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Biomedical subjects

T Tsuchiya

Publications and source records attributed to T Tsuchiya.

At least 199 records · Page 11Linked to original sources
PubMed1998

A stimulation factor for hydrolysis of ATP bound to DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli.

Hydrolysis of ATP bound to DnaA protein by its intrinsic ATPase activity negatively controls chromosomal DNA replication in Escherichia coli. We developed a new in vitro assay system for ATP hydrolysis, which makes feasible a search for factors affecting the ATPase activity of DnaA protein. A crude cell extract enhanced the hydrolysis of ATP bound to DnaA protein, in a dose-dependent manner. Gel-filtration analyses revealed a single entity of the stimulation factor for the ATP hydrolysis and an apparent molecular mass of 170 kDa. The stimulation activity for ATP hydrolysis coeluted with the inactivation activity for DnaA protein initiating an oriC DNA replication, as determined by anion-exchange and gel-filtration column chromatographies. Activity of the stimulation factor required DNA and ATP. These observations suggested that IdaA protein, a previously described negative factor for DnaA protein, inactivated DnaA protein through stimulation of the hydrolysis of ATP bound to DnaA protein.

Adenosine Diphosphate↗
PubMed1998

Effect of environmental factors on the dominant lethality caused by expression of a mutated DnaA protein with decreased intrinsic ATPase activity.

Induction of a mutant DnaA protein (DnaA E204Q) with decreased intrinsic ATPase activity in cells was previously shown to cause overinitiation of chromosomal DNA replication and a dominant lethal phenotype. Here it is shown that the dominant lethality required incubation at high temperatures; cells harboring the expression plasmid of DnaA E204Q showed very weak colony formation ability (less than 1/10(5) that of the wild-type DnaA) at 42 degrees C, whereas they showed a normal colony formation ability at 28 degrees C on LB agar plates. Overinitiation of chromosomal DNA replication caused by expression of DnaA E204Q also required incubation at high temperatures in LB medium. When the incubation was performed in synthetic (Tanaka) medium at 42 degrees C, neither the dominant lethality nor overinitiation caused by expressing DnaA E204Q was observed. These results suggest that the dominant lethality and the overinitiation caused by expressing DnaA E204Q require culture conditions that provide a high potential for cell growth.

Adenosine Triphosphatases↗
PubMed1998

CCAAT/enhancer-binding protein delta gene expression is mediated by autoregulation through downstream binding sites.

CCAAT/enhancer-binding protein delta (C/EBP delta) transcription factor is sharply induced at the early stage of the acute phase response. We previously reported that the C/EBP delta gene expression is induced by the acute-phase response factor/signal transducers and activators of transcription 3 (APRF/STAT3). However, the expression level of the C/EBP delta gene is relatively high up to several hours after the stimulation, whereas APRF/STAT3 is inactivated within one hour. In this report, we identified the two C/EBP delta binding sites at the downstream region of this gene. The binding analysis revealed that both of these sites bound recombinant C/EBP delta protein. A cotransfection analysis identified these sites as the cis-elements for the autoregulation. We conclude that the C/EBP delta gene is activated by APRF/STAT3, and the expression level is then maintained by an autoregulation mechanism.

Animals↗
PubMed1998

Synthesis of 1-N-[(2S,4S)- and (2S,4R)-5-amino-4-fluoro-2-hydroxypentanoyl]dibekacins (study on structure-toxicity relationships).

(2S,4S)- and (2S,4R)-5-azido-2-O-benzyl-4-fluoro-2-hydroxypentanoic acids (15 and 19) have been prepared from L-malic acid (1), and coupled to the H2N-1 group of 3,2',6'-tris(N-benzyloxycarbonyl)-3"-N-(trifluoroacetyl)dibekacin (23), to give, after reduction and deblocking, 1-N-[(2S,4S)- and (2S,4R)-5-amino-4-fluoro-2-hydroxypentanoyl]dibekacins (26 and 27). The fluorinated arbekacin analogs showed almost the same antibacterial activities as that of arbekacin, but lower toxicity. Comparison of the toxicity between 26 (and 27) and the arbekacin analogs (28-30) with change of the 1N-side-chain indicates that the observed decrease in toxicity was a function of the chain length rather than the introduction of fluorine.

