Biomedical subjects

K Shiozaki

Publications and source records attributed to K Shiozaki.

At least 55 records · Page 3Linked to original sources

PubMed1992

Functional dissection of the phosphorylated termini of fission yeast DNA topoisomerase II.

Fission Yeast DNA topoisomerase II (165 kD) consists of an enzymatically active 125-kD core, approximately 10-kD NH2-terminal and 30-kD COOH-terminal domains. The question addressed in the present study is what is the role of the topo II termini. Although deletion of either the NH2 or the COOH terminus is viable, deletion of both termini is lethal; the termini share an essential role for viability. We show here that topo II phosphorylation sites are localized in the terminal domains, but dephosphorylated topo II is still active. The topo II terminal sequences are required for nuclear localization; topo II double terminal deletion mutants are deficient for nuclear targeting, whereas wild-type and single deletion mutant topo IIs are transported into the nucleus with different efficiencies. Functional subdomains in the NH2 terminus are further dissected; we identified a 15 amino acid nuclear localization sequence (NLS) which is essential for viability and nuclear localization when the COOH terminus is deleted. This NLS could be substituted with SV-40 large T-antigen NLS. Two other functional subdomains were found; a non-essential acidic stretch which is phosphorylated and apparently enhances the nuclear localization and an essential hydrophilic stretch of unknown function. Motifs similar to these three NH2-terminal subdomains are also found in the COOH terminus. Our results support the possibility that phosphorylation of topo II does not play an essential role in fission yeast.

Amino Acid Sequence↗

PubMed1991

Identification of two novel GTP-binding protein alpha-subunits that lack apparent ADP-ribosylation sites for pertussis toxin.

Molecular cloning of cDNAs encoding alpha-subunits of guanine nucleotide-binding regulatory proteins (G-proteins) has revealed the existence of nine species of alpha-subunits. We have identified two additional G-protein alpha-subunits, which we refer to as GL1 alpha and GL2 alpha, by isolating bovine liver cDNA clones that cross-hybridized at reduced stringency with bovine Gi1 alpha-subunit cDNA. The deduced amino acid sequences of GL1 alpha and GL2 alpha share 83% identity with each other and show 45-55% identity with those of other known G-protein alpha-subunits. Both GL1 alpha and GL2 alpha lack a consensus site for ADP-ribosylation by pertussis toxin. Messenger RNA corresponding to GL2 alpha was detected in all tissues examined, but GL1 alpha mRNA was detected only in liver, lung, and kidney. Antiserum prepared against a synthetic pentadecapeptide corresponding to the deduced carboxyl terminus of GL2 alpha specifically reacted with a 40-kDa protein in mouse liver, brain, lung, heart, kidney, and spleen. The amount of the 40-kDa protein was highest in brain and lung. We suggest that GL1 alpha and GL2 alpha are new members of a subfamily of pertussis toxin-insensitive G-proteins.

Adenosine Diphosphate Ribose↗

PubMed1991

Characteristics of the principal neutralizing determinant of HIV-1 prevalent in Japan.

The principal neutralizing determinants (PNDs) of 29 human immunodeficiency virus type 1 (HIV-1) isolates in Japan were analyzed using polymerase chain reactions. The viruses were isolated from 16 hemophiliacs, 11 individuals infected by their sexual transmission and 1 patient infected by blood transfusion (total 28 patients). Two virus isolates which were obtained from the same individual at different periods were also analyzed. All individuals were Japanese except one. The results produced 32 different PND sequences. A highly conserved central core sequence (GPG) was present in 27 of 32 patients, similar to the number reported in the United States, despite the marked heterogeneity in flanking regions of PNDs. The PNDs of all the 16 HIV-1 isolates obtained from patients with coagulation disorders had GPG sequences. Secondary structure prediction of PNDs by a joint method suggested that they were composed of coil-beta strand-coil-beta strand-alpha helix. It is suggested that the conserved core sequence has a type I turn. These findings may be useful in planning further clinical trials for passive vaccination.

Adolescent↗

PubMed1991

A functional 125-kDa core polypeptide of fission yeast DNA topoisomerase II.

We purified fission yeast DNA topoisomerase II (topo II) to apparent homogeneity. It consists of a single 165-kDa polypeptide in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and, upon treatment with a bifunctional reagent, doubles its molecular weight. Limited proteolysis of intact topo II by papain produces a 125-kDa core, which lacks the N-terminal 75 and the C-terminal approximately 260 amino acids but still contains regions similar to those of bacterial or phage T4 topo II subunits. The core retains relaxing and unknotting activities. Further digestion inactivates the core, cleaving it at the middle of the GyrB-like domain and at the beginning of the GyrA-like domain. Therefore, papain appears to cleave spatially distinct subdomains of topo II. We made top2 mutant genes deleted of the C-terminal 286 or N-terminal 74 amino acids, which can substitute for the wild-type top2+ gene in mitosis and meiosis. However, a mutant containing deletions of both termini cannot rescue the top2 null mutant, despite the fact that the product is enzymatically active. Therefore, the top2 product of the doubly truncated gene may not fulfill all of the in vivo requirements for top2+ function.

