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M Ujihara

Publications and source records attributed to M Ujihara.

At least 37 records · Page 2Linked to original sources
PubMed1993

Mutagenesis of the 'determinant loop' region of human choriogonadotropin beta.

The hormone-specific beta subunits of the four human glycoprotein hormones are homologous, and mapping studies are underway in many laboratories to delineate the amino acid residues responsible for receptor binding and activation. Results on the human choriogonadotropin beta (hCG beta) subunit, obtained using synthetic peptides, chemically modified derivatives, and mutant forms prepared via site-directed mutagenesis, have suggested that amino acid residues enclosed by the purported disulfide loop between Cys-93 and Cys-100 may contribute to receptor binding and perhaps specificity. Indeed, the 93-100 amino acid sequence is referred to as a determinant loop. We have used site-directed mutagenesis to prepare single amino acid residue replacements at positions not previously investigated in full length beta subunits; these include Arg-95, Ser-96, Thr-97, and Thr-98. In addition, Leu-92 was studied in an effort to determine whether changes immediately adjacent to the determinant loop alter receptor binding. The wild-type and mutant cDNAs for hCG beta were subcloned into a Prsv expression vector and transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for bovine alpha. The concentrations of total expressed hCG beta in heterodimer form with the bovine alpha subunit were determined by radioimmunoassays. The mutant gonadotropins were assayed in vitro using a competitive binding assay with [125I]hCG and progesterone production, both in the transformed murine Leydig cell line, MA-10. Mutant beta subunits containing the replacements Lys-92, Ser-95, Asp-96, and Tyr-97 exhibited normal alpha subunit binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Comparison of biological aspects among ovine luteinizing hormone isoforms with charge heterogeneity.

Ovine luteinizing hormone (oLH), a pituitary hormone with sulfated asparagine-linked oligosaccharides, was examined with regard to how its isoforms having different isoelectric points (pIs) modulate in vivo biological activity. oLH was separated into five fractions by means of an isoelectric focusing (IEF) column, i.e., IEF fr 1 (pI > 10.78), IEF fr 2 (pI 10.78-10.26), IEF fr 3 (pI 10.22-10.07), IEF fr 4 (pI 9.99-9.84), and IEF fr 5 (pI 9.76-9.48). Of these, we studied the three major fractions, IEF fr 2, 3 and 4. The binding of 125I-oLH to rat testis homogenates was inhibited most strongly by IEF fr 4, followed by 2 and 3. The ability to stimulate cyclic AMP release from dispersed rat Leydig cells followed the same sequence. After bolus injections of IEF fractions, the plasma immunoreactive oLH levels of male rabbits were measured. IEF fr 4 and 3 had longer plasma half-lives than 2. As expected, plasma testosterone levels after injections increased with the additive effects of in vitro activity and the rate of clearance from the circulation, i.e., IEF fr 4 > 3 = 2. In vivo renotropic activity, 3H-thymidine incorporation into the renal DNA of hypophysectomized, castrated rats, was found only in IEF fr 4. Our study indicates that an oLH isoform with a lower alkali pI had stronger LH receptor binding and in vitro bioactivity and a longer plasma half-life, resulting in stronger in vivo bioactivity.

Animals↗
PubMed1992

Subunit-specific sulphation of oligosaccharides relating to charge-heterogeneity in porcine lutrophin isoforms.

