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Biomedical subjects

T Kadowaki

Publications and source records attributed to T Kadowaki.

At least 289 records · Page 16Linked to original sources
PubMed1989

Use of polymerase chain reaction catalyzed by Taq DNA polymerase for site-specific mutagenesis.

The polymerase chain reaction catalyzed by Taq DNA polymerase has been used for site-specific mutagenesis. The amplification was primed by two oligodeoxyribonucleotides complementary to insulin receptor cDNA. To direct the synthesis of mutant DNA, mismatches were introduced into one of the primers. Six different mutations were constructed by this technique. Of twelve clones whose sequences were determined, ten (83%) had the correct sequence. This technique, which does not require the use of single-stranded DNA templates, provides a simple and efficient approach to site-specific mutagenesis.

Base Sequence↗
PubMed1989

[Clinical reevaluation of aztreonam (AZT) on urinary tract infections].

The clinical effectiveness and safety of Aztreonam (AZT) were studied in 55 patients with complicated urinary tract infections. AZT was administered at a dose of 4 g/day for 5 days. The results as evaluated by the UTI criteria were excellent in 8 cases, moderate in 26 and poor in 21. The overall effectiveness rate was 61.8%. Out of 64 strains of bacteria isolated before treatment, 50 strains (78.0%) were eradicated by the treatment. Bacteriological eradication rates for gram negative rods and gram positive cocci were 79.6% and 66.7%, respectively. There were no obvious side effects, except for a temporary elevation of glutamic oxaloacetic transaminase, glutamic pyruvic transaminase alkalinephosphatase, and total bilirubin in some patients. The results indicate that AZT tablets are useful and safe for the treatment of complicated urinary tract infections.

Adolescent↗
PubMed1988

Enhanced transcription of mitochondrial genes after growth stimulation and glucocorticoid treatment of Reuber hepatoma H-35.

When a serum-deprived culture of Reuber hepatoma H-35 cells was stimulated by serum, cells proliferated synchronously showing a peak of [3H]thymidine incorporation within 16 h and doubling of cell number within 24 h. A 5-10-fold increase of mRNAs of mitochondrial genes was observed prior to S-phase by Northern hybridization of total cellular RNA with cloned fragments of rat mitochondrial DNA as probes. In organelle transcription by mitochondria isolated from growth-arrested or growth-stimulated cells suggested that the increase of mitochondrial mRNAs was mainly due to enhanced transcription. Less enhanced transcription was observed by treating the cells with dexamethasone, which causes translocation of large amount of protein into mitochondria. The latter effect was counteracted by insulin.

Animals↗
PubMed1988

Two mutant alleles of the insulin receptor gene in a patient with extreme insulin resistance.

Insulin receptor complementary DNA has been cloned from an insulin-resistant patient with leprechaunism whose receptors exhibited multiple abnormalities in insulin binding. The patient is a compound heterozygote, having inherited two different mutant alleles of the insulin receptor gene. One allele contains a missense mutation encoding the substitution of glutamic acid for lysine at position 460 in the alpha subunit of the receptor. The second allele has a nonsense mutation causing premature chain termination after amino acid 671 in the alpha subunit, thereby deleting both the transmembrane and tyrosine kinase domains of the receptor. Interestingly, the father is heterozygous for this nonsense mutation and exhibits a moderate degree of insulin resistance. This raises the possibility that mutations in the insulin receptor gene may account for the insulin resistance in some patients with non-insulin-dependent diabetes mellitus.

Alleles↗
PubMed1988

Alteration in growth, cell morphology, and cytoskeletal structures of KB cells induced by epidermal growth factor and transforming growth factor-beta.

