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Biomedical subjects

H Tsuzuki

Publications and source records attributed to H Tsuzuki.

At least 19 recordsLinked to original sources
PubMed1999

Inhibition of N-linked glycosylation by tunicamycin enhances sensitivity to cisplatin in human head-and-neck carcinoma cells.

Tunicamycin (TM), a naturally occurring antibiotic, blocks the first step in the biosynthesis of N-linked oligosaccharides in cells. In this study, we investigated whether changes in N-linked glycosylation affect the sensitivity of head-and-neck carcinoma cell lines to cis-diaminedichloroplatinum(II) (cisplatin) in vitro and in vivo. In vitro treatment of the IMC-3 and KB cell lines with TM significantly decreased the 50% inhibitory concentration (IC50) of cisplatin, as determined by the MTT assay (24.15 to 10.97 microg/ml, p < 0.05). In addition, TM significantly decreased the IC50 of cisplatin against established cisplatin-resistant IMC-3/CR cells (>100 to 14.4 microg/ml, p < 0.05) to levels similar to those against parental IMC-3 cells. TM treatment decreased the number of Con A- and L-PHA-binding sites on the surface of tumor cells but had no effect on the intracellular platinum concentration. Induction of apoptosis in vitro by TM plus cisplatin in combination was increased compared with that by cisplatin alone. Furthermore, in vivo administration of TM plus cisplatin in combination significantly inhibited local tumor growth in the cisplatin-resistant in vivo C3H/He mouse model as compared with the control group (p < 0.05) and increased in vivo apoptosis of tumor cells. Our results suggest that the manipulation of glycosylation by TM in tumor cells might be a useful therapeutic strategy for successful chemotherapy using cisplatin against head-and-neck cancer.

Animals↗
PubMed1999

Granulocyte-colony-stimulating factor enhances invasive potential of human head-and-neck-carcinoma cell lines.

Granulocyte-colony-stimulating factor (G-CSF), a hematopoietic cytokine, regulates the proliferation and differentiation of granulocytic progenitor cells and functionally activated mature neutrophils. G-CSF also affects nonhematopoietic tumor cells by the binding of G-CSF to its specific receptor (G-CSFR) on the cells. In this study, we investigated the effect of G-CSF on the invasive potential of head-and-neck carcinoma cells, and explored the intracellular events initiated by the binding of G-CSF in tumor cells. In vitro treatment of head-and-neck-carcinoma cell lines, IMC-2, IMC-3, KB, Ca9-22, SCCKN and SCCTF, with recombinant G-CSF (rG-CSF) significantly augmented their invasive potential in dose- and time-dependent manners. Among these cancer cells, IMC-2, IMC-3, KB and Ca9-22 cells produced little G-CSF, while large amounts of G-CSF were produced by SCCKN and SCCTF cell lines. Anti-G-CSF antibody (Ab) abrogated the rG-CSF-enhanced invasiveness to the control level of that in untreated cancer cell lines. Immunocytochemical staining and Western blotting using anti-G-CSFR monoclonal antibody (MAb) revealed the expression of G-CSFR on head-and-neck-cancer cell lines exhibiting the enhancement of invasive activity by rG-CSF. IMC-2 cells, having the highest invasive ability among the cell lines used, showed augmentation of G-CSFR expression on stimulation with rG-CSF. Furthermore, stimulation of IMC-2 cells with rG-CSF induced rapid activation of tyrosine-phosphorylated JAK1, suggesting that the G-CSF signal may be transduced into the cells through G-CSFR. Moreover, the gelatinolytic activity of IMC-2 cells was enhanced by stimulation of rG-CSF, and the enhanced invasiveness was inhibited on addition of the tissue inhibitors of metalloproteinases (TIMPs). These results suggest that exogenous rG-CSF may increase the risk of metastasis and/or local recurrence in patients with G-CSFR-positive head-and-neck squamous-cell carcinoma, via an invasive mechanism.

Antibodies, Monoclonal↗
PubMed1998

Expression of multidrug resistance-associated protein (MRP) in head and neck squamous cell carcinoma.

We investigated the expression of multidrug resistance-associated protein (MRP) in 115 cases of head and neck squamous cell carcinoma (H&NSCC) by immunohistochemistry and examined the relationship between MRP expression and clinical factors. Thirty-four (30%) of 115 cases of H&NSCC had expression of MRP. The clinical stage was inversely associated with the expression of MRP (P = 0.0090), but not with age, sex, tumor size, metastasis, recurrence, death from disease or overall survival rate for 5 years. In vitro chemosensitivity to five chemotherapeutic agents (cis-diamminedichloroplatinum, 5-fluorouracil, peplomycin, mitomycin C and Adriamycin) was tested by ATP assay and no correlation between the sensitivity of tumor cells to the cytotoxicity of any drug and MRP expression was found. These results suggest that the resistance to anticancer drugs is not dependent only on the expression of MRP in H&NSCC.

