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Biomedical subjects

T Kadowaki

Publications and source records attributed to T Kadowaki.

At least 253 records · Page 14Linked to original sources
PubMed1992

Mitogen-induced tyrosine-phosphorylated 41- and 43-kDa proteins are family members of extracellular signal-regulated kinases/microtubule-associated protein 2 kinases.

Two antipeptide antibodies, one against the peptide corresponding to residues 307-327 (alpha Y91) and one against the peptide corresponding to the C-terminal portion (alpha C92) of the deduced amino acid sequence of the extracellular signal-regulated kinase 1 (ERK1), precipitated two 41-kDa and/or two 43-kDa phospho-proteins from mitogen-stimulated Swiss 3T3 cells. Electrophoretic mobilities on two-dimensional gels of the immunoprecipitated 41- and 43-kDa phosphoproteins were similar to those of the 41- and 43-kDa cytosol proteins, whose increased tyrosine phosphorylation we and others had originally identified in various mitogen-stimulated cells (Cooper, J. A., Sefton, B. M., and Hunter, T. (1984) Mol. Cell. Biol. 4, 30-37; Kohno, M. (1985) J. Biol. Chem. 260, 1771-1779); phosphopeptide map analysis revealed that they were respectively identical molecules. All those phosphoproteins contained phosphotyrosine, and the more acidic forms contained additional phosphothreonine. Immunoprecipitated 41- and 43-kDa phosphoproteins had serine/threonine kinase activity toward myelin basic protein (MBP) and microtuble-associated protein 2 (MAP2). With the combination of two-dimensional gel electrophoresis and the kinase assay in MBP-containing polyacrylamide gels of the alpha Y91 immunoprecipitates, with or without phosphatase 2A treatment, we showed that only their acidic forms were active. These results clearly indicate that 41- and 43-kDa proteins, the increased tyrosine phosphorylation of which is rapidly and commonly induced by mitogen stimulation of fibroblasts, are family members of ERKs/MAP2 kinases and that phosphorylation both on tyrosine and threonine residues is necessary for their activation.

3T3 Cells↗
PubMed1992

A conditional yeast mutant deficient in mRNA transport from nucleus to cytoplasm.

Transport of mRNA from nucleus to cytoplasm is critical for eukaryotic gene expression; however, the mechanism of export is unknown. Selection and screening procedures have therefore been used to obtain a family of temperature-sensitive conditional mutants of Saccharomyces cerevisiae that accumulate poly(A)+ RNA in the nucleus when incubated at 37 degrees C, as judged by in situ hybridization. In one such mRNA transport mutant, mtr1-1, RNA synthesis continues, the export of poly(A)+ RNA is inhibited, intranuclear poly(A)+ is remarkably stable, and protein synthesis gradually stops. Thus, there is no tight coupling between RNA synthesis and export. The export lesion is reversible. Although mRNA export is clearly not a default option, neither inhibition of protein synthesis, inhibition of mRNA splicing, nor inhibition of poly(A)-binding protein function blocks export of the average poly(A)+, as judged by in situ hybridization. Further analysis of the family of mtr mutants should help map the path of RNA transport.

Adenosine Triphosphate↗
PubMed1992

Expression of M-CSF receptor encoded by c-fms on smooth muscle cells derived from arteriosclerotic lesion.

Vascular smooth muscle cells in atherosclerotic lesions are phenotypically different from those in the normal arterial wall, and no expression of macrophage colony stimulating factor (M-CSF) receptor encoded by the proto-oncogene c-fms has been demonstrated in normal smooth muscle cells. In the present study, we demonstrated expression of c-fms and high affinity binding of M-CSF in smooth muscle cells isolated from an experimental rabbit model of arteriosclerosis (intimal smooth muscle cells), while no expression of c-fms was shown in medial smooth muscle cells. In the immunocytochemical analysis, both types of smooth muscle cells similarly reacted with an antibody specific to muscle cells (HHF 35) but did not react with an antibody specific to rabbit macrophages (RAM 11). In intimal smooth muscle cells, when cells were incubated with acetylated low density lipoproteins (LDL), the binding of acetylated LDL and foam cell formation were observed. In response to M-CSF, tyrosine-phosphorylation, as analyzed by the detection of anti-phosphotyrosine-reactive proteins, and an increased rate of cell proliferation were observed in intimal smooth muscle cells. These results indicated that intimal smooth muscle cells have the characteristics of monocyte-macrophages such as the expression of c-fms, which may be related to their proliferation and phenotypic conversion into foam cells in atheromatous lesions.