Long-term biological effects of epidermal growth factor (EGF), insulin, insulin-like growth factor-I (IGF-I), and transforming growth factor-beta (TGF-beta) were examined with human epidermoid carcinoma KB cells. EGF inhibited the growth of KB cells in both serum-containing and serum-free synthetic media by reducing the growth rate and by lowering the saturation density. The cells cultured with EGF showed relatively high motility and grew dispersely as single cells, whereas the cells cultured in the absence of EGF grew in clusters. Although TGF-beta itself did not inhibit the growth of KB cells, it augmented the growth inhibition by EGF. TGF-beta also affected the cell morphology. In the presence of TGF-beta, the cells became flattened and actin stress fibers were well developed compared to those cultured in its absence. The effects of EGF on growth, cell motility, and cell morphology were reversible. Tyrosine phosphorylation of EGF receptors was continuously observed for at least 50 h in the presence of EGF. TGF-beta did not increase the phosphorylation induced by EGF. These results suggested that signals continuously transmitted through EGF receptors caused the changes in cell growth and morphology and that TGF-beta did not act on the cells by modulating binding of EGF to its receptors or activation of the receptor kinase. In contrast to EGF and TGF-beta, neither insulin nor IGF-I affected cell morphology or growth, although KB cells express their receptors and the receptor kinases were also continuously activated during exposure of the cells to insulin or IGF-I.

Actin Cytoskeleton↗
PubMed1988

Rapid stimulation of fluid-phase endocytosis and exocytosis by insulin, insulin-like growth factor-I, and epidermal growth factor in KB cells.

Effects of growth factors on fluid-phase endocytosis and exocytosis in human epidermoid carcinoma KB cells were examined by measuring horseradish peroxidase (HRP) as a marker. Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) promoted HRP accumulation. They also stimulated the efflux of the preloaded HRP from the cells. From these results it follows that these growth factors stimulate the influx as well as the efflux of HRP, because the accumulation rate is the sum of the influx rate and the efflux rate. The stimulation of both HRP accumulation and HRP efflux was rapidly induced within 2-4 min of the addition of growth factors and persisted for at least 60 min. The concentrations eliciting half-maximal stimulatory effects of insulin, IGF-I, and EGF were about 5 X 10(-7), 1 X 10(-9), and 5 X 10(-10) M, respectively. aIR-3 (anti-type I IGF receptor antibody) completely blocked the stimulation of HRP accumulation by IGF-I but very slightly inhibited the stimulation by insulin. The 528 IgG (anti-EGF receptor antibody) inhibited the stimulation of HRP accumulation by EGF. These results indicated that each of these growth factors stimulates the HRP accumulation mediated by the corresponding (homologous) growth factor receptors. The rapid stimulation of fluid-phase influx and efflux may constitute one of the common early cellular responses to growth factors.

Antibodies↗
PubMed1988

Stimulation of the phosphorylation of cytoskeletal 350-kDa and 300-kDa proteins by insulin-like growth factor-I, platelet-derived growth factor and phorbol ester in rat 3Y1 cells.

Insulin-like growth factor-I (IGF-I) stimulated the phosphorylation of cytoskeletal 350-kDa and 300-kDa proteins which were immunoprecipitated with antibodies against brain high molecular weight microtubule-associated proteins in quiescent rat 3Y1 cells. The data on the effective concentrations of IGF-I and 125I-labeled IGF-I binding indicated that type I IGF receptors mediate this IGF-I effect. Platelet-derived growth factor (PDGF) as well as phorbol ester (TPA) also stimulated the phosphorylation of these proteins. These proteins, whether immunoprecipitated from cells stimulated by insulin, IGF-I, TPA, PDGF, or epidermal growth factor, produced very similar phosphopeptide mapping patterns irrespective of the stimulant. The results suggest the possibility that these growth factors and phorbol esters may activate a common protein kinase which is responsible for the phosphorylation of the 350-kDa and 300-kDa proteins in cells.

Animals↗
PubMed1988

[A case of vesicosigmoidal fistula].