ATP-Binding Cassette Transporters↗
PubMed1998

Expression of granulocyte colony-stimulating factor receptor correlates with prognosis in oral and mesopharyngeal carcinoma.

Granulocyte colony-stimulating factor receptors (G-CSFRs) have been observed on the surface of not only hematopoietic cells but also several cancer cells. The stimulation of G-CSF has been demonstrated to induce proliferation and activation of G-CSFR-positive cells. In this study, we investigated the expression of G-CSFR on the surface of tumor cells and G-CSF production in oral and mesopharyngeal squamous cell carcinoma (SCC) by an immunohistochemical approach. Of 58 oral and mesopharyngeal SCCs, 31 cases (53.4%) and 36 cases (62.1%) were positive for G-CSFR and G-CSF, respectively. There was no association between G-CSFR expression and G-CSF staining. In the group positive for G-CSFR expression, relapse was significantly more likely after primary treatment (P = 0.0069), whereas there was no association between G-CSFR expression and age, sex, tumor size, lymph node metastasis, and clinical stage. Also, the G-CSFR-positive groups had a significantly lower disease-free and overall survival rate than the G-CSFR-negative groups (P = 0.0172 and 0.0188, respectively). However, none of the clinical markers correlated significantly with G-CSF staining, nor did the status of G-CSF production influence the overall survival. The results imply that assessment of G-CSFR may prove valuable in selecting patients with oral and mesopharyngeal SCC for aggressive therapy.

Aged↗
PubMed1998

Highly selective and orally active inhibitors of type IV collagenase (MMP-9 and MMP-2): N-sulfonylamino acid derivatives.

Various N-sulfonylamino acid derivatives were synthesized and evaluated for their in vitro and in vivo activities to inhibit type IV collagenase (MMP-9 and MMP-2). When the amino acid residue and the sulfonamide moiety were modified, their inhibitory activities were greatly affected by the structure of the sulfonamide moiety. A series of aryl sulfonamide derivatives containing biaryl, tetrazole, amide, and triple bond were found to be potent and highly selective inhibitors of MMP-9 and MMP-2. In addition, these compounds were orally active in animal models of tumor growth and metastasis. These results revealed the potential of the N-sulfonylamino acid derivatives as a new type of candidate drug for the treatment of cancer.

Administration, Oral↗
PubMed1998

Complete nucleotide sequence and genetic organization of Aichi virus, a distinct member of the Picornaviridae associated with acute gastroenteritis in humans.

The complete nucleotide sequence of a novel enteric virus, Aichi virus, associated with nonbacterial acute gastroenteritis in humans was determined. The Aichi virus genome proved to be a single-stranded positive-sense RNA molecule with 8,251 bases excluding a poly(A) tail; it contains a large open reading frame with 7,302 nucleotides that encodes a potential polyprotein precursor of 2,433 amino acids. The genome contains a 5' nontranslated region (NTR) with 712 bases and a 3' NTR with 240 bases followed by a poly(A) tail. The structure of the genome, VPg-5' NTR-leader protein-structural proteins-nonstructural proteins-3' NTR-poly(A), was found to be typical of a picornavirus. The VP0-VP3 and VP3-VP1 cleavage sites were determined to be Q-H and Q-T, respectively, by N-terminal amino acid sequence analyses using purified virion proteins. Possible cleavage sites, Q-G, Q-A, and Q-S, which cleave P2 and P3 polyproteins were found to be similar to those of picornaviruses. A dendrogram based on 3Dpol proteins indicated that Aichi virus is genetically distinct from the known six genera of picornaviruses including entero-, rhino-, cardio-, aphtho-, and hepatovirus and echovirus 22. Considering this together with other properties of the virus (T. Yamashita, S. Kobayashi, K. Sakae, S. Nakata, S. Chiba, Y. Ishihara, and S. Isomura, J. Infect. Dis. 164:954-957, 1991), we propose that Aichi virus be regarded as a new genus of the family Picornaviridae.

Acute Disease↗
PubMed1998

Expression of platelet-derived endothelial cell growth factor in oral and oropharyngeal carcinoma.