Lutrophin (LH) consists of an array of isoforms with different charges and bioactivities. This study was undertaken to clarify specifically how oligosaccharides of alpha and beta subunits contribute to LH isoform charges. Porcine LH (pLH) was separated into four isoforms by isoelectric focusing (IEF), followed by subunit isolation. Their oligosaccharides were released by hydrazinolysis, labelled by reduction with NaB3H4, and fractionated by HPLC with a Mono Q column into five populations differing in the number of sulphate (S) and sialic acid (N) residues, designated as Neutral, N-1, S-1, S-N and S-2. Oligosaccharides were predominantly sulphated (S-1 and S-2) and infrequently sialylated (N-1 and S-N). Further analysis, including concanavalin A (Con A) affinity chromatography, desialylation, desulphation, sequential exoglycosidase digestion and methylation, clarified the structures of the acidic oligosaccharides. All were of the biantennary complex type. Their two peripheral branches were SO4-4GalNAc beta 1-4Glc-NAc and GalNAc beta 1-4GlcNAc or GlcNAc in S-1, SO4-4GalNAc beta 1-4GlcNAc and Sia alpha 2-6Gal beta 1-4GlcNAc in S-N, and (SO4-4GalNAc beta 1-4GlcNAc)2 in S-2 (where GalNAc is N-acetylgalactosamine and GlcNAc is N-acetylglucosamine). Ten percent of S-1 and of S-N had a bisecting GlcNAc residue. Sulphate residues occurred in nearly the same amount for both subunits; however, the alpha and beta subunits were sulphated differently. S-1 predominated in the alpha subunit, while S-1 and S-2 were major components in the beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Plasma aldosterone response to upright posture and angiotensin II infusion in aldosterone-producing adenoma.

Nineteen patients with primary aldosteronism due to surgically confirmed aldosterone-producing adenoma (APA) were examined to evaluate the response of aldosterone to upright posture and angiotensin II infusion. Upright posture reportedly decreases the plasma aldosterone concentration (PAC) in APA but raises it in idiopathic hyperaldosteronism. However, our findings showed the opposite result, in that the upright posture did not change or raised PAC in 15 of 19 cases (79%). Angiotensin II was infused i.v. at doses from 0.5-2 ng/min.kg body weight in six patients in whom the upright posture raised PAC, but did not raise PAC in all cases. This result supports the assumption that APA is functionally insensitive to angiotensin II. A concomitant rise of ACTH, pretreatment with calcium channel blockade, and other modulating factors may be involved in this PAC rise. Whatever the reason, such a high frequency of patients with increased PAC in APA raises some question about the clinical value of the upright posture test. We believe, then, there is reason to check any interpretation concerning increased PAC in the case of the upright posture test in distinguishing between APA and idiopathic hyperaldosteronism.

Adenoma↗
PubMed1992

Clinical significance of adrenal computed tomography in Addison's disease.

Adrenal computed tomographic (CT) scanning was conducted in twelve patients with Addison's disease during the clinical course. In tuberculous Addison's disease (n = 8), three of four patients examined during the first two years after disease onset had bilaterally enlarged adrenals, while one of four had a unilaterally enlarged one. At least one adrenal gland was enlarged after onset in all six patients examined during the first four years. Thereafter, the adrenal glands may atrophy bilaterally, in contrast to adrenal glands in idiopathic Addison's disease, which atrophy bilaterally from disease onset (n = 2). Adrenal calcification was a less sensitive clue in tracing pathogenesis, i.e., adrenal calcification was observed in five of eight patients with tuberculous Addison's disease, but not in idiopathic patients. Thus, adrenal CT scanning could show the etiology of Addison's disease (infection or autoimmunity) and the phase of Addison's disease secondary to tuberculosis, which may be clinically important for initiating antituberculous treatment.

Addison Disease↗
PubMed1991

A case of hypocalciuric hypercalcemia without family history.

Familial hypocalciuric hypercalcemia (FHH) is usually characterized by asymptomatic hypercalcemia, mild hypermagnesemia, and low urinary calcium excretion, and is occasionally associated with pulmonary fibrosis. It is inherited as an autosomal-dominant, and no sporadic case of hypocalciuric hypercalcemia has been heretofore reported. This report describes a patient with hypocalciuric hypercalcemia completely compatible with FHH but with no family history, suggesting that the most likely diagnosis is "nonfamilial" hypocalciuric hypercalcemia. We propose that the urinary excretion of calcium be examined in all patients with hypercalcemia, hypophosphatemia, and increased PTH before neck surgery, even if patients have no family history of hypercalcemia.

