Biomedical subjects

M Homma

Publications and source records attributed to M Homma.

At least 55 records · Page 3Linked to original sources

PubMed1999

Conversion of a bacterial warm sensor to a cold sensor by methylation of a single residue in the presence of an attractant.

The aspartate chemoreceptor (Tar) of Escherichia coli also serves as a thermosensor, and it is very amenable to genetic and biochemical analysis of the thermosensing mechanism. Its thermosensing properties are controlled by reversible methylation of the cytoplasmic signalling/adaptation domain of the protein. The unmethylated and the fully methylated (aspartate-bound) receptors sense, as attractant stimuli, increases (warm sensor) and decreases (cold sensor) in temperature respectively. To learn more about the mechanism of thermosensing, we replaced the four methyl-accepting glutamyl residues with non-methylatable aspartyl residues in all possible combinations. In a strain defective in both methyltransferase (CheR) and methylesterase (CheB) activities, all of the mutant Tar proteins functioned as warm sensors. To create a situation in which all of the remaining glutamyl residues were methylated, we expressed the mutant proteins in a CheB-defective, CheR-overproducing strain. The fully glutamyl-methylated proteins were designed to mimic the full range of methylation states possible for wild-type Tar. Almost all of the methylated mutant receptors, including those with single glutamyl residues, were cold sensors in the presence of aspartate. Thus, binding of aspartate to Tar and methylation of its single glutamyl residue can invert its temperature-dependent signalling properties.

Aspartic Acid↗

PubMed1999

Suppression by the DNA fragment of the motX promoter region on long flagellar mutants of Vibrio alginolyticus.

The axial length of the polar flagellum (Pof) of Vibrio alginolyticus is about 5 microm. We previously isolated mutants that make abnormally long flagella. The swarm sizes of these mutants in a soft agar plate are smaller than that of a wild-type strain. We cloned a DNA fragment into the pMF209 plasmid that restored the swarming ability of the long-Pof strain V10578. The swimming speed and flagellar length of these transformants were almost equal to the wild-type values. The amounts of PF47 flagellin and PF60 sheath-associated protein, which increased in the long-Pof mutants, were retrieved to almost the wild-type level in the transformants. The plasmid pMF209 contained only a 143 bp chromosomal fragment whose sequence is about 80% similar to that of the motX promoter region of V parahaemolyticus. We speculate that this sequence interacts with a regulatory protein that controls Pof expression. The mutation causing the long-Pof phenotype may be in the gene encoding this protein or in the control region of a structural gene that is regulated by this protein.

Bacterial Proteins↗

PubMed1999

High-performance liquid chromatographic determination of ribavirin in whole blood to assess disposition in erythrocytes.

Ribavirin is an antiviral agent used in the treatment of chronic hepatitis C virus infection. One of the limitations associated with the use of ribavirin is a reversible anemia caused by its accumulation in erythrocytes. Therefore, it is of interest to determine ribavirin levels in erythrocytes, as well as in plasma, as these measurements may be predictive of hematotoxicity. In the present study, we describe a high-performance liquid chromatographic (HPLC) assay for ribavirin in whole blood to estimate concentrations of free ribavirin and phosphorylated anabolites in erythrocytes. Since ribavirin exists primarily as phosphorylated anabolites (mono-, di-, and triphosphates) in erythrocytes, whole-blood extracts were initially dephosphorylated with acid phosphatase. The enzyme-treated samples were subjected to phenyl boronic acid column extraction for cleanup. The purified fraction was analyzed by reversed-phase HPLC, which was optimized for determination of ribavirin levels in whole blood. The recoveries of ribavirin from whole blood ranged from 63.1 to 90.7% at concentrations ranging from 1.67 to 40.0 microM. Intra- and interassay variations estimated at these concentrations were 3.2 to 10.4 and 4.7 to 11.7%, respectively. This method was used to quantitate ribavirin in samples both treated and untreated with acid phosphatase to estimate the extent of intracellular phosphorylation in erythrocytes. The method was also used to evaluate the effects of dipyridamole, a nucleoside transporter inhibitor, on ribavirin disposition in erythrocytes in in vitro experiments.

Acid Phosphatase↗

PubMed1999

Functional interaction between PomA and PomB, the Na(+)-driven flagellar motor components of Vibrio alginolyticus.

Four proteins, PomA, PomB, MotX, and MotY, appear to be involved in force generation of the sodium-driven polar flagella of Vibrio alginolyticus. Among these, PomA and PomB seem to be associated and to form a sodium channel. By using antipeptide antibodies against PomA or PomB, we carried out immunoprecipitation to verify whether these proteins form a complex and examined the in vivo stabilities of PomA and PomB. As a result, we could demonstrate that PomA and PomB functionally interact with each other.