Aminoglycosides↗
PubMed1998

Synthesis and antitumor activity of 7-O-(2,6-dideoxy-2-fluoro-alpha-L-talopyranosyl) daunomycinone derivatives modified at C-3' or C-4'.

As a part of a study to exploit anthracycline glycosides effective against resistant tumor cells, the 3'-O-methyl (3), 4'-O-methyl (4), 3'-deoxy (6), 3'-deoxy-3'-fluoro (7), and 3'-deoxy-3'-iodo (8) derivatives of 7-O-(2,6-dideoxy-2-fluoro-alpha-L-talopyranosyl)daunomycinone have been prepared by coupling suitably protected glycosyl bromides with daunomycinone. The doxorubicin-type analog (5) of 4 was also prepared. Among the compounds prepared, 5 showed the highest antitumor activity. Relationships between chemical structures of the synthetic products and antitumor activities, together with the degree of resistance were discussed.

Animals↗
PubMed1998

Synthesis of alpha,alpha-, alpha,Beta-, and Beta,Beta-(dimaltoside)s of ethane-1,2-diol, propane-1,3-diol, and butane-1,4-diol: a proposal for an initial adhesion mode.

Nine dimaltoside derivatives of ethane-1,2-diol, propane-1,3-diol, and butane-1,4-diol having the alpha,alpha, alpha,beta, and beta,beta anomeric configurations at the linkage sites have been synthesized. Suitably protected maltosyl halides or a 1-(phenylthio) derivative were condensed with the foregoing diols and the resulting monomaltosyl derivatives were further condensed with the maltosyl donors to give, after deprotection, the title compounds. Their structures were fully characterized by NMR spectroscopy. Interactions between the three alpha,alpha-(dimaltoside)s and cinnamyl alcohol are briefly discussed.

Carbohydrate Conformation↗
PubMed1998

Attenuation of cell death mediated by membrane depolarization different from that by exogenous BDNF in cultured mouse cerebellar granule cells.

Membrane depolarization accompanying calcium (Ca2+) influx into neurons is thought to play an essential role in controlling the survival and death of cultured mouse cerebellar granule cells (CGCs). In this study, we sequentially controlled the survival and death of CGCs in culture and monitored the expression of several kinds of genes including brain-derived neurotrophic factor (BDNF) gene. Deprivation and subsequent induction of membrane depolarization by lowering and re-elevating the extracellular concentration of potassium chloride, respectively, led to death of CGCs and then to an attenuation of the death process depending upon the Ca2+ influx into CGCs through voltage-dependent calcium channels (VDCCs). De novo protein synthesis was critical for attenuating the death of non-depolarized CGCs. Accompanying this attenuation was an activation of c-fos and BDNF genes and an inactivation of c-jun and neurotrophin-3 (NT-3) genes. The attenuation of cell death mediated by exogenous BDNF was only partial compared to that by membrane depolarization, suggesting that not only BDNF but also other factors could be involved in the membrane depolarization-mediated attenuation of death of CGCs. In good agreement with this observation, the mode of activation of c-fos, c-jun, BDNF and NT-3 genes induced by exogenous BDNF was different from that induced by membrane depolarization. Thus, membrane depolarization effectively attenuates the death of non-depolarized CGCs, the mode of which seems to be different from that mediated by BDNF alone.

Animals↗
PubMed1998

Orchiectomy and response to testosterone in the development of obesity in young Otsuka-Long-Evans-Tokushima Fatty (OLETF) rats.

