Studies of RAS function in the yeast Saccharomyces cerevisiae.
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Biomedical subjects
Publications and source records attributed to T Toda.
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A total of 13 forty-day-old male Japanese quails had free access to a atherogenic diet containing 15% corn oil and 2% cholesterol or commercial basal diet for 3 months. Birds fed basal diet showed no significant intimal lesions. These birds had two types of cells, i.e. smooth muscle cell and fibroblast-like cell, in the tunica media of the ascending aorta. While fat-fed birds showed marked lipid-rich intimal lesions in the ascending aorta but not in the abdominal aorta. Some macrophage-derived foam cells, which were stained for lysozyme and OKM1, were demonstrated in the superficial portion of the thickened intima. The majority of the cells in the lipid-rich thickened intima showed ultrastructural character of fibroblast-like cells with or without lipid droplets. Alpha-1-antichymotrypsin was positive for fibroblast-like cells in the thickened intima but not for those in the tunica media of the ascending aorta. These results suggest that metaplasia of the medial fibroblast-like cells is responsible for the development of atherosclerosis in the quail.
The atherogenicity of corn oil, hydrogenated corn oil, and butter fat was studied using 57 fourty-day-old male Japanese quails. The animals were fed one of the following diets: basal diet, basal diet with corn oil plus cholesterol, basal diet with hydrogenated corn oil plus cholesterol, or basal diet with butter fat plus cholesterol. Each atherogenic diet contained 15% fat and 2% cholesterol. Marked hypercholesterolemia developed in all fat-fed groups after 3 weeks or 3 months, but no significant difference was seen among the groups. The degree of the luminal narrowing of the ascending aorta and brachiocephalic arteries was highest in the corn oil group and lowest in the hydrogenated fat group. Ultrastructurally, all the fat-fed groups showed similar cellular changes in their aortic lesions. The major cell type of the thickened intima was fibroblast-like cells with or without lipid droplets. Immunohistochemical studies disclosed that alpha-1-antichymotrypsin was strongly positive for fibroblast-like cells in the thickened intima, whereas it was negative for those in the tunica media of the ascending aorta and its branches. These data suggest that hydrogenated corn oil has a less potent stimulating effect on the thickening of the arterial intima than corn oil and butter fat.
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A prospective randomized and comparative study of FT-207 and UFT was performed for cases of Stage IV gastric cancer involving long-term cancer chemotherapy after surgery. Immediately after the surgery, the subjects were randomly divided into two groups: A group treated by MMC and FT-207: and B group treated by MMC and UFT. From a total of 54 cases, 8 cases were excluded, so that A group consisted of 24 subjects and B group of 22 subjects. There were no differences in background factors between the two groups. In a comparison of all the cases, B group revealed a significantly higher survival rate than A group. These results indicated that the simultaneous use of MMC and UFT was effective as a long-term cancer chemotherapy after surgery for patients with Stage IV gastric cancer.
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A case of a leiomyosarcoma of the duodenum is reported. A 52-year-old man with a duodenal submucosal tumor underwent a pancreatico-duodenectomy. Histologically, the resected tumor revealed moderate cellular atypism and moderate pleomorphism, but revealed no mitotic figures. The histological diagnosis was leiomyoma. Nine years after operation, the tumor showed a recurrence and metastasis in the liver. Thus, the patient underwent a lobectomy of the liver. The resected liver tumor showed similar histological features seen in the primary duodenal tumor but with high mitotic activity. Histologically, the patient was diagnosed as having a metastatic leiomyosarcoma. This history suggests that a smooth muscle tumor of potential malignancy, as seen in this case, should be followed continuously, even though mitoses may not be recognized.
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We have isolated three genes (TPK1, TPK2, and TPK3) from the yeast S. cerevisiae that encode the catalytic subunits of the cAMP-dependent protein kinase. Gene disruption experiments demonstrated that no two of the three genes are essential by themselves but at least one TPK gene is required for a cell to grow normally. Comparison of the predicted amino acid sequences of the TPK genes indicates conserved and variable domains. The carboxy-terminal 320 amino acid residues have more than 75% homology to each other and more than 50% homology to the bovine catalytic subunit. The amino-terminal regions show no homology to each other and are heterogeneous in length. The TPK1 gene carried on a multicopy plasmid can suppress both a temperature-sensitive ras2 gene and adenylate cyclase gene.
