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Biomedical subjects

M Nakai

Publications and source records attributed to M Nakai.

At least 163 records · Page 9Linked to original sources
PubMed1995

Mitochondrial receptor complex protein. The intermembrane space domain of yeast MAS17 is not essential for its targeting or function.

MAS17 (MAS22) is an essential component of the import receptor complex in the yeast mitochondrial outer membrane. MAS17 consists of three distinct domains: the N-terminal cytosolic domain, the internal membrane-spanning domain, and the C-terminal intermembrane space domain. In the present study, we examined the roles of the C-terminal domain of MAS17, which is rich in acidic amino acids, in protein import into mitochondria both in vivo and in vitro. Cells expressing MAS17 delta 120-152, a mutant MAS17 lacking the C-terminal acidic domain, could grow as fast as those expressing wild-type MAS17, while cells expressing MAS17 delta 97-152, a mutant MAS17 lacking both the intermembrane space and the membrane-spanning domains, stopped growing as soon as wild-type MAS17 was depleted. MAS17 delta 120-152 was correctly integrated into the mitochondrial outer membrane like wild-type MAS17. Mitochondria containing MAS17 delta 120-152 instead of wild-type MAS17 could import both authentic and artificial mitochondrial precursor proteins nearly as efficiently as wild-type mitochondria in vitro. These results suggest that the C-terminal intermembrane space domain of MAS17 is not essential for targeting or functions of MAS17.

Amino Acid Sequence↗
PubMed1995

Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase.

A recombinant adenovirus (Ad) expressing Cre recombinase derived from bacteriophage P1 was constructed. To assay the Cre activity in mammalian cells, another recombinant Ad bearing an on/off-switching reporter unit, where a LacZ-expression unit can be activated by the Cre-mediated excisional deletion of an interposed stuffer DNA, was also constructed. Co-infection experiments together with the Cre-expressing and the reporter recombinant Ads showed that the Cre-mediated switching of gene expression was detected in nearly 100% of cultured CV1, HeLa and Jurkat cells. These results suggest that the recombinant Ad efficiently expressed functional Cre and offers a basis for establishing a powerful on/off switching strategy of gene expression in cultured mammalian cells and presumably in transgenic animals. The method is also applicable to construction of recombinant Ad bearing a gene the expression of which is deleterious to propagation of recombinant Ad.

Adenoviridae↗
PubMed1995

Detection of vanadyl-nitrogen interaction in organs of the vanadyl-treated rat: electron spin echo envelope modulation study.

ESEEM spectroscopy was applied for the first time to organs of an animal, viz. the kidney and liver of the rat treated with vanadyl sulfate. The aim of this study is to investigate the in vivo coordination structure of vanadyl ions administrated, and to gain information concerning the insulin-mimic activity of vanadium. ESEEM measurements for kidney and liver performed at 77 K have established nitrogen coordination to a certain percentage of vanadyl ion in the organs. The rotios of nitrogen-coordinating vanadyl ion were estimated as 70-80% in the liver, and 50-55% in the kidney. Isotropic portions of the 14N HFC were estimated as magnitude of Aiso approximately 5.0 MHz for liver, and approximately 5.2 MHz for kidney, indicating that the coordinating nitrogen is an amino nitrogen. Coordination of the Lys epsilon-amine or the N-terminal alpha-amine of a protein or (a peptide) to vanadyl ion in vivo is suggested.

Animals↗
PubMed1995

Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes.

The nucleotide sequences of four genes encoding Trimeresurus gramineus (green habu snake, crotalinae) venom gland phospholipase A2 (PLA2; phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) isozymes were compared internally and externally with those of six genes encoding Trimeresurus flavoviridis (habu snake, crotalinae) venom gland PLA2 isozymes. The numbers of nucleotide substitutions per site (KN) for the noncoding regions including introns were one-third to one-eighth of the numbers of nucleotide substitutions per synonymous site (KS) for the protein-coding regions of exons, indicating that the noncoding regions are much more conserved than the protein-coding regions. The KN values for the introns were found to be nearly equivalent to those of introns of T. gramineus and T. flavoviridis TATA box-binding protein genes, which are assumed to be a general (nonvenomous) gene. Thus, it is evident that the introns of venom gland PLA2 isozyme genes have evolved at a similar rate to those of nonvenomous genes. The numbers of nucleotide substitutions per nonsynonymous site (KA) were close to or larger than the KS values for the protein-coding regions in venom gland PLA2 isozyme genes. All of the data combined reveal that Darwinian-type accelerated evolution has universally occurred only in the protein-coding regions of crotalinae snake venom PLA2 isozyme genes.