The clinical course of a case of vesicosigmoidal fistula is presented. The patient, a 76-year-old woman, became aware of terminal micturition pain and pollakisuria in February, 1985. She was first treated under the diagnosis of cystitis to be relieved of her subjective symptoms, although there was no improvement of pyuria. She also began to feel lower abdominal pain on April 3, 1985. After various examinations including intravenous pyelography, enteroclysis and cystoscopy the diagnosis of vesicosigmoidal fistula originating from sigmoid diverticulitis was established. Careful observation at operation revealed remarkable adhesion among the sigmoid colon, bladder, uterus and left ovary. The sigmoid colon, was resected followed by end-to-end anastomosis. Because of considerably extensive inflammatory changes over the mucosal membrane of the bladder, the hole of fistula on the vesical wall was simply closed from outside of the bladder without performing partial cystectomy. Histological examination only demonstrated non-specific inflammatory changes without evidence of malignancy. She had a favorable progress postoperatively.

Aged↗
PubMed1988

[Intraperitoneal spontaneous rupture of the bladder subsequent to irradiation of the uterus: a case report].

We report a case of spontaneous intraperitoneal rupture of the bladder. A 54-year-old woman was admitted to our hospital with the chief complaints of severe lower abdominal pain, dysuria and macroscopic hematuria in October, 1985. In 1969, she had had a radical hysterectomy and postoperative irradiation for cancer of the uterus. Two years later she had undergone additional irradiation. On physical examination, the abdomen was tender with guarding and signs of peritonitis. Laboratory data revealed a blood urea nitrogen of 32.8 mg/dl and all electrolytes were normal. Excretory urogram showed normal upper urinary tract but irregularity of the bladder dome. Cystoscopy revealed acute inflammation of the bladder mucosa. Consequently, we made a presumptive diagnosis of radiation cystitis and she was treated with antibiotics and drip infusion. Within a week her general condition was improved and she had discharged. In June, 1986 she was admitted again with the same chief complaints as at her first admission. Cystoscopic findings showed a hole on the postero-superior wall and retrograde cystogram revealed an intraperitoneal rupture of the bladder. At exploration a necrotic bladder wall was resected and closed in 3 layers. The post operative course was uneventful.

Combined Modality Therapy↗
PubMed1987

Tyrosine phosphorylation by the epidermal growth factor receptor kinase induces functional alterations in microtubule-associated protein 2.

We have examined the effect of tyrosine phosphorylation of microtubule-associated protein 2 (MAP2) by the epidermal growth factor (EGF) receptor kinase on its functions. Incubation of MAP2 with the EGF receptor in the presence of ATP resulted in a great decrease in the ability of MAP2 to promote tubulin polymerization. Under a variety of conditions, the decrease in the ability correlated with the extent of phosphorylation of MAP2. Furthermore, another function of MAP2, the actin filament cross-linking activity, was also inactivated by the incubation of MAP2 with the EGF receptor and ATP. The loss of this activity also correlated well with the extent of phosphorylation. These data indicate that tyrosine phosphorylation of MAP2 by the EGF receptor kinase inactivates both the tubulin polymerizing activity and actin filament cross-linking activity of MAP2. Thus, this study has clearly shown that tyrosine phosphorylation could modify the function of a cytoskeletal protein.

Actins↗
PubMed1987

Tyrosine phosphorylation of common and specific sets of cellular proteins rapidly induced by insulin, insulin-like growth factor I, and epidermal growth factor in an intact cell.

KB cells respond to insulin and insulin-like growth factor I (IGF-I) in a closely similar way (induction of membrane ruffling, stimulation of pinocytosis, and amino acid transport) but respond to epidermal growth factors (EGF) in a similar but distinct way. In the KB cells, using phosphotyrosine-specific antibody we have found that: the receptors for insulin (beta subunit), IGF-I (beta subunit), and EGF undergo tyrosine phosphorylation as early as 10 s after addition of their respective ligands; a 185-kDa protein is rapidly (less than 10 s) tyrosine phosphorylated by insulin and IGF-I through their respective receptor kinases but not EGF; tyrosine phosphorylation of a 190-kDa glycoprotein is rapidly (less than 10 s) induced by EGF through EGF receptor kinase; and tyrosine phosphorylation of a 240-kDa protein is stimulated within 30 s by all three growth factors. These patterns of tyrosine phosphorylation could be causally related to biological responses induced by the three growth factors.

