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Biomedical subjects

H Tsuzuki

Publications and source records attributed to H Tsuzuki.

At least 55 records · Page 3Linked to original sources
PubMed1996

[Successful combination chemotherapy for a post-operative gastric cancer patient with multiple liver metastases and elevated CEA].

A 75-year-old male was admitted one year after surgery for advanced gastric cancer. He was diagnosed as having multiple liver metastases with elevated serum CEA level. Combination chemotherapy consisting of THP-ADM, MMC and 5'-DFUR was done in the outpatient clinic. As a result, both multiple metastatic liver tumors and serum CEA level showed a remarkable response.

Aged↗
PubMed1995

A Malignant Phyllodes Tumor of the Breast in a 6-Year Old Girl.

We recently encountered a 6-year-old girl with a malignant phyllodes tumor of the breast. The patient's mother noticed a tumor on the right breast of her first doughter at 8 months after from her birth. The baby was brought to the Second Depertment of Surgery, University of Tokushima, at age 20 months. We recommended removal of the 3.5 x 2.9 cm tumor in the right breast, but it was not done. Next, she visited our department at 6 years of age because the tumor had gradually enlarged, reaching a size of 4.3 &timus; 4.0 cm. She underwent excisional resection of the tumor, and the tumor was diagnosed as a malignant phyllodes tumor of the breast coexisting with a borderline phyllodes tumor. To our knowledge, this is the youngest reported case of a malignant phyllodes tumor of the breast. Moreover, the malignant lesion was positive for estrogen receptor (ER) and showed strong proliferating cell nuclear antigen (PCNA) staining. On the other hand, the borderline part was negative for ER and showed weak PCNA staining. Thus, in the present case, the expression of ER, the exposure to estrogen (for example, the mother's estrogen during gestation) and increase in the proliferation rate may have played important roles in the mechanism of the transformation of the phyllodes tumor.

Journal Article↗
PubMed1995

An improved method for large-scale purification of recombinant human glucagon.

Glucagon was expressed in Escherichia coli as a fusion protein including the glucagon sequence [Ishizaki et al. (1992), Appl. Microbiol. Biotechnol. 36, 483-486]. The high-level expression of a protein in E. coli often results in an insoluble aggregate called an inclusion body containing a fusion protein. In our previous report [Yoshikawa et al. (1992), J. Protein Chem. 11, 517-525], we solubilized this inclusion body by using guanidinium chloride. However, the existence of denaturant caused problems such as a low proteolytic activity for transforming the fusion protein into glucagon and complicated purification methods. We tried to improve the method to enable large-scale purification. At alkaline pH, the inclusion body could be solubilized to a high concentration and cleaved by amino acid-specific endopeptidases. By utilizing isoelectric precipitations as a new economical purification method for glucagon from intermediates, the glucagon obtained was shown to be over 99.5% pure by analytical RP-HPLC. The yield was almost equal that of our previous method, and the glucagon produced was chemically and biochemically equivalent to natural glucagon.

Amino Acid Sequence↗
PubMed1995

[A case of an intrapulmonary lymph node indistinguishable from carcinoma of the lung by CT].

A 64-year-old female was referred to our hospital for further examination of a coin lesion, 8 mm in diameter, superimposed on the right ninth posterior rib. CT revealed a nodule, close to the minor fissure, with spiculation, which seemed to be connected with the pulmonary vein. Since the bronchofiberscopic examination revealed no histological diagnosis, the exploratory thoracotomy was performed. The nodule was found to be a 10 x 7 x 5 mm black mass located subpleural at the minor fissure. Histologically, it was recognized as a lymph node with anthracosis and silicotic nodules.

Diagnosis, Differential↗
PubMed1994

Crystal structure analysis of a serine proteinase from Streptomyces fradiae at 0.16-nm resolution and molecular modeling of an acidic-amino-acid-specific proteinase.

