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Biomedical subjects

M Watanabe

Publications and source records attributed to M Watanabe.

At least 19 recordsLinked to original sources

Diversion of the sign of phototaxis in a Chlamydomonas reinhardtii mutant incorporated with retinal and its analogs.

The blind mutant FN68 of the unicellular flagellate green alga Chlamydomonas reinhardtii is negatively phototactic in the presence of the native chromophore all-trans retinal. In contrast, analog chromophores such as a ring-acyclic retinal and those in which trans/cis isomerization about the C11 = C12 double bond was blocked induced predominantly positive phototaxis in the same strain under the same experimental conditions. These observations can be interpreted by assuming that the negative and the positive phototaxis is mediated distinctively by two rhodopsin species which differ in their affinities with the exogenous chromophores. However, a more reasonable explanation, which requires fewer assumptions, is that the sign of phototaxis depends on a delay in intracellular photosignal transduction. This novel view was deduced directly from the widely accepted hypothesis [1980, Microbiol. Rev. 44, 572-630] on phototaxis mechanisms.

Animals

Site-directed mutation makes rabbit calcyclin dimer.

Unlike human, rat and mouse calcyclin, purified rabbit calcyclin did not form a dimer on Tricine SDS-PAGE under non-reduced conditions. Based on the internal peptide sequence of rabbit calcylin, we isolated and sequenced a cDNA clone encoding calcyclin. The sequence of this clone (pCalC) is 629 bp long and codes 90 amino acid residues of a protein with a molecular mass of 10,153 Da. By Northern blot analysis, a major band of 0.9 kbp and a minor band of 2.6 kbp were detected in the lung. The recombinant calcyclin mutated serine at the third position to cysteine was expressed in E. coli and made dimer formation under non-reduced conditions on SDS-PAGE. Whether or not this type of mutation which prevents dimer formation of calcyclin plays a physiological role in the rabbit lung is the subject of an ongoing study.

Amino Acid Sequence

Synergistic stimulation of nitric oxide hemoglobin production in rats by recombinant interleukin 1 and tumor necrosis factor.

Nitric oxide (NO) is formed from arginine in Escherichia coli lipopolysaccharide (LPS) treated rat; however, none of specific cytokine inducing NO generation is yet determined. We studied the effect of interleukin 1 (IL-1) and tumor necrosis factor (TNF) on NO production in rats by detecting NO-hemoglobin in their blood, using electron spin resonance. Either IL-1 or TNF alone stimulated NO-hemoglobin formation. Combined administration of IL-1 and TNF markedly enhanced NO-hemoglobin generation, demonstrating the synergistic character of both stimuli on NO production. Further, LPS and TNF in combination were more potent stimulator of NO-hemoglobin production in rats than each alone.

Animals

Dopaminergic stimulation up-regulates the in vivo expression of brain-derived neurotrophic factor (BDNF) in the striatum.

We investigated the effect of dopamine on the in vivo expression of brain-derived neurotrophic factor (BDNF) in the striatum of mouse. BDNF mRNA expression in the striatum, which was quantified with the reverse transcriptase polymerase chain reaction, was up-regulated from 2 h after oral administration of levodopa, a precursor of dopamine. The increase was sustained for 16 h. Co-administration of haloperidol partially inhibited dopamine-induced BDNF enhancement. These data suggest that dopaminergic stimulation directly promotes the expression of BDNF in the striatum in vivo.

Amino Acid Sequence

Cloning and expression of the epsilon 4 subunit of the NMDA receptor channel.

The primary structure of a novel subunit of the mouse NMDA (N-methyl-D-aspartate) receptor channel, designated epsilon 4, has been revealed by cloning and sequencing the cDNA. The epsilon 4 subunit shares high amino acid sequence identity with the epsilon 1, epsilon 2 and epsilon 3 subunits of the mouse NMDA receptor channel, thus constituting the epsilon subfamily of the glutamate receptor channel. Expression from cloned cDNAs of the epsilon 4 subunit together with the zeta 1 subunit in Xenopus oocytes yields functional NMDA receptor channels. The epsilon 4/zeta 1 heteromeric channel exhibits high apparent affinities for agonists and low sensitivities to competitive antagonists. The epsilon 4 subunit is thus distinct in functional properties from the epsilon 1, epsilon 2 and epsilon 3 subunits, and contributes further diversity of the NMDA receptor channel.

