Immunohistological localization of smg p25A, a ras p21-like guanosine 5'-triphosphate (GTP)-binding protein in human skin.
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Fluorescent Antibody Technique↗
Biomedical subjects
Y Takai
Publications and source records attributed to Y Takai.
The response of murine B-16 melanoma to fractionated doses of pions.
The relative biological effectiveness (RBE) of pions has been studied in mouse B-16 melanoma transplanted into C57BL/6 mice. To determine the RBE at both high and low doses per fraction, a range of fractionation schedules was used, with 1, 4 and 10 fractions. The reference 250 kV X ray dose rate was 1.5 Gy/min which was much higher than the dose rate of pions (0.25 Gy/min). The RBE varied depending on the number of fractions and, within the same fractionation schedule, also on the dose per fraction. The RBE ranged from 1.15 for single fractions at 12.5 days of growth delay, to 1.80 for 10 fractions at 5 days of growth delay, which was determined by the time taken for the tumors to reach 5 times the average of their original volume. RBEs at the iso-effect level of 10 days growth delay were 1.20, 1.29 and 1.62 for single, 4 fractions and 10 fractions, respectively. RBE values were influenced by both the number of fractions and the dose per fraction, that is, the larger the number of fractions and the smaller the dose per fraction, the larger the value of RBE. In comparison with RBE of normal mouse skin, it was suggested that pion therapy may provide advantage over conventional photontherapy for radioresistant tumors such as this melanoma with the maximum therapeutic gain factor of 1.2. alpha/beta ratios for B-16 melanoma were also obtained from the 10 day growth delay iso-effect curve, and were 10.5 Gy and 32.6 Gy for X ray and pions, respectively.
Animals↗
Small GTP-binding proteins.
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Amino Acid Sequence↗
Immunoreaction of keratin, actin, S-100 protein and rat-EGF in duct-ligated rat salivary glands.
Duct-ligated submandibular and sublingual glands of rats were evaluated immunohistochemically for changes in keratin (MoAb 1164), actin, S-100 protein and rat-EGF (rEGF). Normal salivary glands were reactive for keratin, S-100 protein and rEGF in the granular convoluted tubule (GCT) and duct cells, and for actin in the myoepithelium. Submandibular glands showed a marked reduction of S-100 protein and rEGF staining following duct ligation, and no increased staining of proliferating epithelial cells of the late stage in duct ligated glands. Sublingual glands revealed no marked changes for actin staining in myoepithelial cells, irrespective of atrophic changes occurring in acinar and duct cells after duct ligation. Immunohistochemical patterns differed for each type of gland; changes associated with the obstructive lesion were more prominent in the submandibular gland.
Actins↗
The posttranslational processing of ras p21 is critical for its stimulation of yeast adenylate cyclase.
Mammalian ras genes substitute for the yeast RAS gene, and their products activate adenylate cyclase in yeast cells, although the direct target protein of mammalian ras p21s remains to be identified. ras p21s undergo posttranslational processing, including prenylation, proteolysis, methylation, and palmitoylation, at their C-terminal regions. We have previously reported that the posttranslational processing of Ki-ras p21 is essential for its interaction with one of its GDP/GTP exchange proteins named smg GDS. In this investigation, we have studied whether the posttranslational processing of Ki- and Ha-ras p21s is critical for their stimulation of yeast adenylate cyclase in a cell-free system. We show that the posttranslationally fully processed Ki- and Ha-ras p21s activate yeast adenylate cyclase far more effectively than do the unprocessed proteins. The previous and present results suggest that the posttranslational processing of ras p21s is important for their interaction not only with smg GDS but also with the target protein.
Adenylyl Cyclases↗
RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae.
The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.
Adenylyl Cyclases↗
Microinjection of smg/rap1/Krev-1 p21 into Swiss 3T3 cells induces DNA synthesis and morphological changes.
Microinjection of either Ki-rasVal-12 p21 or the GDP-bound form of Ki-ras p21 plus smg GDP dissociation stimulator (GDS), a stimulatory GDP/GTP exchange protein for Ki-ras p21, smg/rap1/Krev-1 p21, and rho p21, into quiescent Swiss 3T3 cells induced DNA synthesis irrespective of the presence or absence of insulin. The guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of smg p21B or the GDP-bound form of smg p21B plus smg GDS also induced DNA synthesis but only in the presence of insulin. Either the GDP-bound form of Ki-ras p21 or the same form of smg p21B alone was inactive, but smg GDS alone was slightly active only in the presence of insulin. The morphology of the cells was analyzed by scanning electron, phase-contrast, and confocal laser scanning microscopies. Ki-rasVal-12 p21 induced membrane ruffling irrespective of the presence or absence of insulin. The GTP gamma S-bound form of smg p21B showed the same effect only in the presence of insulin. Either the GDP-bound form of Ki-ras p21, the same form of smg p21B, or smg GDS alone was inactive. Upon microinjection of Ki-rasVal-12 p21, stress fibers markedly decreased and the cells became round and piled up. In contrast, upon microinjection of the GTP gamma S-bound form of smg p21B, stress fibers did not markedly decrease and the cells neither became round nor piled up. These results indicate that both ras p21 and smg p21 are mitogenic in Swiss 3T3 cells but that their actions are slightly different.
