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I Wada

Publications and source records attributed to I Wada.

At least 19 recordsLinked to original sources
PubMed1992

The p88 molecular chaperone is identical to the endoplasmic reticulum membrane protein, calnexin.

We previously described a novel molecular chaperone (designated p88) that participates in the assembly of murine class I histocompatibility molecules (Degen, E., and Williams, D. B. (1991) J. Cell Biol. 112, 1099-1115). Our findings suggest that p88 may either promote proper assembly of class I molecules or retain them, probably within the endoplasmic reticulum (ER), until assembly of the ternary complex of heavy chain, beta 2-microglobulin, and peptide ligand is complete. In this report, we compare p88 to calnexin, a calcium-binding 90-kDa phosphoprotein of the ER membrane (Wada, I., Rindress, D., Cameron, P. H., Ou, W.-J., Doherty, J.-J., II, Louvard, D., Bell, A.W., Dignard, D., Thomas, D. Y., and Bergeron, J. J. M. (1991) J. Biol. Chem. 266, 19599-19610). We show that p88 and calnexin share antigenic epitopes defined by a polyclonal anti-calnexin antiserum. Furthermore, both proteins were immunoprecipitated in association with an intracellularly retained variant of the class I H-2Kb molecule. Since p88 and calnexin were also indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, were resistant to digestion with endoglycosidase H, and exhibited virtually identical patterns of peptide fragments following digestion with either V8 protease or trypsin, we conclude that p88 and calnexin represent the same protein. The identification of the p88 chaperone as a phosphorylated, calcium-binding protein of the ER membrane suggests possible means whereby its interaction with class I molecules may be regulated.

Animals↗
PubMed1992

Decrease in beta-subunit phosphotyrosine correlates with internalization and activation of the endosomal insulin receptor kinase.

In a previous study, we showed that the rat hepatic insulin receptor (IR) kinase of endosomes (ENs) was transiently activated to levels exceeding those of plasma membrane (PM) receptors following insulin injection. Phosphatase treatment of EN receptors abolished IR kinase activation implicating beta-subunit autophosphorylation as a mediator of the activation process (Khan, M. N., Baquiran, G., Brule, C., Burgess, J., Foster, B., Bergeron, J. J. M., and Posner, B. I. (1989) J. Biol. Chem. 264, 12931-12940). In the present study, the phosphotyrosine (PY) content of the IR beta-subunit in PM and ENs was estimated by two different methods. In one method, direct in vivo labeling with 32Pi followed by receptor immunoprecipitation was carried out. In the second method, immunoblotting with antibodies against the submembrane domain of the IR beta-subunit, encompassing residue 960 (alpha 960), and with antibodies against PY (alpha PY) was used to determine the content of PY/beta-subunit in PM and ENs following injection of insulin. By both methods, it was found that the PY content of PM IR was significantly greater than that of IR in ENs. With doses of 1.5 micrograms of insulin/100 g body weight (50% receptor occupancy) or 15 micrograms/100 g body weight (receptor saturation), the PY/beta-subunit of PM IR attained a level 2.0 to 2.5-fold of that observed for the IR of ENs. Surprisingly, the IR of ENs incorporated 3 to 5 times more PY/beta-subunit than those of PM consequent to autophosphorylation. Exogenous IR kinase activity (poly(Glu:Tyr)) in PM changed only slightly with insulin dose. In contrast, EN receptors exhibited a dose-dependent increase in kinase activity coincident with the decrease in PY/beta-subunit levels. A comparison of the proportion of receptor and kinase activity immunoprecipitated by alpha PY both before and after autophosphorylation indicated that ENs but not PM contained a small population of lightly phosphorylated but highly activated receptors. Since Thr12-Lys (IR kinase residues 1142-1153) efficiently inhibited IR autophosphorylation of both PM and EN receptors, Tris phosphorylation of beta-subunit regulatory tyrosines was unlikely. These results may be explicable by a dephosphorylation-dependent activation of IR kinase, as seen with the src family of tyrosine kinases.

Amino Acid Sequence↗
PubMed1992

Overlap and discrepancy between tests for anti-C100, anti-GOR and anti-CP9 in patients with chronic liver disease and inhabitants in Saga, Japan.