Animals↗
PubMed1992

Immunoglobulin heavy chain-binding protein binds to misfolded mutant insulin receptors with mutations in the extracellular domain.

Cell-surface proteins are transported through the endoplasmic reticulum and Golgi apparatus en route to the plasma membrane. Previously, we have identified three point mutations in the insulin receptor gene that impair transport of the mutant receptors to the cell surface: Asn15----Lys, His209----Arg, and Phe382----Val. Furthermore, these mutations impair post-translational processing steps that normally occur as the receptors are transported through the endoplasmic reticulum and Golgi apparatus. In this study, we have demonstrated that the unprocessed Arg209 and Val382 mutant proreceptors are bound to the immunoglobulin heavy chain-binding protein (BiP) in the endoplasmic reticulum. This was demonstrated by the fact that monoclonal anti-BiP antibody coimmunoprecipitated the mutant proreceptors. Moreover, when ATP was added to the immunoprecipitates, the mutant proreceptors were released from BiP. In contrast, neither the normal human insulin receptor nor the Lys15 mutant proreceptor was coimmunoprecipitated by anti-BiP antibody. It seems likely that the Lys15 receptor also binds BiP, but that the affinity was too low to resist dissociation during the stringent washing of the immunoprecipitate. In conclusion, these observation are consistent with the hypothesis that binding to BiP explains the impaired transport of mutant receptors through the endoplasmic reticulum and Golgi apparatus to the plasma membrane.

3T3 Cells↗
PubMed1992

Mutations in the insulin receptor gene in patients with genetic syndromes of insulin resistance and acanthosis nigricans.

Mutations of the insulin receptor gene have been identified in patients with genetic syndromes of insulin resistance associated with acanthosis nigricans. These mutations impair insulin responses by reducing the number of insulin receptors on the surface of target cells, or by reducing the receptor's ability to bind insulin or to undergo insulin-stimulated autophosphorylation, an important step in insulin action. Studies of mutant receptors expressed in transfection systems have contributed to our understanding of the structure-function relationships of the insulin receptor.

Acanthosis Nigricans↗
PubMed1992

Detection of mutations in the human insulin gene by single strand conformation polymorphisms.

Single strand conformation polymorphisms (SSCP) is the method by which mutations can be detected in DNA amplified by the polymerase chain reaction (PCR). This method utilizes the fact that the electrophoretic mobility of single stranded DNA under nondenaturing condition depends not only on its size but also on its conformation. We examined whether this technique could detect five mutations that had previously been identified in the insulin genes of patients with abnormal insulin or familial hyperproinsulinemia. Five mutant insulin genes were constructed by site directed mutagenesis, and the cloned mutant insulin genes were amplified by PCR. All five mutations were detected because they were associated with shifts in the electrophoretic mobility of the DNA fragments. In addition, we analyzed amplified genomic DNA from a patient who is heterozygotes for the Insulin Wakayama mutation A3(Val----Leu). This mutation was also detected by the SSCP technique. We conclude that PCR-SSCP method is a simple, fast, and efficient method for detection of mutations in the human insulin gene.

Base Sequence↗
PubMed1992

Structure of the human glucokinase gene and identification of a missense mutation in a Japanese patient with early-onset non-insulin-dependent diabetes mellitus.

