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Biomedical subjects

A Ohta

Publications and source records attributed to A Ohta.

At least 91 records · Page 5Linked to original sources
PubMed1999

Misfolded membrane-bound cytochrome P450 activates KAR2 induction through two distinct mechanisms.

Using the mRNA differential display technique and Western blot analysis, the present study demonstrates that induction of KAR2 occurs when misfolded membrane-bound cytochrome P450, mutated in its cytosolically exposed domain, is expressed in Saccharomyces cerevisiae. Using various KAR2 promoter constructs in front of the Escherichia coli beta-galactosidase reporter gene, we found a fast and strong induction through the heat shock element (HSE), which was enhanced several fold by its adjacent GC-rich region. Additionally, a less pronounced induction was detected for the UPR element (UPRE). As expected, this response was absent in the ire1 disruptant strain. However, the HSE-mediated induction was enhanced upon disruption of IRE1 suggesting that the HSE pathway can compensate for the lack of a functional UPR pathway. Western blotting confirmed that Kar2p levels were increased to the same extent in the ire1 disruptant and in the non-disruptant strain. Removal of the P450 membrane-spanning region also abolished the UPRE-mediated induction of KAR2 transcription, but the HSE-mediated response remained. The data show for the first time that the transcription of KAR2 is significantly induced in response to a misfolded membrane-bound endoplasmic reticulum protein, and identifies the HSE and UPRE regions as KAR2 promoter elements responding to the misfolded cytosolic P450 domain and to the membrane-integrated mutant P450, respectively.

Blotting, Western↗
PubMed1999

Natural killer T cell ligand alpha-galactosylceramide inhibited lymph node metastasis of highly metastatic melanoma cells.

The role of natural killer T (NKT) cells in the prevention of multiple tumor metastasis was examined. The i.v. inoculation of a highly metastatic subline of B16-BL6 (B16-BL6-HM) melanoma cells resulted in the formation of metastatic nodules in lymph nodes in addition to lung, intrapleural cavity, and ovary. However, treatment of the mice with the NKT cell ligand alpha-galactosylceramide (alpha-GalCer) three times from 1 day after B16-BL6-HM melanoma inoculation caused a significant inhibition of multiple metastasis. Lymph node metastasis of B16-BL6-HM was almost completely blocked by alpha-GalCer treatment. This antimetastatic effect of alpha-GalCer was abolished in NKT cell-deficient mice. These results suggest that alpha-GalCer-activated NKT cells played a critical role in the prevention of lymph node metastasis of melanoma cells.

Adjuvants, Immunologic↗
PubMed1999

[An analysis of fever of unknown origin defined by newly proposed practical criteria. A prospective study of 56 cases].

The criteria proposed by Petersdorf has been in use internationally to define the fever of unknown origin (FUO) since 1961 and the research of FUO has progressed with this criteria. Mean-while, new diagnostic methods have been developed and illness behavior of febrile patients has changed considerablly. Accordingly, the definition by Petersdorf is becoming less matched to current clinical situation. Therefore, we have developed a new practical criteria of FUO; i.e., out-patients who are febrile more than 2 weeks, documented temperature higher than 37.5 degrees C at least on one occasion and undetermined diagnosis and in-patients who are febrile more than 1 week with documented temperature higher than 37.5 degrees C, and undetermined diagnosis. Between October 1, 1993, and October 31, 1996, we prospectively collected a series of febrile patients who fulfilled our new criteria. We identified 56 patients by our criteria (23 male and 33 female, age: 49.7 +/- 20.6. range 15 to 88). Of this 56 patients, 19 (32%) were found to have infections, 18 (31%) had collagen disorders, 5 (9%) had malignancy and 6 (10%) had died. Of 56 patients, 38 (68%) were in the newly added group. Of these 38 patients, 4 patients had subacute necrotizing lymphoadenitis, 4 cytomegalovirus infection, 3 polymyalgia rheumatica, 3 tuberculosis. Four patients (10%) died. With our criteria, febrile patients who previously had not been included were found to have as poor a prognosis as with the Petersdorf group.

Adolescent↗
PubMed1999

Proliferation of intrahyphal hyphae caused by disruption of csmA, which encodes a class V chitin synthase with a myosin motor-like domain in Aspergillus nidulans.