Amino Acid Sequence↗
PubMed1995

Isolation and characterization of the cDNA for pea chloroplast SecA. Evolutionary conservation of the bacterial-type SecA-dependent protein transport within chloroplasts.

We report here the isolation of the cDNA for pea chloroplast SecA. Pea SecA encodes a polypeptide of 1,011 amino acids and shows high sequence similarity with cyanobacterial SecA. Pea SecA was synthesized as a larger precursor and was imported into isolated chloroplasts in vitro. The purified pea SecA, which was expressed in Escherichia coli cells, stimulated the in vitro import of the 33 kDa protein of the oxygen-evolving complex into thylakoids. These results indicate that higher plant chloroplasts contain a bacterial-type SecA protein-dependent system for the intraorganellar protein transport into thylakoids.

Adenosine Triphosphatases↗
PubMed1995

Chemical stimulation of the nucleus tractus solitarii decreases spinal cord blood flow in anesthetized rats.

L-Glutamate was microinjected into the nucleus tractus solitarii (NTS) in anesthetized (chloralose and urethane), paralyzed and artificially ventilated rats, and spinal cord blood flow (SCBF) was determined using a combination of labeled microspheres. Unilateral chemical stimulation of the NTS (n = 13) significantly decreased SCBF in the cervical cord from 43 +/- 6 (mean +/- SEM) to 28 +/- 4 (P < 0.05), in the thoracic cord from 35 +/- 3 to 24 +/- 4 (P < 0.01), and in the lumbar cord from 49 +/- 3 to 40 +/- 3 ml min-1 (100 g)-1 (P < 0.05). The decrease in SCBF was not due to the decrease in arterial blood pressure (ABP) because the SCBF during the chemical stimulation of the NTS was significantly smaller (P < 0.05) than the SCBF during controlled hemorrhagic hypotension (n = 11). Chemical stimulation of the NTS did not affect the reactivity of the spinal cord vessels to hypercapnia (n = 5). Microinjection of the vehicle solution into the NTS had no effects on spinal cord circulation (n = 9). These results suggest that the cell bodies within the NTS may play a role in the control of spinal cord circulation.

Animals↗
PubMed1995

Identification of yeast MAS17 encoding the functional counterpart of the mitochondrial receptor complex protein MOM22 of Neurospora crassa.

MOM22 of Neurospora crassa was suggested to be required for the transfer of mitochondrial precursor proteins from the receptors to the protein translocation machinery. We isolated a yeast mutant the viability of which depended on the expression of the introduced N. crassa MOM22 gene. The mutant cells showed defects in protein import into mitochondria when the cells were depleted of MOM22. By screening for suppressor genes for the mutant, we could identify the yeast gene MAS17 encoding the functional counterpart of MOM22. The MAS17 gene was found to be essential for the viability of yeast cells.

Amino Acid Sequence↗
PubMed1995

The post-transcriptional regulator Rex of the human T-cell leukemia virus type I is present as nucleolar speckles in infected cells.

The rex-encoded protein (Rex) of human T-cell leukemia virus type I (HTLV-I) is responsible for the cytoplasmic accumulation of incompletely spliced mRNAs that encode the virion structural proteins. Rex is known to be located predominantly in the cell nucleoli in transient transfections or in the isolated nuclei of HTLV-I-infected cells. However, precise location of Rex under physiological conditions has not been determined unequivocally. Here we report that Rex is primarily located as intranucleolar speckles in HTLV-I-infected cells, except for a few nucleoplasmic speckles. This is in contrast to the more diffuse nucleolar distribution of the rev-encoded protein (Rev) of human immunodeficiency virus type 1 (HIV-1), the functional homologue to Rex, in HIV-1-infected cells. Accumulation of Rev is associated with disruption of nucleolar structure and cell death, whereas Rex does not have these effects. The difference in distribution of Rex and Rev within the nucleoli may reflect the difference of toxicity toward the host cells. Involvement of the nucleolus in processing of certain mRNAs is also discussed.

Animals↗
PubMed1995

Development of a fistula between an internal mammary artery graft and the pulmonary vasculature following coronary artery bypass grafting: report of a case.

We report herein the rare case of a 56-year-old man who gradually developed congestive cardiac failure 6 months after undergoing coronary artery bypass grafting and was found to have a fistula between the internal mammary artery and the pulmonary artery of the upper lobe diagnosed by selective left internal mammary arteriogram. A second sternotomy was performed and demonstrated dense adhesion between the fissure surrounding the internal mammary artery and the upper lobe, and the fistula was resected. We believe that the patient's increasing cardiac failure was almost certainly caused by coronary seal.

