Biomedical subjects

Hideyuki Suzuki

Publications and source records attributed to Hideyuki Suzuki.

At least 19 recordsLinked to original sources

PubMed2006

Crystal structure of the gamma-glutamyltranspeptidase precursor protein from Escherichia coli. Structural changes upon autocatalytic processing and implications for the maturation mechanism.

Gamma-glutamyltranspeptidase (GGT) is an extracellular enzyme that plays a key role in glutathione metabolism. The mature GGT is a heterodimer consisting of L- and S-subunits that is generated by posttranslational cleavage of the peptide bond between Gln-390 and Thr-391 in the precursor protein. Thr-391, which becomes the N-terminal residue of the S-subunit, acts as the active residue in the catalytic reaction. The crystal structure of a mutant GGT, T391A, that is unable to undergo autocatalytic processing, has been determined at 2.55-A resolution. Structural comparison of the precursor protein and mature GGT demonstrates that the structures of the core regions in the two proteins are unchanged, but marked differences are found near the active site. In particular, in the precursor, the segment corresponding to the C-terminal region of the L-subunit occupies the site where the loop (residues 438-449) forms the lid of the gamma-glutamyl group-binding pocket in the mature GGT. This result demonstrates that, upon cleavage of the N-terminal peptide bond of Thr-391, the newly produced C terminus (residues 375-390) flips out, allowing the 438-449 segment to form the gamma-glutamyl group-binding pocket. The electron density map for the T391A protein also identified a water molecule near the carbonyl carbon atom of Gln-390. The spatial arrangement around the water and Thr-391 relative to the scissile peptide bond appears suitable for the initiation of autocatalytic processing, as in other members of the N-terminal nucleophile hydrolase superfamily.

Bacterial Proteins↗

PubMed2006

Autoprocessing of Helicobacter pylori gamma-glutamyltranspeptidase leads to the formation of a threonine-threonine catalytic dyad.

Helicobacter pylorigamma-glutamyltranspeptidase (HpGT) is a glutathione-degrading enzyme that has been shown to be a virulence factor in infection. It is expressed as a 60-kDa inactive precursor that must undergo autocatalytic processing to generate a 40-kDa/20-kDa heterodimer with full gamma-glutamyl amide bond hydrolase activity. The new N terminus of the processed enzyme, Thr-380, is the catalytic nucleophile in both the autoprocessing and enzymatic reactions, indicating that HpGT is a member of the N-terminal nucleophile hydrolase superfamily. To further investigate activation as a result of autoprocessing, the structure of HpGT has been determined to a resolution of 1.9 A. The refined model contains two 40-kDa/20-kDa heterodimers in the asymmetric unit and has structural features comparable with other N-terminal nucleophile hydrolases. Autoprocessing of HpGT leads to a large conformational change, with the loop preceding the catalytic Thr-380 moving >35 A, thus relieving steric constraints that likely limit substrate binding. In addition, cleavage of the proenzyme results in the formation of a threonine-threonine dyad comprised of Thr-380 and a second conserved threonine residue, Thr-398. The hydroxyl group of Thr-398 is located equidistant from the alpha-amino group and hydroxyl side chain of Thr-380. Mutation of Thr-398 to an alanine results in an enzyme that is fully capable of autoprocessing but is devoid of enzymatic activity. Substrate docking studies in combination with homology modeling studies of the human homologue reveal additional mechanistic details of enzyme maturation and activation, substrate recognition, and catalysis.

Binding Sites↗

PubMed2006

RET finger protein enhances MBD2- and MBD4-dependent transcriptional repression.

