development of the DNA-replicating ability depends on new protein synthesis during meiosis I, and that mere ablation of the endogenous c-mos product Mos allows maturing oocytes to enter interphase and replicate DNA just after meiosis I. Moreover, we demonstrate that during normal maturation cdc2 kinase undergoes precocious inactivation in meiosis I and then premature reactivation before meiosis II; importantly, this premature cdc2 reactivation absolutely requires Mos function and its direct inhibition by a dominant-negative cdc2 mutant also results in nuclear reformation and DNA replication immediately after meiosis I. These findings indicate that suppression of DNA replication during meiotic divisions in Xenopus oocytes is accomplished by the Mos-mediated premature reactivation of cdc2 kinase. We suggest that these mechanisms for suppressing DNA replication may be specific for meiosis in animal oocytes, and that the ultimate biological function, including the well known cytostatic factor activity, of Mos during meiotic maturation may be to prevent undesirable DNA replication or parthenogenetic activation before fertilization.

Animals↗
PubMed1994

A mouse B16 melanoma mutant deficient in glycolipids.

Mouse B16 melanoma cell line, GM-95 (formerly designated as MEC-4), deficient in sialyllactosylceramide was examined for its primary defect. Glycolipids from the mutant cells were analyzed by high-performance TLC. No glycolipid was detected in GM-95 cells, even when total lipid from 10(7) cells was analyzed. In contrast, the content of ceramide, a precursor lipid molecule of glycolipids, was normal. Thus, the deficiency of glycolipids was attributed to the first glucosylation step of ceramide. The ceramide glucosyltransferase (EC 2.4.1.80) activity was not detected in GM-95 cells. There was no significant difference of sialyllactosylceramide synthase activity, however, between GM-95 and the parental cells. The deficiency of glycolipids in GM-95 cells was associated with changes of the cellular morphology and growth rate. The parental cells showed irregular shapes and tended to overlap each other. On the other hand, GM-95 cells exhibited an elongated fibroblastic morphology and parallel arrangement. The population-doubling times of GM-95 and the parental cells in serum-free medium were 28 hr and 19 hr, respectively.

Animals↗
PubMed1991

Excitation and inhibition of trigeminal motoneurons by palatal stimulation.

Excitation and inhibition of jaw-closing motoneurons (Masseteric and Temporal Motoneurons, Mass. and Temp. Mns) during transient jaw closing, the so-called jaw-closing reflex, and prolonged jaw opening elicited by palatal stimulation were studied. By pressing the anterior palatal surfaces sustained jaw opening was elicited, suggesting that sustained jaw opening results from inhibition of tonic background activity of jaw-closing motoneurons by inhibitory postsynaptic potentials (IPSPs) elicited by mechanical stimulation of the anterior palatal mucosa. Recordings showed that the onset of IPSPs was 80 ms earlier than the onset of jaw opening. Application of diffuse pressure stimulation to the posterior palatal surfaces elicited bursts of spikes triggered on excitatory postsynaptic potentials (EPSPs), suggesting that mechanosensory receptors from the posterior palatal mucosa send excitatory synaptic inputs to jaw-closing motoneurons. Furthermore, it is suggested that mechanosensory inputs from the posterior palatal mucosa may excite neurons in the central pattern generator and provide the motor patterns responsible for jaw closure during the jaw-closing reflex. We have demonstrated that excitation of Mass. Mns innervating the deep masseter muscle mainly contributed to maintaining the occlusal phase of jaw closure during the jaw-closing reflex. However, the onset of EPSPs was 100 to 160 ms (n = 27) earlier than the onset of jaw closure. In studies on spontaneously occurring jaw closure it was demonstrated that there was a proportional increase in the number of spikes of the Temp. Mn and the mechanical response (jaw closure).

Animals↗
PubMed1990

Spontaneous oscillation of artificial membrane: equivalence in effects of temperature and volatile anesthetic.

Oscillatory phenomenon at an oil-water interface in the presence of hexadecyltrimethylammonium bromide (CTAB) has been studied. The oscillation was attributable to successive formation and destruction of surfactant monolayer. According to the temperature elevation, the frequency of the electrical oscillation increased whereas the amplitude decreased. Addition of diethylether increased the frequency and decreased the amplitude. These effects of temperature and anesthetic were analyzed by the theory of the "N"-shaped relationship between surface-pressure and surface-concentration of the surfactant.

Cetrimonium↗