Arterio-Arterial Fistula↗
PubMed1995

A cell line (MCA-B1) derived from a canine oral acanthomatous epulis.

A canine oral acanthomatous epulis cell line designated MCA-B1 was established in culture. Immunohistochemically the cells reacted intensely with anti-keratin antibody and mildly with anti-vimentin antibody. An ultrastructural examination of the cells confirmed their epithelial nature. The modal chromosome number was 72 and the cloning efficiency in soft agar culture ranged from 3.4 to 16.1 per cent with a population doubling time of nine hours. MCA-B1 has since been passaged more than 70 times and maintained in culture for more than a year. Most of the characteristics of the original canine tumour cells were retained in the isolated cell line.

Animals↗
PubMed1995

Purification and primary structure of a myotoxic lysine-49 phospholipase A2 with low lipolytic activity from Trimeresurus gramineus venom.

Four acidic phospholipase A2 (PLA2) isozymes named PLA2-I, II, III and IV have previously been isolated from Trimeresurus gramineus (green habu snake) venom and sequenced [Oda et al. (1991) Toxicon 29, 157; Fukagawa et al. (1992) Toxicon 30, 1131; Fukagawa et al. (1993) Toxicon 31, 957]. They contain aspartate-49 which is known to bind Ca2+, essential for catalysis. In the present study, a basic PLA2 named PLA2-V containing lysine-49 was newly isolated from the same snake venom. Its isoelectric point was 9.4 and considerably higher than those (c. 4.5) of PLA2-I-IV. PLA2-V was 1.1% as active as PLA2-I toward egg-yolk emulsion but exhibited strong myotoxicity. The amino acid sequence of PLA2-V was determined by sequencing the S-carboxamidomethylated derivative and its peptide fragments produced by enzymatic (clostripain, chymotrypsin, Achromobacter protease I and Staphylococcus aureus V8 protease) cleavages. PLA2-V consists of 122 amino acid residues and is highly homologous (72-78%) to Lys-49 PLA2s so far isolated from Viperidae snake venoms but less homologous (52%) to PLA2-I. The presence of Asn-28, which is characteristic of Lys-49 PLA2s, was confirmed.

Amino Acid Sequence↗
PubMed1995

Comparable sensitivities for detection of HIV-1 reverse transcriptase (RT) and other polymerases by RT assays requiring no radioisotopic materials.

An improved non-radioisotopic (Non-RI) reverse transcriptase (RT) assay with a template-primer-immobilized microtiter plate is described, which has greater sensitivity than the former Non-RI RT assay previously described. Non-RI and commercially available non-radioactive (Non-RA) RT assays were compared for their ability to detect various polymerases. Two RTs from Rous-associated virus 2 (RAV-2) and avian myeloblastosis virus (AMV), one polymerase from Escherichia coli (Pol-I) and one recombinant RT of human immunodeficiency virus type 1 (HIV-1) were assessed. Two HIV-1 samples in a culture supernatant and pelleted virion suspended in Triton X-100 solution were measured. The Non-RI RT assay was one hundred times more sensitive by RAV-2 and Pol-I polymerases, and one thousand times more sensitive by the Non-RA assay than by the AMV RT. The Non-RI RT assay was 10, 16 and 64 times more sensitive than the Non-RA assay for measuring recombinant HIV-1 RT, pelleted virus and virus suspended in culture medium, respectively. To explain the discrepancy, it is shown that free biotin, such as in culture medium, disturbs the assay system of the Non-RA RT assay, but not the Non-RI assay. The present assay can be used to clarify the inhibitory mechanism of an anti-HIV-1 substance.

Animals↗
PubMed1995

Avidin fusion protein as a tool to generate a stable translocation intermediate spanning the mitochondrial membranes.

We have designed a fusion gene encoding a chimeric mitochondrial precursor protein (avidin fusion protein) that consists of the mitochondrial presequence followed by mouse dihydrofolate reductase, a spacer segment, and streptavidin. The avidin fusion protein synthesized in vitro formed a tetramer at the avidin moiety on incubation with biotin during or after the translation reaction. The avidin fusion protein purified from the Escherichia coli overexpresser cells also formed the tetramer on dilution from 6M urea into buffer containing biotin. In in vitro import experiments with isolated yeast mitochondria, the tetramer of the avidin fusion protein became stuck across both mitochondrial membranes, with its N-terminal dihydrofolate reductase moiety in the matrix and its C-terminal avidin moiety exposed on the mitochondrial surface. Accumulation of the translocation intermediate of the fusion protein inhibited the import of a mitochondrial precursor protein, and allowed us to estimate the number of mitochondrial import sites.