We recently demonstrated that MBD4 possesses the ability to repress transcription through methyl-CpG and is associated with methylated promoters in the CDKN2A and MLH1 genes. In order to further investigate the role of MBD4 in methylation-based transcriptional repression, a yeast two-hybrid screening was performed, and the RET finger protein (RFP) was found to be one of the major proteins that interact with the transcriptional repression domain in MBD4. The effect of the MBD4-mediated transcriptional repression in methylated CDKN2A and MLH1 promoters was extremely enhanced by the overexpression of RFP. Furthermore, RFP forms a protein complex not only with MBD4 but also with MBD2 or MBD3 and was shown to enhance transcriptional repression through MBD2. These results suggest that RFP is a mediator connecting several MBD proteins and allowing the formation of a more potent transcriptional repressor complex. Because RFP has been detected at high levels in a variety of tumor cell lines as well as testis, and embryos, RFP may have an important role in the enhancement of transcriptional repression through MBD proteins in tumorigenesis, spermatogenesis, and embryogenesis.

DNA-Binding Proteins↗

PubMed2006

Clarification of pathway-specific inhibition by Fourier transform ion cyclotron resonance/mass spectrometry-based metabolic phenotyping studies.

We have developed a metabolic profiling scheme based on direct-infusion Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). The scheme consists of: (1) reproducible data collection under optimized FT-ICR/MS analytical conditions; (2) automatic mass-error correction and multivariate analyses for metabolome characterization using a newly developed metabolomics tool (DMASS software); (3) identification of marker metabolite candidates by searching a species-metabolite relationship database, KNApSAcK; and (4) structural analyses by an MS/MS method. The scheme was applied to metabolic phenotyping of Arabidopsis (Arabidopsis thaliana) seedlings treated with different herbicidal chemical classes for pathway-specific inhibitions. Arabidopsis extracts were directly infused into an electrospray ionization source on an FT-ICR/MS system. Acquired metabolomics data were comprised of mass-to-charge ratio values with ion intensity information subjected to principal component analysis, and metabolic phenotypes from the herbicide treatments were clearly differentiated from those of the herbicide-free treatment. From each herbicide treatment, candidate metabolites representing such metabolic phenotypes were found through the KNApSAcK database search. The database search and MS/MS analyses suggested dose-dependent accumulation patterns of specific metabolites including several flavonoid glycosides. The metabolic phenotyping scheme on the basis of FT-ICR/MS coupled with the DMASS program is discussed as a general tool for high throughput metabolic phenotyping studies.

Arabidopsis↗

PubMed2006

Reconstructing state spaces from multivariate data using variable delays.

We study two methods for constructing a nonuniform embedding for multivariate data. A nonuniform embedding is a state space reconstruction which is more flexible than the common delay coordinates with fixed delays since it contains variable delays. Using these methods, we can extract causal relationships among many variables in a more suitable way. We demonstrate that the proposed methods can give more precise predictions and simpler models than some previous methods.

Journal Article↗

PubMed2006

Gamma-(monophenyl)phosphono glutamate analogues as mechanism-based inhibitors of gamma-glutamyl transpeptidase.

Gamma-glutamyl transpeptidase (GGT, EC 2.3.2.2) catalyzes the hydrolysis and transpeptidation of extracellular glutathione and plays a central role in glutathione homeostasis. We report here the synthesis and evaluation of a series of hydrolytically stable gamma-(monophenyl)phosphono glutamate analogues with varying electron-withdrawing para substituents on the leaving group phenols as mechanism-based and transition-state analogue inhibitors of Escherichia coli and human GGTs. The monophenyl phosphonates caused time-dependent and irreversible inhibition of both the E. coli and human enzymes probably by phosphonylating the catalytic Thr residue of the enzyme. The inactivation rate of E. coli GGT was highly dependent on the leaving group ability of phenols with electron-withdrawing groups substantially accelerating the rate (Brønsted betalg = -1.4), whereas the inactivation of human GGT was rather slow and almost independent on the nature of the leaving group. The inhibition potency and profiles of the phosphonate analogues were compared to those of acivicin, a classical inhibitor of GGT, suggesting that the phosphonate-based glutamate analogues served as a promising candidate for potent and selective GGT inhibitors.

Escherichia coli↗

PubMed2006

Human VIP-alpha: a long-acting, biocompatible and biodegradable peptide nanomedicine for essential hypertension.