We have determined the three-dimensional structure of a proteinase from Streptomyces fradiae ATCC 14544 (SFase-2) at 0.16-nm resolution. SFase-2, a typical serine proteinase, has broad substrate specificity. The characterization and crystallographic analysis of this enzyme have been reported previously [Kitadokoro, K., Tsuzuki, H., Nakamura, E., Sato, T. & Teraoka, H. (1994) Eur. J. Biochem. 220, 55-61]. In the present study, data were collected to approximately 0.16-nm resolution on a Rigaku R-AXIS IIC imaging plate detector system. Preliminary phases were obtained by molecular replacement methods with a search model derived from the previously determined structure of Streptomyces griseus protease A [Sielecki, A. R., Hendrickson, W. A., Broughton, C. G., Delbaere, L. T., Brayer, G. D. & James, M. N. (1979) J. Mol. Biol. 134, 781-804]. The starting model gave an initial crystallographic R factor of 0.443. Refinement with restrained least-squares converged at a final R factor of 0.182 for 16128 observed reflections. The final model includes 86 water molecules. The crystal structure showed that the enzyme consists of two domains, each of which is comprised of a beta barrel with six-stranded beta sheets and two alpha helices. The overall tertiary structure of SFase-2 is similar to the structures of other chymotrypsin-like proteinases from S. griseus, namely proteinase A and proteinase B. The essential residues of the catalytic triad are located on the cleft between the two domains. These two domains have different sequences, but possess similar three-dimensional structures, indicating that a gene duplication event has occurred to produce these two domains. We predicted the tertiary structure of an acidic-amino-acid-specific proteinase on the basis of the crystal structure of SFase-2, and compared the active-site conformations of these two enzymes. We found a characteristic histidine cluster of three histidine residues in the active site of the acidic-amino-acid-specific proteinase. The substrate recognition mechanism of SFase-2 may be mediated through the hydrophobic amino acid residues. However, in the acidic-amino-acid-specific proteinase, the positive charge of this histidine cluster would attract the negative charges of glutamic acid and aspartic acid.

Amino Acid Sequence↗
PubMed1994

Purification, characterization, primary structure, crystallization and preliminary crystallographic study of a serine proteinase from Streptomyces fradiae ATCC 14544.

A proteinase having wide substrate specificity was isolated from Streptomyces fradiae ATCC 14544. This proteinase, which we propose to call SFase-2, was purified from the culture filtrate by S-Sepharose chromatography. The purified enzyme showed an apparent molecular mass of 19 kDa on SDS/PAGE. When synthetic peptides were used as substrates, SFase-2 showed broad substrate specificity. It also hydrolyzed keratin, elastin and collagen as proteinaceous substrates. It was completely inhibited by diisopropylfluorophosphate and chymostatin, but not by tosylphenylalaninechloromethane, tosyllysinechloromethane or EDTA, indicating that it can be classified as a serine proteinase. The matured protein sequence of SFase-2 was determined by a combination of amino acid sequencing and the DNA sequencing of the gene. SFase-2, consisting of 191 amino acids, is a novel proteinase. It showed 76% similarity in the amino acid sequence with Streptomyces griseus proteinase A [Johnson P. and Smillie L. B. (1974) FEBS Lett. 47, 1-6]. For insight into the three-dimensional structure of SFase-2, we obtained single crystals by the vapor diffusion method using sodium phosphate as a precipitant. These crystals belonged to the orthorhombic, space group P2(1)2(1)2(1) with cell dimensions a = 6.92 nm, b = 7.28 nm, c = 2.99 nm; one molecule was present in the asymmetric unit.

Amino Acid Sequence↗
PubMed1994

Ultrastructural observation of capillary sprouts in the dental organs of rat molars.

