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Biomedical subjects

H Tsuzuki

Publications and source records attributed to H Tsuzuki.

At least 73 records · Page 4Linked to original sources
PubMed1992

Production of recombinant human glucagon in the form of a fusion protein in Escherichia coli; recovery of glucagon by sequence-specific digestion.

Recombinant human glucagon was successfully produced with a high level of expression in Escherichia coli as a fusion protein with human interferon gamma. The synthetic gene was designed to release glucagon, which does not contain glutamic acid residues, from fusion protein with the Staphylococcus aureus strain V8 protease that specifically cleaves the peptide bond on the carboxyl side of the glutamic acid residue. The resulting glucagon was purified to homogeneity by a combination of C18 reverse-phase HPLC and ion-exchange HPLC. The yield of intact glucagon obtained from 11 of culture was approximately 12 mg. The structure of recombinant human glucagon was confirmed by HPLC and amino acid composition/sequence analyses.

Amino Acid Sequence↗
PubMed1992

Recombinant human glucagon: large-scale purification and biochemical characterization.

Recombinant glucagon was expressed in Escherichia coli as a fusion protein including the glucagon sequence therein as previously reported [Ishizaki et al. (1992). Appl. Microbiol. Biotechnol. 36, 483-486]. We developed a large-scale method for the isolation and purification of recombinant glucagon. After cell disruption, the resultant pellets were solubilized with 2 M guanidine-HCl, to which Staphylococcus aureus V8 protease had been added, and were digested into intermediates composed of 53- and 60-residue peptides containing the glucagon moiety. After the digestion came to an end, the solution was desalted, and the remaining V8 protease was allowed to resume digestion of the intermediates into glucagon, followed by partial purification by S-Sepharose and Sephacryl S-100 chromatographies. The glucagon obtained was found to be not less than 99.5% pure by analytical HPLC. One liter of culture produced about 180 mg of pure glucagon. The amino acid composition and the sequence agreed well with the theoretical values. Radioreceptor assay gave an affinity constant similar to that of pancreatic glucagon, and similar activities in cAMP production and glycogenolysis were also observed. Thus, the recombinant glucagon was confirmed to be biochemically identical with pancreatic glucagon.

Amino Acid Sequence↗
PubMed1992

The characteristics of interval breast cancer in mass screening.

To investigate the characteristics of interval breast cancer in mass screening, comparisons were made of the following three groups: interval group (21 interval breast cancer cases), mass screening group (87 breast cancer cases detected by mass screening) and outpatient group (266 breast cancer cases diagnosed at outpatient clinics). There were no differences among the three groups in terms of the case distribution by age or obesity, but significant differences in the case distribution according to nodal involvement and tumor size. Histological grading of the malignancy of the primary tumors disclosed that the incidence of breast cancer showing frequent mitoses was high in the interval group compared to the mass screening and outpatient groups. The 7-year cumulative disease-free survival rate was 75.3% in the interval group, 90.0% in the mass screening group and 83.1% in the outpatient group. The mean tumor size of the interval cases at the time of mass screening, back-calculated on the basis of the estimated tumor doubling time, was 1.5 cm in diameter, smaller than that of the mass screening group. It is surmised that interval breast cancer is characterized by marked proliferation of the tumor cells and has a poorer prognosis than the other group cases. These findings might be due to the marked proliferation of interval breast cancer rather than because of cases having been overlooked at the time of the last screening.

Adult↗
PubMed1991

Immunohistochemical analysis of pulpal innervation in developing rat molars.

The beginning of pulpal innervation was examined in these developing teeth. Mandibular first molars removed from newborn to 7-day-old rats were cryosectioned and nerve fibres were localized by the peroxidase-antiperoxidase technique using a neuropeptide-specific (200 kDa) antibody. Some pulps from 5- and 7-day-old rats were also examined by conventional electron microscopy. In newborn to 4-day-old rats, molars in the initial stages of the dentine deposition were innervated in the follicles but not in the pulps. In molars from 5-day-old rats, nerve fibres were found in the pulps of 11 out of 16 samples. The fibres were mainly located along the blood vessels in the basal part of the pulp, with some arborizations. In rats of 6 and 7 days old, nerve fibres were found in all the pulps examined. These had gradually extended deep into the cuspal area and were increasingly arborized with increasing age. Nerve fibres were also found along the basal laminae of blood vessels in some dental pulps from 5-day-old rats when examined by electron microscopy. At 7 days, nerve fibres were composed of bundles of axons, some of which were covered with Schwann cell processes and basal laminae. These observations indicate that the innervation of rat molar dental pulps begins after the start of the deposition of enamel, in animals of around 5 days of age, which is at the same stage as in mouse molars, as others have shown by a silver-staining method.