The authors evaluated the clinical significance of anti-C100, anti-GOR and anti-CP9 in hepatitis C virus (HCV)-related liver disease in two populations: 459 healthy subjects and 385 patients with chronic liver disease (CLD). Previously we reported high rates of mortality and morbidity (5.3%) of CLD in subjects in Saga, Japan. This was ascribed to the high prevalence (10.8%) of anti-HCV among randomized populations, as detected by the C100 ELISA test system, as compared with a finding of 2-3% in Japanese blood donors in the same decade. The incidence of anti-C100, anti-GOR and anti-CP9 detected by ELISA test system in the healthy population currently surveyed was 17.0%, 19.2% and 32.0% respectively, as compared with 75.3%, 60.3% and 73.0% respectively, in those with CLD. The incidence of positivity for at least one of the three antibodies was high (36.4%) among healthy subjects, and even higher (86.5%) among the patients with CLD. In the healthy subjects, incidence of positivity increased with age. The healthy and CLD populations differed in the proportion of cases positive for all three antibodies vs. those positive for at least one antibody: healthy subjects, 52/167, 31.1%, vs. CLD patients, 197/333, 59.2%; P less than 0.01. Among the anti-C100-positive healthy cases, these was a significantly high level of AST, ALT, ZTT and gamma GTP compared with negative cases, with or without anti-GOR and anti-CP9 (P less than 0.01-0.05). These observations suggest that the presence of anti-C100 may be related to the active state of HCV-related liver disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Association of the tyrosine phosphorylated epidermal growth factor receptor with a 55-kD tyrosine phosphorylated protein at the cell surface and in endosomes.

After the intraportal injection of EGF, the EGF receptor (EGFR) is rapidly internalized into hepatic endosomes where it remains largely receptor bound (Lai et al., 1989. J. Cell Biol. 109:2751-2760). In the present study, we evaluated the phosphotyrosine content of EGFRs at the cell surface and in endosomes in order to assess the consequences of internalization. Quantitative estimates of specific radioactivity of the EGFR in these two compartments revealed that tyrosine phosphorylation of the EGFR was observed at the cell surface within 30 s of ligand administration. However, the EGFR was also highly phosphorylated in endosomes reaching levels of tyrosine phosphorylation significantly higher than those of the cell surface receptor at 5 and 15 min after EGF injection. A 55-kD tyrosine phosphorylated polypeptide (pyp55) was observed in association with the EGFR at the cell surface within 30 s of EGF injection. The protein was also found in association with the EGFR in endosomes as evidenced by coprecipitation studies using a mAb to the EGFR as well as by coelution with the EGR in gel permeation chromatography. Limited proteolysis of isolated endosomes indicated that the tyrosine phosphorylated domains of the EGFR and associated pyp55 were cytosolically oriented while internalized EGF was intraluminal. The identification of pyp55 in association with EGFR in both hepatic plasma membranes and endosomes may be relevant to EGFR function and/or trafficking of the EGFR.

Amino Acid Sequence↗
PubMed1992

Outcome of treatment subsequent to the elective cryopreservation of all embryos from women at risk of the ovarian hyperstimulation syndrome.

From 1st June 1989 to 31st May 1991, 78 women with a serum oestradiol level greater than 3500 pg/ml on the day of the ovulatory trigger, following pituitary suppression with buserelin and ovarian stimulation with human menopausal gonadotrophins (HMG), had all their embryos electively cryopreserved at the pronucleate stage to minimize the risk of developing ovarian hyperstimulation syndrome (OHS). Treatment with buserelin was continued in the luteal phase. A median of 19 oocytes (range 7-43) was obtained and 12 embryos (range 1-37) frozen per cycle. Twenty-one (27%) women developed OHS (six severe). Women developing OHS had higher (P less than 0.05) serum oestradiol concentrations on the 7th day after oocyte retrieval, compared to those who did not. No differences were found for any of the following criteria: aetiology of infertility, age, total dose of HMG, number of oocytes, fertilization rate or freeze-thaw survival of embryos. Subsequently, 125 frozen-thawed embryo replacements have been undertaken, using buserelin and hormone replacement therapy (HRT) (n = 93) or natural cycles (n = 32). The overall freeze-thaw survival and implantation rates per embryo were 71.8 and 11.7%, respectively. The pregnancy rates in natural cycles (19%) and buserelin/HRT cycles (29%) were not significantly different.

