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H Tsuzuki

Publications and source records attributed to H Tsuzuki.

At least 19 recordsLinked to original sources
PubMed1992

Purification, characterization, cloning, and expression of a glutamic acid-specific protease from Bacillus licheniformis ATCC 14580.

A glutamic acid-specific protease has been purified to homogeneity from Bacillus licheniformis ATCC 14580 utilizing Phe-Leu-D-Glu-OMe-Sepharose affinity chromatography and crystallized. The molecular weight of the protease was estimated to be approximately 25,000 by SDS-polyacrylamide gel electrophoresis. This protease, which we propose to call BLase (glutamic acid-specific protease from B. licheniformis ATCC 14580), was characterized enzymatically. Using human parathyroid hormone (13-34) and p-nitroanilides of peptidyl glutamic acid and aspartic acid, we found a marked difference between BLase and V8 protease, EC 3.4.21.9, although both proteases showed higher reactivity for glutamyl bonds than for aspartyl bonds. Diisopropyl fluorophosphate and benzyloxycarbonyl Leu-Glu chloromethyl ketone completely inhibited BLase, whereas EDTA reversibly inactivated the enzyme. The findings clearly indicate that BLase can be classified as a serine protease. To elucidate the complete primary structure and precursor of BLase, its gene was cloned from the genomic DNA of B. licheniformis ATCC 14580, and the nucleotide sequence was determined. Taking the amino-terminal amino acid sequence of the purified BLase into consideration, the clones encode a mature peptide of 222 amino acids, which follows a prepropeptide of 94 residues. The recombinant BLase was expressed in Bacillus subtilis and purified to homogeneity. Its key physical and chemical characteristics were the same as those of the wild-type enzyme. BLase was confirmed to be a protease specific for glutamic acid, and the primary structure deduced from the cDNA sequence was found to be identical with that of a glutamic acid-specific endopeptidase isolated from Alcalase (Svendsen, I., and Breddam, K. (1992) Eur. J. Biochem. 204, 165-171), being different from V8 protease and the Glu-specific protease of Streptomyces griseus which consist of 268 and 188 amino acids, respectively.

Amino Acid Sequence↗
PubMed1992

Purification, characterization and gene cloning of a novel glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600.

Twenty strains of Staphylococcus aureus from ATCC type cultures and strains found in clinical studies were cultivated, and their endopeptidase activity specific for glutamic acid was surveyed using benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. The activity was found in two of the strains, ATCC 12600 and ATCC 25923. A glutamic acid-specific proteinase, which we propose to call SPase, was purified from the culture filtrate of S. aureus strain ATCC 12600 by a series of column chromatographies on DEAE-Sepharose twice and on Sephacryl S-200. A single band was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified SPase. The molecular weight of the proteinase was estimated to be 34000 by SDS-PAGE. When synthetic peptides and oxidized insulin B-chain were used as substrates, SPase showed the same substrate specificity as V8 proteinase, EC 3.4.21.9, which specifically cleaves peptide bonds on the C-terminal side of glutamic acid and aspartic acid. Examination with p-nitroanilides of glutamic acid and aspartic acid as substrates, however, revealed that both proteinases are highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure of SPase, its gene was cloned from genomic DNA of S. aureus ATCC 12600, and the nucleotide sequence was determined. Taking the amino acid sequence of SPase from the NH2-terminus to the 27th residue into consideration, the clones encode a mature peptide of 289 amino acids, which follows a prepropeptide of 68 residues. SPase was confirmed to be a novel endopeptidase specific for glutamic acid, being different from V8 proteinase which consists of 268 amino acids.

Amino Acid Sequence↗
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↗
PubMed1979

Protease and elastase of Pseudomonas aeruginosa: inactivation of human plasma alpha 1-proteinase inhibitor.

The present study indicates that crystalline elastase of Pseudomonas aeruginosa is a very potent inactivator of human plasma alpha 1-proteinase inhibitor, the enzyme (E) inactivated the inhibitor (I) almost completely within 1 h at 25 degrees C at a molar ratio of E/I = 1:100. The crystalline P. aeruginosa protease also inactivated the inhibitor, but 100-fold less. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the alpha 1-proteinase inhibitor inactivated by the elastase and protease showed decreases in molecular weight of approximately 5,000 and 10,000, respectively. Regeneration of trypsin was negligible even when bovine trypsin-alpha 1-proteinase inhibitor complex (E/I = 1.0) was treated with the elastase. The affinity of alpha 1-proteinase inhibitor to trypsin was much higher than that to elastase. It was suggested that, assuming the pseudomonal proteases are produced and can inactivate alpha 1-proteinase inhibitor in vivo during pseudomonal diseases, the loss of alpha 1-proteinase inhibitor activity may permit the endogenous serine proteases to cause tissue destruction.

