Biomedical subjects
Publications and source records attributed to T Toda.
A 20-month-old phenotypically male child with ambiguous external genitalia came to this hospital. Chromosomal and DNA analyses revealed 46/XX karyotype and absence of SRY gene, respectively. Two gonadal masses in the labioscrotal folds histologically showed both ovarian and testicular tissues. Clinicopathological features of the patient suggested true hermaphroditism with bilateral ovotestes.
We have isolated a gene, pmk1+, a third mitogen-activated protein kinase (MAPK) gene homolog from the fission yeast Schizosaccharomyces pombe. The predicted amino acid sequence shows the most homology (63 to 65% identity) to those of budding yeast Saccharomyces Mpk1 and Candida Mkc1. The Pmk1 protein contains phosphorylated tyrosines, and the level of tyrosine phosphorylation was increased in the dsp1 mutant which lacks an attenuating phosphatase for Pmk1. The level of tyrosine phosphorylation appears constant during hypotonic or heat shock treatment. The cells with pmk1 deleted (delta pmk1) are viable but show various defective phenotypes, including cell wall weakness, abnormal cell shape, a cytokinesis defect, and altered sensitivities to cations, such as hypersensitivity to potassium and resistance to sodium. Consistent with a high degree of conservation of amino acid sequence, multicopy plasmids containing the MPK1 gene rescued the defective phenotypes of the delta pmk1 mutant. The frog MAPK gene also suppressed the pmk1 disruptant. The results of genetic analysis indicated that Pmk1 lies on a novel MAPK pathway which does not overlap functionally with the other two MAPK pathways, the Spk1-dependent mating signal pathway and Sty1/Spc1/Phh1-dependent stress-sensing pathway. In Saccharomyces cerevisiae, Mpk1 is involved in cell wall integrity and functions downstream of the protein kinase C homolog. In contrast, in S. pombe, Pmk1 may not act in a linear manner with respect to fission yeast protein kinase C homologs. Interestingly, however, these two pathways are not independent; instead, they regulate cell integrity in a coordinate manner.
We analyzed DNA from 153 Japanese men with azoospermia or severe oligozoospermia whose Y chromosomes were cytogenetically normal. A total of 23 loci on the Y chromosome were examined: 15 loci within interval 6 including YRRM1 and DAZ, and 8 loci outside interval 6. Microdeletions were observed in 20 individuals. All deletions involved at least one locus within interval 6. The YRRM1 gene deletion was found in 4 patients, of whom 2 also lacked the SMCY gene. The remaining 16 individuals shared the absence of 10 loci between DYS7C and DYS239 including the DAZ gene. Among them, 13 were azoospermic while 3 showed a little sperm production, indicating that the common deletion resulted in phenotypic diversity. Since there is no region commonly deleted in all patients, azoospermia caused by deletion of the Y chromosome long arm may be genetically heterogeneous.
Cell morphogenesis is a fundamental phenomenon that involves understanding a number of biological processes including the developmental program, polarity and cell division. Fission yeast sts5 mutant cells are round rather than cylindrical with cortical actin randomly dispersed. Genetic analyses demonstrate that the sts5+ gene is required for maintenance of cell shape during interphase when the cell normally exhibits polarised growth. The sts5 mutant is not defective in cell wall integrity. Deletion of ppe1+, which encodes a type 2A-like protein phosphatase, shows similar phenotypes to the sts5 mutant and these two mutations are synthetically lethal. Multicopy plasmids containing either the protein kinase C-like gene pck1+ or the protein tyrosine phosphatase pyp1+, an inhibitor of an osmosensing Sty1/Spc1 MAP-kinase, are capable of suppressing the sts5 mutation. Consistent with this, we have found that the wis1 mutation, which is defective in a MAP-kinase kinase of the pathway, suppresses the sts5 mutation. The predicted sts5+ gene product exhibits sequence similarity to two yeast proteins, Dis3 and Ssd1 and a nematode protein, F46E8.6, where the former two yeast proteins have been shown to be involved in cell cycle control and cell morphogenesis. The sts5+ gene is not essential for cell viability, but is absolutely required for polarised growth as the gene disruption showed the same phenotypes as those of the original mutants. Overexpression of the sts5+ gene resulted in altered cell morphology and, cortical actin in these overproducing cells was also abnormal, fainter and often dispersed. Anti-Sts5 antibody specifically detected a 130 kDa protein by western blotting. A green fluorescent protein-Sts5 fusion protein localised in the cytoplasm with a discrete punctate pattern, suggesting that the Sts5 protein is a component of a novel structure. These results have indicated that the Sts5 protein is a crucial determinant of polarised growth and that it functionally interacts with the serine/threonine phosphatase, protein kinase C, and an osmosensing MAP-kinase to maintain cell morphology.
