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Biomedical subjects

M Takagi

Publications and source records attributed to M Takagi.

At least 433 records · Page 24Linked 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

Interleukin-3 (IL-3) poor-responsive inbred mouse strains carry the identical deletion of a branch point in the IL-3 receptor alpha subunit gene.

Interleukin-3 (IL-3) stimulates colony formation of multiple lineages of hematopoietic cells. Bone marrow cells of A/J mice are nonresponsive to IL-3, and this observation has recently been correlated with aberrant mRNA splicing and impaired expression of the IL-3 receptor alpha subunit (IL-3R alpha), a binding component of the high-affinity receptors. We examined the IL-3R alpha gene in 27 inbred mouse strains and found the identical mutation, a 5-bp deletion at the branch point of intron 7, in 10 of these mouse strains. Bone marrow cells isolated from these 10 mouse strains did not express IL-3R alpha on the cell surface and did not form colonies in response to IL-3. Because the defective IL-3R alpha gene was found in several distantly related mouse strains, it appears to be a recessive allele rather than a sporadic mutation. In contrast, only 1 of 21 wild-derived mouse strains carried the 5-bp deletion in the IL-3R alpha gene. This study suggests that IL-3 function is not required for normal hematopoiesis in mice, but the retention of the IL-3 and IL-3R system may be of some selective advantage in wild populations.

Alleles↗
PubMed1995

Impaired interleukin-3 (IL-3) response of the A/J mouse is caused by a branch point deletion in the IL-3 receptor alpha subunit gene.

Interleukin-3 (IL-3) alone does not support hematopoietic colony formation of bone marrow cells from the A/J mouse. To elucidate the molecular lesion in A/J mice, we examined expression of the alpha and beta subunits of the IL-3 receptor (IL-3R). While IL-3R beta was normally expressed, IL-3R alpha was not detectable on the surface of A/J-derived cells by antibody staining. Genetic linkage analysis using recombinant inbred mouse strains between A/J and IL-3-responsive C57BL/6 indicated that the IL-3R alpha gene locus was responsible for the impaired IL-3 response in A/J mice. Molecular cloning and characterization of A/J-derived IL-3R alpha cDNA revealed that it lacked the sequence corresponding to exon 8, which encodes 10 amino acid residues in the extracellular domain. The aberrant splicing was due to a 5 base pair deletion at the branch point in intron 7 and was reproduced in heterologous cells by transfecting with an IL-3R alpha minigene carrying the deleterious intron. The A/J-specific abnormal form of IL-3R alpha was localized inside the cells, but not on the cell surface, providing the molecular basis for the impaired IL-3 response in the A/J mouse.

Amino Acid Sequence↗
PubMed1995

Multi-colony stimulating activity of interleukin 5 (IL-5) on hematopoietic progenitors from transgenic mice that express IL-5 receptor alpha subunit constitutively.

The interleukin 3 (IL-3), IL-5, and granulocyte/macrophage colony-stimulating factor receptors consist of a cytokine-specific alpha subunit and the common beta subunit. Whereas IL-3 stimulates various lineages of hematopoietic cells, including multipotential progenitors, IL-5 acts mainly as an eosinophil lineage-specific factor. To investigate whether the lineage specificity of IL-5 is due to restricted expression of the IL-5 receptor alpha subunit (IL-5R alpha), we generated transgenic mice that express the mouse IL-5R alpha constitutively by phosphoglycerate kinase promoter. The transgenic mouse expressed IL-5R alpha ubiquitously, and the bone marrow cells formed various types of colonies, including multi-lineage colonies, in response to IL-5. IL-5 also supported formation of both multi-lineage and blast cell colonies from dormant progenitors of the 5-fluorouracil-treated transgenic mice. The cells composing the blast cell colony gave rise to many colonies including multi-lineage colonies when they were replated in secondary culture containing either Il-5 or IL-3. There was no significant difference in replating efficiency or in types of secondary colonies between IL-5- and IL-3-stimulated cultures. Conversely, the cells from the IL-3-induced blast cell colonies of the transgenic mice proliferated in response to either IL-3 or IL-5. Thus, the development of the progenitors can be equally supported by either IL-5 or IL-3, suggesting that intracellular signals from the IL-3R can be replaced by those from IL-5. These results strongly suggest that the lineage specificity of IL-5 is mainly due to the restricted expression of IL-5R alpha.