OBJECTIVE: Withdrawal of testosterone prevents the development of hyperglycaemia in male Otsuka-Long-Evans-Tokushima Fatty (OLETF) rats, a model of non-insulin-dependent diabetes mellitus (NIDDM), but the exact mechanism has not been established. The present studies were undertaken to examine a possible role of testosterone in the development of obesity in young OLETF rats who have not shown marked hyperphagia. METHODS: Body weight, food intake and circulating concentrations of metabolic factors including immunoreactive leptin (IRL) were measured at five weeks of age in young male OLETF rats and their lean controls, Long-Evans-Tokushima-Otsuka (LETO) rats. At six weeks of age, both LETO and OLETF rats were bilaterally orchiectomized (Orchx) and half of each group implanted with a silastic tube containing testosterone. After a three week observation period, all animals were killed and circulating concentrations of metabolic factors and the ob gene expression in retroperitoneal white adipose tissues were measured. RESULTS: Body weight and 24h food intake were already increased in OLETF rats at five weeks of age. Serum testosterone concentrations were significantly lower in OLETF rats than in LETO rats. Expression of the ob gene was significantly decreased in the retroperitoneal white adipose tissue of OLETF rats, and their serum IRL concentrations were lower. Food intake and body weight gain for three weeks after the operation were significantly lower in the Orchx group of OLETF rats than in the sham-operated group. Hyperglycaemia, accompanied by hyperinsulinaemia, was attenuated by orchiectomy in OLETF rats. Circulating IRL concentrations were significantly higher in OLETF rats than in LETO rats and decreased by orchiectomy. Testosterone supplement reversed all of the changes caused by orchiectomy in OLETF rats. In contrast, the changes, which were observed after orchiectomy in OLETF rats, were not obvious in LETO rats. CONCLUSION: The present data indicate that testosterone plays a role in the development of obesity and NIDDM in young OLETF rats, but that changes of leptin production in white adipose tissue may not be important in the development of obesity in young OLETF rats.

Animals↗
PubMed1998

An increase of circulating leptin in patients with liver cirrhosis.

OBJECTIVE: Leptin, the ob gene product, is an anorexigenic peptide secreted from adipose tissue. However, the mechanism of leptin clearance/degradation has not been well determined in humans. The present study was undertaken to examine a possible involvement of liver in determining circulating leptin concentrations in humans. SUBJECTS: In the present study 58 healthy control subjects and 68 patients with liver cirrhosis (LC) without any renal dysfunction were randomly included. METHOD: The serum immunoreactive leptin (IRL) concentrations relative to the body mass index (BMI) were determined. Serum IRL and estradiol (E2) concentrations were assayed by radioimmunoassay (RIA). RESULTS: The correlations between the BMI and circulating IRL concentrations were all significant in male healthy controls (M-C), male patients with LC (M-LC), female healthy controls (F-C) and female patients with LC (F-LC). Circulating IRL concentrations were significantly higher than control in F-LC but not M-LC groups. The ratio of circulating IRL concentrations to the BMI was significantly higher in the M-LC group than in the M-C group and also significantly higher in the F-LC group than in the F-C group. The correlation between the IRL/BMI ratio and serum total bilirubin concentrations was significant (r = 0.417, P < 0.05) in the M-LC group, but not in the F-LC group. There was no significant correlation of the IRL/BMI ratio to serum E2 or albumin concentrations in either M-LC or F-LC groups. CONCLUSION: The present data demonstrated that the rate of increase in circulating IRL concentrations with the BMI was higher in LC patients of both genders. Liver may play a role in determining circulating leptin levels.

Aged↗
PubMed1998

Determination of the pH difference across a cell membrane using a methylammonium-selective membrane electrode.

A method was developed for determining pH differences across cell membranes using a methylammonium-selective membrane electrode, based on monitoring of the pH gradient-induced uptake of methylammonium in situ. The methylammonium electrode was constructed using calix[6]arene-hexaacetic acid hexaethyl ester as a neutral carrier and bis(2-ethylhexyl) sebacate as a membrane solvent in a poly(vinyl chloride) membrane matrix. This electrode exhibited a near-Nernstian response to methylammonium in the concentration range 2 x 10(-5)-1 x 10(-2) M with a slope of 58 mV per concentration decade in a buffer solution of 150 mM choline chloride-10 mM TRIS-HCl (pH 7.5). The limit of detection was 5 x 10(-6) M. In experiments using liposomes, the uptake of methylammonium into liposomes occurred effectively when the pH of the outside suspension medium was alkaline, and the determination of changes in methylammonium concentrations in the outer medium was quantitatively related to changes in the pH differences across the liposomal membrane. The transmembrane pH differences in Escherichia coli cells were also determined by this method.