We have found that the Aspergillus nidulans csmA gene encodes a novel protein which consists of an N-terminal myosin motor-like domain and a C-terminal chitin synthase domain (M. Fujiwara, H. Horiuchi, A. Ohta, and M. Takagi, Biochem. Biophys. Res. Commun. 236:75-78, 1997). To clarify the roles of csmA in fungal morphogenesis, we constructed csmA null mutants. The growth rate of the mutant colonies was almost the same as that of the wild-type strain, but hyphal growth was severely inhibited when a chitin-binding reagent, Calcofluor white or Congo red, was added to the medium. Moreover, morphological abnormalities in tip growth and septum formation were identified microscopically. Proliferation of intracellular new hyphae, called intrahyphal hyphae, which behaved as intrinsic hyphae, was the most striking phenotypic feature among them. These phenotypes were not suppressed when the only chitin synthase domain of csmA was expressed under the control of the alcA promoter, whereas they were suppressed when the intact form of csmA was expressed. Therefore, it was concluded that the product of csmA (CsmA) has important roles in polarized cell wall synthesis and maintenance of cell wall integrity and that the myosin motor-like domain is indispensable for these functions.

Aspergillus niger↗
PubMed1999

Effect of 3,6-dimethylpyrazine-2-thiol on androstenedione-induced increase of uterine weight in female rats.

3,6-Dimethylpyrazine-2-thiol administered at 10-70 mg/kg, p.o. was found to suppress androstenedione-induced increase of uterine weight in female rats. This action was weaker than that of aminoglutethimide (3-30 mg/kg, p.o.). After administration of androstenedione, increased plasma estradiol levels were reduced by 3,6-dimethylpyrazine-2-thiol. Moreover, in vitro, production of estradiol in the pregnant mare serum gonadotropin (PMSG)-treated ovary was inhibited by 3,6-dimethylpyrazine-2-thiol. These results suggest that 3,6-dimethylpyrazine-2-thiol has an inhibitory action on aromatase activity.

Aminoglutethimide↗
PubMed1999

Polyangiitis overlap syndrome with eosinophilia associated with an elevated serum level of major basic protein.

Polyangiitis overlap syndrome is a new disease entity and the reported cases in the literature are still limited. We describe a female patient presenting with finger ulcers, skin eruptions, pleural effusion, interstitial pneumonia and eosinophilia. Skin biopsy showed systemic small-sized angiitis and thrombosis. She was diagnosed as having polyangiitis overlap syndrome and was successfully then treated with corticosteroid. It is also of interest that the disease activity was correlated with the number of eosinophils in peripheral blood. The measurement of the serum level of major basic protein released from eosinophils functioning as a coagulant indicated the possible association of eosinophilia with thrombosis and polyangiitis.

Aged↗
PubMed1999

[Relationship between regular exercise and life style, social network, education and subjective symptoms in Japanese middle aged and elderly residents].

OBJECT AND METHODS: A community-based survey of 12,630 residents of Gunma Prefecture aged 40 to 69 years was conducted to investigate the relationship between the habit of regular exercise and life style, social network, and subjective physical and mental symptoms in particular. The subjective physical and mental symptoms were measured using the Todai Health Index (THI). The response rate was 91.6%. A total of 6,736 responses were analyzed after excluding respondents with chronic diseases from the analysis. On the question about the habit of regular exercise ("Do you exercise regularly?"), the responses were divided into three categories: 1. Often, 2. Sometimes, 3. Hardly ever or Never. RESULTS AND CONCLUSION: The results are summarized as follows: 1. The proportions of residents who exercise "often" were 14.2% and 11.1% for males and females, respectively, while those of "hardly ever or never" were 52.3% and 59.4% for males and females, respectively. More males than females had the habit of regular exercise. Respondents were divided into three age categories: 40-49, 50-59 and 60-69 years. Among the female respondents, it was shown that the older the age category the greater the habit of regular exercise. 2. Both male and female respondents who had regular exercise habits scored significantly lower on the THI scales for vague complaints, depression, and irregularity of life, while they scored higher on the aggression and lie (social desirability) scales. 3. Both male and female respondents who had regular exercise habits also tended to have be better perceived health and social networking, i.e. having close friends, having a hobby and involvement in community social activities, as well as taking care of pets. This group also had a higher average education level and less of a smoking habit. This study suggests that regular exercise habit correlates not only with physical health and fitness levels but with mental health status as well.

Adult↗
PubMed1999

Ingestion of guar-gum hydrolysate partially restores calcium absorption in the large intestine lowered by suppression of gastric acid secretion in rats.