Buserelin↗
PubMed1992

Is continuation of a gonadotrophin-releasing hormone agonist (GnRHa) necessary for women at risk of developing the ovarian hyperstimulation syndrome?

A total of 28 women scheduled for in-vitro fertilization used buserelin and human menopausal gonadotrophin (HMG) for ovarian stimulation. One group (I) of 17 women was given human chorionic gonadotrophin (HCG 10,000 IU) to trigger ovulation, but the resulting embryos were electively cryopreserved because of the risk (serum oestradiol greater than or equal to 3500 pg/ml) of developing the ovarian hyperstimulation syndrome (OHSS). Six women continued the buserelin therapy in the luteal phase and eleven did not. In group II (n = 11), the HMG injections were discontinued because of an exaggerated ovarian response and the HCG was omitted. Six of these women continued the buserelin injections until the onset of menses and five did not. In both groups, the ovarian response to induction of ovulation (serum oestradiol concentrations and number of follicles) was similar for those who did or did not continue buserelin therapy. There was no difference in the rate of ovarian quiescence (weekly fall in serum oestradiol concentration following the stimulation) between those women who did or did not continue the buserelin therapy in either group. The serum luteinizing hormone concentrations remained low in all women in both groups. We conclude that the omission of buserelin therapy after discontinuing the HMG in women at risk of developing OHSS does not affect subsequent ovarian quiescence.

Adult↗
PubMed1991

SSR alpha and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane.

GTP phosphorylation of rough microsomes in vitro is limited to four integral membrane proteins. Two of these, a phosphoprotein (pp90) and a phosphoglycoprotein (pgp35) were purified as a complex with two nonphosphorylated membrane glycoproteins, gp25H and gp25L. The authenticity of this complex was confirmed using two different purification procedures and by coimmunoprecipitation. By immunofluorescence a reticulated cytoplasmic network was revealed for the proteins which was similar to that for Louvard et al. (Louvard, D., Reggio, H., and Warren, G. (1982) J. Cell Biol. 92, 92-107) marker antisera which also recognized purified pp90 on immunoblots. Amino acid sequencing of peptides derived from pgp35 identified this protein as SSR alpha, an endoplasmic reticulum constituent as identified by cross-linking of translocating nascent chains (Görlich, D, Prehn, S., Hartmann, E., Herz, J., Otto, A., Kraft, R., Wiedmann, M., Knespel, S., Dobberstein, B., and Rapoport, T. A. (1990) J. Cell Biol. 111, 2283-2294). The sequence of gp25H was determined from cDNA clones and was identical with SSR beta identified by Görlich et al. (1990) as being tightly bound to SSR alpha. Sequencing of gp25L revealed no similarity of the deduced sequence with other proteins. However, pp90 revealed a high degree of sequence identity with the Ca(2+)-binding protein, calreticulin. 45Ca2+ overlay studies indicated that pp90 bound Ca2+ and the name calnexin is proposed. Surprisingly, pgp25 (SSR alpha) also bound Ca2+ although gp25H (SSR beta) and gp25L did not. Triton X-114 partitioning of the integral membrane proteins of rough microsomes suggested that pgp35 (SSR alpha) and calnexin were major Ca(2+)-binding proteins of the endoplasmic reticulum membrane. We propose that the function of the complex is to regulate Ca(2+)-dependent retention mechanisms for luminal proteins of the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1991

[Clinical evaluation of severe toxicity in two patients with acute lymphoblastic leukemia receiving outpatient methotrexate therapy].