We have previously shown that self-association of human vasoactive intestinal peptide with sterically stabilized liposomes (VIP-alpha) alters peptide conformation from random coil in aqueous solution to alpha-helix. This, in turn, protects the peptide from hydrolysis and amplifies and prolongs its bioactivity. The purpose of this study was to determine whether a single, intravenous injection of low-dose human VIP-alpha normalizes systemic arterial pressure in anesthetized spontaneously hypertensive hamsters for a prolonged period of time in a selective fashion. We found that intravenous injection of human VIP-alpha, VIP alone (each, 1.0 nmol) and empty liposomes had no significant effects on mean arterial pressure (MAP) in normotensive hamsters. By contrast, human VIP-alpha (0.01-1.0 nmol) evoked a significant concentration-dependent decrease in MAP to the normative range in spontaneously hypertensive hamsters that lasted throughout the observation period (6 h; p<0.05). VIP alone and empty liposomes had no significant effects on MAP in these animals. We conclude that a single, low-dose intravenous injection of human VIP-alpha normalizes systemic arterial pressure in spontaneously hypertensive hamsters for a prolonged period of time in a selective fashion. We suggest that human VIP-alpha should be further developed as a long-acting, biocompatible and biodegradable peptide nanomedicine for essential hypertension.

Animals↗

PubMed2006

Crystal structures of gamma-glutamyltranspeptidase from Escherichia coli, a key enzyme in glutathione metabolism, and its reaction intermediate.

Gamma-glutamyltranspeptidase (GGT) is a heterodimic enzyme that is generated from the precursor protein through posttranslational processing and catalyzes the hydrolysis of gamma-glutamyl bonds in gamma-glutamyl compounds such as glutathione and/or the transfer of the gamma-glutamyl group to other amino acids and peptides. We have determined the crystal structure of GGT from Escherichia coli K-12 at 1.95 A resolution. GGT has a stacked alphabetabetaalpha fold comprising the large and small subunits, similar to the folds seen in members of the N-terminal nucleophile hydrolase superfamily. The active site Thr-391, the N-terminal residue of the small subunit, is located in the groove, from which the pocket for gamma-glutamyl moiety binding follows. We have further determined the structure of the gamma-glutamyl-enzyme intermediate trapped by flash cooling the GGT crystal soaked in glutathione solution and the structure of GGT in complex with l-glutamate. These structures revealed how the gamma-glutamyl moiety and l-glutamate are recognized by the enzyme. A water molecule was seen on the carbonyl carbon of the gamma-glutamyl-Thr-391 Ogamma bond in the intermediate that is to be hydrolyzed. Notably the residues essential for GGT activity (Arg-114, Asp-433, Ser-462, and Ser-463 in E. coli GGT) shown by site-directed mutagenesis of human GGT are all involved in the binding of the gamma-glutamyl moiety. The structure of E. coli GGT presented here, together with sequence alignment of GGTs, may be applicable to interpret the biochemical and genetic data of other GGTs.

Amino Acid Sequence↗

PubMed2006

Plant lanosterol synthase: divergence of the sterol and triterpene biosynthetic pathways in eukaryotes.

Sterols, essential eukaryotic constituents, are biosynthesized through either cyclic triterpenes, lanosterol (fungi and animals) or cycloartenol (plants). The cDNA for OSC7 of Lotus japonicus was shown to encode lanosterol synthase (LAS) by the complementation of a LAS-deficient mutant yeast and structural identification of the accumulated lanosterol. A double site-directed mutant of OSC7, in which amino acid residues crucial for the reaction specificity were changed to the cycloartenol synthase (CAS) type, produced parkeol and cycloartenol. The multiple amino acid sequence alignment of a conserved region suggests that the LAS of different eukaryotic lineages emerged from the ancestral CAS by convergent evolution.

Amino Acid Sequence↗

PubMed2006

Cytochrome P450 CYP710A encodes the sterol C-22 desaturase in Arabidopsis and tomato.