Aged↗
PubMed1991

Downward regulation of plasma LH by LHRH agonist, leuprolide acetate, resulting in inhibited renal growth and function in the castrated male rat.

We previously reported that ovine and porcine luteinizing hormone (LH) stimulated kidney growth in castrated hypophysectomized rats. Our present study focuses on the physiological role of the renotropic activity of LH isoforms. Plasma LH levels were decreased to 10% of that of castrated control rats by injections of a slow-releasing LHRH agonist, leuprolide acetate, from microcapsules. Compared to controls, which were injected with microcapsules only, the kidney weight in leuprolide-treated castrated rats decreased 12%. Renal protein and DNA contents decreased significantly. Body, liver and spleen weights were not changed by the treatment, however. This effect on the kidney was not observed in castrated hypophysectomized rats, suggesting that leuprolide affected the kidneys indirectly, rather than directly, by suppressing LH secretion. In leuprolide-treated castrated rats, urinary fractional excretion of sodium (FENa) increased, indicating suppressed renal function at the proximal tubules. We concluded that the secretion of renotropically active LH isoforms was regulated at least partially by LHRH and played a physiological role in growth and the function of the proximal tubules.

Animals↗
PubMed1991

Renotropic and gonadotropic activity in homologous and heterologous hybrids of ovine luteinizing hormone and human chorionic gonadotropin subunits.

We previously demonstrated the renotropic activity of ovine and porcine LH, i.e. the stimulation of [3H]thymidine incorporation into the renal DNA of castrated hypophysectomized rats. We conducted this study to determine which condition is required to promote renotropic activity. We prepared homologous (oLH alpha.oLH beta, hCG alpha.hCG beta) and heterologous (oLH alpha.hCG beta, hCG alpha.oLH beta) hybrids from isolated oLH- and/or hCG-derived subunits. Native oLH and all hybrids showed gonadotropic activity when examined for cyclic AMP generation in cultured Leydig tumour cells (MA-10). Native oLH and the hybrids which showed renotropic activity were all found to contain at least one of the oLH subunits. Our findings point out the important, special role of oLH subunits in promoting renotropic activity. The condition required is suggested to be presence of a common structure in the two oLH subunits. Such a structure should consequently not be present in hCG. A carbohydrate moiety of oLH containing a sulphate group is proposed as a candidate, but the assumption remains to be demonstrated.

Animals↗
PubMed1990

Mast cells contain spleen-type prostaglandin D synthetase.

Prostaglandin D synthetase activity in the cytosol (100,000 x g, 1-h supernatant) fraction of peritoneal mast cells of adult rats (105.0 nmol/min/mg protein) was the highest among such activities in various rat tissues and cells. As judged by the absolute requirement for glutathione for the reaction (Km = 300 microM), the Km value for prostaglandin H2 (200 microM), and insensitivity of the activity to 1 mM 1-chloro-2,4-dinitrobenzene, the enzyme in mast cells was similar to rat spleen prostaglandin D synthetase and differed from rat brain prostaglandin D synthetase or glutathione S-transferase, all of which catalyze the isomerase reaction from prostaglandin H2 to prostaglandin D2. In immunotitration analyses, the activity in mast cells showed a titration curve exactly identical with that of the purified spleen-type enzyme and almost completely absorbed by an excess amount of antibody against this enzyme, but it remained unchanged after incubation with antibodies against the brain-type enzyme and glutathione S-transferase isozymes thus far purified. In Western blot after two-dimensional electrophoresis of crude extracts of mast cells, a single immunoreactive spot was observed with antibody against the spleen-type enzyme at the same position as that of the purified enzyme (Mr = 26,000, pI = 5.2). Furthermore, the immunoreactive protein obtained from mast cells showed the same peptide fingerprints as those of the purified spleen-type enzyme, after partial digestion with Staphylococcus aureus V8 protease or trypsin. In immunoperoxidase staining, the immunoreactivity of the spleen-type enzyme was found in the cytosol of tissue mast cells in various organs such as thymus, intestine, stomach, and skin of adult rats. These findings indicate that prostaglandin D2 is produced by the spleen-type synthetase in mast cells of various tissues.