3T3 Cells↗
Distribution of smg p25A and smg p21s, ras p21-like guanine nucleotide-binding proteins, in the rat stomach.
Existence and distribution of small guanine nucleotide binding proteins (G proteins) such as smg p25A, smg p21s, and ras p21s were examined in the rat gastric mucosa. By immunoblot analysis using the specific monoclonal antibodies against smg p25A and ras p21 and the anti-smg p21 antiserum, smg p25A and smg p21s were detected in the gastric mucosa, while ras p21s were not detected. In immunocytochemical studies, moderate fluorescence of smg p25A was homogenously seen in the cytoplasmic portions of chief cells and surface epithelial cells. smg p21 immunoreactivity was detected in glandular cells and submucosal muscle layer in the mucosa. On the other hand, the cells constituting gastric glands were unstained with the anti-ras p21 monoclonal antibody, RASK-4. These results suggest that smg p25A and smg p21s exist as in other tissues and might exert their specific actions in the rat gastric mucosa.
Animals↗
Intraoperative radiation therapy combined with hyperthermia against pancreatic carcinoma.
Fourteen patients with pancreatic carcinoma were treated by intraoperative radiation therapy (IORT) combined with hyperthermia (hyperthermia group). Their treatment results were compared with those of fifty five patients treated by IORT without hyperthermia (control group). Most of patients underwent some kind of chemotherapy for the carcinoma and some of them received post-operative irradiation. Although there was no significant difference in pain relief between hyperthermia group and control group, the local tumor control rate of the former group was a little better. The survival rate of all patients was 14.5% at one year, 2.9% at two years, 2.9% at three years and 0% at four years after surgery. The survival rate of the hyperthermia group was 21.4% at one year and 7.1% at two years and that of the control group was 12.7% at one year and 1.8% at two years. The survival of the hyperthermia group was a little better than that of control group, but the difference was not significant. Only 36% of patients of hyperthermia group were successfully heated, and if hyperthermia were given successfully to all patients, their prognoses would be possibly improved.
Adenocarcinoma↗
Treatment results by uneven fractionated irradiation, low-dose rate telecobalt therapy as a boost, and intraoperative irradiation for malignant glioma.
The prognosis of malignant glioma is extremely poor. We applied conventionally fractionated irradiation combined with 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), uneven fractionated irradiation with ACNU, low dose rate telecobalt therapy as a boost, and intraoperative irradiation against 110 malignant gliomas to investigate the efficacy of these methods as alternative treatments for malignant glioma. Although local tumor control by uneven fractionated irradiation was better than that by the other methods, no significant improvement was obtained in survival rates. As a result of multiple regression analysis, age and histology were major factors for survival rates, and the difference of treatment methods was not important. Both low-dose rate telecobalt therapy as a boost and intraoperative irradiation showed little advantage because of the high risk of brain necrosis associated with them.
Adolescent↗
Radioresponse and prognosis of malignant glioma.
Radioresponse and prognosis of 91 malignant gliomas were studied to examine the efficacy of radiotherapy. There was no case of complete response. No statistically significant difference was observed among the prognoses of patients with various radiation methods. General survival rate was significantly higher than relapse-free survival rate both in astrocytoma grade III and in glioblastoma. This means that the retreatment after relapse is exceedingly important in any malignant glioma. In comparison with reported resection alone data, the efficiency of radiotherapy was evident in astrocytoma grade III and a part of glioblastoma; cases with minimal or no contrast enhanced area (CEA) in CT scans prior to irradiation. Poor radioresponders of glioblastoma with CEA should be reoperated.
Adolescent↗
A new technique for retrieving catheter fragments in the pulmonary artery.
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Catheterization, Central Venous↗
Prognostic factors in radiation-treated esophageal carcinoma.
Prognostic factors in esophageal carcinoma treated with irradiation were examined. The prognosis of 111 patients without metastasis who had received more than 60 Gy was analyzed. Significant associations were found between survival rates and tumor length, stage, radioresponse of the primary tumor and the s.c. X-P classification based on barium contrast radiography; superficial type (tumor limited to the surface of the esophageal wall), tumorous type (solid mass without ulceration), Ul-A type (tumor with shallow ulceration with regular margin), Ul-B type (tumor with deep ulceration or irregular ulcer margin), and funneled type (tumor invading the esophageal wall in a scirrhous pattern). In multiple regression analysis, the X-P classification had the strongest correlation with survival and the survival rates of patients with the superficial type, the tumorous type and the s.c. Ul-A type were significantly higher than those of patients with the other tumor types (p < 0.001).
Aged↗
The small G protein superfamily--possible functions and modes of action.