Aging↗
PubMed1991

[Studies on metoclopramide (MCP) loading test during pregnancy and puerperium for the evaluation of lactating capacity].

To evaluate the correlation between prolactin (PRL) secreting capacity and puerperal lactation, 66 women who had delivered normally without any complications during pregnancy were studied. They were divided into two groups depending on the lactating state a month after the delivery: The breast feeding group (group A, 40 cases), and the supplementary feeding group (group B, 26 cases), respectively. 10 mg of metoclopramide (MCP) loading was carried out in the 36th week of gestation and in the third day of the puerperium, and serum levels of PRL (at 0, 30, 60 min. after the MCP loading) were measured by RIA. Serum levels of insulin, glucose, triglyceride, free fatty acid, cholesterol and lipoproteins were also examined. There was no significant change in the serum levels of the various, substances during pregnancy and puerperium. Also, no significant change could be seen in the MCP loading test in the 36th week of gestation. However, on the third day of puerperium, serum PRL after 30 min. of the MCP loading showed a significant increase in group B compared with group A (587.1 +/- 202.9 vs. 431.2 +/- 196.6 ng/ml, p less than 0.02, mean +/- standard deviation). These results suggested that the MCP loading test during the early stage of puerperium might be an important index to use in determining whether the mother was ready to breast feed.

Female↗
PubMed1990

Isolation and characterization of human reg protein produced in Saccharomyces cerevisiae.

reg was originally identified as a gene expressed during the regeneration of insulin-producing pancreatic beta-cells of the rat. We built an expression vector containing human reg cDNA to drive Saccharomyces cerevisiae to synthesize the reg protein, and purified it from the culture medium. The 144-amino acid sequence of the recombinant protein was consistent with that deduced from the cDNA and genomic DNA sequence except that the signal sequence of 22 amino acids was eliminated, and the amino-terminal residue of the protein was pyroglutamic acid. The secondary structure of the reg protein was predicted by determination of the intramolecular cystine linkage and of alpha-helix and beta-sheet contents.

Amino Acid Sequence↗
PubMed1990

[Comprehensive approach to clinical background and effect of bromocriptine administration in patients with endocrinological polycystic ovarian disease].

Present study was performed to elucidate the clinical features and the treatment of so-called endocrinological polycystic ovarian disease (PCO). 36 cases out of 189 infertile patients who had various ovulatory disturbances were subjected during past four years. They were selected by the definitions as follows; 1) serum LH levels greater than or equal to 30 mIU/ml and serum FSH levels less than or equal to 15 mIU/ml, 2) hyper-response of LH secretion by LH-RH (100 micrograms) loading test; maximum values greater than or equal to 250 mIU/ml. Serum androstenedione (ASD), dehydroepiandrosterone-sulfate (DHA-S), estrone (E1), estradiol (E2), progesterone (P, in the mid-luteal phase) and testosterone (T) levels were examined by RIA method for the purpose of the evaluation of the endocrinological background of the PCO in Japanese women. The same examinations were also done in 8 volunteer women who had normal ovulatory menstrual cycles for the control study. In 6 cases of the PCO patients who showed biphasic BBT charts, the endometrial biopsy was done in the mid-luteal phase at the same time of the blood sampling. And the correlation between various serum hormone levels and the endometrial morphology in the PCO patients was also discussed. Then bromocriptine (5 mg/day) was administered in these patients for more than 30 days and the effect of the treatment was investigated. Serum ASD levels in the PCO patients were significantly higher than those in the control (2.52 +/- 1.30 vs. 1.43 +/- 1.21 ng/ml, M +/- S.D., p less than 0.05), while serum E2 and P levels in the patients were significantly lower than those in the control (E2: 118.6 +/- 39.5 vs. 192.5 +/- 53.9 pg/ml, p less than 0.005. P: 7.26 +/- 5.08 vs. 124.4 +/- 4.6 ng/ml, p less than 0.005, respectively). There were no significant differences in serum levels of the other hormones. By the administration of bromocriptine, serum ASD levels decreased (1.62 +/- 1.34 ng/ml, p less than 0.05), and E2 (177.9 +/- 48.6 pg/ml, p less than 0.025) and P (11.8 +/- 4.3 ng/ml, p less than 0.005) levels increased significantly into the levels of control. Serum LH levels of the patients were also suppressed by the treatment of bromocriptine (42.5 +/- 13.7 vs. 27.4 +/- 12.0 mIU/ml, p less than 0.005), however there was no change in serum FSH levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1989

Determination of prolactin, growth hormone, beta-endorphin, and cortisol in both maternal plasma and amniotic fluid during human gestation.