Cell Membrane↗
PubMed1998

Immobilization-induced and crowded environment-induced stress delay barrier recovery in murine skin.

To examine the effect of stress on skin homeostasis, cutaneous barrier recovery was measured in rate exposed to immobilization stress after tape stripping or sodium dodecyl sulphate treatment. The barrier function was evaluated by measuring transepidermal water loss. Barrier recovery was delayed in rats exposed to stress in comparison with untreated controls. This tendency was observed in both male and female animals. The delay in barrier recovery was blocked by application of the sedative drugs diazepam and chlorpromazine. The barrier recovery rate in mice which were kept at a high population density (10 animals per cage) for 2 weeks was slower than that in mice kept at lower population densities (five animals or one animal per cage). These animal models could be useful for objectively quantifying the influence of stress on the cutaneous function.

Animals↗
PubMed1998

Alteration in the contents of unsaturated fatty acids in dnaA mutants of Escherichia coli.

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, has a high affinity for acidic phospholipids containing unsaturated fatty acids. We have examined here the fatty acid composition of phospholipids in dnaA mutants. A temperature-sensitive dnaA46 mutant showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) at 42 degrees C (non-permissive temperature) and at 37 degrees C (semi-permissive temperature), but not at 28 degrees C (permissive temperature), compared with the wild-type strain. Plasmid complementation analysis revealed that the dnaA46 mutation is responsible for the phenotype. Other temperature-sensitive dnaA mutants showed similar results. On the other hand, a cold-sensitive dnaAcos mutant, in which over-initiation of DNA replication occurs at low temperature (28 degrees C), showed a higher level of unsaturation of fatty acids at 28 degrees C. Based on these observations, we discuss the role of phospholipids in the regulation of the activity of DnaA protein.

Bacterial Proteins↗
PubMed1998

Exposure to a dry environment enhances epidermal permeability barrier function.

Previous studies have suggested that transepidermal water movement may play an important role in epidermal homeostasis and barrier repair. Here we analyzed cutaneous barrier function, epidermal morphology, and lipid content of the stratum corneum in hairless mice maintained in a high relative humidity (RH > 80%) versus low humidity (RH < 10%) environment for 2 wk. Basal transepidermal water loss was reduced by 31% in animals maintained in a dry versus humid environment. Moreover, the number of lamellar bodies in stratum granulosum cells, the extent of lamellar body exocytosis, and the number of layers of stratum corneum increased in animals kept in a dry environment. Furthermore, the dry weight of the stratum corneum and the thickness of the epidermis also increased in a dry environment. In addition, total stratum corneum lipids increased but lipid analysis revealed no significant differences in lipid distribution. Lastly, barrier recovery following either acetone treatment or tape stripping was accelerated after prolonged prior exposure to a dry environment, while conversely, it was delayed by prior exposure to a humid environment. These studies demonstrate that environmental conditions markedly influence epidermal structure and function, and suggest mechanisms by which the environment could induce or exacerbate various cutaneous disorders.

Animals↗
PubMed1998

Low humidity stimulates epidermal DNA synthesis and amplifies the hyperproliferative response to barrier disruption: implication for seasonal exacerbations of inflammatory dermatoses.

Although seasonal changes in humidity are thought to exacerbate various skin diseases, whether these flares can be attributed to prolonged exposure to extremes in environmental humidities has not been studied systematically. We recently showed that prolonged exposure to high versus low humidities induced profound changes in epidermal structure and permeability barrier homeostasis. Therefore, we asked here whether comparable extremes in humidity could initiate not only homeostatic, but also potentially pathophysiologic alterations. We showed first that exposure to low humidity increases epidermal DNA synthesis in normal murine epidermis. Moreover, exposure to a low humidity for 48 h further amplifies the DNA synthetic response to barrier disruption, resulting in marked epidermal hyperplasia. Additionally, exposure to a dry environment for 48 h prior to barrier disruption results in dermal mast cell hypertrophy, degranulation, as well as histologic evidence of inflammation. To demonstrate the role of changes in external moisture on these phenomena, we applied either an occlusive, water-impermeable plastic membrane, Petrolatum, or a nonocclusive humectant, both to nonperturbated and to perturbed skin. All three forms of treatment prevented the epidermal hyperplasia and dermal mast cell hypertrophy and degranulation induced by exposure to low humidity. These studies indicate that (i) exposure to changes in environmental humidity alone induces increased keratinocyte proliferation and markers of inflammation, and (ii) that these changes are attributable to changes in stratum corneum moisture content. Finally, these studies provide evidence that changes in environmental humidity contribute to the seasonal exacerbations/amelioration of cutaneous disorders, such as atopic dermatitis and psoriasis, diseases which are characterized by a defective barrier, epidermal hyperplasia, and inflammation.