Antibodies, Bacterial↗

PubMed1999

Hybrid motor with H(+)- and Na(+)-driven components can rotate Vibrio polar flagella by using sodium ions.

The bacterial flagellar motor is a molecular machine that converts ion flux across the membrane into flagellar rotation. The coupling ion is either a proton or a sodium ion. The polar flagellar motor of the marine bacterium Vibrio alginolyticus is driven by sodium ions, and the four protein components, PomA, PomB, MotX, and MotY, are essential for motor function. Among them, PomA and PomB are similar to MotA and MotB of the proton-driven motors, respectively. PomA shows greatest similarity to MotA of the photosynthetic bacterium Rhodobacter sphaeroides. MotA is composed of 253 amino acids, the same length as PomA, and 40% of its residues are identical to those of PomA. R. sphaeroides MotB has high similarity only to the transmembrane region of PomB. To examine whether the R. sphaeroides motor genes can function in place of the pomA and pomB genes of V. alginolyticus, we constructed plasmids including both motA and motB or motA alone and transformed them into missense and null pomA-paralyzed mutants of V. alginolyticus. The transformants from both strains showed restored motility, although the swimming speeds were low. On the other hand, pomB mutants were not restored to motility by any plasmid containing motA and/or motB. Next, we tested which ions (proton or sodium) coupled to the hybrid motor function. The motor did not work in sodium-free buffer and was inhibited by phenamil and amiloride, sodium motor-specific inhibitors, but not by a protonophore. Thus, we conclude that the proton motor component, MotA, of R. sphaeroides can generate torque by coupling with the sodium ion flux in place of PomA of V. alginolyticus.

Amiloride↗

PubMed1999

The polar flagellar motor of Vibrio cholerae is driven by an Na+ motive force.

Vibrio cholerae is a highly motile bacterium which possesses a single polar flagellum as a locomotion organelle. Motility is thought to be an important factor for the virulence of V. cholerae. The genome sequencing project of this organism is in progress, and the genes that are highly homologous to the essential genes of the Na+-driven polar flagellar motor of Vibrio alginolyticus were found in the genome database of V. cholerae. The energy source of its flagellar motor was investigated. We examined the Na+ dependence and the sensitivity to the Na+ motor-specific inhibitor of the motility of the V. cholerae strains and present the evidence that the polar flagellar motor of V. cholerae is driven by an Na+ motive force.

Flagella↗

PubMed1999

Glycyrrhetinic acid-induced apoptosis in thymocytes: impact of 11beta-hydroxysteroid dehydrogenase inhibition.

It has been proposed that glycyrrhetinic acid (GA) enhances endogenous glucocorticoid (GC) action by suppressing the metabolism of the steroid. We show here that marked involution of the thymus occurred within 24 h of a single intraperitoneal administration of GA in mice. Thymocytes from mice treated with GA exhibited DNA cleavage and mitochondrial transmembrane potential disruption, as demonstrated with agarose gel electrophoresis and flow cytometric analysis. Immunocytochemical staining revealed that CD4(+)CD8(+) double positive cells markedly decreased after GA treatment. In contrast to GA in vivo, GA in vitro did not induce apoptosis of cultured thymocytes. These findings suggest that the apoptosis-inducing effect of GA on thymocytes is due to its indirect action. Because GA has been known to inhibit 11beta-hydroxysteroid dehydrogenase (11beta-HSD), we measured the enzyme activity in major organs and endogenous corticosterone concentration after GA treatment. The results showed a significant decrease of 11beta-HSD activity (P < 0.0001) and an increase in serum corticosterone concentration (P < 0.005). We concluded that the inhibition of hepatic 11beta-HSD activity by GA has a serious effect on GC metabolism, which results in a significant elevation of systemic GC levels. Apoptosis of thymocytes occurred as a consequence of the elevation in the level of endogenous corticosterone.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1999

Monoamine oxidase inhibitory naphthoquinone and/or naphthalene dimers from Lemuni Hitam (Diospyros sp.), a Malaysian herbal medicine.

From the extract of a Malaysian herbal medicine, Lemuni Hitam (Diospyros sp.), which exhibited monoamine oxidase (MAO) inhibition, three new naphthoquinone and/or naphthalene dimers (lemuninols A-C, 1-3) were isolated together with 4,6-dihydroxy-5-methoxy-2-methyl-naphthalene (8) and six known monomers (4-7, 9 and 10). The structures were determined by spectroscopic methods including 2D-NMR techniques. Among them, lemuninol A showed 45% inhibition of MAO (mouse liver) at 5.0 x 10(-6) g/ml, and lemuninols B and C and a naphthoquinone (9) indicated weak activity. Some related quinones were also tested for their MAO inhibitory activities.