The morphological events associated with the formation of capillary sprouts were examined in the dental organs to elucidate the morphological processes of angiogenesis in epithelial tissues. Mandibular first molars of 2-day-old male rats were processed for routine observation by transmission electron microscopy. In angiogenic regions, capillaries were located in the caves delineated by outer enamel epithelium, and extravasated erythrocytes were often observed in the stellate reticulum. At the capillary sprout, endothelial cells were amoeboidal in shape, and extended their filopodia to the fluffy extracellular matrix. On the other hand, each endothelial cell interdigitated with each process to form a tubule, and bulbous cell processes filled the luminal side of capillaries. Coated vesicles or pits 50-90 nm in diameter lined the luminal plasma membrane and that comprising the base of filopodial processes. Some endothelial sprouting tips (30 of 47 sprouts) were associated with pericyte-like cells. However, pericyte-like cells were observed in most of the caves where capillaries were located or destined to be located. We investigated the dynamic status of endothelial cells via parameters such as cell elongation and spreading which were supported by the additional observation of membranous components such as coated vesicles at the capillary sprouting region. It was also suggested that pericyte-like cells participated in angiogenesis of epithelial tissues by guiding the sprouts and sealing labile junctions between endothelial cells.

Animals↗
PubMed1994

Early progression stage of malignancy as revealed by immunohistochemical demonstration of DNA instability; II, Otorhinolaryngeal border-line neoplastic lesions.

The degree of DNA-instability as revealed by the immunohistochemical staining with anti-single-stranded DNA antibody after acid hydrolysis (DNA-instability test) was used as a marker of malignancy. This was applied to benign, border-line, and malignant neoplastic lesions found in the otorhinolaryngeal regions (31 cancer, 22 leukoplakia, 10 nasal inverted papilloma, 33 salivary gland pleomorphic adenoma, and 7 Warthin's tumor cases). Proliferative activity and polarity of the proliferative cell distribution were evaluated by PCNA-immunohistochemistry, and the quantitative analyses of the number, mean size, largest size, and maximum shape-irregularity of AgNORs in a nucleus were performed for all these cases. As the results, 31 cancer (100%), 20 leukoplakia (90.1%), 10 nasal inverted papilloma (100%), and 21 pleomorphic adenoma (63.6%) cases were positively stained by the DNA-instability test diffusely or sporadically, indicating their malignancy. Reflecting the malignant character, these cases showed a remarkable increase in the PCNA-index with the loss of polarity of PCNA-positive cell distribution, and also increased number, mean and largest sizes, and maximum shape-irregularity of AgNORs. These results indicate that all nasal inverted papillomas are malignant in nature, namely, in situ carcinoma, and the majority of leukoplakia is also regarded as in situ cancer, although a certain percentage of simple hyperplasia may be included. Furthermore, the pleomorphic adenoma of the salivary gland can be regarded as an "unstable tumor" which often contains or predisposes to bear malignant subclones with occasional capsular or extracapsular invasion, reflecting the progression of malignancy. In the present study, no sign of malignancy was detected in Warthin's tumor.

Antibodies, Monoclonal↗
PubMed1994

[Successful neoadjuvant chemotherapy in a patient with advanced gastric cancer with multiple liver metastases].

We described a case of advanced gastric cancer with multiple liver metastases, who was placed on neoadjuvant chemotherapy using CDDP and 5-FU (FP therapy) with a marked reduction in tumor load. The case was a 67-year-old male, who was admitted with a Borrmann III type advanced gastric cancer with multiple liver metastases. FP chemotherapy was carried out two times as neoadjuvant chemotherapy. As a result, both primary cancer and the metastatic tumors showed a remarkable reduction. Then, total gastrectomy with combined resections of spleen and transverse colon was done, and a reservoir was inserted into the hepatic artery. Postoperatively, intrahepatic arterial infusion of CDDP with oral administration of 5-FU was done in the outpatient clinic for about eleven months. But thirteen months later, he died from the rapid recurrence of the tumor.

Adenocarcinoma↗
PubMed1993

[A study with diagnostic standard of occult hyperprolactinemia (OHP) and the effect of bromocriptine administration].