We examined the effects of feeding guar-gum hydrolysate (GGH), a highly fermentable form of dietary fibre with low viscosity, on Ca absorption in the small and large intestines in rats under conditions in which gastric acid secretion was suppressed by a proton pump inhibitor, omeprazole. We also examined the role of the caecum in influencing these effects. The study was designed in a 2 x 2 x 2 factorial arrangement with two diet (GGH-containing (50 g/kg diet) and GGH-free diets) groups, two injection (omeprazole and vehicle) groups and two operation (sham and caecectomy) groups. Apparent Ca absorption was lower in rats administered omeprazole (30 mg/kg body weight per d) for 8 d than in rats administered the vehicle. Ingestion of GGH led to partial restoration of Ca absorption decreased by omeprazole treatment. However, this increment in Ca absorption was not sufficient to meet requirements because the dietary Ca level (3.0 g/kg diet) was the minimum requirement for the intact rats. The small increment in Ca absorption caused by the GGH diet was completely abolished by caecectomy. Soluble Ca pools in the caecal and colonic contents were increased by feeding GGH, and the soluble Ca concentrations were much higher than the Kt values of the Ca active transport system in the large intestine or the serum Ca concentration. These findings suggest that Ca solubilization is not a limiting factor for Ca absorption in the large intestine. Apparent Mg absorption was clearly lower in caecectomized rats than in sham-operated rats, and higher in the GGH-fed groups than in the groups fed on the GGH-free diet, even in the case of caecectomized rats. We conclude that Ca absorption lowered by inhibition of gastric acid secretion is partially restored in rats fed with GGH, but the increment is not sufficient to meet requirements.

Animals↗
PubMed1998

Studies on cycloheximide-sensitive and cycloheximide-resistant ribosomes in the yeast Candida maltosa.

Cycloheximide sensitivity or resistance in yeast is under the control of genes encoding different forms of ribosomal protein L41. In our previous studies, we have shown by isolating L41-Q1a, L41-P1a and their respective allelic genes, L41-Q1b and L41-P1b, from the partial diploid genome of C. maltosa, that this species, which is inducibly resistant to CYH, has both types of the L41 genes and that the expression of at least one of the L41-Q genes is induced by CYH, whereas L41-P genes are constitutively expressed. Here, we have identified another L41 (L41-Q2a), its allelic gene (L41-Q2b) and a third gene (L41-Q3) from the genome of C. maltosa. By gene disruption experiments, we now show that L41-Q1a and L41-Q1b are not responsible for the resistance to CYH and that the DeltaL41-Ps strain, which has only functional L41-Q genes, shows constitutive resistance to CYH, but grows more slowly than the DeltaL41-Qs strain, which has only functional L41-P genes, in the absence of CYH. Our results also show that in vitro, ribosomes containing L41-Q-type are less active in translation than those containing L41-P-type, although only the former ribosomes are active in the presence of CYH. These data suggest that ribosomes containing L41-Q-type are less active under normal growth conditions, but that this activity is not affected in the presence of CYH. We discuss the possible multi-step evolutionary event(s) by which C. maltosa has acquired the property of inducible resistance to CYH.

Amino Acid Sequence↗
PubMed1998

Isolation of a class IV chitin synthase gene from a zygomycete fungus, Rhizopus oligosporus.

We found the presence of DNA sequence which shows sequence similarity to the class IV chitin synthase gene (CHS3) of Saccharomyces cerevisiae in the genome of 14 Rhizopus species which belong to zygomycetes. We cloned a gene (chs3), which might correspond to one of these homologous sequences, from Rhizopus oligosporus by low stringency plaque hybridization probed with CHS3. The deduced amino acid sequence of this gene showed highest similarity to the class IV chitin synthase of Neurospora crassa (46.7% identity over 1087 amino acids), showing that this gene encodes a class IV chitin synthase. Northern analysis revealed the differential expression pattern of this gene in the asexual life cycle with highest expression in the early stage of asexual spore formation. This is the first report of the isolation and analysis of a class IV chitin synthase gene from zygomycete fungi.

Amino Acid Sequence↗
PubMed1998

Relation between evolutionary distance and enzymatic properties among the members of the CYP52A subfamily of Candida maltosa.

The CYP52A subfamily of the alkane-assimilating yeast Candida maltosa consists of six structurally related isoforms. Four of them (CYP52A3, 4, 5, and 9) are strongly induced by alkanes and play an important role for the conversion of various alkanes and fatty acids. Taking advantage of a homologous overexpression system, we found in the present study that both of the two other CYP52A forms, CYP52A10 and CYP52A11, represent specialists for the hydroxylation of lauric acid suggesting their preference for short-chain fatty acids. At the same time, they hydroxylated palmitic acid only moderately and failed to convert hexadecane. Based on the now completed knowledge about the principal substrate specificities of all members of the CYP52A subfamily of C. maltosa, it became apparent that evolutionarily more distantly related P450 forms developed either to alkane or to fatty acid hydroxylases, whereas P450 forms which retained the ability to convert both types of substrates were also found to be evolutionarily related to both alkane and fatty acid hydroxylases.