Humans↗
PubMed1979

Pepstatin-insenstive acid proteases from Scytalidium lignicolum. Kinetic study with synthetic peptides.

A kinetic study was conducted on the acid proteases A-1 and A-2 from Scytalidium lignicolum using synthetic peptides as substrates. Almost maximum activity was attained with N-acylated tetrapeptides as the molecular size of substrates was increased. Suitable amino acid residues were required at the P1-P2 and P1'-P2' positions [notation of Schechter and Berger (14)]. Hydrophobic or bulky residues such as leucine were specifically required at the P1 and P1' positions, with the specificity at the latter position being considerably lower than that at the former. For catalysis, the presence of certain amino acid residues at the P2 and P2' positions was essential, mainly in relation to kcat. An inhibition study supported this view. Stringent stereospecificity was observed at the P2 and P2' positions, but the side chain specificity was low. Study of the B enzyme from the same organism was very difficult owing to its low activity against the peptides used. The Scytalidium acid proteases A-1, A-2, and B showed considerably different behavior against peptide substrates in comparison with usual acid proteases, which are senstive to pepstatin.

Aspergillus niger↗
PubMed1978

Paroxysmal discharges triggered by hearing spoken language.

We examined the modality of EEG activation by various kinds of acoustic stimulation in a middle-aged Japanese female with epilepsy. Paroxysmal discharges were triggered in the right frontal area (F4) by verval stimulation. For the activation of EEG, concentration of attention on the stimulation was essential; therefore paroxysmal discharges were triggered most easily by verbal stimuli when someone spoke to the patient directly. Stronger responses than usual were triggered by specific words, and apparently reflected the interest and concern of the patient. The latency from stimulation to paroxysmal discharges ranged from 230 to 1,300 msec, suggesting that the responses may have been a function of the perception and recognition of acoustic stimuli. "Heard-word epilepsy" or "Angesprochene Epilepsie" is suggested in this case.

Acoustic Stimulation↗
PubMed1978

Effectiveness of immunization with single and multi-component vaccines prepared from a common antigen (OEP), protease and elastase toxoids of Pseudomonas aeruginosa on protection against hemorrhagic pneumonia in mink due to P. aeruginosa.

Toxoids of protease and elastase of Pseudomonas aeruginosa were successfully prepared by treatment with 8% formalin plus 0.2 m lysine and by 4% formalin respectively. The two toxoids proved sufficiently potent to elicit high antibody titers as estimated by both the enzyme-neutralizing and passive hemagglutination tests. The effectiveness of immunizing minks with a single component-vaccine consisting of the common antigen (OEP) of P. aeruginosa or the protease toxoid (PT) or the elastase toxoid (ET) and with the two (PT and ET) or the three (OEP, PT and ET) component-mixed vaccines, on hemorrhagic pneumonia in minks due to the bacteria was investigated. Female Sapphire minks, 3.5 months old, were used in two experiments performed in 1975 and 1976. Minks were immunized three times in one month with a total of 1 mg of each of the three antigens in the case of a single component vaccine and with a total of 2 or 3 mg (equal amounts of each component) in the case of the two or three component vaccines. Two or three weeks after the last immunization, challenge exposure with strain No. 5 was carried out by intranasally inoculating an inoculum containing serial dilutions of 10(3) -10(10) of live bacteria. Summarizing the results of the two experiments, in the case of controls, nonimmunized minks and minks immunized with potassium aluminum sulfate (potash alum) alone, the LD50 values were approximately 10(3) -10(4) with no significant difference between the two. In the case of OEP-vaccinated minks, the LD50 value was about 10(6) and thus clearly differed from those of the controls. In minks immunized with the three-component-vaccine, however, the LD50 value was about 10(8) -10(9), which indicated that the three-component-mixed vaccine was remarkably more effective than the single OEP vaccine component. In minks immunized with either PT or ET or both, the LD50 values were about 10(8) -10(9). The effectiveness of the vaccine made with ET or PT alone is discussed in the text. The pathological findings of the minks which died or survived are described.

Animals↗