Animals↗
PubMed1995

A micro-suspension-test for evaluation of disinfectants against human immunodeficiency virus.

We devised a micro-suspension-test to evaluate disinfectants against human immunodeficiency virus type-1 (HIV-1) and confirmed its reliability. Suspensions of persistently HIV-1-infected Molt-4 cells were used as targets of disinfectants and residual infectivity was measured by an infectivity assay: after cocultivation with uninfected Molt-4 cells reverse transcriptase activity (RTA) in the supernatant and giant cell formation (GCF) were monitored. Our new infectivity assay consists of a short-term assay, that is RTA and GCF monitoring on the second day of co-culture, and a long-term assay, that is RTA monitoring up to the 28th day of co-culture. The sensitivity of the short-term assay was 1 x 10(3) infected cells and that of the long-term assay 1 x 10(1) infected cells. All the chemical disinfectants examined in this study showed dose- and time-dependent inactivation of HIV-1. By 5-minute contact with ethanol, glutaraldehyde, formalin, sodium hypochlorite and povidone-iodine, HIV-1 was effectively inactivated at concentrations of 20, 0.01, 5, 0.05 and 0.1%, respectively. Since the micro-suspension-test is easy and sensitive, we recommend it as a method for evaluating disinfectants against HIV-1.

Cells, Cultured↗
PubMed1995

Effects of aging and chronic hypertension on cerebral blood flow and cerebrovascular CO2 reactivity in the rat.

We measured regional cerebral blood flow (rCBF) in young and old Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats by a minimally invasive microsphere technique. Blood flows to the cerebrum, diencephalon, mesencephalon, cerebellum and pons-medulla during normocapnia were determined. To test the ability of the vessels to dilate, rCBF was also measured during hypercapnia. Reactivity to CO2 was calculated as delta rCBF/delta paCO2. In the old SHR, blood flow to the pons-medulla (88 +/- 8 ml/min/100 g, mean +/- SEM) was markedly lower than that in the young SHR (107 +/- 4 ml/min/100 g, p < 0.05), whereas the difference of those values in the old and young WKY rats was slight (0.05 < p < 0.1). There were no differences in the values of blood flow to the cerebrum, diencephalon, mesencephalon or cerebellum between the young and old rats in both species. Cerebrovascular CO2 reactivity was markedly impaired in the old SHR (p < 0.05) compared to that in the young SHR, but the difference in reactivity in the WKY rats was not significant. The results indicate that blood flow to the pons-medulla is reduced in association with age, particularly in the hypertensive animals, and that the ability of the cerebral vessels to dilate is impaired homogeneously by the combination of chronic hypertension and aging.

Aging↗
PubMed1995

Mechanism on insulin-like action of vanadyl sulfate: studies on interaction between rat adipocytes and vanadium compounds.

When rats with streptozotocin (STZ)-induced diabetes were given a daily intraperitoneal (i.p.) injection of VOSO4 (+4 oxidation state of vanadium), their serum glucose dropped from hyperglycemic level to normal level within 2d and serum free fatty acid (FFA) level also dropped to normal level. Vanadium was incorporated in most organs as well as in the adipose tissues, as detected by neutron activation analysis (NAA). The mechanism for the insulin-like action vanadium in terms of FFA release from isolated rat adipocytes was investigated: (1) Vanadyl (IV) and vanadic (III) ions normalize the FFA release in the adipocytes treated with epinephrine; (2) vanadate (V) ion treated with ascorbic acid, cysteine or glucose is effective in normalizing the FFA release but vanadate ion alone has no effect on FFA release; (3) vanadyl ion is incorporated into the adipocytes, while vanadate ion is not, as indicated by ESR spectroscopy; and (4) vanadyl ion can act on the glucose transporter, as indicated by experiments using cytochalasin B which is an inhibitor of this transporter. From these results, the normalization of both serum glucose and FFA levels by vanadyl ion was concluded to be due to the incorporation of vanadyl ion into the adipocytes, in which the metal ion acts on the glucose transporter and induces both the promotion of glucose uptake and the decrease of FFA release form peripheral adipocytes. The vanadyl state was suggested to be a possible pharmacologically active form of vanadium allowing the insulin-like action.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipocytes↗