Biomedical subjects

T Shibata

Publications and source records attributed to T Shibata.

At least 181 records · Page 10Linked to original sources

PubMed2000

[Three long surviving patients with gastric cancer metastasizing to the liver under interdisciplinary therapy].

In 30 patients with gastric cancer metastasizing to the liver over the past 15 years at our hospital the primary foci in the stomach could be resected in a curative manner. The authors report herein three long surviving patients in this series. [Case 1] A 49-year-old male. Distal gastrectomy was performed in November 1984. Metastasis to the liver occurred in June 1986. The right lobe of the liver was resected in November 1987 after transcatheter arterial embolization (TAE). Although hepatic arterial infusion chemotherapy was conducted, the cancer metastasized to the whole body, and the patient died in December 1991. [Case 2] A 65-year-old female. Distal gastrectomy was performed in July 1994. The left hepatic lobe and segment 5 in the right lobe were resected in June 1995. Although TAE was performed six times starting in December 1996, the patient died of hepatic failure in July 1999. [Case 3] A 73-year-old male. This patient simultaneously received distal gastrectomy and extended resection of the posterior hepatic segments in September 1997. Cancer recurred in the remaining liver in July 1998. Although microwave coagulation therapy (MCT) and TAE were performed, the patient died of hepatic failure in January 2000. In these patients who survived for a long period, the primary focus was well-differentiated adenocarcinoma under sufficient local control with metastasis limited to the nearest regional lymph nodes (group 1 lymph nodes). The patients could undergo interdisciplinary therapy, including hepatectomy, MCT, TAE, and hepatic arterial infusion chemotherapy.

Adenocarcinoma↗

PubMed2000

[A study of reduction of NF 2 protein, mRNA and loss of 22 q in sporadic meningiomas].

Neurofibromatosis 2(NF 2) is one of the tumor suppressor genes, and its disorder has been reported in sporadic meningiomas. We investigated some reduction of expression of NF 2 protein and mRNA in 33 sporadic meningiomas, using immunohistochemistry and in situ hybridization, respectively. Seventeen of 33 (51.5%) meningiomas demonstrated significantly reduced or absent NF 2 protein expression. Its mRNA was also reduced or absent in 11 of 24(45.8%) meningiomas, and some statistically significant correlation was shown between reductions of NF 2 protein and its mRNA. Furthermore, we studied about loss of 22 q in 5 meningiomas with fluorescence in situ hybridization. In the cases with reduction of NF 2 protein and/or mRNA, 22 q telomere signals were observed as loss of heterozygosity. On the other hand, in the cases with expression of NF 2 protein and mRNA, 22 q telomere signals were normal. In conclusion, reduction or absence of NF 2 protein and mRNA may play an important role in the tumorigenesis of about half number of sporadic meningiomas.

Chromosomes, Human, Pair 22↗

PubMed2000

Use of A heparin-coated circuit for extracorporeal circulation with selective cerebral perfusion.

The use of selective cerebral perfusion with a heparin-coated extracorporeal circuit has not been reported. We developed a unique extracorporeal circulation system that can be used for selective cerebral perfusion based on an existing heparin-coated circuit with a centrifugal pump for general open-heart surgery and a heparin-coated oxygenator without venous drainage by gravity. Between July 1994 and December 1999, this system was used for 12 patients undergoing surgery for true aortic arch aneurysm. The dose of heparin used was reduced to maintain minimum activated clotting time above 300 sec during extracorporeal circulation. One patient suffered a stroke, and one patient had transient postoperative neurologic deficit. One patient died suddenly of ventricular arrhythmia on the 11th postoperative day. The circuit remained thrombus-free. Selective cerebral perfusion with a heparin-coated bypass circuit was safe and very easy to perform after a simple modification of a standard circuit for open-heart surgery.

Aged↗

PubMed2000

Binding of alkyl- and alkoxy-substituted simple phenolic compounds to human serum proteins.