We developed a method that allows rapid isolation of mutant Bacillus subtilis 168 carrying an inversion of a specific DNA segment of the genome. Two incomplete neomycin resistance gene cassettes were integrated at both ends of the 1652-kb segment to be inverted. Reciprocal recombination within the 590-bp homologous region of these two cassettes created an intact neomycin resistance gene with concomitant inversion of the 1652-kb segment flanked by the two cassettes. Structure of the mutant genome was verified by analyzing the physical map for rare cutting endonucleases, SfiI, NotI, I-CeuI, and I-SceI. The inversion rate was estimated to be 6.9 +/- 1.4 x 10(-8)/cell/cell division at 37 degrees C. The method should be in principle applicable not only to other regions of the B, subtilis genome but also to other bacterial genomes.
To compare the atherogenecity of different fats and oils, a total of forty, 40-day-old male Japanese quails were fed one of the following diets for three months: basal diet (control), a diet-containing 15% corn oil (CO) and 2% cholesterol (CH), a diet-containing 15% oleic acid (OL) and 2% CH, a diet-containing 15% perilla oil (PE) and 2% CH, a diet-containing 15% evening [corrected] primrose oil (PR) and 2% CH. A higher plasma cholesterol concentration was found in the birds in the CO and OL groups, whereas the PE and PR groups showed a much lower level of plasma cholesterol than the CO and OL groups. In proportion to the increased plasma cholesterol, both CO and OL groups showed narrowing of the lumen of the ascending aorta and its large branches due to marked lipid-rich intimal thickening. Ultrastructural changes in the ascending aorta and its large branches were correlated with the degree of intimal thickening. The major foam cell types were macrophages and fibroblastic cells. The PE and PR groups showed the fewest lipid-rich intimal thickening lesions in their ascending aorta and its large branches. These findings suggest that the alpha-linolenic acid contained in perilla oil is less atherogenic than oleic and linoleic acid, and gamma-linolenic acid contained in evening [corrected] primrose oil has a tendency to decrease the plasma lipid level.
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The hyperlipidemia- and atherosclerosis-prone (HAP) Japanese quail is a strain developed for the study of atherosclerosis by genetic selection from the commercially available (CA) Japanese quail. To delineate the characteristics of cholesterol metabolism in this strain, concentrations of serum lipids as well as hepatic enzyme activities were compared between HAP and CA quail. The hepatic enzymes studied are involved in the key step reaction in cholesterol metabolism: HMG-CoA reductase, ACAT, and cholesterol 7 alpha-hydroxylase. The animals were fed ad libitum with either 1% cholesterol or cholesterol-free semipurified diet for 28 days. Although a significant increase (p < 0.01) in serum cholesterol was observed in both strains on elapse of cholesterol feeding, formation of atheroma was seen exclusively in HAP quail of the cholesterol-fed group. The serum and liver cholesterol levels of HAP quail fed the cholesterol diet were significantly higher (p < 0.01) than those of CA quail. No significant differences were seen in the rate of cholesterol biosynthesis (HMG-CoA reductase activity), cholesterol ester formation (ACAT activity) and cholesterol catabolism (7 alpha-hydroxylase activity) between CA and HAP quail. Furthermore, the fecal excretions of acidic and neutral sterol showed no significant difference between strains. Although the formation of atheroma in HAP quail may be presumably due to the contribution of the marked increase in serum cholesterol level, the rate of cholesterol catabolism and synthesis in HAP quail compared well with those of CA quail. These observations suggest that the retarded rate of cholesterol biosynthesis or catabolism is not responsible for hypercholesterolemia in HAP quail.
