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M Ohkuma

Publications and source records attributed to M Ohkuma.

At least 55 records · Page 3Linked to original sources
PubMed1995

Proliferation of intracellular membrane structures upon homologous overproduction of cytochrome P-450 in Candida maltosa.

In an alkane-assimilating yeast, Candida maltosa, a cultivation on alkane causes both induction of endoplasmic reticulum (ER)-resident membrane proteins, such as cytochrome P-450, and proliferation of ER. In this study, individual genes for alkane-inducible forms of cytochrome P-450 (P-450alk) were homologously overexpressed in C. maltosa using a galactose-inducible expression system developed in this yeast. Immunoelectron microscopy revealed that, upon the overexpression, a dramatic proliferation of ER occurred, in which overproduced P-450alk protein accumulated. The proliferated membranes were mainly tubular forms and stacks of paired membranes were also observed after prolonged expression. The tubular forms were morphologically very similar to the proliferated ER in alkane-induced C. maltosa cells. The observed proliferation of ER membranes by homologous overproduction of P-450alk, here depicted, will provide a unique opportunity for investigating the mechanisms by which cells regulate ER biogenesis, in comparison with the intrinsic form of ER proliferation.

Candida↗
PubMed1995

In vivo evidence for non-universal usage of the codon CUG in Candida maltosa.

An alkane-assimilating yeast Candida maltosa had been studied in order to establish systems suitable for biotransformation of hydrophobic compounds. However, functional expression of heterologous genes tested for this purpose had not been successful in several cases. On the other hand, it had been reported that the codon CUG, a universal leucine codon, is read as serine in C. cylindracea. The same altered codon usage had also been suggested by in vitro experiments in some Candida yeasts which are phylogenetically closely related to C. maltosa. In this study we have shown that the failure in functional expression of a heterologous gene is due to the fact that the codon CUG is read as serine in C. maltosa. This conclusion was drawn from the following experimental results: (1) when a cytochrome P450 gene of C. maltosa containing a CTG codon was expressed in C. maltosa, the corresponding amino acid was found to be serine, and not leucine; (2) a tRNA gene with an almost identical structure to that of the tRNASerCAG gene of C. albicans could be isolated from the genome of C. maltosa; (3) the Saccharomyces cerevisiae URA3 gene, which has one CTG codon, could not complement the ura3 mutation of C. maltosa as itself, but when the CTG codon was changed to another leucine codon, CTC, the mutated gene could complement the ura3 mutation. The last result is the first example of succeeding in functional expression of a heterologous gene in Candida species having an altered codon usage by changing the CTG codon in the gene to another codon.

Amino Acid Sequence↗
PubMed1995

Expression of the Escherichia coli fabA gene encoding beta-hydroxydecanoyl thioester dehydrase and transport to chloroplasts in transgenic tobacco.

The fabA gene of Escherichia coli encodes beta-hydroxydecanoyl thioester dehydrase (HDDase), a pivotal enzyme in the biosynthesis of the unsaturated fatty acid cis-vaccenic acid, through the anaerobic pathway. This enzyme is specific to bacterial fatty acid biosynthetic pathways, although other enzymes for fatty acid synthesis are very similar in plants and bacteria. We constructed chimaeric plant expression vectors, pfab21 and pfab22, carrying the fabA gene under the transcriptional control of the cauliflower mosaic virus (CaMV) promoter of 35S RNA. In pfab21, fabA was placed directly under the control of the CaMV 35S promoter; whereas in pfab22, the DNA sequence coding for the chloroplast-targeting transit peptide (TP) of the pea ribulose-1,5-bisphosphate carboxylase (RuBisCo) small subunit was fused to the fabA gene in order to allow transport of HDDase to the chloroplast, the organelle responsible for de novo fatty acid biosynthesis in plants. Transgenic plants of Nicotiana tabacum were obtained by Agrobacterium-mediated transformation with pfab21 or pfab22. Expression of fabA transcripts of sizes expected from the chimaeric constructs was shown by RNA blot hybridization. The HDDase protein derived from pfab22 was correctly processed and transported to chloroplasts in transformed plants. The enzymatic activity of HDDase was also detected in chloroplasts isolated from the transformants derived from pfab22 (but not pfab21) and in total leaf protein of all transformants. However, no significant changes were observed in the fatty acid compositions, including cis-vaccenic acid, of leaf chloroplasts and self-fertilized seeds. These results are discussed in relation with the possible structural organization of plant fatty acid synthase.