Methotrexate (MTX) is now widely used for the treatment of acute leukemia and non-Hodgkin lymphoma in the pediatric oncology field and is thought to be one of the key drugs for this treatment. A regimen utilizing high dose MTX (HD-MTX) with leucovorin rescue is being investigated as effective chemotherapy in the patients with these kinds of cancer. Relatively large amounts of MTX (225 mg/m2) are given to such outpatients by intravenous push as a course of maintenance therapy. It is said that those amounts will infuse safely. However, we experienced two serious cases-patients T.H. and M.Y.--which developed into severe side effects after this treatment. Both patients showed acute renal failure, severe myelosuppression, erosion around the oral and anal region, and continuous diarrhea. Judging from the serum concentration of MTX, patient T. H. was exposed to more than the maximum allowance serum MTX level for 9.6 days, patient M. Y. for 6.5 days. This suggests physicians must pay attention to the clinical symptoms even after treatment using MTX without HD-MTX.

Acute Kidney Injury↗
PubMed1990

Use of condoms as contraceptive and disease preventive measures among residents of Jos, northern Nigeria.

The effectiveness, acceptability and protective aspects of condoms as both contraceptive and disease control measures was evaluated in 168 male residents of Jos in Nigeria. They were aged between 15 and 40 years and were attending the Family Planning Clinic of the Jos University Teaching Hospital. The results showed that the majority (87.5%) of our patients were in the 21 to 30 years age group and most of the clients had used condoms for between 1 to 5 years. A good number of our clients (40.5%) used condom because it protects them against venereal disease while others felt it was safe and effective. The Family Planning Clinic was the main source of knowledge of the condom among our clients (42.9%), and the condom was rated as good or excellent by 137 (81.5%) clients. The only side effect of condom usage which featured prominently among our clients was reduced sexual excitement. The results were compared with studies carried out elsewhere. It was concluded that the condom could be an acceptable and effective form of contraception in this population. The users must, however, be highly motivated so as to use it during every act of coitus for optimal efficacy.

Adolescent↗
PubMed1990

Changes in circulating inhibin levels during pregnancy and early lactation in the Japanese monkey.

Changes in immunoreactive (ir-) inhibin concentrations in serum throughout pregnancy and early lactation up to one month after parturition were characterized in 6 Japanese monkeys (Macaca fuscata fuscata) by a heterologous radioimmunoassay (RIA) based on a bovine RIA. Serum levels of FSH, LH/monkey chorionic gonadotropin (mCG), estradiol-17 beta, and progesterone were also monitored for the entire period. Ir-inhibin levels in the serum were low (under 0.5 ng/ml) before conception. Three marked increases in serum ir-inhibin levels were found during pregnancy. The first increase was noted during early pregnancy, with a peak (2.2 +/- 0.2 ng/ml) at Day 22 of pregnancy (Day 0 = day of LH surge). The second increase was noted after Day 38 until Day 72 of pregnancy, when a peak value was noted (19.0 +/- 1.4 pg/ml). Plateau levels were maintained until late pregnancy, and a final rise was evident near the term with a peak (36.7 +/- 3.8 ng/ml) at Day 158 of pregnancy, 5 days before parturition. After parturition, ir-inhibin levels in the serum plummeted to nonpregnant levels within one day, and were maintained during early lactation. The first rise in serum inhibin during pregnancy was parallel to the rise of mCG and estradiol-17 beta, and the second and third rise were well correlated with serum estradiol-17 beta. Serum FSH was maintained at low levels throughout pregnancy, followed by a slight increase after parturition when serum inhibin decreased abruptly. Both bioactivity and immunoreactivity of inhibin were detected in the placental homogenates obtained at 120 days of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Ligand-mediated internalization, recycling, and downregulation of the epidermal growth factor receptor in vivo.

EGF receptor internalization, recycling,a nd downregulation were evaluated in liver parenchyma as a function of increasing doses of injected EGF. The effect of ligand occupancy in vivo on the kinetics and extent of internalization was studied with changes in the receptor content of isolated plasmalemma and endosome fractions evaluated by direct binding, Scatchard analysis, and Western blotting. For all doses of injected EGF, receptor was lost from the plasmalemma and accumulated in endosomes in a time- and dose-dependent fashion. However, at doses of injected EGF equivalent to less than or equal to 50% surface receptor occupancy (i.e., less than or equal to 1 microgram/100 g body weight), receptor levels returned by 120 min to initial values. This return was resistant to cycloheximide and therefore did not represent newly synthesized receptor. Neither was the return due to replenishment by an intracellular pool of low-affinity receptors as such a pool could not be detected by Scatchard analysis or Western blotting. Therefore, receptor return was due to the recycling of previously internalized receptor. At doses of injected EGF greater than 50% receptor occupancy, net receptor loss-i.e., downregulation-was observed by evaluating the receptor content of total particulate fractions of liver homogenates. At the higher saturating doses of injected EGF (5 and 10 micrograms/100 g body weight), the majority of surface receptor content was lost by 15 min and remained low for at least an additional 105 min. As the kinetics of ligand clearance from the circulation and liver parenchyma were similar for all doses of EGF injected, then the ligand-mediated regulation of surface receptor content and downregulation were not a result of a prolonged temporal interaction of ligand with receptor. Rather, the phenomena must be a consequence of the absolute concentrations of EGF interacting with receptor at the cell surface and/or in endosomes.