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The effects of chronic ethanol feeding on lipid composition of rat liver plasma membrane were studied by following ethanol loading or its withdrawal in vivo. Male Wistar rats were pair-fed by a liquid diet containing ethanol as 36% of energy or an isocarolic amounts of glucose for 6 weeks. Chronic ethanol feeding resulted in an increase of cholesterol content and the cholesterol/phospholipid molar ratio in liver plasma membrane compared with pair-fed control rats. Acute ethanol (3 g/kg body weight) orally loading to rats fed ethanol chronically did not change any lipid composition of liver plasma membrane. However, withdrawal of ethanol for 2 days led cholesterol content and the cholesterol/phospholipid molar ratio of liver plasma membrane to be normal values. These data indicated that chronic ethanol feeding produced membrane alterlation that induced resistance to ethanol-induced membrane structural disordering (membrane tolerance), and that this alterlation resulted in a homeoviscous adaptation of liver plasma membrane.
It is generally considered that we have already been through with problems caused by various kinds of parasites which had once raged throughout the country. On the contrary to our common concept, we occasionally encounter some kinds of parasites in a laboratory as well as in clinical fields, which have become unfamiliar to us in these days. Parasitic diseases are in the first place, proper and limited to certain local regions, but the present situation has been greatly changed. Nowadays, specific parasites are no longer limited to polluted areas, but also they can be detected in any part of the world owing to facilitated transportations and a promoted international exchange of people. A recent gourmet boom is also one of the causes of infection as seen in anisakiasis. This study was conducted on the investigation of parasites detected from clinical specimens in our laboratory during the period from 1989 to 1993. 1) The following parasites were detected : (1) Strongyloides stercoralis, (2) Giardia lamblia, (3) Diphyllobothrium latum, (4) Schistosoma mansoni, (5) Entamoeba histolytica, (6) Necator americanus, (7) Isospora belli. 2) Strongyloides stercoralis was detected at the highest frequency. This result gives an account of high prevalence of the parasite among the inhabitants in Okinawa. In addition, the agar plate medium method which has been newly adopted has definitely led to far-advanced results for detection of this parasite. 3) Schistosoma mansoni and Necator americanus were found from foreigners one of whom was a Tanzanian and the other was a Dominican. 4) Isospora belli was found from those compromised cases such as ATL and AIDS.
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Intravascular ultrasound (IVUS) imaging was used to measure internal luminal area immediately after percutaneous transluminal coronary angioplasty (PTCA) in 83 patients (59 males, 24 females, mean age 63 +/- 12 years) with angina pectoris to determine the need for additional intervention. The effectiveness of these interventions to prevent restenosis was also studied. Thirty-five patients (42%) with insufficient dilatation revealed an internal luminal area less than 5 mm2 or luminal stenosis greater than 60% as evaluated by IVUS imaging following the procedure. The luminal area increased from 4.5 +/- 1.1 to 7.9 +/- 2.8 mm2 and the percentage luminal stenosis improved from 66 +/- 9% to 54 +/- 9% in patients who underwent further dilatation with a larger size balloon, longer dilatation time, directional coronary atherectomy, or stenting. The insufficient dilatation group exhibited hard plaque and calcification more frequently than in the other group (48 patients, 58%) in whom sufficient dilatation of the target lesion was achieved. The incidence of restenosis in the sufficient dilatation group was 25%, compared to 33% of the patients receiving additional treatment after IVUS imaging and 42% in the 192 patients who underwent PTCA without IVUS imaging. IVUS imaging is a useful method for evaluation of complex luminal morphology to decrease the incidence of restenosis and for determination of the end point and the extent of dilatation required.
Fukuyama-type congenital muscular dystrophy (FCMD), the second most common form of muscular dystrophy in Japan, is an autosomal recessive severe muscular dystrophy associated with brain anomalies. After our initial mapping of the FCMD locus to chromosome 9q31-33, we have further defined the locus within a approximately 5-cM region between D9S127 and D9S2111 and have found linkage disequilibrium between FCMD and D9S306 in this candidate region on 9q31. The high prevalence of FCMD among the Japanese, who are a relatively isolated population, provides an opportunity to utilize linkage-disequilibrium mapping. We developed three new microsatellites, near D9S306, from the FCMD YAC contig, determined their positions on YACs, and performed linkage-disequilibrium mapping with these markers and other newly published loci. The maximum value of p(excess), which represents the strength of linkage disequilibrium, was obtained at D9S2107; and this value showed a relatively steady rise and fall across the region that is likely to contain FCMD. Distances between FCMD and each marker were presumed to be approximately 1 Mb, approximately 350 kb, approximately 140 kb, approximately 20 kb, approximately 280 kb, approximately 450 kb, and approximately 740 kb for D9S306, A107XF9, D9S2105, D9S2107, D9S172, D9S299, and D9S2109, respectively. Haplotype analysis using the three closest markers D9S2105, D9S2107, and D9S172 indicated that most FCMD-bearing chromosomes are derived from a single ancestral founder and suggested that these markers can be used for the diagnosis of sporadic FCMD. Thus, the FCMD gene is most likely to lie within a region of <100 kb containing D9S2107.