Amino Acid Sequence

A new glycolipid from Mycobacterium avium--Mycobacterium intracellulare complex.

From a nonpolar lipid fraction of Mycobacterium avium--Mycobacterium intracellulare complex cell mass, a new glycolipid was obtained, which was shown to be 5-mycoloyl-beta-arabinofuranosyl-(1-->2)-5-mycoloyl-alpha-ar abinofuranosyl- (1-->1')-glycerol. When examined by TLC, all the 12 strains of this species tested, including clinical isolates, were found to contain this glycolipid. But the glycolipid was not detected in Mycobacterium bovis BCG or Mycobacterium tubrculosis H37Rv.

Chromatography, Thin Layer

[Pregnancy in Cushing's syndrome].

Pregnancy in cases of Cushing's syndrome is rare. A pregnant patient with Cushing's syndrome due to an adrenal adenoma who was diagnosed in the third trimester is described. She underwent conservative treatment for Cushing's syndrome and delivered a normal infant by Caesarean section. Currently, 121 pregnancies in 97 patients have been reported, but a principle for the treatment of the mother and fetus has not yet been established. We reviewed pregnancy in Cushing's syndrome based on the world literature and evaluated the choice of treatment to take. In the first trimester of pregnancy, therapeutic abortion or surgical treatment, such as adrenalectomy or resection of the pituitary tumor in Cushing's syndrome, is recommended for patients with severe hypercorticism (plasma cortisol > or = 30 micrograms/dl, urinary 17-OHCS > or = 15 mg/day, urinary free cortisol > or = 1000 micrograms/day), while conservative treatment is recommended for patients with mild hypercorticism (plasma cortisol < 30 micrograms/dl, urinary 17-OHCS < 15 mg/day, urinary free cortisol < 1000 micrograms/day). In the second trimester of pregnancy, surgical treatment is recommended for patients with severe hypercorticism, while conservative treatment is recommended for patients with mild hypercorticism. In the third trimester of pregnancy, Caesarean section is recommended for most cases. Drug treatments such as with metyrapone should be limited to patients showing severe hypercorticism or a maternal high risk who have contraindications to surgical treatment.

Adenoma

Establishment of the human BSMZ breast cancer cell line, which overexpresses the erbB-2 and c-myc genes.

A new cell line, designated BSMZ, was established from a malignant pleural effusion from a woman with breast cancer. This line has a doubling time of 27 h and has now been cultured for over 120 passages. The large, rounded BSMZ cells grow as both a monolayer and as aggregations in suspension. Intracytoplasmic lumen, a finding consistent with results from cells derived from mammary tissue, was detected on ultrastructural analysis. Injection of BSMZ cells into nude mice resulted in the growth of solid tumors 4 weeks after inoculation. The solid tumor was identical to the original BSMZ cells in microscopic and electron microscopic studies. These cells possess an average of 80 chromosomes. Expression of erbB-2 and c-myc genes was increased by 10-fold, while there was no detectable overexpression of the N-ras and c-myb genes. Southern analysis has revealed amplification of the erbB-2 and c-myc loci. The BSMZ cell line may therefore provide a useful model for the study of human breast cancer and overexpression of the erbB-2 gene.

Adult

Characterization of human brain cDNA encoding the general isoform of beta-spectrin.

The complete primary structure of the general form of human beta-spectrin (beta G) has been deduced from cDNAs isolated from human brain. beta G-Spectrin is encoded by a gene located on human chromosome 2. beta G-Spectrin and erythrocyte beta-spectrin (beta R) share identical domain organization, with sequence identity of 60% and sequence similarity of 77%. beta-Spectrins have closely related N-terminal domains implicated in binding to actin, and 17 copies of a 106-residue repeat motif with consensus residues that are highly conserved between beta-spectrins as well as alpha-spectrins. C-terminal domains of beta G and the 270-kDa beta R-spectrins are candidate regions to associate with alpha-spectrin, and exhibit 75% similarity. beta G- and beta R-spectrins exhibit different patterns of expression in tissues and follow different developmental programs in those tissues where they are co-expressed. beta G-Spectrin is present in all tissues examined except for erythrocytes, while beta R-spectrin could be detected only in erythrocytes, brain, and heart. beta G- and beta R-Spectrins are both expressed in brain, but beta R appeared later in post-natal development and was highly enriched in cerebellum in contrast to the broad regional distribution of beta G-spectrin. beta-Spectrins are likely to perform related but distinct functions, with beta G in a general, constitutive role and beta R-spectrin involved in more specialized activities of differentiated cells.