Amino Acid Sequence↗

PubMed1991

Protein phosphatases in cell division: how vital are they?

In contrast to the wealth of information on cellular function of protein kinases, many of which are known to be the products of proto-oncogenes, little is known about how protein dephosphorylation is involved in growth control of normal and malignant cells. In the present study, roles of protein phosphatases in cell division cycle control were examined by molecular genetic approaches using a lower eukaryote, the fission yeast Schizosaccharomyces pombe. Nine protein phosphatase genes have been so far identified and characterized in this organism. Each of two (dis2+, sds21+, and ppa1+, ppa2+) gene products is highly similar to mammalian type 1 and 2A ser/thr phosphatases, respectively. The ppx1+ product is an intermediate of type 1 and 2A, while the ppb1+ product is similar to Ca(2+)-dependent type 2B. At least two protein tyrosine phosphatase genes (pyp1+ and pyp2+) exist. The cdc25 protein is now established to be a tyrosine phosphatase that activates cdc2 kinase. Some of these phosphatase genes are interrelated but have distinct, essential functions in cell cycle control. Missense mutations, deletions or high dosage expression of these phosphatase genes affect entry into and exit from mitosis, mitotic chromosome disjunction, cell size and cell shape. They seem to interact with the main regulators of mitosis, cdc2, cdc13/cyclin, cdc25 and weel, or with mitotic structural components, such as condensed chromosomes or the spindle apparatus. We show that the product of an essential gene, sds22+, is an important, positive factor in controlling the expression and modulating the activity of dis2 phosphatase.

Amino Acid Sequence↗

PubMed1991

[Significance of umbilical plasma concentrations of catecholamines and their metabolites in patients with term vaginal deliveries].

In an attempt to further clarify the physiological significance of Catecholamine (CA) s in the feto-placental unit, umbilical plasma concentrations of CAs and CA metabolites, 3-methoxy-4-hydroxy-phenylglycol (MHPG), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured in cases of full-term vaginal deliveries by HPLC-ECD. Results were as follows. 1. Norepinephrine (NE) and epinephrine (E) levels were significantly higher in arterial than in venous plasma, but no significant arterio-venous differences were found with dopamine (DA) concentrations. 2. High concentrations of MHPG, DOPAC and HVA were demonstrated in both arterial and venous plasma. 3. DOPAC and HVA levels were significantly higher in arterial than in venous plasma, but no significant arterio-venous differences were noted with MHPG. 4. A significant positive linear relations hip was demonstrated between arterial levels of NE and MHPG as well as DA and DOPAC, DA and HVA. 5. Arterial plasma MHPG levels were correlated significantly with arterial plasma pH and pO2. These results suggest that, in the feto-placental unit, NE, E and DA turnover is increased at term vaginal deliveries, and NE and E secretion sensitively responds to fetal respiratory and metabolic states. In addition, it is suggested that the mechanism which controls the DA turnover is different from that of NE and E.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1987

Cloning and sequencing of Schizosaccharomyces pombe DNA topoisomerase I gene, and effect of gene disruption.

We cloned the structural gene topl+ for Schizosaccharomyces pombe DNA topoisomerase I (topo I) by hybridization. An eight-fold increase of topo I relaxing activity was obtained in S. pombe cells transformed with multicopy plasmid with topl+ insert. Nucleotide sequence determination showed a hypothetical coding frame interrupted by two short introns, encoding a 812 residue polypeptide (M.W. 94,000), 43 residues longer than and 47% homologous to Saccharomyces cerevisiae topo I. We show that the topl (null) strain made by gene disruption is viable, although its generation time is 20% longer than that of wild type. The topl locus is mapped in the long arm of chromosome II, using the Leu+ marker integrated with the cloned topl+ sequence. We constructed a double mutant topl (null) top2 (ts) and found its defective phenotype similar to that of previously obtained topl (heat sensitive) top2 (ts). The other double mutant topl (null) top2 (cs), however, was lethal. Our results suggest that topl+ gene of S. pombe is dispensable only if topo II activity is abundant.

Amino Acid Sequence↗

PubMed1987

DNA topoisomerase II is required for condensation and separation of mitotic chromosomes in S. pombe.

We show that DNA topoisomerase II (topo II) is continuously required for mitotic chromosome changes in Schizosaccharomyces pombe. We constructed cold-sensitive (cs) or temperature-sensitive (ts) strains mutated in the genes coding for topo II (top2) and beta-tubulin (nda3). The ATP-dependent activity of the top2cs gene product is cs in vitro. The cloned top2cs gene sequence predicts an amino acid substitution. A cs top2-cs nda3 double mutant at 20 degrees C shows long, entangled chromosomes, which condense and separate upon the shift to permissive temperatures. If spindle formation is prevented at permissive temperatures, the chromosomes condense but do not separate. Thus topo II is required for final chromosome condensation; moreover, pulse-shift experiments show that topo II is required for chromatid disjuction. Experiments with ts top2-cs nda3 cells show that topo II is also required for chromosome separation in anaphase: inactivation of topo II and activation of beta-tubulin allow normal spindle formation but result in "streaked" chromosomes.

Adenosine Triphosphate↗

PubMed1987