It is well known that the transient excessive increase of serum prolactin level is harmful for the mechanism of ovulation or the steroidogenesis of the ovaries. The pathogenesis of latent or occult hyperprolactinemia (OHP) has been investigated recently. The present study was conducted to determine the diagnostic standard of OHP, and to elucidate the efficacy of bromocriptine administration for the treatment of OHP and other ovulatory disturbances. 110 cases of hypothalamic anovulations were selected from 385 cases of infertile patients by the LH-RH and TRH loading tests. Bromocriptine (5mg/day) was administered to all of the subjects for more than three months, and the efficacy of the bromocriptine administration was investigated. Follicular development was observed by transvaginal ultrasonography (mature follicular diameters > or = 20mm), and also luteal function was estimated by the duration of the luteal phase in the BBT charts (high phase > or = 12 days), the mid-luteal serum estradiol (> or = 200pg/ml) and progesterone (> or = 10ng/ml) levels. The subjects were divided into two groups: group A, bromocriptine effective patients (63 cases) and group B, bromocriptine non effective patients (47 cases). The results of the LH-RH and TRH loading tests were compared between these two groups. Serum prolactin levels at 30 min. after TRH loading (PRL30) in group B (61.5 +/- 28.3 vs. 38.0 +/- 19.3ng/ml, p < 0.01). At the cut-off points of 50, 60 and 70ng/ml over in the values of the PRL, the efficacies of the bromocriptine administration were 77.4, 78.9 and 88.5%, respectively. From these facts, it was thought suitable that the diagnostic standard of OHP was PRL30 > or = 70ng/ml, and values of PRL30 from 50 to 70ng/ml were borderline cases of OHP. The efficacy of the bromocriptine administration in the cases without OHP (n = 57) was also investigated. Serum LH levels at 30 min. after LH-RH loading (LH30) were compared between the cases of the bromocriptine effective (n = 22) and non effective (n = 35). As a result, the LH30 of the former was significantly higher than that of the latter (96.5 +/- 64.2 vs. 45.1 +/- 31.5mIU/ml, p < 0.005). In conclusion, the diagnostic standard of OHP was determined as PRL30 > or = 70ng/ml (borderline: 50 > 70ng/ml), and bromocriptine administration was effective not only in cases of OHP, but also in cases of hyperreactivities of LH (so-called endocrinological PCOD).

Anovulation↗
PubMed1993

Purification, characterization and molecular cloning of an acidic amino acid-specific proteinase from Streptomyces fradiae ATCC 14544.

We have isolated a novel acidic amino-acid-specific proteinase from Streptomyces fradiae ATCC 14544, using benzyloxycarbonyl-L-Phe-L-Leu-L-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. A proteinase, which we propose to call SFase, was purified from the culture filtrate by salting out, repeated S-Sepharose chromatography, and affinity chromatography (CH-Sepharose-Phe-Leu-D-Glu-OMe). The purified enzyme showed a single band having an apparent molecular weight of 19,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. When synthetic peptides were used as substrates, SFase showed high specificity for Z-Phe-Leu-Glu-pNA. Comparison with nitroanilides of glutamic acid and aspartic acid as substrates revealed that the reactivity was about 10-fold higher for a glutamyl bond than an aspartyl bond. SFase selectively hydrolyzed the -Glu-Ala-bond of two glutamyl bonds in the oxidized insulin B-chain within the initial reaction time until the starting material was completely digested. Diisopropylfluorophosphate and benzyloxycarbonyl-Phe-Leu-Glu chloromethylketone completely inhibited SFase, while metalloproteinase inhibitors, such as EDTA and o-phenanthrolin, did not inhibit the enzyme. The findings indicate that SFase can be classified as a serine proteinase, and is highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure and precursor of SFase, its gene was cloned from genomic DNA of the producing strain, and the nucleotide sequence was determined. Consideration of the N- and C-terminal amino-acid sequences of the mature protein of SFase indicates that it consists of 187 amino acids, which follows a prepropeptide of 170 residues. In comparison with the acidic amino-acid-specific proteinase from Streptomyces griseus (Svendsen, I., Jensen, M.R. and Breddam, K. (1991) FEBS Lett. 292, 165-167), SFase had 82% homology in the amino acid sequence. The processing site for maturation of SFase was a unique sequence (-Glu-Val-), so that the propeptide could be released by cleavage of the peptide bond between Glu and Val.