Wood creosote, primarily a mixture of simple alkyl- and/or alkoxy-substituted phenolic compounds with closely related structures, has long been used as an oral antidiarrheal agent. The use of wood creosote as a parenteral antidiarrheal agent was investigated, and for basic pharmacokinetic data we measured the extent of equilibrium binding of its six major constituent phenolic compounds to human serum proteins using an ultrafiltration method. The percent binding of these major constituent phenolic compounds, namely phenol, guaiacol, p-cresol, o-cresol, creosol and 4-ethylguaiacol, bound to 40-mg/ml human serum albumin was 15.5+/-0.9, 28.0+/-1.5, 37.2+/-0.7, 52.3+/-5.3, 36.8+/-2.0 and 56.7+/-2.4%, respectively, while percent binding to human serum (68 mg protein/ml) was 41.3+/-0.7, 42.6+/-0.5, 64.8+/-0.4, 70.1+/-1.6, 65.7+/-0.2 and 83.1+/-0.1% (mean +/- standard deviation, n = 4), respectively, when tested individually at a concentration of 500 micromol/l. Saturation of binding was not observed for the phenolic compounds up to a concentration of 50 mmol/l. Phenolic compounds with a lipophilic substituent showed higher percent binding to proteins than those without it. We conclude that simple phenolic compounds having alkyl- and/or alkoxy-substituents bind to serum proteins to a considerable extent and that the binding is hydrophobic and nonspecific.

Blood Proteins↗

PubMed2000

Mechanisms of proton transport in isolated rat osteoclasts attached to bone.

Osteoclasts resorb bone by transporting protons (H+) into the space between the cell and the bone. To investigate the roles of a sodium-hydrogen exchanger (NHE) and a vacuolar type H+-ATPase (V-ATPase) in H+ extrusion of osteoclasts attached to bone, changes in intracellular pH (pHi) were monitored in osteoclasts isolated on glass coverslips and on bone slices using a fluorescent pHi indicator. Acid-loaded osteoclasts on glass coverslips recovered their pHi in the presence of Na+. The pHi recovery was inhibited by 5-(N, N-hexamethylene) amiloride (HMA), an inhibitor of NHE, or by removal of Na+. 7-chloro-4-nitrobenz-2-oxa-1, 3-diazol (NBD-CI), a blocker of V-ATPase, did not block the H+ transport in osteoclasts on glass coverslips. Acid-loaded osteoclasts on bone slices recovered their pHi in the absence of Na+, and this recovery was blocked by NBD-Cl. In addition, neither HMA nor NBD-Cl inhibited the pH, recovery in the presence of Na+. These results indicated that osteoclasts attached to bone extrude H+ by both V-ATPase and NHE and that osteoclasts attached to glass mainly extrude H+ by NHE.

4-Chloro-7-nitrobenzofurazan↗

PubMed2000

Metabolic engineering study on the direct fermentation of 2-keto-L-gulonic acid, a key intermediate of L-ascorbic acid in Pseudomonas putida IFO3738.

We have achieved production of 2-keto-L-gulonic acid (2-KLGA) in recombinant Pseudomonas putida IFO3738. Firstly, the genes for sorbose dehydrogenase (SDH)/sorbosone dehydrogenase (SNDH) were introduced into P. putida. The recombinant P. putida/pBBR-SDH produced 0.7 mg/ml of 2-KLGA in a culture broth containing 5% L-sorbose. Replacement of the native SNDH promoter by the Escherichia coli tufB promoter (pBBR-SDH-tufB) improved the productivity of 2-KLGA up to 11.4 mg/ml. Secondly, the sorbitol dehydrogenase (SLDH) gene was also introduced into P. putida. The recombinant P. putida/pUCP19-3DH carrying the genes for SDH, SNDH and SLDH had the ability to produce 2-KLGA (7.5 mg/ml) in a 5% d-sorbitol broth. The productivity of 2-KLGA was improved up to 9.8 mg/ml by changing to an expression system with two plasmids, pBBR-SDH-tufB (for SDH/SNDH) and pUCP19-SLDH (for SLDH), respectively. Moreover, the replacement of the native SLDH promoter by the E. coli tufB promoter (pUCP19-SLDH-tufB) improved the 2-KLGA productivity up to 11.6 mg/ml. Optimization of cultivation conditions increased the conversion yield of 2-KLGA to 32% and that of l-idonate, a metabolite of 2-KLGA, to 40%. These results indicate P. putida IFO3738 is one of the candidate strains for direct fermentation of 2-KLGA.