Delta22-unsaturated sterols, containing a double bond at the C-22 position in the side chain, occur specifically in fungi and plants. Here, we describe the identification and characterization of cytochrome P450s belonging to the CYP710A family as the plant C-22 desaturase. Recombinant proteins of CYP710A1 and CYP710A2 from Arabidopsis thaliana and CYP710A11 from tomato (Lycopersicon esculentum) were expressed using a baculovirus/insect system. The Arabidopsis CYP710A1 and tomato CYP710A11 proteins exhibited C-22 desaturase activity with beta-sitosterol to produce stigmasterol (CYP710A1, K(m) = 1.0 microM and kinetic constant [k(cat)] = 0.53 min(-1); CYP710A11, K(m) = 3.7 microM and k(cat) = 10 min(-1)). In Arabidopsis transgenic lines with CYP710A1 and CYP710A11 overexpression, stigmasterol levels increased by 6- to 32-fold. Arabidopsis CYP710A2 was able to produce brassicasterol and stigmasterol from 24-epi-campesterol and beta-sitosterol, respectively. Sterol profiling analyses for CYP710A2 overexpression and a T-DNA insertion event into CYP710A2 clearly demonstrated in planta that CYP710A2 was responsible for both brassicasterol and stigmasterol production. Semiquantitative PCR analyses and promoter:beta-glucuronidase transgenic approaches indicated strict tissue/organ-specific regulation for each CYP710A gene, implicating differential tissue distributions of the Delta(22)-unsaturated sterols in Arabidopsis. Our results support the possibility that the CYP710 family may encode P450s of sterol C-22 desaturases in different organisms.

Amino Acid Sequence↗

PubMed2006

Triple therapy of interferon and ribavirin with zinc supplementation for patients with chronic hepatitis C: a randomized controlled clinical trial.

AIM: To study the therapeutic effect of interferon (IFN) and ribavirin with zinc supplement on patients with chronic hepatitis C viral (HCV) infection. METHODS: A total of 102 patients confirmed histologically to have chronic HCV infection with genotype 1b and more than 100 KIU/mL of HCV were randomly assigned to each arm of the study and each received 10 million units of pegylated interferon (IFN-alpha-2b) daily for 4 wk followed by the same dose every other day for 20 wk plus ribavirin (600 or 800 mg/d depending on body weight), with or without polaprezinc (150 mg/d) orally for 24 wk. The primary endpoint was sustained virological response (SVR) defined as negative HCV-RNA in the serum 6 mo after treatment. RESULTS: There were no differences in the clinical background between the two groups except for more females in the dual therapy group than in the other group (P<0.05). SVR was observed in 33.3% of the triple therapy group and 33.3% of the dual therapy group. The side effects were almost the same in both groups except for gastrointestinal symptoms, which were less in the triple therapy group (P=0.019). CONCLUSION: Considered together, triple therapy of zinc plus IFN and ribavirin for HCV infection patients with genotype 1b and high viral load is not better than dual therapy except for lower incidence of gastrointestinal side effects.

Adult↗

PubMed2006

Randomized controlled trial of consensus interferon with or without zinc for chronic hepatitis C patients with genotype 2.

AIM: The beneficial effect of zinc supplementation on the efficacy of interferon as a treatment for chronic hepatitis C had been demonstrated in hepatitis virus genotype 1b of high viral load. This study focused on patients with genotype 2, which is more sensitive to interferon than genotype 1b, and used consensus interferon (CIFN) with or without zinc. METHODS: We randomized 83 patients with chronic hepatitis C to CIFN at 18 MIU six times/wk for 4 wk, followed by CIFN at 18 MIU six times/wk for another 20 wk, in combination with polaprezinc 300 mg (regimen A, n=41) or as monotherapy (regimen B, n=42). Thirty-one patients in regimen A and 33 patients in regimen B completed the clinical trial; the remaining patients withdrew because of side effects or a transfer to another hospital. RESULTS: Sustained biochemical response, defined as a normal aminotransferase level at the end of the 6-mo post-treatment observation, was 68% and 69%, and sustained virological response, defined as undetectable HCV-RNA at the end of the 6-mo post-treatment observation, was 54% and 67% for regimens A and B, respectively. CONCLUSION: CIFN treatment combined with zinc did not enhance the effect of CIFN as shown by biochemical, virological criteria. No side effects related to polaprezinc were noted.