This study focuses on PRL, GH, beta-endorphin and cortisol in maternal blood and amniotic fluid during human pregnancy. Maternal blood and amniotic fluid samples were obtained from 18 normal pregnant women in the second trimester, 12 full-term gravidas having spontaneous delivery, and 10 full-term gravidas having elective cesarean section. Two gravidas bearing anencephalic fetuses in the third trimester were also studied. In the second trimester women, levels of PRL (3215.9 +/- 458.9 micrograms/l), GH (19.1 +/- 1.7 micrograms/l) and beta-endorphin (11.1 +/- 0.9 pmol/l) were significantly higher in the amniotic fluid than in maternal plasma. In addition, PRL was significantly correlated with beta-endorphin (r = 0.670) and with GH (r = 0.547) in the amniotic fluid. However, amniotic fluid cortisol levels (0.27 +/- 0.18 nmol/l) were significantly lower than plasma cortisol levels. The amniotic fluid of the women with anencephalic fetuses had normal levels of PRL, GH and beta-endorphin. In full-term gravidas, plasma PRL levels were significantly lower in women with vaginal delivery than in those with elective cesarean section, and there was a significant negative correlation between plasma PRL and beta-endorphin, and between plasma PRL and cortisol levels. Plasma GH levels in women with vaginal delivery showed no significant difference from those in women with cesarean section. Examination of amniotic fluid yielded no significant differences in the levels of PRL, beta-endorphin and GH between these two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1988

[Combined therapy with bromocriptine, clomiphene and gonadotropin for polycystic ovary syndrome patients failing to respond to clomiphene alone].

The purpose of this study is to investigate the ovulatory effect of the combined therapy with bromocriptine (Brc), clomiphene (Cl) and HMG. We investigated 25 patients with polycystic ovary syndrome (PCOS) who had anovulatory normoprolactinemia and failed to respond to Cl therapy alone. The results were as follows: 1) The ovulation rate with Method I(Brc/Cl) was 64.0% (16/25) in 25 cases, and 59.8% in 122 cycles. 2) Resting levels of serum dehydroepiandrosterone sulfate in the effective group with Method I were significantly lower than those in the non-effective group. 3) With Method I, serum prolactin (PRL), LH and testosterone were significantly decreased, while estradiol and progesterone concentrations were significantly increased in the effective group. However, there were no significant changes in the hormone levels except PRL in the non-effective group. 4) Out of the 9 nonrespondent Method I cases, 5 additional cases ovulated with both Method II(Brc/Cl+ a small dose of HMG) and Method III(Brc/Cl+ an increased dose of HMG-HCG). The total ovulatory rate was 84.0% (21/25) in 25 cases and 59.4% (85/143) in 143 cycles. 5) The total pregnancy rate was 43.8% (7/16). Six cases were normal pregnancies, one case was a twin pregnancy and there was no case of abortion. Our results suggested that the combined therapy with Brc, Cl and HMG had almost the same therapeutic effect as the HMG-HCG therapy in severe cases of PCOS.

Adolescent↗
PubMed1987

Cholinergic traits in rat mandibular processes observed by electron microscopy.

Cholinergic traits in rat mandibular processes were examined histochemically, under the electron microscope, at early developmental stages (Stages 20 to 23, by Christie's nomenclature). The histochemical reaction for detection of enzymes was performed by the thiocholine method. Nonspecific cholinesterase (EC 3.1.1.8) activity was found in ectomesenchymal cells, vascular endothelial cells, and in some epidermal cells at stages 20 and 21. The enzymatic activity was localized in the perinuclear and endoplasmic reticular cisternae. At stage 22, the number of cells with enzymatic activity decreased gradually, except in the case of the capillary endothelial cells. At stage 23, when the trigeminal nerve fiber was obvious in the mandibular processes, nonspecific cholinesterase activity was restricted to some of the endothelial cells and trigeminal ganglionic cells. In contrast, acetylcholinesterase activity was found on the membrane of trigeminal nerve fiber. Thus, the transient, nonspecific, cholinesterase activity, found in rat mandibular processes, may serve some functions in transmission, lipid metabolism or destruction of toxic cholinesters during the period that precedes organogenesis.

Animals↗
PubMed1987

[Study on the neuroendocrinological control of prolactin release in early puerperium].