Animals↗
PubMed1995

Analysis of the 3-phosphoglycerate kinase 2 promoter in Rhizopus niveus.

Promoter analysis was performed on the Rhizopus niveus 3-phosphoglycerate kinase 2-encoding gene (pgk2), one of the two pgk genes (pgk1 and pgk2) from this filamentous fungus sequenced so far. Deletion mutants of the promoter region were fused to the Escherichia coli uidA gene (which codes for beta-glucuronidase; GUS), and introduced into R. niveus to measure the intracellular GUS activities of the transformants. Deletion of the sequence between nt -174 to -133 (numbers indicate the position from the putative translation start codon) caused a significant decrease in the ratio of the GUS activity of the transformant cultured in glucose medium compared to that in glycerol medium. In this region, a 21-nt sequence which is well conserved between pgk1 and pgk2 is present. When it was inserted into the promoter region of the uninducible gene encoding RNase Rh of R. niveus, ligated in front of uidA and introduced into R. niveus, the GUS activity of the transformant was greatly induced by glucose, but less by glycerol. We therefore suggest that the 21-nt sequence is a glucose-inducible transcriptional activator of R. niveus. This is the first report on a transcriptional activator in zygomycetes.

Base Sequence↗
PubMed1995

Elastase activity, uninhibited by alpha 1-antitrypsin, in the periprosthetic connective matrix around loose total hip prostheses.

To clarify the proteolytic cascade in the loosening of total hip prostheses, the presence, tissue localization, and content of the serine proteinase, elastase, and its endogenous inhibitor, alpha 1-antitrypsin, were studied in periprosthetic tissues around 12 loose hip prostheses by immunohistochemistry, spectrophotometric enzyme assay, and immunoblot analysis, and the results were compared with those in control synovial tissue samples from eight knees. Increased numbers of elastase-immunoreactive cells and elevated elastase activity, inhibited by the addition of native alpha 1-antitrypsin, were observed both in the interface tissues between the bone and implants and in the pseudocapsular tissues from around loose hip prostheses. Elevated elastase activity, uninhibited by alpha 1-antitrypsin in situ and inhibited by the addition of native inhibitor, suggests a proteinase-inhibitor imbalance that contributes to the weakening of periprosthetic tissues and thus causes the loosening of hip prostheses.

Adult↗
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

The influence of polyvinylpyrrolidone on freezing of bovine IVF blastocysts following biopsy.

A study was conducted to develop a better freezing protocol for in vitro developed biopsied bovine blastocysts. Biopsied blastocysts were exposed to 1.8 M ethylene glycol (EG) + 0.05 M trehalose (T) and different concentration (5, 10, and 20%) of polyvinylpyrrolidone (PVP). Exposure to the solutions alone did not affect their in vitro development (Experiment 1). Experiments 2, 3, and 4 tested the viability of biopsied blastocysts cryopreserved in 1.8 M EG + different concentrations of T (0, 0.05, 0.1, and 0.3 M), 1.8 M EG + different concentrations of PVP (0, 5, 10, and 20%), and 1.8 M EG + 0.05 M T + different concentrations of PVP (0, 5, 10, and 20%), respectively. The proportion of biopsied blastocysts that reexpanded following cryopreservation in 1.8 M EG + 0.05 M T (38.5%) and 1.8 M EG + 0.1 M T (36.1%) was significantly (P < 0.05) higher than the proportion that reexpanded in 1.8 M EG + 0.3 M T (13.9%) (Experiment 2). The viability and the percentage of embryos that developed to > 250 microns in diameter in the 5, 10, and 20% PVP groups (77.8 and 50.0%, 78.1 and 43.8%, 76.9 and 65.4%, respectively) were significantly higher than those that developed cryopreserved without PVP (55.1 and 20.7%) (Experiment 3). Optimum development of in vitro culture of frozen-thawed biopsied blastocysts was obtained using 1.8 M EG + 0.05 M T and 20% PVP. Analysis of blastocysts > 250 microns in diameter showed that the number of ICM cells of biopsied blastocysts cryopreserved in 1.8 M EG + 0.05 M T with or without PVP was not different from the number of unfrozen biopsied blastocysts. These results indicate that PVP has some beneficial effect on freezing of biopsied bovine blastocysts.