Adult↗

PubMed2006

Hepatocellular carcinoma in extremely elderly patients: an analysis of clinical characteristics, prognosis and patient survival.

AIM: To identify the clinical and prognostic features of patients with hepatocellular carcinoma (HCC) aged 80 years or more. METHODS: A total of 1310 patients with HCC were included in this study. Ninety-one patients aged 80 years or more at the time of diagnosis of HCC were defined as the extremely elderly group. Two hundred and thirty-four patients aged > or = 50 years but less than 60 years were regarded as the non-elderly group. RESULTS: The sex ratio (male to female) was significantly lower in the extremely elderly group (0.90:1) than in the non-elderly group (3.9:1, P < 0.001). The positive rate for HBsAg was significantly lower in the extremely elderly group and the proportion of patients negative for HBsAg and HCVAb obviously increased in the extremely elderly group (P < 0.001). There were no significant differences in the following parameters: diameter and number of tumors, Child-Pugh grading, tumor staging, presence of portal thrombosis or ascites, and positive rate for HCVAb. Extremely elderly patients did not often receive surgical treatment (P < 0.001) and they were more likely to receive conservative treatment (P < 0.01). There were no significant differences in survival curves based on the Kaplan-Meier methods in comparison with the overall patients between the two groups. However, the survival curves were significantly worse in the extremely elderly patients with stage I/II, stage I/II and Child-Pugh grade A cirrhosis in comparison with the non-elderly group. The causes of death did not differ among the patients, and most cases died of liver-related diseases even in the extremely elderly patients. CONCLUSION: In the patients with good liver functions and good performance status, aggressive treatment for HCC might improve the survival rate, even in the extremely elderly patients.

Adult↗

PubMed2006

Intraoperative thermal imaging in esophageal replacement: its use in the assessment of gastric tube viability.

We examined the use of intraoperative thermal imaging to assess the gastric vascularization and gastric tube viability during esophagectomy. The surface temperatures of the intact stomach, devascularized stomach, and gastric tube were measured in 13 patients from the proximal end to the pylorus longitudinally along the greater curvature or along the entire gastric tube during esophagectomy. Thermal images clearly demonstrated a surface temperature decline in the proximal region of the gastric tube. The mean decline rate in the surface temperature in the proximal region of the gastric tube in comparison to the intact stomach was 17.7% (P < 0.001). One patient who developed gastric tube necrosis exhibited a prominent drop in the surface temperature in the proximal region of 20.6% in comparison to that in the distal region, compared to that of 12.5% in other patients. Intraoperative thermal imaging is a noninvasive and reliable technique for the assessment of the gastric tube viability.

Aged↗

PubMed2006

Preoperative oral immune-enhancing nutritional supplementation corrects TH1/TH2 imbalance in patients undergoing elective surgery for colorectal cancer.

PURPOSE: Recent studies have shown that the type 1/2 CD4+ T cell (Th1/Th2) balance shifts toward Th2 dominance in cancer-bearing state or by surgical stress. Perioperative immunonutrition is reported to improve the outcome in patients with gastrointestinal cancer. This study was designed to investigate whether preoperative immunonutrition corrects the impaired Th1/Th2 balance in the perioperative period. METHODS: Thirty-six patients with colorectal cancer were prospectively divided into two groups as follows: preoperative oral intake supplementation with a formula enriched with arginine, omega-3 fatty acids and ribonucleic acid for five days (supplemented group; n = 19); and (control group; n = 17). Blood sampling was performed before supplementation, on the morning of surgery, and 3, 7, and 14 days postoperatively. The proportions of CD4+ T cells producing intracellular cytokines (interferon-gamma and interleukin-4) were measured by flow cytometry. RESULTS: In the preoperative period, the proportions of CD4+ T cells producing interleukin-4 significantly decreased and Th1/Th2 ratio significantly increased on the morning of surgery compared with those before supplementation. In the postoperative period, the proportions of CD4+ T cells producing interferon-gamma in both groups maintained the preoperative level. The proportions of CD4+ T cells producing interleukin-4 in the control group showed a gradual increase from the preoperative level, which implies Th2 dominant shift. In contrast, the supplemented group maintained the preoperative level of Th1/Th2 ratio. CONCLUSIONS: Preoperative immunonutrition corrects impaired Th1/Th2 balance in both cancer-bearing state and the postoperative period. This correction may be one of the important determinants of the clinical benefits of immunonutrition.