The apical canal sealing ability of newly developed calcium phosphate cement (TDM-C), an equimolar mixture of tetracalcium and dicalcium phosphate dihydrate kneaded with McIlvain's buffer solution containing sodium carboxymethyl cellulose, was evaluated in a relative comparison with several recognized available products. The latter included BONETRIX (alpha-tricalcium phosphate mixed with citric acid and tannic acid), ARS (alpha-tricalcium phosphate and hydroxyapatite mixed with polyacrylic acid), and zine oxide eugenol sealer (ZOE). Canals of forty extracted human maxillary central incisors were prepared to the file size #70, divided into four groups, and obturated with each of the four above materials. Setting of all sealers took place either in 30% bovine serum solution or in phosphate buffered saline solution. Teeth were then decalcified and cleared to measure linear penetration of India ink from the apex into the canal. Results were statistically analyzed by Student's t-test. Surfaces of all materials, after setting, were also observed by scanning electron microscopy (SEM). We found that TDM-C, BONETRIX, and ZOE all showed excellent apical sealing ability whereas ARS was less effective in this regard. SEM analysis revealed micropores on the surface of every apatitic product, but ZOE was free of such porosity. It was concluded that TDM-C and BONETRIX may prove clinically useful as root canal sealant materials.
We describe the identification and characterization of a transcription factor encoded by the atf1+ gene of the fission yeast Schizosaccharomyces pombe. The factor Atf1, contains a bZIP domain at its C-terminus with strong homology to members of the ATF/CREB family of mammalian factors and in vitro binds specifically to ATF/CRE recognition sites. Furthermore the ATF-like binding activity detected in extracts from fission yeast cells is entirely lost upon deletion of the atf1+ gene. Upon growth to saturation, fission yeast cells exit the mitotic cycle and enter a G0-like stationary phase. However, on rich medium, entry of atf1- cells into stationary phase is restricted and they rapidly lose viability; this does not occur on minimal medium unless cAMP levels are raised. Thus stationary phase entry appears to be regulated negatively by cAMP and positively by Atf1. atf1- cells are also sterile and this sterility appears to be due to a combination of two defects: first, upon nitrogen starvation the majority of atf1- cells fail to arrest in the G1 phase of the cell cycle and second, the induction of ste11+ expression is lost. Thus expression of ste11+ represents a second example of an event that is negatively regulated by the cAMP pathway and positively regulated by Atf1. Despite their close association however, these two regulatory pathways function independently and Atf1 activity is not directly modulated by cAMP levels or mutations that alter the activity of components of the cAMP signalling pathway. Thus Atf1 is a transcription factor that plays an important role in the response of cells to adverse environmental conditions, which is to exit the mitotic cell cycle and either sexually differentiate or enter a resting state.
Temperature-sensitive suppressor mutants were isolated from two fission yeast mutants defective in cell shape control: ppe1, encoding a type 2A-like protein phosphatase, and sts5, one of 11 staurosporine-supersensitive mutants. Complementation tests showed that suppression was due to two chromosomal loci, ssp1 and ssp2. Cells of the ssp1 mutant grown at the restrictive temperature arrested uniformly with an elongated cell body and a 2C content of DNA. Interestingly, these mutant cells grew only in a monopolar manner. At a specific point in the G2 phase of the cell cycle, wild-type cells exhibit a drastic alteration in growth polarity, from mono- to bipolar. This change coincides with the distribution of cortical actin from one end of the cell to both ends. In the ssp1 mutant cells, cortical actin was localized only at one end, suggesting that the mutant fails to change growth polarity. Nucleotide sequence determination showed that ssp1+ encodes a novel protein kinase. Ectopic overexpression of ssp1+ resulted in an altered cell morphology and cortical actin was randomly dispersed within the cells. Immunocytological analysis revealed that the protein was primarily localized in the cytoplasm and that half of the protein existed in an insoluble fraction. These results show that the dynamics of actin-based growth polarity during the cell cycle are regulated, at least in part, by a novel set of protein kinases and phosphatases.
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