Animals↗
PubMed1990

Uninephrectomy inducing quantitative and qualitative changes in LH isoforms in the male rat.

Recent findings that LH stimulates renal growth have prompted us to examine whether LH exercises renotropic effects on the growing kidney following uninephrectomy. Intact (not castrated) male rats were used for sham uninephrectomy to induce surgical stress suppressing plasma LH and testosterone levels. Compared to sham surgery, uninephrectomy caused a transient 240% increase in plasma LH with a transient increase in plasma testosterone (80%) and in prostate weight on day 2. Although pituitary LH content did not change, chromatofocusing of pituitary extract revealed changed distribution patterns among seven LH isoforms, suggesting selective synthesis or secretion of LH isoforms. Castrated rats were then used so that surgical stress did not affect LH. Uninephrectomy caused sustained increases in plasma LH levels (60-110% increase) for up to six days. Three days after uninephrectomy, pituitary LH content increased significantly (30% increase) with a change in chromatofocusing profiles of LH isoforms. Plasma and pituitary FSH levels did not change. In summary, uninephrectomy changed LH isoforms quantitatively and qualitatively. These changes imply a change in gonadotropic and renotropic activities of LH after uninephrectomy.

Animals↗
PubMed1989

The major source of endogenous prostaglandin D2 production is likely antigen-presenting cells. Localization of glutathione-requiring prostaglandin D synthetase in histiocytes, dendritic, and Kupffer cells in various rat tissues.

The cellular localization of glutathione-requiring PGD synthetase, which catalyzes the predominant formation of PGD2 in various peripheral tissues, was investigated in adult rats by immunoperoxidase-staining with a polyclonal antibody specific for this enzyme. Although the 25 N-terminal amino acid residues of synthetase are 56% identical and 76% similar to those of several rat glutathione S-transferase subunits, the antibody cross-reacted only with synthetase in dot blotting and was nearly completely inactive with all transferase isozymes thus far purified. In Western blotting after SDS-PAGE of crude extracts of rat spleen, the antibody showed a single positive band at the same position as that of the purified enzyme (Mr = 26,000). The positive immunocytochemical stain was found in a number of histiocytes and/or dendritic cells in spleen, thymus, and Peyer's patch of intestine. The immunostain was also observed in such cells in lamina propria of the villus in small intestine and colon, in submucosal layer of stomach, and in Kupffer cells in liver. Immunoelectron microscopy confirmed that immunoreactivity of this enzyme was distributed in cytoplasm of those cells. Such immunoreactive cells were not observed in brain, spinal cord, kidney, heart, testis, and skeletal muscle. These observations suggest that PGD2 is produced by glutathione-requiring PGD synthetase localized in these types of APC in various tissues and may play a critical role in dictating the progression of immune responses.

Amino Acid Sequence↗
PubMed1989

Inhibitory effect of tranilast on prostaglandin D synthetase.

The effect of Tranilast [N-(3,4-dimethoxycinnamoyl) anthranilic acid] on the synthesis of prostaglandin D2 (PGD2) by homogenates of rat peritoneal mast cells was investigated. The major cyclooxygenase product formed by mast cell homogenates was PGD2, smaller quantities of PGE2 and PGF2 alpha were also formed. Tranilast suppressed the production of PGD2 in a dose-dependent manner with an IC50 of 0.1 mM. This suppression was due to inhibition of PGD synthetase, but not cyclooxygenase, since the formation of PGE2 and PGF2 alpha were unchanged at a 0.1 mM concentration. In addition, the glutathione-dependent conversion of [14C]PGH2 to PGD2 by PGD synthetase (PGH-D isomerase, EC 5.3.99.2) was inhibited by Tranilast, with 50% inhibition achieved at 0.08 mM in broken cell preparations of rat peritoneal mast cells. Tranilast also inhibited purified rat spleen and brain PGD synthetases. Furthermore, Tranilast prevented the PGD2 generation from intact mast cells stimulated by the calcium ionophore A23187. These results suggest that Tranilast exerts some of its therapeutic effects by prevention of PGD2 generation in mast cells and some other tissues.