Base Sequence↗
PubMed1995

CYP52 (cytochrome P450alk) multigene family in Candida maltosa: identification and characterization of eight members.

Previously, we characterized three genes and presented evidence for an n-alkane-inducible cytochrome P450 (P450alk) multigene family in an n-alkane-assimilating and diploid-type yeast, Candida maltosa. In the present report, we isolated and characterized additional members of this gene family, including a total of thirteen P450alk-related sequences (eight genes and five of their alleles). Two sets, each consisting of two genes, were tandemly arranged in the genome. A gene replacement experiment showed that at least one gene had only a single allele in the genome. The determined nucleotide and the deduced amino acid sequences indicated that all had a characteristic constituent for P450s and exhibited amino acid identities from 94% to 37% to each other. Six genes showed relatively higher similarities to each other than to the other two genes and were thus classified into a subfamily. All the members of this subfamily were assigned to the same single chromosome, showing a good correlation between sequence similarity and chromosomal linkage. Although all the genes except for one were induced by n-alkane, their inducibilities by some other aliphatic carbon sources showed variabilities.

Alleles↗
PubMed1995

Evidence that the expression of the gene for NADPH-cytochrome P-450 reductase is n-alkane-inducible in Candida maltosa.

A gene coding for NADPH-cytochrome P-450 reductase of an n-alkane-assimilating yeast, Candida maltosa, was isolated and sequenced. Northern analysis and assay of the expression of the reporter gene under the control of the promoter of this gene showed that the transcriptional level was induced 4 to 8-fold in cells grown on n-alkane relative to cells grown on glucose.

Alkanes↗
PubMed1994

Expression of an endogenous and a heterologous gene in Candida maltosa by using a promoter of a newly-isolated phosphoglycerate kinase (PGK) gene.

A gene encoding phosphoglycerate kinase (PGK) was isolated from the genomic library of C. maltosa to construct an expression vector for this yeast. The PGK gene had an open reading frame of 1,251 base pairs encoding approximately 47-kDa polypeptide of 417 amino-acid residues. Expression of this gene assayed by Northern-blot analysis was significantly induced in cells grown on glucose but not in cells grown on n-tetradecane, n-tetradecanol, or oleic acid. By using the promoter region of this gene, an expression vector (termed pMEA1) for C. maltosa was constructed and expression of an endogenous gene (P450alk1 encoding one of cytochrome P450s for n-alkane hydroxylation in C. maltosa) and a heterologous gene (LAC4 encoding Kluyveromyces lactis beta-galactosidase) was tested. Expression of P450alk1 gene was confirmed at both mRNA and protein levels. LAC4 gene expression was confirmed by determining beta-galactosidase activity. The activity in cells grown on various carbon sources correlated very well with the expression levels of PGK mRNA in these cells.

Alkanes↗
PubMed1993

Cloning of the C-URA3 gene and construction of a triple auxotroph (his5, ade1, ura3) as a useful host for the genetic engineering of Candida maltosa.

The C-URA3 gene of the n-alkane assimilating-yeast Candida maltosa was cloned by complementation of the ura3 mutation of Saccharomyces cerevisiae. The nucleotide sequence of C-URA3 and its deduced amino-acid sequence showed significant homology to those of the orotidine 5'-phosphate decarboxylases of other fungal species. To construct a useful host for genetic engineering of C. maltosa using C-URA3 as a marker, one allele of C-URA3 in a double auxotroph (his5, ade1) was disrupted by C-ADE1, and subsequently two kinds of ura3 mutants were isolated by selecting for spontaneous 5-fluoro-orotic acid (5FOA) resistance. One of the mutants was homozygous for the disruption (ura3::C-ADE1/ura3::C-ADE1); the other was heterozygous (ura3::C-ADE1/ura3). The ura3::C-ADE1 allele in the latter strain was re-substituted by C-URA3 to rescue the adenine auxotroph (his5, ade1, C-URA3/ura3). Finally, by selecting a 5FOA-resistant mutant, a triple auxotroph (his5, ade1, ura3/ura3) was isolated.