Animals↗

PubMed1999

Hypoglycemic coma masquerading thyrotoxic storm.

A 59-year-old woman was hospitalized in hypoglycemic coma. Although hypoglycemia was promptly reversed, she was in a somnolent, restless state with tachycardia, tremor, profuse sweating, and high body temperature. Thyrotoxic storm was highly suspected and vigorous antithyroid regimens gradually brought her up to normal mental and cardiovascular states in several days. However, profound generalized myopathy necessitated the maintenance with a respirator. One month later, an episode of angina pectoris was followed by generalized convulsion, coma, and death in a few days. Neuroimaging study disclosed posterior leukoencephalopathy syndrome. This case is instructive in that hypoglycemic coma may masquerade the major symptomatology of thyrotoxic storm, and that profound myopathy and angiopathic or angiospastic processes of the brain and the heart may interfere with the outcome.

Brain↗

PubMed1999

[The role of virulence factor of elastolytic proteinase from Aspergillus fumigatus].

The role of virulence factor of elastolytic proteinase produced by Aspergillus fumigatus was investigated in mice immunocompromised with cyclophosphamide (CY) to block neutrophil and lymphocyte functions or with carrageenan (CA) to block the phagocytic activity of macrophages. These mice were infected through inhalation with the spores of elastolytic proteinase producing or nonproducing strains of A. fumigatus, and then were observed daily for 15 days or until death. Compared to CY-immunocompromised mice treated with the spores of proteinase nonproducing strains, CY-immunocompromised mice treated with the spores of proteinase producing strains had a significantly shorter survival rate. Furthermore, when treated with the spores of proteinase producing strains, CA-immunocompromised mice survived significantly longer than did CY-immunocompromised mice. Assessment of the effect of elastolytic proteinase on rat lung showed that this enzyme induced hemorrhage of the lung, alveolar septal edema and alveolar hypertrophy due to blood neutrophil infiltration. Neither fusion nor denaturation of the elastic fiber was observed, however. These studies have suggested that when infected with A. fumigatus, a severe lesion in bronchioli or alveoli is induced by the proteinase producing strains, and that these strains are more virulent than the proteinase nonproducing strains.

Animals↗

PubMed1999

High-affinity efflux transport system for glutathione conjugates on the luminal membrane of a mouse brain capillary endothelial cell line (MBEC4).

Cumulative evidence suggests that several organic anions are excreted from the brain to the blood across the blood-brain barrier. In the present study, we carried out a kinetic investigation of the transport activity in MBEC4, an immortalized cell line established from BALB/c mouse cerebral microvessel endothelial cells. The presence of an efflux system in intact cells was examined by using monochlorobimane (MCB), which is conjugated with glutathione intracellularly to produce glutathione bimane (GS-B). The efflux of GS-B was inhibited by ATP depletion and also by 1-chloro-2, 4-dinitrobenzne, a precursor of 2,4-dinitrophenyl-S-glutathione, in a concentration-dependent manner. Using this MBEC4 monolayer, we investigated the direction of this transport activity. Although the efflux of GS-B was observed on both luminal and abluminal sides of MBEC4 monolayer, the profile differed for the two sides with respect to the concentration dependence of MCB; the analysis suggested the presence of high-affinity transport system on the luminal side. To investigate the mechanism for the transport, we examined the ATP-dependent uptake of GS-B into the membrane vesicles prepared from MBEC4. ATP-dependent uptake systems with high (Km = 35 nM) and low (Km = 14 microM) affinities were identified. These results suggested that this high-affinity transport system of glutathione conjugates is expressed on the luminal side of the blood-brain barrier and is involved in the detoxification of xenobiotics.

Animals↗

PubMed1998

Intersubunit interaction between transmembrane helices of the bacterial aspartate chemoreceptor homodimer.