Animals↗
PubMed1989

Changes of the activities of enzymes involved in prostaglandin synthesis in rat skin during development and aging.

The developmental changes of enzymes involved in prostaglandin (PG) synthesis were investigated in rat skin from birth to 1.5 years old. In all stages of development, the activities of PG-synthesizing enzymes were found in 100,000 x g supernatants of homogenates of rat skin, and PGD2 was the major PG among those formed from PGH2 in the presence of 1 m zeta glutathione (GSH). The PGD synthetase activity in rat skin at birth was 2.14 nmol/min per mg protein, increasing to the highest level (3.69 nmol/min per mg protein) at 3 weeks after birth and then gradually decreasing up to 1.5 years old. The activities of PGE2 and PGF2 alpha synthetases in rat skin were almost unchanged during development and aging. In contrast, the activity of GSH-S-transferase was at its lowest level at birth and gradually increased, reaching a plateau at 3 weeks after birth and remaining relatively constant during the development. The increase of PGD synthetase activity in 3-week-old rats was mainly due to the increase of specific activity of PGD synthetase in the epidermis, which was separated from the dermis by heat treatment (55 degrees C, 30 s). Immunohistochemical study, using (rat spleen PGD synthetase)-specific antibody, revealed that the number of immunopositive cells, which were identified as Langerhans cells, increased in the epidermis in 3-week-old rats. These results suggest that a change of PGD2 synthetase activity during aging of the skin is closely related to the development of ATPase+ Langerhans cells in the epidermis.

Aging↗
PubMed1989

Porcine luteinizing hormone isoform(s): relationship between their molecular structures, and renotropic versus gonadotropic activities.

We recently demonstrated the renotropic activity in ovine LH isoform(s). In this study we purified porcine (p) LH isoforms and analyzed their structures and bioactivities. Purified pLH preparation (G-100 fraction 3) was dissociated into alpha- and beta-subunits, followed by isolation with reverse phase HPLC. Six components were isolated and analyzed for their amino acid compositions and amino-terminal amino acid sequences. alpha-Subunits were found to have heterogeneous N-terminal sequences, which started at Phe-1, Gly-4, Phe-6, or Thr-7. This N-terminal heterogeneity and the presence of the initial 6 amino acids has not been reported previously to the best of our knowledge. Moreover, the first 10 residues, Phe-Pro-Asp-Gly-Glu-Phe-Thr-Met-Gln-Gly, were identical with those of the ovine LH alpha subunit. At least 3 different beta-subunits were identified as heterogeneous in their carboxyl-terminal amino acids. The pLH preparation (G-100-fraction 3) was then chromatographed by an isoelectrofocusing (IEF) column. Four IEF fractions were obtained. Their amino acid structures appeared to be identical, as judged by the identical elution profiles on reverse phase HPLC, but their carbohydrate compositions were slightly different, especially in N-acetylgalactosamine in alpha-subunits. Thus, fractionation on IEF depended on the heterogeneity in carbohydrate structures. Each IEF fraction had different potencies in terms of its gonadotropic activity (in vitro) and renotropic activity (in vivo and in vitro). The discrepancy was observed not only between gonadotropic activity and renotropic activity, but also between in vivo and in vitro renotropic activity. This study identified the structural heterogeneity of pLH isoforms and demonstrated their biological heterogeneity. We concluded that the carbohydrate structure of the pLH isoform is important for expressing its biological heterogeneity (gonadotropic and renotropic activity.