Carcinoma, Squamous Cell↗
PubMed1987

A Mr = 190,000 glycoprotein phosphorylated on tyrosine residues in epidermal growth factor stimulated KB cells is the product of the c-erbB-2 gene.

In human epidermoid carcinoma KB cells, a glycoprotein of Mr = 190,000 (gp190) has been shown to be phosphorylated on tyrosine residues upon EGF stimulation (Kadowaki et al., 1987, J. Biol. Chem. in press). Using a specific antibody to the c-terminal portion of the human c-erbB-2 gene product, we have found that gp190 is the human c-erbB-2 gene product which is structurally closely related to the epidermal growth factor (EGF) receptor. Since monoclonal antibody specific for the EGF receptor abolished both EGF binding to its receptor and tyrosine phosphorylation of the c-erbB-2 gene product, we have concluded that activation of EGF receptor tyrosine kinase activity upon EGF binding leads to the phosphorylation of the c-erbB-2 gene product on its tyrosine residues.

Epidermal Growth Factor↗
PubMed1987

Insulin-like growth factor I rapidly stimulates tyrosine phosphorylation of a Mr 185,000 protein in intact cells.

The type I insulin-like growth factor (IGF) receptor, like the insulin receptor, contains a ligand-stimulated protein-tyrosine kinase activity in its beta-subunit. However, in vivo, no substrates have been identified. We used anti-phosphotyrosine antibodies to identify phosphotyrosine-containing proteins which occur during IGF-I stimulation of normal rat kidney and Madin-Darby canine kidney cells labeled with ortho[32P]phosphate. Both cells provide a good system to study the function of the type I IGF receptors because they contain high concentrations of these receptors but no insulin receptors. In addition, physiological levels of IGF-I, but not insulin, stimulated DNA synthesis in growth-arrested cells. IGF-I stimulated within 1 min of tyrosine phosphorylation of two proteins. One of them, with a molecular mass between 97 and 102 kDa, was supposed to be the beta-subunit of the type I IGF receptor previously identified. The other protein had an approximate molecular mass of 185 kDa, which resembled, by several criteria, pp 185, originally identified during the initial response of Fao cells to insulin binding (White, M. F., Maron, R., and Kahn, C. R. (1985) Nature 318, 183-186). These data suggest that tyrosine phosphorylation of pp 185 may occur during activation of both the type I IGF receptor and the insulin receptor, and it could be a common substrate that transmits important metabolic signals during ligand binding.

Animals↗
PubMed1986

Insulin-like growth factors, insulin, and epidermal growth factor cause rapid cytoskeletal reorganization in KB cells. Clarification of the roles of type I insulin-like growth factor receptors and insulin receptors.

Insulin-like growth factor (IGF) I (greater than or equal to 10(-10)M, insulin-like growth factor II (greater than or equal to 10(-9) M), insulin (greater than or equal to 10(-9) M, and epidermal growth factor (EGF, greater than or equal to 10(-11) M) caused rapid membrane ruffling in KB cells. The morphological change was observed within 1 min after the addition of these growth factors and was accompanied by microfilament reorganization, but not by microtubule reorganization. IGF-I, IGF-II, and insulin induced morphologically very similar or identical membrane ruffles with the order of potency IGF-I greater than IGF-II greater than insulin, whereas EGF-induced membrane ruffles were morphologically different. KB cells possessed EGF receptors, type I IGF receptors, and insulin receptors, but few or no type II IGF receptors. Monoclonal antibody against type I IGF receptors, which completely inhibited the binding of 125I-IGF-I to the cells but did not inhibit the binding of 125I-insulin, caused marked inhibition of IGF-I (10(-8) M)-stimulated membrane ruffling. IGF-II (10(-8) M)-stimulated membrane ruffling was partially inhibited in the presence of this antibody, but insulin (10(-7) M)-stimulated membrane ruffling was only slightly inhibited. In contrast, monoclonal antibody against insulin receptors blocked insulin (10(-7) M) stimulation, but not IGF-I (10(-8) M) stimulation, of membrane ruffling. Thus, this study provides evidence that IGF-I and insulin act mostly through their own (homologous) receptors and that IGF-II acts by cross-reacting with both type I IGF and insulin (heterologous) receptors in causing rapid alterations in cytoskeletal structure.