Animals↗
PubMed1989

[Primary osteogenic sarcoma of the breast. Report of a case].

Primary osteogenic sarcoma (POS) of the breast is very rare. Only eleven cases have been previously reported in Japan. In this paper, we report an additional case of POS. A 30 year old female was admitted to our hospital with chief complaint of rapidly growing mass of the right breast. She was diagnosed carcinoma of the right breast and underwent a radical mastectomy (Br + Ax + Mj + Mn). Histological findings of the resected tissues revealed POS of the breast. After three months from the operation, local recurrence was demonstrated by an incisional biopsy. One more months later, chest X-P revealed bilateral lung metastases. Irradiation with 60Co for recurrence and chemotherapy using vincristine, methotrexate for distant metastases were not effective. However, cisplatin caused the necrosis of local recurrent tumor. She died nine months after operation. At autopsy, extended metastasis of tumor cells diffusely to the pleura, lungs, liver, adrenal glands, pancreas, peritoneum, fifth lumbar vertebra, skull, dura mater and left pulmonary hilar lymph nodes was observed.

Adult↗
PubMed1989

[A case of congenital retinoschisis in a 34-week gestation female infant].

At age one month a female infant was referred for a fundus examination because she was delivered after a 34-week gestation and her birth weight was 1908 g. Fundus examination revealed a brown granular pigmentary deposition surrounding the foveola in the macular region and a large highly ballooning retinoschisis occupying almost the lower half of the fundus in each eye. There was slight hemorrhage in the vitreous body and the retinoschisis cavity, and no retinal hole. The anterior border of the retinoschisis did not extend to the ora serrata. The almost negative ERG response showed an abnormal b-wave amplitude. Over the course of about one year, the vitreous hemorrhage developed and cleared. The ballooning feature of the retinoschisis also developed and disappeared. On examination of the infant's family her father and elder sister had no abnormal findings but her mother had chorioretinal degeneration with macular yellow pigmentary deposition, visual defect and negative ERG. These abnormal findings of her mother were suggested to have been derived from congenital retinoschisis. Therefore, this is a very rare case because of the different hereditary form, the common sex-linked recessive inheritance, the 34-week gestation and female infant.

Female↗
PubMed1987

5'-Nucleotidase in rat liver lysosomes.

5'-Nucleotidase was found in purified rat liver tritosomes. When tritosomes were subfractionated into the membrane and soluble contents fractions, 73% of the total 5'-nucleotidase activity was found in the membrane fraction and 24% in the soluble contents fraction. Immunoblotting using specific polyclonal antibodies against the rat liver plasma membrane 5'-nucleotidase showed that the mobilities on SDS-polyacrylamide gel electrophoresis of both 5'-nucleotidases in the membrane and contents fractions were identical to that of the enzyme in the plasma membranes (Mr = 72,000). 5'-Nucleotidases in the membrane and contents fractions were sensitive to neuraminidase and converted into a form that was 4 kDa smaller after digestion, as observed in the case of plasma membrane enzyme. 5'-Nucleotidases, both from the membrane and contents fractions, were purified using immunoaffinity chromatography, and the isoelectric points, heat stability, and oligomeric structure of the purified enzymes were compared. Isoelectric focusing and the heat stability test indicated the resemblance of the soluble enzyme to the membrane-bound enzyme. However, the membrane-bound enzyme aggregated in the absence of Triton X-100, whereas the soluble enzyme behaved as a dimer. The topography of 5'-nucleotidase in the tritosomal membranes was studied using antibodies against 5'-nucleotidase and neuraminidase treatment. The inhibition of 5'-nucleotidase were not observed in the intact tritosomal fraction until the tritosomes had been disrupted by osmotic shock. These results show that the active sites and the oligosaccharide chains of 5'-nucleotidase are located on the inside surface of the tritosomal membranes.