Journal Article↗

PubMed1999

An NMR analysis of ubiquitin recognition by yeast ubiquitin hydrolase: evidence for novel substrate recognition by a cysteine protease.

Yeast ubiquitin hydrolase 1 (YUH1), a cysteine protease that catalyzes the removal of ubiquitin C-terminal adducts, is important for the generation of monomeric ubiquitin. Heteronuclear NMR spectroscopy has been utilized to map the YUH1 binding surface on ubiquitin. When YUH1 was titrated into a sample of ubiquitin, approximately 50% of the (1)H-(15)N correlation peaks of ubiquitin were affected to some degree, as a result of binding to YUH1. It is noteworthy that the amide resonances of the basic residues (Arg42, Lys48, Arg72, and Lys74) were highly perturbed. These positively charged basic residues may be involved in direct interactions with the negatively charged acidic residues on YUH1. In addition to the electrostatic surface, the hydrophobic surfaces on ubiquitin (Leu8, Ile44, Phe45, Val70, Leu71, and Leu73) and YUH1 are also likely to contribute to the binding interaction. Furthermore, the amide resonances of Ile13, Leu43, Leu50, and Leu69, the side chains of which are not on the surface, were also highly perturbed, indicating substrate-induced changes in the environments of these residues as well. These large changes, observed from residues located throughout the five-stranded beta-sheet surface and the C-terminus, suggest that substrate recognition by YUH1 involves a wider area on ubiquitin.

Amino Acid Sequence↗

PubMed1999

Stable association of 70-kDa heat shock protein induces latent multisite specificity of a unisite-specific endonuclease in yeast mitochondria.

The multisite-specific endonuclease Endo.SceI of yeast mitochondria is unique among endonucleases because its 50-kDa subunit forms a stable dimer with the mitochondrial 70-kDa heat shock protein (mtHSP70), which otherwise fulfills a chaperone function by binding transiently to unfolded proteins. Here we show that the mtHSP70 subunit confers broader sequence specificity, greater stability, and higher activity on the 50-kDa subunit. The 50-kDa subunit alone displayed weaker activity and highly sequence-specific endonuclease activity. The 50-kDa protein exists as a heterodimer with mtHSP70 in vivo, allowing Endo.SceI to cleave specifically at multiple sites on mitochondrial DNA. Endo.SceI may have evolved from a highly specific endonuclease that gained broader sequence specificity after becoming a stable partner of mtHSP70.

Deoxyribonucleases, Type II Site-Specific↗

PubMed1999

Substance P regulates the function of rabbit cultured osteoclast; increase of intracellular free calcium concentration and enhancement of bone resorption.

The effects of substance P on cultured rabbit osteoclasts were investigated. The intracellular Ca(2+) concentration ([Ca(2+)](i)) which was monitored by the microfluorometric technique using fura-2, in osteoclasts elevated by the addition of substance P (0.3-5 microM). The EC(50) value of substance P was about 200 nM. This substance P-evoked [Ca(2+)](i) elevation was not observed in the Ca(2+)-free medium. Simultaneous application of spantide, a substance P receptor antagonist, blocked the Ca(2+) response. The addition of substance P (0.1-10 microM) to cultured osteoclasts enhanced their bone resorption activity which was evaluated by the pit formation assay. Maximum enhancement of the pit formation by substance P (5 microM) peaked at about 170% of the control level. The addition of substance P receptor antagonists also inhibited the enhancement of the bone resorption by substance P addition. Substance P possibly stimulates the bone remodeling by osteoclasts via the [Ca(2+)](i) elevation.

Analysis of Variance↗

PubMed1999

Human Rad51 amino acid residues required for Rad52 binding.