Antibodies, Monoclonal↗
PubMed1986

Substrate specificities of tyrosine-specific protein kinases toward cytoskeletal proteins in vitro.

We have previously reported that fodrin (beta subunit), tubulin (alpha subunit) and microtubule-associated proteins (MAPs; MAP2 and tau) are good substrates for the purified insulin receptor kinase (Kadowaki, T., Nishida, E., Kasuga, M., Akiyama, T., Takaku, F., Ishikawa, M., Sakai, H., Kathuria, S., and Fujita-Yamaguchi, Y. (1985) Biochem. Biophys. Res. Commun. 127, 493-500 and Kadowaki, T., Fujita-Yamaguchi, Y., Nishida, E., Takaku, F., Akiyama, T., Kathuria, S., Akanuma, Y., and Kasuga, M. (1985) J. Biol. Chem. 260, 4016-4020). In this study, to investigate the substrate specificities of tyrosine kinases, we have examined the actions of the purified epidermal growth factor (EGF) receptor kinase and Rous sarcoma virus src kinase on purified microfilament- and microtubule-related proteins. Among microfilament-related proteins examined, the purified EGF receptor kinase phosphorylated the beta subunit, but not the alpha subunit, of fodrin on tyrosine residues with a Km below the micromolar range. The fodrin phosphorylation by the EGF receptor kinase was markedly inhibited by F-actin. In contrast, the purified src kinase preferentially phosphorylated the alpha subunit of fodrin on tyrosine residues. Fodrin phosphorylation by the src kinase was not inhibited by F-actin. Among microtubule proteins examined, MAP2 was the best substrate for the EGF receptor kinase. By contrast, src kinase favored phosphorylation of tubulin as compared to MAP2. The peptide mapping of MAP2 phosphorylated by the EGF receptor kinase and by the insulin receptor kinase produced very similar patterns of phosphopeptides, while that of MAP2 phosphorylated by the src kinase gave a distinctly different pattern. When the phosphorylation of the tubulin subunits was examined, the EGF receptor kinase preferred beta subunit to alpha subunit, but the src kinase phosphorylated both alpha and beta subunits to a similar extent. These results, together with our previous results, indicate that the substrate specificities of the EGF receptor kinase and the insulin receptor kinase are very similar, but not identical, while that of the src kinase is distinctly different from that of these growth factor receptor kinases.

Amino Acids↗
PubMed1986

Purified tyrosine kinases, the EGF receptor kinase and the src kinase, can catalyze the phosphorylation of the band 3 protein from human erythrocytes.

The band 3 glycoprotein from human erythrocytes was found to be phosphorylated on tyrosine residues by the purified EGF receptor kinase and the purified src kinase in vitro. Kinetic analysis revealed that Km of the band 3 protein phosphorylation by the EGF receptor kinase was 0.17 microM and 0.65 microM in the absence and presence of EGF (3 X 10(-7)M), respectively, and that in the case of the src kinase it was 0.4 microM. From these data the band 3 protein can be regarded as one of the best substrates common for the EGF receptor kinase and the src kinase in vitro.

Anion Exchange Protein 1, Erythrocyte↗