The purpose of this study is to investigate the neuroendocrinological control mechanism of prolactin (PRL) acting on the hypothalamo-pituitary axis during early puerperium. The puerperal women consisted of three groups: the breast-feeding group (n = 39), the bromocriptine (BRC)-treated group (5 mg/day, n = 17) and naloxone-treated group (1 mg iv, n = 16). In each group, 10 mg metoclopramide (MCP), 500 micrograms TRH or 400 mg cimetidine was given intravenously. 1) The plasma PRL levels increased significantly after the injection of MCP, TRH and cimetidine. The peak values of delta PRL levels were 447.0 +/- 62.3 ng/ml after MCP, 278.3 +/- 65.1 ng/ml after TRH and 86.5 +/- 27.3 ng/ml after cimetidine. 2) This PRL increase after the injection of MCP and cimetidine was suppressed significantly by pretreatment with BRC. However, the PRL increase after TRH was not suppressed by pretreatment with BRC. 3) Naloxone had no significant effect on PRL response to MCP and TRH, since the plasma PRL levels rose significantly after the injection of MCP and TRH in the naloxone-treated group. These results revealed that there were different mechanisms of PRL release in MCP and TRH. Furthermore, the PRL releasing mechanism was influenced by histamine H2-receptor, but was not influenced by opioid peptide in early puerperium.

Bromocriptine↗
PubMed1985

Epithelial-mesenchymal interface in the rat mandibular process observed by electron microscopy.

Epithelial-mesenchymal interfaces in the stomodeal side of the rat mandibular processes were observed at Theiler's stages 14 and 15, when no epithelial organogenesis could be detected by electron microscopy. At stage 14, the basal lamina was discontinuous. Disintegrated basal lamina-like materials were seen in the epithelial-mesenchymal interface. A number of coated vesicles and smooth vesicles were seen in epithelial cell processes adjacent to the disintegrated basal lamina-like materials. Some epithelial cell processes penetrated into the mesenchymal space through the basal lamina and touched the ectomesenchymal cells. Other epithelial cell processes or satellite cell bodies protruded into the mesenchymal space, but were covered with basal laminae. At stage 15, the basal lamina was smooth and continuous with partial duplications. Ruthenium red-positive anionic sites were distributed in the basal lamina and on thin filaments underneath the basal lamina. At stage 15, the separation among anionic sites became more regular than that at stage 14. In this study we found some stage-specific morphological events in the epithelial-mesenchymal interface in the stomodeal side of the mandibular process at stages preceding the initiation of epithelial organogenesis.

Animals↗
PubMed1985

Fate of recombinants of the rat mandibular epithelium with cranial ectomesenchymal cells of different sources in vitro.

The recombinants of the mandibular molar bud epithelia with cranial ectomesenchymal cell groups from several different sources--mandibular molar area, tongue anlagen, and lateral nasal process--were cultured. Dental laminalike buds were developed in each of the recombinants (incidence of development 38-86%). In the heterotrophic recombinants, heterotypic differentiation of mandibular epithelium was also induced. However, the foreign ectomesenchymal cells were not induced heterotypically by the epithelial genetic factor, but the mesenchymal genetic factor is maintained. It is suggested that mandibular molar bud epithelia have potency to proliferate into mesenchyme under non-organ-specific influences of ectomesenchymal cells and that presumptive mandibular mucosal epithelia have multipotency for differentiation sensitive to inductive influences by the heterotypic cranial ectomesenchymal cells but that the mandibular molar bud epithelia have no heterotypic inductive activity for the differentiation of cranial ectomesenchymal cells.

Animals↗
PubMed1984

Purification of human plasma alpha 1-proteinase inhibitor and its inactivation by Pseudomonas aeruginosa elastase.

Human alpha 1-proteinase inhibitor was purified according to a modification of the method of Kurecki et al. (Anal. Biochem. 99, 415 (1979) ), with Affi-Gel Blue treatment before Zn-affinity column chromatography. The inhibitor was inactivated in the presence of Pseudomonas aeruginosa elastase (1/2,000 molar ratio) for 2 h at pH 7.5 and 25 degrees C. The inactivated inhibitor was purified by column chromatography on Sephadex G-75 and DE-52. Little or no difference was observed between the native and inactive inhibitors in immunological response, amino acid composition or far-ultraviolet CD spectrum. On the other hand, a considerable difference was observed in the near-ultraviolet CD spectrum. Two amino-terminal sequences were found in the inactive inhibitor in almost the same ratio; one was the same as that of the intact inhibitor and the other was Met-Ser-Ile-Pro-. The two components were separated by high-performance liquid chromatography using 0.1% trifluoroacetic acid containing 30-70% CH3CN (gradient) as the eluent. Amino acid analysis and N- and C-terminal amino acid sequence analyses indicated that one fraction corresponded to the sequence of 1-357 of the alpha 1-proteinase inhibitor and the other to 358-394. We concluded that P. aeruginosa elastase can inactivate human alpha 1-proteinase inhibitor by splitting the peptide bond of Pro357-Met358, leading to local change near the active site but little change in the structure as a whole. The split carboxy-terminal fragment binds tightly to the rest of the inhibitor.

Amino Acids↗