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GTP-Binding Proteins↗
smg/rap1/Krev-1 p21s inhibit the signal pathway to the c-fos promoter/enhancer from c-Ki-ras p21 but not from c-raf-1 kinase in NIH3T3 cells.
smg/rap1A/Krev-1 p21 cDNA is known to inhibit v-Ki-ras p21-induced cell transformation in NIH3T3 cells, but the inhibitory mechanism is not clear at present. In the present study, we examined the effect of smg p21s on the c-fos promoter/enhancer linked to the luciferase reporter gene (c-fos-luciferase). After transfection of c-fos-luciferase into NIH3T3 cells constitutively expressing c-Ki-ras(val-12) p21 or activated c-raf-1 kinase, expression of c-fos-luciferase was much higher than after transfection into control NIH3T3 cells. Addition of platelet-derived growth factor (PDGF), 12-O-tetradecanoyl phorbol 13-acetate (TPA) or dibutyryl cyclic AMP (Bt2cAMP) to the control NIH3T3 cells stimulated c-fos-luciferase expression. Transfection of the smg p21 cDNAs inhibited the activated ras p21-, PDGF- or TPA-stimulated c-fos-luciferase expression, but did not inhibit the activated c-raf-1 kinase- or Bt2cAMP-stimulated reaction. These results indicate that smg p21s inhibit the signal pathways from the PDGF receptor, protein kinase C, and ras p21s to the c-fos promoter/enhancer, but not those from c-raf-1 kinase and cyclic AMP-dependent protein kinase to the c-fos promoter/enhancer.
3T3 Cells↗
Immunoreactivity of calmodulin, S-100 protein alpha and beta subunits in rat submandibular glands.
The calcium-binding proteins calmodulin and S-100 protein alpha and beta subunits were studied in Rat submandibular salivary gland using monoclonal antibodies and immunoperoxidase staining. Histologically, Rat submandibular gland consists of acinar and ductal components. The latter include intercalated, granular convoluted tubule, striated and excretory ducts. Ultrastructural studies of adult Rat granular convoluted tubule show granular cells, pillar cells and transition cells. Immunostaining for S-100 alpha and calmodulin was observed in granular convoluted tubule pillar cells and transition cells, whereas cells of striated ducts and intercalated ducts stained positive for calmodulin. S-100 beta staining in acinar cells was visible as a fine granular pattern. Although immunohistochemical localization in rat submandibular gland differed for S-100 alpha and beta subunits, expression was similar for S-100 alpha and calmodulin in the granular convoluted tubule segment. No immunodeposition of calmodulin, S-100 alpha or S-100 beta was found in granular cells, which contain growth factors such as EGF and NGF.
Animals↗
Post-irradiation protective effect of irsogladine maleate on intestinal crypt stem cells in mice.
Radioprotective effect of irsogladine maleate, an anti-ulcer drug, on the intestinal crypt stem cell survival was studied in mice using a crypt microcolony assay. Irsogladine maleate was injected intraperitoneally immediately after irradiation and then, daily for three days. A successive administration of the drug following 10 Gy of irradiation increased the survival of intestinal stem cells with a clear dose-related trend. In order to estimate the D0, survival curves were determined for X-ray plus placebo and X-ray plus 10 mg/kg of irsogladine maleate. The D0, for X-ray plus the drug was 2.2 Gy while it was 1.9 Gy for X-ray plus placebo. These findings suggest that isogladine maleate can be applied for the alleviation of intestinal damages in heavily irradiated people by radiation accidents.
Animals↗
The functional domain of the stimulatory GDP/GTP exchange protein (smg GDS) which interacts with the C-terminal geranylgeranylated region of rap1/Krev-1/smg p21.
rap1/Krev-1/smg p21 (smg p21), a member of the small GTP-binding protein (G protein) superfamily, has a geranylgeranylated cysteine residue and clustered basic amino acids in the C-terminal region. The GDP/GTP exchange reaction of smg p21 is regulated by smg GDS, which is also active on Ki-ras p21 and rho p21. The C-terminal region of smg p21 is essential for its interaction with smg GDS. Moreover, smg p21 is phosphorylated by cyclic AMP- and cyclic GMP-dependent protein kinases at the serine residue between the polybasic region and the prenylated cysteine residue, and this phosphorylation initiates the smg GDS-induced smg p21 activation. Thus, the C-terminal cationic and hydrophobic region is important for the regulation of the smg p21 activity. In the present study, we attempted to determine the functional domain of smg GDS which interacts with the C-terminal region of smg p21 by use of a cross-link method and a site-directed mutagenesis method. The region of smg GDS cross-linked with the C-terminal region of smg p21B was residues 444-492, which is located at the C-terminal fifth of smg GDS. On deletion of these residues, smg GDS became inactive on smg p21B, Ki-ras p21 and rhoA p21. These results indicate that residues 444-492 of smg GDS are at least one of the domains which interact with the C-terminal region of its substrate small G proteins.
Amino Acid Sequence↗