Acetone↗
PubMed1998

Ku antigen binds to Alu family DNA.

The GC-rich segment containing GGAGGC (Alu core) is conserved within the RNA polymerase III (pol III) promoters of Alu family sequences. We have shown that the GGAGGC motif functions as a modulator of DNA replication as well as of transcription, and identified the proteins binding to the motif in human HeLa cells. In this study, the Alu core binding proteins were partially purified from human Raji cells by using an Alu core DNA affinity column. Both the proteins thus purified were implied to be subunits of Ku antigen based on the following criteria: The molecular weights of the proteins estimated on gel electrophoreses were 70 and 85 kDa, respectively, under denaturing conditions, while under non-denaturing conditions only one band was observed for the same sample at 150 kDa, probably representing hetero-dimer formed between the 70 and 85 kDa proteins. The sizes and the hetero-dimer formation are reminiscent of the 70 and 80 kDa subunits of Ku antigen (Ku-p70 and Ku-p80). Moreover, the purified proteins were immunoreactive with anti-Ku antibodies, and the specific DNA-protein complex on the Alu core element was cancelled by the anti-Ku antibodies. The nucleoprotein complex showed the same clipping patterns as those of the complex between the Alu core element and an authentically purified Ku antigen after proteolytic cleavage with trypsin and chymotrypsin.

Antigens, Nuclear↗
PubMed1998

Effect of glycerol on the affinity of DnaA protein for ATP in the presence of cardiolipin.

Acidic phospholipids, such as cardiolipin, decrease the affinity of DnaA protein for adenine nucleotides and can activate the inactive form of DnaA protein in vitro. In this study, we examined the effect of glycerol on the affinity of DnaA protein for ATP in the presence of cardiolipin. High concentrations of glycerol (34%) restored the affinity of DnaA protein for ATP, which was decreased by cardiolipin. Glycerol inhibited the binding of cardiolipin with DnaA protein. Glycerol had little effect on membrane fluidity, which is essential for the interaction between cardiolipin and DnaA protein, whereas it increased the Kd value of DnaA protein for ATP in the absence of cardiolipin. These results suggest that glycerol causes DnaA protein to become insensitive as to the interaction with cardiolipin by changing the conformation of the protein without altering the physical nature of the phospholipid.

Adenosine Triphosphate↗
PubMed1998

Evidence for chloramphenicol/H+ antiport in Cmr (MdfA) system of Escherichia coli and properties of the antiporter.

We detected chloramphenicol/H+ antiport activity in membrane vesicles of Escherichia coli and cloned a gene for the antiporter from chromosomal DNA of E. coli. Introduction of the gene into E. coli cells conferred resistance to chloramphenicol and ethidium. A slight increase in resistance to acridine orange was also observed. Elevated chloramphenicol efflux and ethidium efflux were observed in cells harboring a plasmid carrying the gene. Addition of chloramphenicol to the assay mixture reduced the efflux of ethidium. Elevated chloramphenicol/H+ antiport activity was observed in membrane vesicles prepared from cells harboring the plasmid. The pH optimum for the activity was 6.5. We sequenced the gene and deduced the amino acid sequence of its product. A sequence homology search revealed that it was same as that of Cmr (or MdfA). Thus, it became clear that Cmr (MdfA) is the chloramphenicol(and ethidium)/H+ antiporter.

Bacterial Proteins↗