Glucokinase is thought to play a glucose-sensor role in the pancreas, and abnormalities in its structure, function, and regulation can induce diabetes. We isolated the human glucokinase gene, and determined its genomic structure including exon-intron boundaries. Structure of the glucokinase gene in human was very similar to that in rat. Then, by screening Japanese diabetic patients using polymerase chain reaction--single strand conformation polymorphism (PCR-SSCP) and direct-sequencing strategies, we identified a missense mutation substituting arginine (AGG) for glycine (GGG) at position 261 in exon 7 of the glucokinase gene in a patient with early-onset non-insulin-dependent diabetes (NIDDM).

Base Sequence↗
PubMed1992

[Persistent müllerian duct syndrome with seminoma: report of a case].

A case of persistent Müllerian duct syndrome associated with seminoma is reported. A 22-year-old man was admitted with the chief complaint of left flank colicky pain. Physical examination revealed huge abdominal mass and absence of left intrascrotal contents. Right hydrothorax and left ureteral calculi were pointed out with radiography. The abdominal tumor was reduced by effective chemotherapy. Extirpation confirmed the presence of seminoma and persistent Müllerian duct structure. This case showed left testicular transverse ectopia. In our review of the Japanese literature, 16 cases of transverse testicular ectopia accompanied with testicular tumor are reported.

Adult↗
PubMed1992

Bipolar prosthetic replacement for the treatment of avascular necrosis of the femoral head.

Eighty-three hips in 66 patients with nontraumatic avascular necrosis of the femoral head (ANFH) showing evidence of severe collapse or secondary osteoarthritic changes were treated with surgical implantation of bipolar hip prostheses either with or without cement fixation of femoral stems. The cases were observed for more than three years (range, three to ten years seven months; average, five years seven months) and assessed in terms of functional and roentgenographic results. These data were compared with the results of classic hemiarthroplasties using fixed head prostheses (Austin-Moore-type with curved or straight stem) that were performed at the authors' institutions before 1980. The results confirm that the clinical outcome is improved with use of the bipolar prosthesis. Satisfactory results (a score of 80 or greater on a hip function scoring system proposed by the Japanese Orthopaedic Association) were maintained throughout the follow-up period in most cases (71 of 83 hips) with the bipolar prosthetic replacements. In a group of patients with unsatisfactory results (12 hips), proximal migration of prosthetic head was seen in two cases. The incidence of proximal migration of bipolar heads, including those exhibiting minimal movement, was significantly lower when compared with that observed in an Austin-Moore-type head-fixed prosthesis group (7/83 versus 12/19) during a comparable postoperative follow-up period. It is noteworthy that the proximal migration of the bipolar head was not progressive, and, in most cases observed more than five years, the cartilaginous spaces of acetabulum were preserved, whereas in the Austin-Moore-replaced group, the migration was evident and progression occurred within three years of surgery.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Insulin and 12-O-tetradecanoylphorbol-13-acetate activation of two immunologically distinct myelin basic protein/microtubule-associated protein 2 (MBP/MAP2) kinases via de novo phosphorylation of threonine and tyrosine residues.