Amino Acid Sequence↗
PubMed1993

Cancerous residue in breast-conserving surgery.

Local tumor extension was studied using a continuous series of multiple blocks of mastectomy specimens to assess malignancy remaining after breast-conserving surgery for early-stage breast cancer. In this study, 183 cases were chosen, consisting of 6 noninvasive ductal carcinoma cases and 177 invasive ductal carcinoma cases. The histopathology in 59 (32%) of the 183 cases corresponded to that showing extensions of more than 2.6 cm from the tumor margin. These wide extensions were also seen in 17% of breast cancers with a tumor size of less than 2 cm. The incidence of wide extension was higher in younger patients with cases of noninvasive ductal carcinoma. Extension to the nipple-areola was seen in 14% of cases with a tumor size of less than 2 cm. Breast cancers with multicentric development accounted for 3% of those with a tumor size under 2 cm. These findings suggest that if lumpectomy is performed with a margin of 2 cm for tumors with a size of 2 cm or less, a cancerous residue would be found in the surgical margin of 15-20% of the cases. The actual incidence was 23% of cases after breast-conserving treatment in our study. On the basis of the data, breast-conserving treatment with only local resection of the primary lesion showed cancerous residue such as intraductal cancerous extension in about 20% of cases. Therefore, it was concluded that, as part of breast-conserving therapy of early-stage breast cancer, radiation therapy of the whole breast should be performed after surgery with clear margins to control local recurrence.

Adult↗
PubMed1993

Tumor progression accompanied by increase in proliferation rate in breast cancer: a preliminary report.

Forty consecutive cases with invasive ductal carcinoma of the breast (samples of both the primary invasive area and the involved node were available for 19 cases, multiple sections of breast tissue including both the primary invasive area and forefront intraductal cancerous area were available for 15 cases; and multiple sections of breast cancerous tissue and the involved node were available for six cases) were examined by immunohistochemical analysis for evaluation of their proliferation rate. In this study, we selected the PCNA index as a marker of the proliferation rate of the tumor cells. We demonstrated that the PCNA index of the primary invasive area was significantly higher than that of the forefront intraductal area (P = 0.0017), and the PCNA index of the involved nodes was significantly higher than that of the primary invasive area (P = 0.0004). These preliminary findings suggest that the progression from the tumor cells of the intraductal cancerous area to the metastatic tumor cells must be accompanied by two phases of increase in the proliferation rate.

Breast Neoplasms↗
PubMed1993

Fluorescent staining of nucleolar organizer regions for three-dimensional display by confocal laser scanning microscope.

A new method of fluorescent staining of nucleolar organizer regions (F1NORs) is described. Aluminum ammonium sulfate was used instead of silver as the cationic metal ion for binding with NORs-associated proteins, and fluorescent morin was successively bound to aluminum by chelating with modification of the method developed by Malinin (1978). After bleaching the fluorescent staining of NORs by washing water, ordinary AgNORs staining was performed on the same section, and both images of F1NORs and AgNORs were found to coincide with each other. F1NORs staining of human malignant and benign tumors, and colorectal adenomas of borderline malignancy were examined by three-dimensional analysis of the fluorescence images under confocal laser scanning microscope (CLSM). A remarkable increase of F1NORs was found, not only in number but also in volume, with bizarre configuration in the process of tumor progression, and the F1NORs-CLSM technique may be helpful for daily pathological diagnosis of malignancy.

Evaluation Studies as Topic↗
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↗