The Rad51 protein, a homologue of the bacterial RecA protein, is an essential factor for both meiotic and mitotic recombination. The N-terminal domain of the human Rad51 protein (HsRad51) directly interacts with DNA. Based on a yeast two-hybrid analysis, it has been reported that the N-terminal region of the Saccharomyces cerevisiae Rad51 protein binds Rad52;S. cerevisiae Rad51 and Rad52 both activate the homologous pairing and strand exchange reactions. Here, we show that the HsRad51 N-terminal region, which corresponds to the Rad52-binding region of ScRad51, does not exhibit strong binding to the human Rad52 protein (HsRad52). To investigate its function, the C-terminal region of HsRad51 was randomly mutagenized. Although this region includes the two segments corresponding to the putative DNA-binding sites of RecA, all seven of the mutants did not decrease, but instead slightly increased, the DNA binding. In contrast, we found that some of these HsRad51 mutations significantly decreased the HsRad52 binding. Therefore, we conclude that these amino acid residues are required for the HsRad51.HsRad52 binding. HsRad52, as well as S. cerevisiae Rad52, promoted homologous pairing between ssDNA and dsDNA, and higher homologous pairing activity was observed in the presence of both HsRad51 and HsRad52 than with either HsRad51 or HsRad52 alone. The HsRad51 F259V mutation, which strongly impaired the HsRad52 binding, decreased the homologous pairing in the presence of both HsRad51 and HsRad52, without affecting the homologous pairing by HsRad51 alone. This result suggests the importance of the HsRad51.HsRad52 interaction in homologous pairing.

Amino Acid Sequence↗

PubMed1999

Ubiquitin binding interface mapping on yeast ubiquitin hydrolase by NMR chemical shift perturbation.

The interaction between the 26 kDa yeast ubiquitin hydrolase (YUH1), involved in maintaining the monomeric ubiquitin pool in cells, and the 8.5 kDa yeast ubiquitin protein has been studied by heteronuclear multidimensional NMR spectroscopy. Chemical shift perturbation of backbone (1)H(N), (15)N, and (13)C(alpha) resonances of YUH1, in a YUH1-ubiquitin mixture and in a 35 kDa covalent complex with ubiquitin (a stable analogue of the tetrahedral reaction intermediate), was employed to identify the ubiquitin binding interface of YUH1. This interface mapped on the secondary structure of YUH1 suggests a wide area of contact for ubiquitin, encompassing the N-terminus, alpha1, alpha4, beta2, beta3, and beta6, coincident with the high specificity of YUH1 for ubiquitin. The presence of several hydrophobic clusters in the ubiquitin binding interface of YUH1 suggests that hydrophobic interactions are equally important as ionic interactions in contacting ubiquitin. The residues in the binding interface exhibit a high percentage of homology among the members of the ubiquitin C-terminal hydrolase family, indicating the well-conserved nature of the ubiquitin binding interface reported in this study. The secondary structure of YUH1, from our NMR studies, was similar to the recently determined structure of its human homologue ubiquitin C-terminal hydrolase L3 (UCH-L3), except for the absence of the helix H3 of UCH-L3. This region in YUH1 (helix H3 of UCH-L3) was least perturbed upon ubiquitin binding. Therefore, the binding interface was mapped onto the corresponding residues in the UCH-L3 crystal structure. A model for ubiquitin binding to YUH1 is proposed, in which a good correlation was observed for the lateral binding of ubiquitin to UCH-L3 (YUH1), stabilized by the electrostatic and hydrophobic interactions.

Amino Acid Sequence↗

PubMed1999

The N-terminal domain of the human Rad51 protein binds DNA: structure and a DNA binding surface as revealed by NMR.