Two site-specific antibodies have been prepared by immunizing rabbits with chemically synthesized peptides derived from the partial cDNA-predicted amino acid sequence of extracellular signal-regulated kinase 1 (ERK1), which has been proposed to encode the microtubule-associated protein 2 (MAP2) kinase (Boulton, T. G., Yancopoulos, G. D., Gregory, J. S., Slauer, C., Moomaw, C., Hsu, J., and Cobb, M. H. (1990) Science 249, 64-67). With immunoprecipitation in the presence of sodium dodecyl sulfate (SDS) and Western blotting, an antibody to the peptide containing triple tyrosine residues (alpha Y91) resembling one of the insulin receptor autophosphorylation sites specifically recognized 42- and 44-kDa proteins. On the other hand, an antibody to the peptide corresponding to the COOH terminus portions (alpha C92) of the ERK1 cDNA gene product recognized the 44-kDa protein much more efficiently than the 42-kDa protein. With immunoprecipitation in the absence of SDS, alpha Y91 could barely recognize these two proteins and alpha C92 recognized the 44-kDa protein but failed to recognize the 42-kDa protein. Kinase assays in myelin basic protein (MBP)-containing gel, after SDS-polyacrylamide gel electrophoresis, revealed that insulin or 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated MBP kinase activity in alpha Y91 immunoprecipitates comigrated at molecular mass 42 and 44 kDa. On the other hand, the stimulated MBP kinase activity in alpha C92 immunoprecipitates comigrated only at molecular mass 44 kDa. Insulin stimulated the MBP kinase activity in gels and phosphorylation of these two proteins by greater than 10-fold with a maximal level at 5 min. Insulin and TPA rapidly stimulate the phosphorylation of the 42- and 44-kDa proteins via de novo threonine and tyrosine phosphorylation. Tryptic phosphopeptide mapping analysis of the 42- and 44-kDa proteins, respectively, revealed a single major phosphopeptide containing phosphothreonine and phosphotyrosine, which was common to both insulin- and TPA-stimulated phosphoproteins. Protein phosphatase 2A treatment of these two phosphoproteins caused a complete loss of kinase activity with selective dephosphorylation of phosphothreonine. These data strongly suggest that these two proteins are highly related to the mitogen-activated protein (MAP) kinase with an apparent molecular mass of 42 kDa (Ray, L. B., and Sturgill, T. W. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3753-3757) and that these two immunologically similar but distinct MBP/MAP2 kinases may represent isozymic forms of MBP/MAP2 kinases. These data also demonstrate that insulin and TPA activate MBP/MAP2 kinase activity by de novo phosphorylation of threonine and tyrosine residues via a very similar pathway.

Amino Acid Sequence↗
PubMed1991

Substitution of arginine for histidine at position 209 in the alpha-subunit of the human insulin receptor. A mutation that impairs receptor dimerization and transport of receptors to the cell surface.

Receptors for insulin and epidermal growth factor contain cysteine-rich domains in the extracellular portion of the molecule. His209 (insulin receptor numbering system) is 1 of 2 amino acid residues that are identically conserved in the cysteine-rich domains of insulin receptors, epidermal growth factor receptors, and other homologous receptors. Previously, we have identified a patient with leprechaunism who is homozygous for a mutation substituting Arg for His209 in the insulin receptor gene (Kadowaki, T., Kadowaki, H., Rechler, M. M., Serrano-Rios, M., Roth, J., Gorden, P., and Taylor, S. I. (1990) J. Clin. Invest. 86, 254-264). In this investigation, the Arg209 mutant receptor was expressed by transfection of mutant cDNA into NIH-3T3 cells. The mutation impairs several steps in the post-translational processing of the insulin receptor:dimerization of 190-kDa proreceptors into a disulfide linked species, proteolytic cleavage of the proreceptor into alpha- and beta-subunits, and terminal processing of the high mannose form of N-linked oligosaccharide into complex carbohydrate. In addition, the defects in post-translational processing within the endoplasmic reticulum and Golgi apparatus are associated with a marked inhibition in transport of receptors to the plasma membrane. Nevertheless, a small number (approximately 10%) of the receptors are transported to the cell surface. These receptors on the cell surface bind insulin with normal affinity and have normal tyrosine kinase activity.

3T3 Cells↗
PubMed1991

Insulin and platelet-derived growth factor stimulate phosphorylation of the c-raf product at serine and threonine residues in intact cells.