Amino Acid Sequence↗
PubMed1993

[Clinical study of patients with deep-seated fungal infection associated with hematological diseases].

An assessment has been made regarding usefulness of measuring beta-D-glucan (beta-glucan) as fungal serodiagnosis in 50 cases of fungal infection with hematological diseases. Further, an assessment has been made regarding relation between hematological findings and therapeutic effect by administering miconazole, an antifungal agent (MCZ: Florid, clinically to the subjects. Positivity of beta-glucan (beta-glucan > or = 10 pg/ml) was observed in 54.5% (24/44), and the effective rate of MCZ in the positive cases was 75.0% (18/24). In the cases in whom fungus was detected, beta-glucan-positive rate was 50.0% (8/16), and MCZ-effective rate in beta-glucan-positive cases was 62.5% (5/8). The total effective rate of MCZ was 80% (40/50). Side effects were observed in 3 cases, but continual administration of MCZ was possible in all of the 3 cases. By the assessment regarding the relation between hematological findings and therapeutic effect of MCZ, it was found that the effective rates in the cases who underwent a transition with neutrophil and lymphocyte counts less than 500/microliters during the period of MCZ administration were 64.7% (11/17) and 50% (5/10), respectively, and large effects were observed in the cases who underwent a transition with the neutrophil and lymphocyte counts more than 500/microliters was 86.7% (19/22) and 91.7% (22/24), respectively. These results suggested that lymphocytes rather than neutrophils had an important role in the morbidity of fungal infection. It was noteworthy that MCZ was effective for the treatment of deep seated mycosis and significant effective rate was obtained in the group of patients who had neutrophils and lymphocytes less than 500/microliters.

Adolescent↗
PubMed1993

Evolutionary position of n-alkane-assimilating yeast Candida maltosa shown by nucleotide sequence of small-subunit ribosomal RNA gene.

We clarified the evolutionary position of Candida maltosa, an n-alkane-assimilating yeast, by sequencing the nucleotides of the small-subunit ribosomal RNA gene. Phylogenetic analyses showed the close evolutionary relationships of C. maltosa with C. tropicalis, C. viswanathii, C. albicans, C. parapsilosis, and C. guilliermondii, forming a sub-group within this genus.

Alkanes↗
PubMed1993

Enzyme-histochemical staining of dermal lymphatic capillaries by guanylate cyclase.

Human foreskins were examined for enzyme-histochemical staining of microvessels using guanylate cyclase, an enzyme similar to adenylate cyclase. Like 5'-nucleotidase (which hydrolyzes 5'-adenosine monophosphate to adenosine), and adenylate cyclase (which converts adenosine triphosphate to cyclic AMP), guanylate cyclase selectively stains positive for lymphatic capillaries and therefore may be another useful histochemical marker to differentiate dermal lymph from blood capillaries.

Adult↗
PubMed1993

Lipoperoxide in the dermis of patients with lymph stasis.

Lipoperoxide has been detected in the thoracic duct lymph of the dog. This finding suggests that lipoperoxides are normally transported in lymph and with impaired lymph drainage may be deposited in the skin and contribute to the soft tissue changes characteristic of chronic lymphedema. Accordingly, after obtaining skin specimens taken from 8 patients (7 with obstructive lymphedema) with lower extremity lymph stasis we determined dermal malondialdehyde (MDA) content (after conversion to fluorescent thiobarbituric acid or TBA), a marker of lipoperoxide. In all 7 patients with obstructive lymphedema, the MDA levels were increased compared to control dermis (p < 0.05). We suggest that inability to clear lipoperoxides from the dermis with lymphatic insufficiency may contribute to the pathogenesis and structural skin derangements of chronic lymphedema.

Adult↗
PubMed1993

[Chronic daily oral low-dose etoposide therapy for an aged patient with aggressive lymphoma: a successful case report].