Human Rad51 protein (HsRad51) is a homolog of Escherichia coli RecA protein, and functions in DNA repair and recombination. In higher eukaryotes, Rad51 protein is essential for cell viability. The N-terminal region of HsRad51 is highly conserved among eukaryotic Rad51 proteins but is absent from RecA, suggesting a Rad51-specific function for this region. Here, we have determined the structure of the N-terminal part of HsRad51 by NMR spectroscopy. The N-terminal region forms a compact domain consisting of five short helices, which shares structural similarity with a domain of endonuclease III, a DNA repair enzyme of E. coli. NMR experiments did not support the involvement of the N-terminal domain in HsRad51-HsBrca2 interaction or the self-association of HsRad51 as proposed by previous studies. However, NMR tiration experiments demonstrated a physical interaction of the domain with DNA, and allowed mapping of the DNA binding surface. Mutation analysis showed that the DNA binding surface is essential for double-stranded and single-stranded DNA binding of HsRad51. Our results suggest the presence of a DNA binding site on the outside surface of the HsRad51 filament and provide a possible explanation for the regulation of DNA binding by phosphorylation within the N-terminal domain.

BRCA2 Protein↗

PubMed1999

Triglyceride-lowering effect of a novel insulin-sensitizing agent, JTT-501.

JTT-501, 4-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]benzyl]-3, 5-isoxazolidinedione, is a novel insulin-sensitizing agent. We investigated the triglyceride-lowering activity of JTT-501 in a high-fat (HF) rat model. The HF rats showed insulin resistance with elevation of fasting insulin levels and reduction of insulin-stimulated glucose oxidation. There was also a tendency towards increased basal insulin and triglyceride levels. Oral administration of JTT-501 (3-30 mg kg(-1) day(-1) for 7 days) reduced basal triglyceride levels dose dependently with a minimum effective dose of 3 mg kg(-1) day(-1). Furthermore, regarding triglyceride metabolism, JTT-501 (30 mg kg(-1) day(-1) for 15 days, p.o.) decreased hepatic triglyceride output rate and serum triglyceride half-life (T1/2). In contrast, pioglitazone (30 mg kg(-1) day(-1) for 15 days, p.o.) reduced T1/2, but did not affect hepatic triglyceride output rate. We conclude that JTT-501 possesses potent triglyceride-lowering activity due to its inhibition of triglyceride secretion from the liver and enhancement of triglyceride disposal in peripheral tissues.

Animals↗

PubMed1999

Competitive inactivation of a double-strand DNA break site involves parallel suppression of meiosis-induced changes in chromatin configuration.

In Saccharomyces cerevisiae, DNA double-strand breaks (DSBs) initiate meiotic recombination at open sites in chromatin, which display a meiosis-specific increase in micrococcal nuclease (MNase) sensitivity. The arg4 promoter contains such a DSB site. When arg4 sequences are placed in a pBR322-derived insert at HIS4 (his4 :: arg4 ), the presence of strong DSB sites in pBR322 sequences leads to an almost complete loss of breaks from the insert-borne arg4 promoter region. Most of the MNase-sensitive sites occurred at similar positions in insert-borne and in normal ARG4 sequences, indicating that hotspot inactivation is not a consequence of changes in nucleosome positioning. However, a meiosis-specific increase in MNase hypersensitivity was no longer detected at the inactive insert-borne arg4 DSB site. Elimination of pBR322 sequences restored DSBs to the insert-borne arg4 promoter region and also restored the meiotic induction of MNase hypersensitivity. Thus, the meiotic induction of MNase hypersensitivity at the DSB sites is suppressed and activated in parallel to DSBs themselves, without changes in the underlying DNA sequence or nucleosome positioning. We suggest that meiosis-specific changes in chromatin at a DSB site are a signal reflecting a pivotal step in DSB formation.

Argininosuccinate Lyase↗

PubMed1999

The mutant RecA proteins, RecAR243Q and RecAK245N, exhibit defective DNA binding in homologous pairing.