Amino Acid Sequence

Localization of mRNA for beta-adrenergic receptor kinase in the brain of adult rats.

A cDNA encoding rat beta-adrenergic receptor kinase (beta-ARK) was cloned and sequenced. By in situ hybridization histochemistry of adult brain, beta-ARK mRNA was expressed intensely in the cerebellar granule cell layer and moderately in the hippocampal pyramidal cells and dentate granule cells. The neocortex and piriform cortex expressed it moderately to weakly, whereas the thalamus and hypothalamus expressed it weakly to faintly. No significant expression of the mRNA was detected in the caudate-putamen. Weak expression of beta-ARK mRNA was detected in several nuclei of the brainstem and in the spinal gray matter.

Animals

Neurocalcin, a novel calcium binding protein with three EF-hand domains, expressed in retinal amacrine cells and ganglion cells.

Neurocalcin (molecular weight 23,000 and 24,000) is a newly identified Ca2+ binding protein with three EF-hand domains and has a strong amino acid sequence homology with visinin and recoverin (Terasawa, M., Nakano, A., Kobayashi, R., and Hidaka, H. J. Biol. Chem. In press). We produced antibody against neurocalcin. Immunoblotting showed the presence of neurocalcin in bovine retina as well as brain, suggesting that neurocalcin was a neuron specific Ca2+ binding protein. Immunohistochemistry revealed the expression of neurocalcin in retinal amacrine cells and ganglion cells but not in the photoreceptor layer. This distribution of neurocalcin was quite different from that of visinin and recoverin. Our results suggest that neurocalcin may play an important role in a Ca2+ signal pathway of the nervous system.

Animals

Augmented expression of HSP72 protein in normal human fibroblasts irradiated with ultraviolet light.

Normal human fibroblasts synthesized heat shock protein (HSP) 72 constitutively and its expression was augmented 6 hours after UV irradiation. Maximum induction of HSP72 was obtained at 12 hours and HSP72 showed a punctuated distribution in nucleus. While unscheduled DNA synthesis was almost completed 12 hours after UV irradiation, the S phase fraction decreased immediately and recovered after 6 hours. Thus, HSP72 augmentation was occurred coincidentally with the recovery of S phase, and suggested that HSP72 had some function during the recovery of DNA replication inhibited after UV irradiation.

Blotting, Western

cDNAs and deduced amino acid sequences of subunits in the binding component of mouse bactericidal factor, Ra-reactive factor: similarity to mannose-binding proteins.

The complement-dependent bactericidal factor, Ra-reactive factor, binds specifically to Ra polysaccharide, which is common to some strains of Gram-negative enterobacteria, and its is a complex of proteins composed of a polysaccharide-binding component and a component that is presumably responsible for the complement activation. The former component consists of two different 28-kDa polypeptides, P28a and P28b. We determined the partial amino acid sequences of P28a and P28b, and the results indicated that these polypeptides were similar to two species of mannose-binding protein, MBP-C and MBP-A (alternative names, liver and serum mannan-binding proteins, respectively), which have been isolated from rat liver and/or serum [Drickamer, K., Dordal, M. S., & Reynolds, L. (1986) J. Biol. Chem. 261, 6878-6887; Oka, S., Itoh, N., Kawasaki, T., & Yamashina, I. (1987) J. Biochem. 101, 135-144]. Thus, we cloned the respective cDNAs, using as probes synthetic oligonucleotides for which the sequences had been deduced from the amino acid sequences of P28a and P28b and of rat MBP cDNAs. The primary structures of P28a and P28b deduced from the cloned cDNAs are homologous to one another. They have three domains, a short NH2-terminal domain, a collagen-like domain, and a domain homologous to regions of some carbohydrate-binding proteins, as has been reported for rat MBPs. Southern and Northern blotting analyses using these cDNAs indicated that the P28a and P28b polypeptides are the products of two unique mouse genes which are expressed in hepatic cells.

Amino Acid Sequence

Specific binding of CAP-50 to calcyclin.