Administration, Oral↗

PubMed2006

Gamma-glutamyl-gamma-aminobutyrate hydrolase in the putrescine utilization pathway of Escherichia coli K-12.

gamma-Glutamyl-gamma-aminobutyrate hydrolase (PuuD) was purified and the properties of the enzyme were characterized. The active center of PuuD was identified as Cys-114 by site-directed mutagenesis. The expression of PuuD was induced by putrescine and O2 (substrates of the Puu pathway), while the addition of succinate or NH4Cl (products of the Puu pathway) to the medium reduced the expression of PuuD. The findings that the puuD-deficient strain accumulated gamma-glutamyl-gamma-aminobutyrate (gamma-Glu-GABA) and could not grow on putrescine as a sole nitrogen source indicate that PuuD is physiologically important as a gamma-Glu-GABA hydrolase.

Aldehyde Oxidoreductases↗

PubMed2006

Outcome of surgical treatment of synchronous liver metastases from colorectal cancer.

BACKGROUND: We retrospectively identified the prognostic factors in cases of synchronous liver metastases from colorectal cancer and established a clinical strategy at our institution. METHODS: One hundred eight patients with hepatic metastases from colorectal cancer underwent a first radical hepatic resection. Of these, 67 were diagnosed with hepatic synchronous metastases from colorectal primaries (S group) and 41 were diagnosed with metachronous metastases (M group). Hepatic lesions were diagnosed concurrently with the primary lesions in 45 of the 67 patients in the S group. Of these 45 patients, 37 underwent synchronous hepatectomy (SH group) and 8 underwent metachronous hepatectomy (MH group). RESULTS: The overall 3-, 5- and 10-year survival rates were 51.4%, 41.6%, and 30.9%, respectively. There were no significant differences between the S and M groups in overall survival. Univariate analysis of the S group revealed significant differences in survival based on tumor factor, pathological lymph node metastases of the primary tumor, and the tumor-free margin. There were no significant differences between the SH and MH groups in overall survival. CONCLUSIONS: Patients with synchronous liver metastases from colorectal cancer should undergo radical resection of the primary lesion and simultaneous hepatectomy with an adequate tumor-free margin as a standard surgical course.

Adult↗

PubMed2006

Ubiquitin-specific protease 14 expression in colorectal cancer is associated with liver and lymph node metastases.

Ubiquitin-specific protease 14, also known as the 60 kDa subunit of tRNA-guanine transglycosylase (USP14/TGT60 kD), belongs to the ubiquitin-specific processing protease (UBP) family. USP14/TGT60 kD expression in leukemic and colorectal cancer cell lines, and the suppression of such an expression after the induction of cell differentiation have been reported. In the present study, we attempted to clarify whether USP14/TGT60 kD overexpression affects the clinicopathological features of colorectal cancer. Immunohistochemically, USP14/TGT60 kD was absent or weakly localized in the cytoplasm of normal colorectal epithelial cells. In 18 of 99 (18.2%) colorectal cancer patients, USP14/TGT60 kD was strongly detected in the cytoplasm of cancer cells. USP14/TGT60 kD expression correlated with pathological stage (P=0.03), and lymph node (P=0.03) and liver (P=0.03) metastases. Furthermore, the percentage of patients strongly positive for USP14/TGT60 kD expression increased with pathological stage. The overall survival rate was worse in patients with a high USP14/TGT60 kD expression level than in those with a low USP14/TGT60 kD expression level. Our results suggest that USP14/TGT60 kD also controls the fate of proteins that regulate tumor invasion and metastasis.

Adenocarcinoma↗