We have examined the phosphorylation of the serine threonine kinase, the product of c-raf proto-oncogene in response to insulin or platelet-derived growth factor in intact cells. Both insulin and platelet-derived growth factor stimulated phosphorylation of the c-raf protein about 2- to 3-fold. The phosphorylation occurred exclusively on serine and threonine residues; phosphotyrosine was not detected. In immune-complex kinase assays, treatment with insulin, and platelet-derived growth factor increased autophosphorylation of the c-raf kinase, suggesting activation of its kinase activity. To investigate whether the phosphorylation of the c-raf protein in intact cells results from an autophosphorylation event or from the phosphorylation by other cellular kinase(s), we replaced lysine 375 in the putative ATP-binding domain of the c-raf protein with alanine using oligonucleotide site-directed mutagenesis and expressed the mutated protein in NIH3T3 cells. The substitution resulted in the inactivation of the serine/threonine-specific autophosphorylation in immune-complex kinase assays. In intact cells, however, although phosphorylation of the mutant protein in response to insulin and platelet-derived growth factor occurred to a lesser extent than that of the wild-type protein, the phosphopeptide maps were indistinguishable. These results suggest that serine threonine phosphorylation might be responsible for the activation of c-raf kinase upon treatment of cells with insulin and platelet-derived growth factor, and most of the phosphate associated with the c-raf protein results from its phosphorylation by as yet uncharacterized cellular serine/threonine kinase(s).

Base Sequence↗
PubMed1991

Hypoxic depression of mitochondrial mRNA levels in HeLa cell.

Steady-state levels of 12 S and 16 S mitochondrial (mt) rRNAs and four mRNAs (NADH dehydrogenase subunits 3 and 4/4L, cytochrome c oxidase subunit III, H(+)-ATPase subunits 6/6L) were estimated by hybridization of cellular RNA with cloned human mt DNA fragments during hypoxia of HeLa cells. When the partial pressure of oxygen (pO2) was shifted from 135 to 15 Torr, the level of all mRNAs coordinately decreased more than 95% in 48 h, while that of rRNAs remained virtually unchanged. mRNA levels recovered within 4-6 h of reexposure to normoxia. The depression was observed at pO2 less than 40 Torr, the physiological pO2 in peripheral tissues. During these transitions, the growth rate of HeLa cells and the copy number of mt DNA per cell remained unchanged. The degradation rate of mt mRNAs in the presence of cordycepin was not affected by pO2. In contrast to the in vivo results, the potential activity of transcription in isolated mitochondria assayed under optimum conditions was not affected by previous hypoxic exposure of the cells. These observations provide evidence for the existence of a new mechanism controlling mitochondrial gene transcription.

Adenosine Triphosphate↗
PubMed1991

Activation of MAP kinase and enhanced phosphorylation of the 350-kDa protein by mitogenic stimuli in quiescent Balb/c 3T3 cells.

In quiescent Balb/c 3T3 cells, competence factors such as platelet-derived growth factor and 12-O-tetradecanoylphorbol-13-acetate (TPA) activated MAP kinase, whereas progression factors such as insulin did not. Insulin was, however, capable of activating MAP kinase in cells pretreated with TPA. Moreover, TPA plus insulin activated MAP kinase more strongly and for a longer time period than did TPA alone. Treatment of Balb/c 3T3 cells with competence factors stimulated phosphorylation of the 350-kDa protein which was immunoprecipitated with antibodies against brain high-molecular-weight microtubule-associated protein MAP1, whereas insulin treatment did not stimulate the phosphorylation. Insulin could induce, however, further increase in the phosphorylation of the 350-kDa protein, when added simultaneously with TPA or added to the TPA-treated cells. The enhanced phosphorylation of the 350-kDa protein thus correlated with the MAP kinase activation. As insulin acts synergistically with TPA to induce initiation of DNA synthesis in the quiescent Balb/c 3T3 cells, it seems that activation of MAP kinase and enhanced phosphorylation of the 350-kDa protein are accompanied by the initiation of DNA synthesis.

Animals↗
PubMed1991

Unusual forms of insulin resistance.

Insulin resistance is an important feature in the pathogenesis of non-insulin-dependent diabetes mellitus. Some rare patients have syndromes associated with a severe form of insulin resistance, usually involving acanthosis nigricans and, in premenopausal females, hyperandrogenism. This review discusses two of the causes of insulin resistance: mutations in the insulin receptor gene and autoantibodies directed against the insulin receptor.

Acanthosis Nigricans↗