The regulatory (R) subunit of cAMP-dependent protein kinase from the yeast Saccharomyces cerevisiae was expressed in Escherichia coli by engineering the gene for yeast R, BCY1, into an E. coli expression vector that contained a promoter from phage T7. Oligonucleotide-directed mutagenesis was used to create an NdeI restriction site at the natural ATG of the yeast R. This facilitated construction of the T7 expression vector so that the sequence of the protein produced was identical to the natural R subunit. Yeast R was highly expressed in a soluble form. 20 mg of purified yeast R was obtained from 4 liters of E. coli. N-terminal amino acid sequencing revealed that the expressed protein began with the natural sequence. 60% of the molecules contained an N-terminal methionine, and 40% initiated with valine, the second amino acid of yeast R. The protein produced in E. coli migrated on a sodium dodecyl sulfate-polyacrylamide gel with an Mr of 52,000. The yeast R bound 2 mol of cAMP/mol of R monomer with a Kd of 76 nM. The protein was treated with urea to remove bound cAMP. Sedimentation values before and after the urea treatment were identical (s20,w = 5.1). Addition of purified R subunit to a preparation of yeast C subunit (TPK1) rendered catalytic activity cAMP-dependent with an activity ratio of 4.6. The yeast R was autophosphorylated by yeast C to a level of 0.8 mol of phosphate/mol of R monomer. By these criteria, the R subunit produced in E. coli was structurally and functionally identical to the natural yeast R subunit and similar to mammalian type II R subunits.
The gene corresponding to the S. cerevisiae cell division cycle mutant cdc25 has been cloned and sequenced, revealing an open reading frame encoding a protein of 1589 amino acids that contains no significant homologies with other known proteins. Cells lacking CDC25 have low levels of cyclic AMP and decreased levels of Mg2+-dependent adenylate cyclase activity. The lethality resulting from disruption of the CDC25 gene can be suppressed by the presence of the activated RAS2val19 gene, but not by high copy plasmids expressing a normal RAS2 or RAS1 gene. These results suggest that normal RAS is dependent on CDC25 function. Furthermore, mutationally activated alleles of CDC25 are capable of inducing a set of phenotypes similar to those observed in strains containing a genetically activated RAS/adenylate cyclase pathway, suggesting that CDC25 encodes a regulatory protein. We propose that CDC25 regulates adenylate cyclase by regulating the guanine nucleotide bound to RAS proteins.
Syngeneic sera modified with seven agents were used as the immunogens to induce lymph-node cell proliferation in C57BL/6 mice. Immunogenicity could be conferred upon tolerated syngeneic serum by modification with diluted tincture of iodine (J) or formocresol (FC). However, the synergistic action of formalin and cresol appeared to be an indispensable condition in acquiring immunogenicity of FC-modified syngeneic serum (FC-MS). Moreover, immunological tolerance to native serum was not terminated after injection of J-MS or FC-MS. These proliferative functions were dependent on Lyt-1 cells. The cells directed against J- or FC-modified syngeneic serum were specific for the immunizing hapten J or FC. However, they did not recognize the hapten per se, but rather, the conjugate-specific determinants, i.e. these contributed by both the hapten and the self-carrier.
We synthesized the 13S mRNA-encoded protein of the early region 1a (E1a) of human adenovirus in Saccharomyces cerevisiae under the control of the yeast GAL7 gene promoter. Similar to the case in HeLa cells, the E1a protein in yeast was phosphorylated and formed multiple bands on sodium dodecylsulfate-polyacrylamide gel electrophoresis. These bands migrated more slowly than expected from the Mr calculated on the basis of the nucleotide sequence of the gene. Synthesis of the E1a protein caused induction of a specific family of heat-shock proteins (Hsp70), which, however, did not confer heat resistance to the yeast. In addition, the E1a production resulted in an elongation of the generation time of yeast from 2.4 h to 3.9 h, which was attributed specifically to elongation of the G1 interval in the cell cycle. In the light of these findings, we suggest that the E1a protein synthesized in yeast exerts a specific function.
Elastase has been reported to have a multiplicity of activities which may be protective against atherosclerosis. The data reported here support those reports and show a clear antiatherosclerotic effect in hyperlipidemic chickens. We have shown here that a reduction in calcium and lipid accumulation in aortic tissue and possibly a plasma cholesterol lowering effect can be seen in the chicken when treated with elastase. The most important effect in this animal is probably to aid removal of lipid bound to elastin in the intima. This was achieved at a dose of only 600 U elastase while reductions in tissue calcium required 1800 U elastase.
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We have isolated and characterized normal and mutant alleles of many of the genes of the RAS/adenylyl cyclase pathway of the yeast Saccharomyces cerevisiae. Manipulation of those genes has revealed a system for feedback control that can modulate cAMP levels over at least a 10,000-fold range. The feedback control depends upon the activity of the cAMP-dependent protein kinases and requires the presence of the CDC25 and RAS proteins. The capacity for such dramatic control of cAMP levels raises fundamental questions about the normal mechanism of action of the cAMP signaling system in yeast.