Candida↗
PubMed1998

Incorporation of extracellular phospholipids and their effect on the growth and lipid metabolism of the Saccharomyces cerevisiae cho1/pss mutant.

The cho1/pss mutant of Saccharomyces cerevisiae, which is auxotrophic for choline or ethanolamine because of the deficiency in phosphatidylserine synthesis, grew in the presence of 0.05 mM phosphatidylcholine (PC) with octanoic acids (diC8PC) or decanoic acids (diC10PC), but not in the presence of PC with longer acyl residues. It did not grow in the presence of the soluble hydrolytic products of PC, phosphorylcholine or glycerophosphorylcholine, at comparable concentrations. Addition of 10 mM hemicholinium-3, a choline transport inhibitor, or disruption of the CTR gene, which encodes a choline transporter, inhibited the growth of the cho1/pss mutant in the presence of choline, but not in the presence of 0.1 mM diC8PC. Under diC8PC-supported growth conditions, octanoic acid was barely detectable in the cellular phospholipid fraction, but was recovered in the culture medium as the free acid, and the phosphatidylethanolamine (PE) content was low in comparison to the choline-supported conditions. These results suggest that PCs with short acyl residues were taken up by the cho1/pss mutant and remodeled as they were used, and that PCs with short acyl residues do not inhibit conversion of PE to PC. The current results provide a new direction in the analysis of intracellular phospholipid movement and metabolism in yeast.

Caprylates↗
PubMed1998

Spontaneous and artificial lesions of magnocellular reticular formation of brainstem deteriorate avoidance learning in senescence-accelerated mouse SAM.

The role of the magnocellular reticular formation (MGRF) of the brainstem on learning and memory was examined in memory-deficient mice with spontaneous spongy degeneration in the brainstem (senescence-accelerated mouse, SAMP8) and control mice (accelerated-senescence resistant mouse, SAMR 1). SAMP8 showed spontaneous age-related impairment of learning and memory, as determined by passive and active avoidance responses. The deficits of learning and memory function in passive avoidance performances began at two months of age and increased with ageing. In the brains of SAMP8 at one month of age and older, spongy degeneration was mainly observed in the brainstem, while no vacuoles were evident in SAMR1 control (normal ageing mouse) brains in the age range tested (up to 12 months). The vacuolization in SAMP8 was marked in the MGRF, especially in the dorsomedial MGRF. Quantitative analysis of the vacuolization showed that the total area and number of vacuoles in the MGRF increased with age, and they were affected by the degree of deficits in learning and memory. The latency 24 h after footshock in passive avoidance tests decreased with the increase in total area and number of vacuoles in MGRF. The number of shocks in active avoidance tests increased with the increase in total number and area of vacuoles. Thus, learning and memory ability in passive and active avoidance responses deteriorated with enlargement in the vacuolated area in MGRF, and it was assumed that MGRF (especially, the dorsomedial part) possesses functions related to learning and memory. To confirm this notion, behavior and memory tests (passive avoidance and active avoidance tests, open field tests and shock sensitivity measurements) were carried out in SAMR1 mice, whose bilateral dorsomedial MGRF was destroyed electrolytically (MGRF-lesioned mice). The MGRF-lesioned mice showed no difference from sham mice in sensory threshold or open field activity; however, there was severe deterioration in passive avoidance behavior and impairment in the active avoidance performances. From the results in SAMP8 and MGRF-lesioned mice, it was confirmed that MGRF (especially the dorsomedial part) has functions related to learning and memory, and is one part in the learning and memory system of the brain. Thus, SAMP8 can serve as a model of RF-lesioned mice with impaired learning and memory functions.

Aging↗
PubMed1998

Isozyme function of n-alkane-inducible cytochromes P450 in Candida maltosa revealed by sequential gene disruption.