In homologous pairing, the RecA protein sequentially binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), aligning the two DNA molecules within the helical nucleoprotein filament. To identify the DNA binding region, which stretches from the outside to the inside of the filament, we constructed two mutant RecA proteins, RecAR243Q and RecAK245N, with the amino acid substitutions of Arg243 to Gln and Lys245 to Asn, respectively. These amino acids are exposed to the solvent in the crystal structure of the RecA protein and are located in the central domain, which is believed to be the catalytic center of the homologous pairing activity. The mutations of Arg243 to Gln (RecAR243Q) and Lys245 to Asn (RecAK245N) impair the repair of UV-damaged DNA in vivo and cause defective homologous pairing of ssDNA and dsDNA in vitro. Although RecAR243Q is only slightly defective and RecAK245N is completely proficient in ssDNA binding to form the presynaptic filament, both mutant RecA proteins are defective in the formation of the three-component complex including ssDNA, dsDNA, and RecA protein. The ability to form dsDNA from complementary single strands is also defective in both RecAR243Q and RecAK245N. These results suggest that the region including Arg243 and Lys245 may be involved in the path of secondary DNA binding to the presynaptic filament.

Base Pairing↗

PubMed1999

Attention changes the peak latency of the visual gamma-band oscillation of the EEG.

To investigate the physiological role of visual gamma-band oscillation (GBO), we calculated the event-related dynamics of the EEG power-spectrum for paired visual stimuli (S1 and S2) with or without attention in 12 subjects. The visual stimuli elicited transient increases in the GBO power (around 40 Hz), which were maximal over the parietal area. The peak GBO increase appeared around 300 ms after stimulus onset, but its latency was shorter after S1 and longer after S2 under the 'with attention' than under the 'without attention' condition. This transient increase in the visual GBO is thought to reflect attention and to reset the activity of the visual system in preparation for a new stimulus.

Adult↗

PubMed1999

A DNA unwinding factor involved in DNA replication in cell-free extracts of Xenopus eggs.

BACKGROUND: Alteration of chromatin structure is a key step in various aspects of DNA metabolism. DNA unwinding factors such as the high mobility group (HMG) proteins are thought to play a general role in controlling chromatin structure and a specific role in controlling DNA replication. For instance, in the in vitro simian virus 40 replication system, minichromosomes containing HMG-17 replicate more efficiently than those without it, suggesting that HMG-17 enhances the rate of replication of a chromatin template by unfolding the higher-order chromatin structure. At present, however, only limited data suggest an involvement of DNA unwinding factors in DNA replication. RESULTS: We purified from Xenopus eggs a novel heterodimeric factor, termed DNA unwinding factor (DUF), that consists of 87 kDa and 140 kDa polypeptides. DUF unwinds closed-circular duplex DNA in the presence of topoisomerase I, but it does not possess a DNA gyrase activity: it does not introduce negative supercoils into DNA at the expense of ATP hydrolysis. Cloning and sequencing of the cDNAs encoding the two polypeptides revealed that the 87 kDa polypeptide is homologous to a mammalian HMG protein, T160/structure-specific recognition protein. The 140 kDa polypeptide is homologous to yeast Cdc68, a protein that controls the expression of several genes during the G1 phase of the cell cycle by modulating chromatin structure. Immunodepletion of DUF from Xenopus egg extracts drastically reduced the ability of the extract to replicate exogenously added sperm chromatin or plasmid DNA. CONCLUSIONS: We propose that DUF plays a role in DNA replication in Xenopus egg extracts.

Amino Acid Sequence↗

PubMed1999

A mitochondrial DNA mutation cosegregates with the pathophysiological U wave.

In a family with long QT syndrome (LQT2), some individuals who did not harbor the HERG mutation had a prolonged QTU interval on electrocardiograms after exercise. It may be determined or modified by other gene(s) or factor(s). The sequence analysis of mtDNA in these individuals of this family showed a candidate pathogenic mutation at 3394 in the ND1 gene. The cybrids (mutation at 3394) showed significantly reduced NADH-CoQ reductase (complex I) activity and O2 consumption to normal levels. These inhibitory effects on respiratory function may result in the depletion of ATP and could possibly produce an increase in Ca2+ concentration in cytosol, and it may lead to the prolongation of the QTU intervals on electrocardiograms. Therefore, we stated that the 3394 mutation in the ND1 gene is pathogenic and could be the cause of prolongation of the QTU intervals or modification of the phenotypes of not only congenital but also so-called "acquired drug-induced long QT syndrome."

Adolescent↗