Biomedical subjects

H Mitsuzawa

Publications and source records attributed to H Mitsuzawa.

25 records · Page 2Linked to original sources

PubMed1993

Responsiveness to exogenous cAMP of a Saccharomyces cerevisiae strain conferred by naturally occurring alleles of PDE1 and PDE2.

The Saccharomyces cerevisiae strain P-28-24C, from which cAMP requiring mutants derived, responded to exogenously added cAMP. Upon the addition of cAMP, this strain showed phenotypes shared by mutants with elevated activity of the cAMP pathway. Genetic analysis involving serial crosses of this strain to a strain with another genetic background revealed that the responsiveness to cAMP results from naturally occurring loss-of-function alleles of PDE1 and PDE2, which encode low and high affinity cAMP phosphodiesterases, respectively. In addition, P-28-24C was found to carry a mutation conferring slow growth that lies in CYR1, which encodes adenylate cyclase, and the slow growth phenotype caused by the cyr1 mutation was suppressed by the pde2 mutation. Therefore P-28-24C is fortuitously a pde1 pde2 cyr1 triple mutant. Responsiveness to cAMP conferred by pde mutations suggests that S. cerevisiae cells are permeable to cAMP to some extent and that the apparent absence of effect of exogenously added cAMP on wild-type cells is due to immediate degradation by cAMP phosphodiesterases.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1992

Characterization of a staurosporine- and temperature-sensitive mutant, stt1, of Saccharomyces cerevisiae: STT1 is allelic to PKC1.

Staurosporine is an antibiotic that specifically inhibits protein kinase C. Fourteen staurosporine- and temperature-sensitive (stt) mutants of Saccharomyces cerevisiae were isolated and characterized. These mutants were divided into ten complementation groups, and characterized for their cross-sensitivity to K-252a, neomycin, or CaCl2. The STT1 gene was cloned and sequenced. The nucleotide sequence of the STT1 gene revealed that STT1 is the same gene as PKC1. The STT1 gene conferred resistance to staurosporine on wild-type cells, when present on a high copy number plasmid. STT1/stt1::HIS3 diploid cells were more sensitive to staurosporine than STT1/STT1 diploid cells. Analysis of temperature-sensitive stt1 mutants showed that the STT1 gene product functioned in S or G2/M phase. These results suggest that a protein kinase (the STT1 gene product) is one of the essential targets of staurosporine in yeast cells.

Alkaloids↗

PubMed1990

The adenylate cyclase/protein kinase cascade regulates entry into meiosis in Saccharomyces cerevisiae through the gene IME1.

Entry into meiosis in Saccharomyces cerevisiae cells is regulated by starvation through the adenylate cyclase/cAMP-dependent protein kinase (AC/PK) pathway. The gene IME1 is also involved in starvation control of meiosis. Multicopy IME1 plasmids overcome the meiotic deficiency of bcy1 and of RASval19 diploids. Double mutants ime1 cdc25 and ime1 ras2 are sporulation deficient. These results suggest that IME1 comes after the AC/PK cascade. Furthermore, the level of IME1 transcripts is affected by mutations in the AC/PK genes CDC25, CYR1 and BCY1. Moreover, the addition of cAMP to a cyr1-2 diploid suppresses IME1 transcription. The presence in a bcy1 diploid of IME1 multicopy plasmids does not cure the failure of bcy1 cells to arrest as unbudded cells following starvation and to enter the G0 state (thermotolerance, synthesis of unique G0 proteins). This indicates that the pathway downstream of the AC/PK cascade branches to control meiosis through IME1, and to control entry into G0 and cell cycle initiation, independently of IME1.

Adenylyl Cyclases↗

PubMed1989

Isolation and characterization of temperature-sensitive mutations in the RAS2 and CYR1 genes of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae contains two ras homologues, RAS1 and RAS2, whose products have been shown to modulate the activity of adenylate cyclase encoded by the CYR1 gene. To isolate temperature-sensitive mutations in the RAS2 gene, we constructed a plasmid carrying a RAS2 gene whose expression is under the control of the galactose-inducible GAL1 promoter. A ras1 strain transformed with this plasmid was subjected to ethyl methanesulfonate mutagenesis and nystatin enrichment. Screening of approximately 13,000 mutagenized colonies for galactose-dependent growth at a high temperature (37 degrees) yielded six temperature-sensitive ras2 (ras2ts) mutations and one temperature-sensitive cyr1 (cyr1ts) mutation that can be suppressed by overexpression or increased dosage of RAS2. Some ras2ts mutations were shown to be suppressed by an extra copy of CYR1. Therefore increased dosage of either RAS2 or CYR1 can suppress the temperature sensitivity caused by a mutation in the other. ras1 ras2ts and ras1 cyr1ts mutants arrested in the G1 phase of the cell cycle at the restrictive temperature, and showed pleiotropic phenotypes to varying degrees even at a temperature permissive for growth (25 degrees), including slow growth, sporulation on rich media, increased accumulation of glycogen, impaired growth on nonfermentable carbon sources, heat-shock resistance, impaired growth on low concentrations of glucose, and lithium sensitivity. Of these, impaired growth on low concentrations of glucose and sensitivity to lithium are new phenotypes, which have not been reported for mutants defective in the cAMP pathway.