An n-alkane-assimilating yeast Candida maltosa contains multiple n-alkane-inducible forms of cytochromes P450 (P450alk), which can be assumed to catalyze terminal hydroxylation of n-alkanes in the assimilation pathway. Eight structurally related P450alk genes have been identified. In the present study, the function of four major isoforms of P450alk (encoded by ALK1, ALK2, ALK3, and ALK5 genes) was investigated by sequential gene disruption. Auxotrophic markers used for the selection of disrupted strains were regenerated repeatedly through either mitotic recombination between heterozygous alleles of the diploid genome or directed deletion of the marker gene, to allow sequential gene disruptions within a single strain. The strain depleted of all four isoforms could not utilize n-alkanes for growth, providing direct evidence that P450alk is essential for n-alkane assimilation. Growth properties of a series of intermediate disrupted strains, plasmid-based complementation, and enzyme assays after heterologous expression of single isoforms revealed (i) that each of the four individual isoforms is alone sufficient to allow growth on long chain n-alkane; (ii) that the ALK1-encoding isoform is the most versatile and efficient P450alk form, considering both its enzymatic activity and its ability to confer growth on n-alkanes of different chain length; and (iii) that the ALK5-encoding isoform exhibits a rather narrow substrate specificity and thus cannot support the utilization of short chain n-alkanes.

Alkanes↗
PubMed1998

Cloning and characterization of an n-alkane-inducible cytochrome P450 gene essential for n-decane assimilation by Yarrowia lipolytica.

A gene encoding cytochrome P450 involved in n-alkane utilization was cloned from an n-alkane assimilating yeast, Yarrowia lipolytica CX161-1B. The RT-PCR was performed on the mRNA prepared from the cells grown on n-alkane as a template using degenerated PCR primers designed for the conserved amino acid sequences of the CYP52 family. The RT-PCR amplified fragment was then used as a probe to isolate genes coding for P450 of the CYP52 family from the genomic DNA library of the strain CX161-1B. The nucleotide sequence of one of the positive clones was determined. An open reading frame which had the same nucleotide sequence as the RT-PCR-amplified fragment was identified. It was of 523 amino acid residues, 60.2 kDa in molecular mass, and had 30-45% sequence identity with the other members of the CYP52 family of Candida species so far analysed. The expression of the P450 gene that was named as YlALK1 was induced by n-tetradecane and repressed by glycerol. A YlALK1 gene disruptant did not grow well on n-decane, but grew on longer-chain n-alkanes such as hexadecane as a sole carbon source. Introduction of YlALK1 on a plasmid to the disruptant restored the decane assimilation. These results suggest that the YlALK1 gene product is the major P450A1k to metabolize short-chain n-alkanes such as decane and dodecane in Y. lipolytica.

Alkanes↗
PubMed1998

Accumulation of IL-12-activated antitumor effector cells into lymph nodes of tumor-bearing mice.

Simultaneous administration of high dose of IL-12 into tumor-inoculated mice resulted in a marked reduction of tumor growth in parallel with the augmented generation of cytotoxic T-cells, natural killer (NK) cells and IFN-gamma-producing Th cells. We found that these IL-12-activated antitumor effector cells preferentially accumulated in peripheral lymph nodes concomitantly with lymphadenopathy. However, IL-12 rather induced disappearance of antitumor effector cells including CD4+ T, CD8+ T and NK cells from spleen in spite of inducing splenomegaly. Lymph node cells obtained from IL-12-treated B16F0-bearing mice showed a marked IFN-gamma production in response to not only IL-2, IL-12, anti CD3 mAb but also B16F0 melanoma cells. Moreover, they could lyse B16F0 melanoma cells in a long-term cytotoxicity assay. It was also confirmed that IL-12-activated IFN-gamma producing Th1 cells were accumulated in tumor local site. Thus, IL-12 appeared to have a capability of stimulating selective migration of antitumor cells into lymph nodes and tumor local sites.

Animals↗
PubMed1998

The restraint stress drives a shift in Th1/Th2 balance toward Th2-dominant immunity in mice.

When mice were physically restrained in 50-ml tubes for 24 h, a marked decrease of NK activity was demonstrated in parallel with the elevation of serum corticosterone levels. The release of mice from restraint stress resulted in the recovery of NK activity, with a decrease of serum corticosterone levels within 48 h. Using this stress model, we also investigated the influence of restraint stress on mouse Th1/Th2 balance. Consistent with the decrease of NK activity, IFN-gamma production of mouse spleen cells greatly reduced after suffering from restraint stress. In contrast, the IL-4 producing ability of spleen cells was not so much affected by restraint stress. These results initially indicated that stress may induce the skewing of the Th1/Th2 balance toward Th2-dominant immunity, which stimulates the occurrence of infectious diseases and allergic disorders.

Animals↗