Amino Acid Sequence↗
PubMed1989

Effect of ultraviolet irradiation on the activity of rat skin prostaglandin D synthetase.

The effect of ultraviolet light-B (UVB) irradiation on the activity of prostaglandin (PG) D synthetase was investigated in adult rat skin. Rats were irradiated with 500 mJ/cm2 of UVB, and PGD synthetase activity was determined in 100,000 g supernatant of the homogenate of rat skin in the presence of glutathione (GSH) before and 3, 6, 12, and 24 h after irradiation. The PGD synthetase activity was decreased time dependently, and within 24 h after UVB irradiation it had dropped to 50% of the control level before irradiation. In contrast, the synthesizing activities of PGE2 and PGF2 alpha were unaffected by UVB irradiation. The reduction of PGD synthetase activity after UVB irradiation was much more prominent in the epidermis than in the dermis, which was separated by heat treatment (55 degrees C, 30 sec). Immunohistochemical studies, using anti-(rat spleen PGD synthetase) antibody, revealed that the number of immunopositive cells, which were identified as Langerhans cells, decreased in the basal layer of the epidermis 24 h after UVB irradiation. These results, together with the reduction of ATPase positive cells in the epidermis after UVB irradiation, suggest that the decrease of PGD synthetase activity in rat skin by UVB irradiation is, at least in part, due to the reduced Langerhans cell population in the basal layer of the epidermis.

Adenosine Triphosphatases↗
PubMed1988

Biochemical and immunological demonstration of prostaglandin D2, E2, and F2 alpha formation from prostaglandin H2 by various rat glutathione S-transferase isozymes.

Glutathione S-transferase isozymes purified from normal rat liver (1-1, 1-2, 2-2, 3-3, 3-4, and 4-4), liver with hyperplastic nodules (7-7), brain (Yn1Yn1), and testis (Yn1Yn2) all had prostaglandin H2-converting activity. The prostaglandin H2 E-isomerase activity was high in 1-1 (1400 nmol/min/mg protein), 1-2 (1170), and 2-2 (420), moderate in 3-3, 3-4, 4-4, Yn1Yn1, and Yn1Yn2 (52-100), and weak but significant in 7-7 (33). The prostaglandin H2 D-isomerase activity was relatively high in 1-1 (170) and 1-2 (200), moderate in 2-2 (60) and Yn1Yn2 (43), and weak but marked in 3-3 (16), 4-4 (16), and 7-7 (14). The prostaglandin H2 F-reductase activity was remarkable in 1-1 (1250), 1-2 (920), and 2-2 (390), and weakly detected in 3-3 (24), 4-4 (28), and 7-7 (14). Glutathione was absolutely required for these prostaglandin H2-converting reactions, and its stoichiometric consumption was associated with F-reductase activity but not E- and D-isomerase activities. The Km values for glutathione and prostaglandin H2 were about 200 and 10-40 microM, respectively. By immunoabsorption analyses with various antibodies specific for each isozyme, we examined its contribution to the formation of prostaglandins D2, E2, and F2 alpha from prostaglandin H2 in 100,000g supernatants of rat liver, kidney, and testis. In the liver, about 90% of the F-reductase activity (9.8 nmol/min/mg protein) was shown to be catalyzed by the 1-2 group of isozymes. The E-isomerase activity (16.5) was catalyzed about 60 and 40% by the 1-2 and 3-4 groups, respectively; and the D-isomerase activity (3.7) was catalyzed by the 1-2 group (50%) and the 3-4 group and Yn1Yn2 (15-25%). In the kidney, the E-isomerase activity (9.4) was catalyzed by 1-1, 1-2 (40%), 2-2, 3-4 group, and 7-7 (10-20%). The F-reductase activity (3.3) was mostly catalyzed by the 1-2 group (75%). In the testis, the E-isomerase activity (3.9) was catalyzed by the 1-2 group (20-30%), the 3-4 group, and Yn1Yn2 (30-60%).

Animals↗