Adenylyl Cyclases↗

PubMed1987

Identification of the domain of Saccharomyces cerevisiae adenylate cyclase associated with the regulatory function of RAS products.

Various truncated CYR1 genes of Saccharomyces cerevisiae were fused to efficient promoters and expressed in Escherichia coli and S. cerevisiae cells with or without the RAS genes. The catalytic domain of adenylate cyclase encoded by the 3'-terminal 1.3 kb region of the open reading frame of the CYR1 gene produced cyclic AMP, irrespective of the presence of RAS genes. The product of the 3'-terminal 2.1 kb region of CYR1 showed guanine nucleotide-dependent adenylate cyclase activity and produced a large amount of cAMP in the presence of the RAS gene. Thus, the domain encoded by the 0.8 kb region adjacent to the catalytic domain is associated with the regulatory function of the RAS products. The cyr1 RAS1 RAS2 cells carrying the 3'-terminal 1.3 kb region of CYR1 were unable to respond to environmental signals such as sulfur starvation and temperature shift, but the cyr1 cells carrying the 2.1 kb region and at least one RAS gene were able to respond to these signals. The environmental signals may be transferred to the adenylate cyclase system through the RAS products.

Adenylyl Cyclases↗

PubMed1985

Reconstitution of the GTP-dependent adenylate cyclase from products of the yeast CYR1 and RAS2 genes in Escherichia coli.

Plasmids carrying the CYR1 gene of yeast Saccharomyces cerevisiae, which encodes adenylate cyclase, were introduced into the cya mutant strain of Escherichia coli. The transformants had a GTP-independent adenylate cyclase activity but did not produce cAMP. The E. coli transformant carrying the yeast RAS2 or RAS2val19 gene had no adenylate cyclase activity. Transformant cells carrying both CYR1 and RAS2 produced GTP-dependent adenylate cyclase and cAMP, and those carrying CYR1 and RAS2val19 produced GTP-independent adenylate cyclase and a large amount of cAMP. Production of cAMP in the transformant carrying CYR1 and either RAS2 or RAS2val19 was confirmed by staining colonies on maltose-MacConkey plates and by measuring induction of beta-galactosidase by isopropyl beta-D-thiogalactopyranoside. Mixing a crude extract from the E. coli transformant carrying CYR1 with a crude extract from cells carrying RAS2 reconstituted the GTP-dependent adenylate cyclase. Reconstitution of the GTP-dependent adenylate cyclase was observed by mixing the plasma membrane fraction of yeast CYR1 ras1 ras2 bcy1 mutant and a crude extract from the E. coli transformant carrying RAS2 or by mixing a crude extract from the E. coli transformant carrying CYR1 and the membrane fraction of yeast cyr1 RAS1 RAS2 BCY1 mutant. The data suggest that the yeast GTP-dependent adenylate cyclase consists of catalytic and regulatory subunits encoded by the CYR1 and RAS2 genes, respectively.

Adenylyl Cyclases↗

PubMedDate not supplied

Middle ear imaging using virtual endoscopy and its application in patients with ossicular chain anomaly.

Virtual endoscopy (VE) is a recently developed technique to provide a realistic surface rendering of various organs, which can be applied to the use of three-dimensional (3D) studies of several lesions. However, its advantages in otological disease have not been well investigated. In this study, we evaluated the application of VE in patients with ossicular chain anomalies. Virtual middle ear endoscopy was a time-saving method, however, we needed the appropriate technical procedures of algorithm and reconstruction spacing to generate accurate 3D images of ossicles. We obtained virtual surgical views of middle ear structures and related anomalies, and confirmed by intraoperative findings that these images were mostly compatible with the actual lesions of ossicles. VE allowed an identification of the anatomy of the ossicles and adjacent structures simultaneously. The elements of the stapedial crura were clearly visualized with VE images in 93.3% of normal ears. Pathological ossicular chain findings such as malleus or incus fixation, dislocation and disruption, except footplate fixation were investigated successfully. One possible procedure, using alterable CT value in the obtained VE images on the monitor, is proposed for further detection of fine lesions of the ossicles. These observations suggest that virtual middle ear simulations accurately represent major intraoperative findings. This technique may have an important role in preoperative planning, surgical training, and/or postoperative evaluation in otology.

Adult↗