Animals↗
PubMed1995

Cloning of the Rhizopus niveus pyr4 gene and its use for the transformation of Rhizopus delemar.

We have cloned a pyr4 gene encoding orotidine-5'-monophosphate decarboxylase of the filamentous fungus Rhizopus niveus. The pyr4 gene of R. nivens has an open reading frame composed of 265 amino-acid residues and has two putative introns. We have also isolated a pyr4 mutant of Rhizopus delemar from 5-fluoroorotic acid-resistant mutants and transformed it with the pyr4 gene of R. niveus as a selectable marker. Introduced DNA was integrated into the chromosome in a multiple tandem array. The mitotic stability of the introduced DNA was increased by a repeated sporulation process. The expression of the Escherichia coli beta-glucuronidase gene in R. delemar was successfully obtained under the control of the pgk2 gene promoter of R. niveus by co-transformation with the pyr4 gene.

Amino Acid Sequence↗
PubMed1995

Characterization of two Japanese encephalitis virus strains isolated in Thailand.

Two strains of Japanese encephalitis (JE) virus were isolated from a pool of Culex tritaeniorhynchus captured in 1992 and another pool of Cx. vishnui captured in 1993, in Chiang Mai Area, Northern Thailand. These two strains, ThCMAr44/92 and ThCMAr67/93, could not be identified either as Nakayama or JaGAr01 subtype by the hemagglutination-inhibition (HI) and the neutralization (N) tests using immune sera raised against these standard JE virus strains. Reverse transcription-polymerase chain reaction showed the presence of JE-specific conserved sequences in these strains. Sequencing of 240 nucleotides in their PrM gene region identified that these two strains belong to the genotype 1 of JE virus. Nucleotide and encoded amino acid sequences of their envelope glycoprotein gene revealed 98.8 and 99.8% identity, respectively. These two strains shared 77.8 to 87.7% homology in the nucleotide sequence and 90.0 to 98.8% homology in the amino acid sequence with other reported JE strains. Five strain-specific amino acid changes were noted in ThCMAr44/92 strain, while one in ThCMAr67/93. In addition, four common amino acid changes were found in both strains. Thus, the findings indicated that these two strains were structurally different from each other as well as different from all the reported strains which was in agreement with the serological tests by hemagglutination-inhibition and neutralization.

Animals↗
PubMed1995

Comparison of the entire nucleotide and deduced amino acid sequences of the attenuated hog cholera vaccine strain GPE- and the wild-type parental strain ALD.

We have determined the complete nucleotide sequences of a live attenuated hog cholera virus (HCV) and its progenitor strain. The viral RNA of each strain consisted of 12,298 nucleotides including untranslated regions of 373 and 228 bases at the 5' and 3' end, respectively. There was a single large open reading frame spanning 11,697 nucleotides which could encode a large protein of 3,899 amino acids with a calculated molecular weight of 438-kDa. We have found 225 nucleotide difference between the two strains, of which six were located in the untranslated region. Four-sixths of these differences resulted in amino acid substitutions.

Amino Acid Sequence↗
PubMed1995

Myocardial recovery during post-ischemic reperfusion: optimal concentrations of Na+ and Ca2+ in the reperfusate and protective effects of amiloride added to cardioplegic solution.