The transmembrane domain that connects the extracellular and intracellular domains of cell-surface receptors must play a critical role in signal transduction. Here, we report studies of the interaction between the transmembrane helices (TM1 and TM2) of the Escherichia coli aspartate chemoreceptor (Tar). Tar exists as a homodimer regardless of its state of ligand occupancy. A particular residue substitution in TM1 (A19K) abolishes the signaling ability of Tar. This signaling defect can be suppressed by single residue substitutions in TM2 (W192R, A198E, V201E, and V202L). We have found that these suppressors can be divided into two groups. A198E and V201E (class 1) almost completely suppress the defects caused by A19K, and this suppression occurs between two subunits of the Tar dimer. In contrast, W192R and V202L (class 2) fail to suppress some signaling defects, and their suppression does not occur between subunits. Because disulfide-crosslinking studies predict that residues 198 and 201 point toward residue 19 of the partner subunit, we propose that the class 1 suppressors form an intersubunit salt bridge with Lys-19. Indeed, A19K was suppressed by several additional aspartate or glutamate substitutions on the same face of TM2 occupied by residues 198 and 201. None of these intersubunit salt bridges perturb signaling function, suggesting that the mechanism of transmembrane signal propagation does not involve large displacements (such as extensive rotation) of the TM1 and TM2 helices relative to each other.

Amino Acid Substitution↗

PubMed1998

Individual variations in lymphocyte-responses to glucocorticoids in patients with bronchial asthma: comparison of potencies for five glucocorticoids.

Glucocorticoids (GCs) are known to be effective for bronchial asthma, however, a considerable number of asthma patients fail to respond to GC despite the onset of serious side effects. Here we examined individual sensitivities to five clinically-used GCs in 40 asthma patients and 100 healthy subjects. Peripheral-blood mononuclear cells (PBMCs) were isolated from these subjects, and their in vitro sensitivities to hydrocortisone, prednisolone, methylprednisolone, dexamethasone, and betamethasone were determined with a mitogen-assay procedure. The number of PBMCs positive to IL-2 receptors (IL-2Rs) as well as soluble IL-2R (sIL-2R) levels in serum were concomitantly measured in asthma patients, and relationships between these cytokine indices and PBMC-sensitivities to GCs were also examined. Large individual variations in GC IC50s have been observed in PBMCs from asthma subjects, especially in prednisolone IC50s (ranged from 1 to 10,000 ng/ml). When compared with healthy subjects, asthma patients tend to show PBMC-resistance to prednisolone (p < 0.05). Moreover, potencies of methylprednisolone on PBMC-blastogenesis were > 10 times higher than those of prednisolone in both healthy subjects and asthmatics (p < 0.01). In asthma patients, IC50s of hydrocortisone, prednisolone and betamethasone against PBMC-blastogenesis were significantly correlated with elevated percentages of IL-2R-positive PBMCs (p < 0.05), while the IC50 of methylprednisolone showed no such correlation. sIL-2R levels did not correlate with IC50s of any of the GCs examined. Thus, the results showed that a part of asthma patients exhibited PBMC-resistance to GCs, especially to prednisolone. Methylprednisolone potency was unexpectedly higher (> 10 times) than prednisolone potency. Our results also raised the possibility that PBMC-resistance to prednisolone in asthma may correlate with an increase in IL-2R positive PBMCs.

Adult↗

PubMed1998

Arthropod-borne tularemia in Japan: clinical analysis of 1,374 cases observed between 1924 and 1996.

Sixteen cases of tularemia transmitted by arthropods are used to characterize arthropod-borne tularemia in Japan. Arthropod-borne tularemia accounted for 1.2% of a total of 1,374 cases of tularemia observed between 1924 and 1996. The number of cases reported in Japan was low when compared with the number of cases reported in the United States. Arthropod-borne tularemia, however, is increasing and has reached 10.1% incidences during the last 16 yr. No arthropod-borne cases were reported prior to 1951. The occurrence of tularemia infection caused by contact with diseased hares was diphasic with the higher peak occurring during the winter, whereas the occurrence of arthropod-borne tularemia was common from spring to autumn. Among the 16 cases that we studied, 5 were initiated by tick-bites, 5 by the crushing of ticks found on domestic dogs, and 1 by an unidentified insect. In the remaining 5 cases, the vectors were not identified although arthropod bites were confirmed. These arthropod-borne cases were observed exclusively in the northeastern area of Honshu, the main island of Japan, and the age of patients ranged from 23 to 74 yr. The arthropod vectors associated with tularemia and factors influencing incidence of arthropod-borne tularemia in Japan are discussed.

Age Factors↗

PubMed1998

Flagellin-containing membrane vesicles excreted from Vibrio alginolyticus mutants lacking a polar-flagellar filament.