Platelet-derived endothelial cell growth factor (PD-ECGF) was isolated as an endothelial cell mitogen from platelets. In this study, we investigated the expression of PD-ECGF and counted microvessels in 58 oral and oropharyngeal squamous cell carcinoma (SCC) specimens by an immunohistochemical technique to examine their prognostic significance and performed tumor in vitro sensitivity to 5-fluorouracil (5-FU) and cisplatin as determined by a bioluminescence assay of the ATP values of tumor cells after continuous exposure. The percentage of PD-ECGF-positive tumor cells (PD-ECGF score) was correlated with the frequency of the recurrence of disease (P=0.0043) but not with sex, tumor size, metastasis, or clinical stage. Overall survival of the high PD-ECGF expression group (>40% PD-ECGF score) was shorter than the low expression (<40%) group (P=0.0365). Vessel count was correlated with lymph node metastasis and clinical stage. The survival of patients with hypervascularity (more than the median of intratumor vessel counts, >82) was shorter than that of those with hypovascularity (vessel count <81, P=0.0446). However, there was no association between PD-ECGF expression and vessel count. Cox proportional multivariate analysis showed that PD-ECGF expression was the most significant independent prognostic indicator for overall survival. The susceptibility to 5-FU cytotoxicity in the extremely high PD-ECGF expression groups (>70% of PD-ECGF score) was significantly higher than that in the low group, whereas there was no difference in their sensitivity to cisplatin. These results showed that carcinoma cells with high PD-ECGF expression were sensitive to 5-FU in spite of poor prognosis. These data provide further information when deciding on adjuvant therapy for oral and oropharyngeal SCCs.

Analysis of Variance↗
PubMed1997

Expression of P-glycoprotein in inner ear capillary endothelial cells of the guinea pig with special reference to blood-inner ear barrier.

Expression of P-glycoprotein (P-gp) was detected immunohistochemically in the guinea pig inner ear using mouse anti-P-gp monoclonal antibody C219. P-gp was found only in the capillary endothelial cells of the cochlea and vestibule. The pattern of P-gp immunostaining in the inner ear was similar to that of the brain. The present investigation suggested that P-gp expression in inner ear capillary endothelial cells might play an important role in the blood-inner ear barrier by acting as an extrusion pump.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Purification and characterization of a glutamic-acid-specific endopeptidase from Bacillus subtilis ATCC 6051; application to the recovery of bioactive peptides from fusion proteins by sequence-specific digestion.

Screening cultures of nonpathogenic microorganisms led us to a glutamic-acid-specific endopeptidase from Bacillus subtilis ATCC 6051, which we purified and named BSase. The nucleotide sequence encoding BSase, with a molecular mass of 23,894 Da, completely agreed with that of the mpr gene, which had been reported by Rufo Jr. and Sloma et al. to encode a metalloprotease [J Bacteriol (1990) 172: 1019-1023 and 1024-1029 respectively]. However, enzymatic characterization revealed it to have the catalytic triad of a serine protease and not the consensus sequence of a metalloprotease, and it was inhibited by diisopropylfluorophosphate. We therefore consider BSase (mpr) to be a serine protease. In the alignment of the acidic-amino-acid-specific proteases, the proteases from bacilli have a highly conserved histidine residue, which is most important in the histidine triad in the proteases from streptomycetes. Furthermore, Ca2+ was necessary for its activity and stability. BSase cleaved the C-terminal glutamic acid with high specificity and was very stable over a wide pH range. On the basis of these properties, we tried to retrieve a bioactive peptide from a fusion protein by sequence-specific digestion, and succeeded in obtaining the bioactive peptide. BSase was found to be very useful as a tool for selective cleavage.

Amino Acid Sequence↗
PubMed1996

A dose-response relationship between the frequency of p53 mutations and tobacco consumption in lung cancer patients.

Mutations of the p53 tumor suppressor gene are frequent in lung cancers. It is suggested that p53 mutations are associated with smoking-induced lung carcinogenesis. We examined p53 mutations in 53 lung cancers by analyzing reverse transcription-polymerase chain reaction-single strand conformation polymorphism (RT-PCR-SSCP) to ascertain the association between p53 mutations and smoking. Twenty-five (47%) of 53 lung cancers carried p53 mutations. A discriminant analysis showed that the Brinkman index (0.156) and gender (0.140) significantly influenced p53 mutations. Furthermore, there was a dose-response relationship between the quantity of cigarettes consumed and the frequency of p53 mutations in lung cancer patients (P < 0.001). In patients with adenocarcinoma, the frequency of p53 mutations correlated with the amount of the tobacco smoked (P < 0.05). We suggest that the p53 gene is a target of particular carcinogen in tobacco smoke.

Adenocarcinoma↗
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

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↗