A 72-year-old female from Fukuoka Prefecture was admitted to our hospital complaining of puffy face and general malaise in April, 1992. Physical examinations revealed generalized lymphadenopathy and hepatosplenomegaly complicated with superior vena cava syndrome. A histological diagnosis of diffuse large cell-type malignant lymphoma was made by cervical lymph node biopsy according to the Working Formulation. Immunohistochemical staining showed the lymphoma cells were of T cell lineage (CD43+). Though anti-human T lymphotropic virus type I (HTLV-I) antibody was positive, southern blot analysis of the cells did not reveal monoclonal integration of HTLV-I proviral DNA. Her liver and renal function were almost normal but there was slight elevation of LDH. Considering her age, oral administration of low-dose etoposide (25 mg/day) was started. In 4 days, her lymph nodes decreased in size with subjective improvements. Ten weeks later, examinations of the hand, ultrasonogram and CT scan showed lymphadenopathy and hepatosplenomegaly had disappeared completely. The neutrophil count was consistently above 1,500/microliters, with no remarkable adverse effects and only a moderate degree of alopecia. Thus, low-dose etoposide therapy was considered to be very useful in such a case of malignant lymphoma in the elderly.

Administration, Oral↗
PubMed1992

The third molecule associated with interleukin 2 receptor alpha and beta chain.

It is known that the affinity cross-linking study of the human high-affinity Interleukin 2 (IL-2) receptor reveals triplet bands consisting of 70 kDa alpha chain(Tac)-IL-2 and the 90/80 kDa doublet. We found the cell lines lacking the lower band of the doublet in spite of the expression of both alpha and beta chains. No IL-2 binding was detectable in the presence of anti-Tac antibody in these cells. Immunoprecipitation from the cell extract of [125 I] IL-2-cross-linked T cells with anti-beta chain polyclonal IgG detected the upper band, but not lower band of the doublet. These data suggest that the lower band of the doublet represents an unknown IL-2-binding protein (p65) distinct from the beta chain and this molecule may be involved in the intermediate-affinity IL-2 binding together with the beta chain.

3T3 Cells↗
PubMed1992

Lymphedema treated by microwave and elastic dressing.

For lymphedema, surgical treatment is contraindicated and other conservative treatments including oral medicine are not sufficient. Treatment with microwave and elastic dressing has been evaluated in 30 cases of lymphedema with 11 control patients who received only one of the two treatments. Twenty-four patients were effectively treated. In six, edema was not decreased as compared with reduction due to daily physical activity. It was observed that the earlier the patient was treated after onset of edema, the better the results. Six of 10 control patients who received only elastic dressing showed slight decrease in edema. A patient who received only microwave did not show any improvement. There was no recurrence in half of the treated patients. Treatment of lymphedema by microwave and elastic dressing appears satisfactory, particularly when given at an early stage of the disease.

Adult↗
PubMed1992

Enzyme-histochemical identification of the human lymphatic capillary by adenylate cyclase.

This investigation was performed in order to establish a new histochemical method for the identification of lymphatic capillaries. The microvasculature in specimens from human foreskin was examined for adenylate cyclase and alkaline phosphatase activity by light and electron microscopy. Lymphatic capillaries showed positive adenylate cyclase reactivity and negative alkaline phosphatase reactivity whereas the blood capillaries showed a positive reaction for alkaline phosphatase and a negative one for adenylate cyclase. The presence of adenylate cyclase activity in the endothelium of the lymphatic capillary may relate to microvascular function including the transcapillary exchange of water and solutes.

Adenylyl Cyclases↗
PubMed1991

CYP52 (cytochrome P450alk) multigene family in Candida maltosa: molecular cloning and nucleotide sequence of the two tandemly arranged genes.

Southern blot analysis under low-stringency conditions using a previously isolated n-alkane-inducible cytochrome P450 (P450alk) gene as a probe revealed the presence of multiple P450alk-related genes in the genome of Candida maltosa. Nine P450alk-related genes (one reported previously and eight in the present report) were isolated from a genomic library constructed from this strain, and these were classified on the basis of sequence similarities into three pairs of putative allelic genes and three nonallelic genes. Two pairs of these alleles were tandemly arranged in the genome. The complete nucleotide sequences of one of these pairs were determined and compared to other members of this P450 family (CYP52) in C. maltosa and C. tropicalis. Northern blot analysis further showed that these genes were regulated by carbon sources. These results provide evidence for a P450alk (CYP52) multigene family in C. maltosa.

Amino Acid Sequence↗