Polar flagellum-defective mutants (Pof- Laf-) have been isolated from a lateral flagella-defective mutant (Pof+ Laf-). Among these Pof- Laf- mutants, polar-filamentless mutants, which have the hook structure but not the filament, were identified by electron microscopy. Their hooks were covered with a sheath structure which is contiguous to the outer membrane. The filament proteins, flagellins, were shed into the culture medium of these mutants. These flagellins could be sedimented by high-speed centrifugation even after heat or low pH treatment whereas the depolymerized flagellin of the Pof+ strain was degraded by these treatments. After Triton X-100 treatment, most flagellin of the filamentless mutants could no longer be sedimented, and was degraded. We observed vesicle-like structures on the tips of the hooks and in the flagellin fraction sedimented by high speed centrifugation. These results suggest that flagellin of the filamentless mutants is not assembled into the tip of the hook, but is excreted together with a membrane structure which is probably the sheath of polar flagella.

Cytoplasmic Granules↗

PubMed1998

Activation of 11 beta-hydroxysteroid dehydrogenase by dehydroepiandrosterone sulphate as an anti-hypertensive agent in spontaneously hypertensive rats.

The anti-hypertensive properties of dehydroepiandrosterone sulphate (DHEAS) have been investigated by studying its effects on blood pressure, on serum concentrations of corticosterone and dehydrocorticosterone, and on 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) activity in spontaneously hypertensive rats (SHR). SHR were given intraperitoneal injections of DHEAS (10 mg day-1 for 70 days) from six to 16 weeks of age. The blood pressure-time curve was significantly (P < 0.05) suppressed immediately after administration of DHEAS. There was no difference between the heart rates of control and DHEAS groups. Serum concentrations of corticosterone and dehydrocorticosterone in the DHEAS group were significantly (P < 0.05) lower than those of the control group. The dehydrocorticosterone/corticosterone concentration ratio was, however, significantly (P < 0.05) higher in the DHEAS group, suggesting that treatment with DHEAS enhanced the overall interconversion of corticosterone to dehydrocorticosterone. The activity of 11 beta-HSD in specific organs of the DHEAS group was affected, characteristic changes being increases in the kidney (14-58%), decreases in the liver (11-27%) and no change in the testis. Direct addition of DHEAS to 11 beta-HSD preparations from the kidneys of control SHR had the same effect as that observed in the in-vivo experiments. The fall in serum corticosterone in the DHEAS group is considered to be related, at least partly, to increased activity of kidney 11 beta-HSD. The inverse correlation of kidney 11 beta-HSD activity with serum corticosterone and blood pressure (-r = 0.628, P < 0.01, and -r = 0.478, P < 0.05, respectively) suggest that DHEAS delayed the development of hypertension in SHR by selective promotion of kidney 11 beta-HSD activity which in turn resulted in lower serum concentrations of corticosterone and its minimal aldosterone-like activity.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1998

Chemotactic adaptation is altered by changes in the carboxy-terminal sequence conserved among the major methyl-accepting chemoreceptors.

In Escherichia coli and Salmonella typhimurium, methylation and demethylation of receptors are responsible for chemotactic adaptation and are catalyzed by the methyltransferase CheR and the methylesterase CheB, respectively. Among the chemoreceptors of these species, Tsr, Tar, and Tcp have a well-conserved carboxy-terminal motif (NWET/SF) that is absent in Trg and Tap. When they are expressed as sole chemoreceptors, Tsr, Tar, and Tcp support good adaptation, but Trg and Tap are poorly methylated and supported only weak adaptation. It was recently discovered that CheR binds to the NWETF sequence of Tsr in vitro. To examine the physiological significance of this binding, we characterized mutant receptors in which this pentapeptide sequence was altered. C-terminally-mutated Tar and Tcp expressed in a receptorless E. coli strain mediated responses to aspartate and citrate, respectively, but their adaptation abilities were severely impaired. Their expression levels and attractant-sensing abilities were similar to those of the wild-type receptors, but the methylation levels of the mutant receptors increased only slightly upon addition of attractants. When CheR was overproduced, both the adaptation and methylation profiles of the mutant Tar receptor became comparable to those of wild-type Tar. Furthermore, overproduction of CheR also enhanced adaptive methylation of wild-type Trg, which lacks the NWETF sequence, in the absence of any other chemoreceptor. These results suggest that the pentapeptide sequence facilitates effective adaptation and methylation by recruiting CheR.

Adaptation, Physiological↗

PubMed1998

Detection of measles virus mRNA from autopsied human tissues.

By reverse transcription-PCR, measles virus (MV) mRNA was detected in the brain, kidney, spleen, liver, and lung tissues obtained from 23 (45.1%) of 51 autopsy subjects, with the detection rates of each tissue ranging from 8 to 20%. Sequence analysis revealed frequent mutations in the corresponding viral protein. These results suggest that MV mutants commonly persist in apparently healthy individuals.

Adolescent↗