CAP-50, a calcyclin-associated protein with an apparent molecular mass of 50 kDa, was purified and proved to be a novel annexin [Tokumitsu, H. et al. (1992) J. Biol. Chem. 267, 8919-8924]. We examined the binding of CAP-50 to other Ca(2+)-binding proteins which have two of four EF-hand structures, by a co-precipitation assay with phospholipid (phosphatidylserine). Among nine Ca(2+)-binding proteins (calcyclin, S-100 proteins, p11, calgizzarin, calvasculin, calmodulin and troponin C) examined, only calcyclin interacted with CAP-50. These results clearly show that the interaction of CAP-50 to calcyclin is specific, i.e. other Ca(2+)-binding proteins with the EF-hand structure could not substitute for calcyclin, thereby suggesting the possible role in specific regulation of the function of CAP-50 by Ca2+/calcyclin.

Animals

Influence of membrane fluidity on the assembly of Staphylococcus aureus alpha-toxin, a channel-forming protein, in liposome membrane.

By use of multilamellar phosphatidylcholine (PC) liposomes of different acyl composition and cholesterol content as model membranes, we studied whether or not membrane fluidity affects the assembly process of Staphylococcus aureus alpha-toxin. Under conditions using fluid and solid membranes, we assayed accessibility (or hemolytic activity) of liposome-bound alpha-toxin to rabbit erythrocytes added, hexamerization of membrane-bound toxin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nondenaturating conditions, and susceptibility of liposome-bound toxin to trypsin digestion. Our data indicated 1) that alpha-toxin bound to PC membrane as a hemolytically active monomer (or reversibly bound state); 2) that when the membrane was fluidized either by phase transition of PC or by inclusion of cholesterol over 20 mol %, the hemolytically active monomer of the toxin was irreversibly converted to nonhemolytic monomer (and/or unstable oligomer) in a first-order kinetics with a t1/2 of about 1 min, and thereafter hexamerization of the toxin gradually proceeded in the following 60-90 min; 3) that alpha-toxin might have different topology and/or conformation in PC membrane, depending on the presence or absence of cholesterol in the PC membrane; and 4) that coexistence of unsaturated acyl chain-carrying PC and cholesterol was a prerequisite for efficient hexamerization of alpha-toxin in membrane. Thus, increase in membrane fluidity promoted the assembly process of S. aureus alpha-toxin.

Animals

Generation of a small cell lung cancer variant resistant to lymphokine-activated killer (LAK) cells: association with resistance to a LAK cell-derived, cytostatic factor.

Cells of OS2-RA, a human small cell lung cancer line sensitive to lymphokine-activated killer (LAK) cells, were repeatedly cocultured with human LAK cells. Fourteen cycles of the coculture produced a variant, termed OS2-RA-R, capable of growing successfully in the presence of LAK cells. OS2-RA-R showed a moderate resistance to lysis by LAK cells in 4-h 51Cr release assays. OS2-RA-R acted positively as a cold target for lysis of OS2-RA by LAK cells, suggesting no loss of the binding site for LAK cells on the cell surface of the variant. On the other hand, LAK cells were shown to produce a factor capable of suppressing the proliferation of OS2-RA and certain other cell lines but not lymphocytes. Interestingly, OS2-RA-R exhibited a substantial resistance to the cytostatic activity of LAK cell supernatants. The cytostatic factor, eluted at the 57-kDa fraction in gel filtration, showed no activity of interleukin 1, gamma-interferon, transforming growth factor beta, or tumor necrosis factor. These results suggest that LAK cells exhibit antitumor activity through not only rapid cytolysis but also slow-acting cytokine production, and the successful growth of OS2-RA-R in a coculture with LAK cells is the result of acquiring resistance to these two different LAK cell phenomena.

Animals

Chimpanzees immunized with recombinant soluble CD4 develop anti-self CD4 antibody responses with anti-human immunodeficiency virus activity.

In view of the efficiency with which human immunodeficiency virus replication can be blocked in vitro with anti-CD4 antibodies, the elicitation of an anti-CD4 antibody response through active immunization might represent a useful therapeutic strategy for AIDS. Here we demonstrate that immunization of chimpanzees with recombinant soluble human CD4 elicited an anti-CD4 antibody response. The elicited antibody bound self CD4 on digitonin-treated but not freshly isolated lymphocytes. Nevertheless, this antibody blocked human immunodeficiency virus replication in chimpanzee and human lymphocytes. These observations suggest that immunization with recombinant soluble CD4 from human immunodeficiency virus-infected humans may be feasible and therapeutically beneficial.

Animals