The effects of Na+ and Ca2+ concentrations in the reperfusate on post-ischemic myocardial recovery were examined. Also, the myocardial protective effects of amiloride, an inhibitor of the Na+/Ca2+ and Na+/H+ exchange systems, added to cardioplegic solutions were assessed, using an isolated working rat heart perfusion system. Global myocardial ischemia was induced by 30-min normothermic cardioplegic arrest, using St. Thomas' solution. The concentration of Na+ in the reperfusate varied, stepwise, from 75 to 145 mM/l, and that of Ca2+, from 0.1 to 2.5 mM/l. In this study post-ischemic functional recovery was best at 110mM/l Na+ and 1.2-1.8 mM/l Ca2+ in the reperfusate. A significantly greater post-ischemic functional recovery and a lower creatine kinase release were observed when amiloride was added to the cardioplegic solution. Ca2+ overload via Na+/Ca2+ and Na+/H+ exchange systems would, thus, appear to be due, at least in part, to post-ischemic reperfusion injury.

Amiloride↗
PubMed1995

Gap-overlap effects on latencies of saccades, vergence and combined vergence-saccades in humans.

We examined the effect of gap-overlap stimuli on the distribution of latencies for pure saccades, pure vergence and combined saccades and vergence in three normal subjects. With the gap stimulus, a distinct peak of "express saccades" occurred, both with and without associated vergence, but a distinct "express vergence" response was not identified. Nevertheless, with the gap stimulus there was a decrease in vergence latencies (17 msec), but less so than for saccades (41 msec). In the combined paradigm the gap effects on saccades and vergence resembled those for each component made alone. In addition, the latencies of the saccade and vergence components were linearly correlated with an average slope of 0.5. To explain these results we suggest that there is common signal processing at an early stage of saccade and vergence initiation, which is followed by activity that builds in separate trigger mechanisms that can be influenced by the conditions of fixation.

Adult↗
PubMed1995

Dysembryoplastic neuroepithelial tumor: report of three cases.

BACKGROUND: Dysembryoplastic neuroepithelial tumor (DNT) is one of the mixed neuronal-glial tumors that should be differentiated from low-grade gliomas. The recognition and correct diagnosis of DNT is important because this tumor is curable by excision. METHODS: We report three cases of histologically proven DNTs with various image studies performed preoperatively and describe characteristic findings of them, as well as those of previous reports. RESULTS: In our three cases, computed tomography (CT) showed a well-defined, nonenhancing hypodense area. Magnetic resonance imaging (MRI) demonstrated a cortical mass of hypointensity on T1- and of marked hyperintensity on T2-weighted images. I-123-IMP or Tc-99m-HMPAO single-photon emission computed tomography (SPECT) images showed marked hypoperfusion of the lesion. Thallium-SPECT showed no uptake. CONCLUSIONS: I-123-IMP, Tc-99m-HMPAO, and Thallium-SPECT images, as well as CT and MRI, may be helpful in distinguishing DNT from low-grade gliomas.

Adult↗
PubMed1995

Differentiation between glycoprotein III gene-deleted vaccine and wild-type strains of pseudorabies virus by polymerase chain reaction (PCR).

One of the attenuated and genetically recombinant modified-live viral (MLV) vaccine strains currently used contains a deletion in its glycoprotein III (gIII) gene, while prototypic wild-type pseudorabies (WT-PR) viruses contain an intact gIII gene. A polymerase chain reaction (PCR) system differentiating, based on this difference, between the vaccine virus and prototypic WT-PR viruses was investigated. This PCR system utilized two consecutive stages. Primers for the first-stage PCR were designed so as to amplify of DNA fragments lengths in respect to the vaccine and WT-PR viruses. The second-stage PCR amplification for improving the sensitivity and specificity and for confirming of the sites deleted from the first-stage PCR products produced an all-or-none result: internal DNA fragments were derived from only WT-PR viruses but not from the vaccine virus. These PCR-amplified fragment length polymorphisms clearly distinguished the vaccine virus from WT-PR viruses. The vaccine and WT-PR viruses in mixtures were each identified in this PCR system. This PCR system may permit rapid and sensitive detection of PR viral gIII gene, analysis of the genotype of PR virus isolates, and also examination of the isolates for purity and identity.

Base Sequence↗