5'-Nucleotidase↗
PubMed1987

Characterization of alpha-adrenoceptor subtypes involved in regulation of ureteral fluid transport.

Experiments were carried out in the dog by the use of experimental procedure which permits to assess independently changes in uretheral peristaltic frequency, bolus volume and intraluminal pressure and flow volume in order to characterize alpha-adrenoceptor subtypes involved in regulation of ureteral urine transport. Norepinephrine caused an increase in ureteral peristaltic frequency, an elevation in intraureteral baseline and contractile pressure and a decrease in bolus volume, with a resultant decrease in the rate of fluid transport. Phenylephrine (alpha 1-agonist) and clonidine (alpha 2-agonist) caused the effects similar to those of norepinephrine on peristaltic frequency, intraureteral baseline and contractile pressure, and bolus volume. Phentolamine (non-selective alpha-antagonist) and prazosin (alpha 1-antagonist) caused a decrease in ureteral peristaltic frequency, and a fall in intraureteral baseline and contractile pressure, and yohimbine (alpha 2-antagonist) abolished peristalsis and bolus formation. These changes were accompanied by an increase in the rate of fluid transport. These data suggest that the ureteral urine transport is controlled by activation of both alpha 1- and alpha 2-adrenoceptors through regulation of peristaltic frequency and bolus volume.

Animals↗
PubMed1986

Biosynthesis and intracellular transport of rat liver 5'-nucleotidase.

To investigate biosynthesis and intracellular transport of 5'-nucleotidase, we purified this enzyme from rat liver and prepared antibodies. In immunoblot analysis, 5'-nucleotidase from the plasmalemmal, Golgi, and tritosomal fractions migrated as a single band of 72 kDa. The immunoreactive 68-kDa band was detected in the rough microsomal fraction and only the 72-kDa polypeptide contained sialic acids. The single polypeptide of 61 kDa immunoprecipitated from the translation products with the membrane-bound polysomal RNAs by the cell-free system converted to the 68-kDa form associated with membranes, when translated in the presence of microsomal vesicles. 5'-Nucleotidase from cultured primary hepatocytes pulse-labeled with [35S]methionine for 20 min migrated as a 68-kDa polypeptide. Within 45 min of chase, the 68-kDa form was converted to the 72-kDa form. Upon treatment with tunicamycin, a new immunoreactive polypeptide of 59 kDa was obtained. These results suggest that 5'-nucleotidase is translated on the membrane-bound polysomes as a 61-kDa precursor and that the cleavage of the 2-kDa signal peptide and the core glycosylation co-translationally convert the precursor to the 68-kDa form, which is subsequently processed in the Golgi complex to the 72-kDa form.

5'-Nucleotidase↗
PubMed1986

Electron microscopic localization of 5'-nucleotidase in rat salivary glands. Comparative enzyme- and immunohistochemical studies.

The localization of 5'-nucleotidase in rat parotid and submandibular glands was investigated at the electron microscope level by an immunohistochemical technique using a highly specific antibody, and the results were compared with those obtained using the newley developed cerium method for enzyme histochemistry. Both methods demonstrated that 5'-nucleotidase is located on the external surface of the luminal plasma membranes of acinar cells as well as on intercalated and striated ductal cells. In the basolateral membranes of these cells, the portions adjacent to myoepithelial cells exhibited intense reaction products, but the other areas of plasma membranes contained only trace amounts of the reaction products. Both cerium-based enzyme histochemistry and immunohistochemistry showed that myoepithelial cells retain the enzyme on their plasma membranes. Neither method produced reaction products in the intracytoplasmic structure of constitutive cells of the salivary glands. We discuss the usefulness of the cerium-ion method for the demonstration of 5'-nucleotidase activity and compare it with the traditional lead-ion method.

5'-Nucleotidase↗