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Biomedical subjects

T Kawabe

Publications and source records attributed to T Kawabe.

At least 55 records · Page 3Linked to original sources
PubMed2000

Proximity of periplasmic loops in the metal-Tetracycline/H(+) antiporter of Escherichia coli observed on site-directed chemical cross-linking.

Our previous study on second-site suppressor mutations of the Tn10-encoded metal-tetracycline/H(+) antiporter suggested that Leu(30) and Ala(354), located in periplasmic loop 1-2 and 11-12, respectively, are conformationally linked to each other (Kawabe, T., and Yamaguchi, A. (1999) FEBS Lett. 457, 169-173). To determine the spatial proximity of these two residues, cross-linking gel-shift assays of the L30C/A354C double mutant were performed after the mutant had been oxidized with Cu(2+)/o-phenanthroline. The results indicated that Leu(30) and Ala(354) are close to each other but that Gly(62), which is located in cytoplasmic loop 2-3, and Ala(354) are distant from each other, as a negative control. Then, a single Cys residue was introduced into each of the six periplasmic loop regions (P1-P6), and eleven double mutants were constructed. Of these eleven double Cys mutants, the L30C/A354C and L30C/T235C mutants showed a mobility shift on oxidation, indicating that P1 is spatially close to P4 as well as P6. In contrast, the other nine mutants, L30C/S92C, L30C/S156C, L30C/S296C, S92C/S296C, S92C/T235C, S92C/A354C, S156C/T235C, S156C/S296C, and S156C/A354C, showed no mobility shift under oxidized conditions on intramolecular cross-linking. The S92C and S296C mutants showed dimerization on intermolecular cross-linking, indicating that P2 and P5 are located at the periphery of the helix bundle.

Amino Acid Sequence↗
PubMed2000

Target cells of apoptosis in the adult murine dentate gyrus and O4 immunoreactivity after ionizing radiation.

The occurrence of radiation-induced apoptosis and the determination of target cells were investigated by using the TdT-mediated dUTP-biotin nick end labeling assay and immunohistochemical analyses. The O4 immunoreactivity, an oligodendrocytes surface antigen, was also evaluated by using western blotting analysis. C57BL/6J adult female mice were subjected to single dose irradiation of 10 Gy. Eight hours after irradiation, the most significant increase of apoptotic cells was detected in the subgranular zone and the hilus of the dentate gyrus. The target cells of radiation-induced apoptosis are the subgranular progenitor cells and the oligodendrocytes in the hilus. The amount of the O4 immunoreactivity, a marker for premature oligodendrocytes, was unchanged until 8 h but enhanced after 12 h of irradiation. These results are the first to show the increase of the O4 immunoreactivity after irradiation and may be associated with the pathogenesis of radiation injury.

Animals↗
PubMed2000

Abolition of anti-glomerular basement membrane antibody-mediated glomerulonephritis in FcRgamma-deficient mice.

Several recent studies have demonstrated the central role of Fc receptors (FcR) rather than the complement system in triggering hypersensitivity reactions. We investigated the role of FcR for IgG (FcgammaR) using a murine model of accelerated anti-glomerular basement membrane (GBM) antibody-mediated glomerulonephritis as a representative of type II hypersensitivity diseases. Intravenous injection of rabbit anti-GBM antibody after preimmunization with normal rabbit IgG induced proteinuria and azotemia in wild-type C57BL/6 and CD40(+/-) mice but not in FcR gamma chain (FcRgamma)(-/-) mice or CD40(-/-) mice. Light microscopic findings revealed marked tissue damage in the glomeruli of wild-type C57BL/6 and CD40(+/-) mice. However, no tissue damage except polymorphonuclear cell infiltration was observed in the glomeruli of FcRgamma(-/-) mice. The glomeruli of CD40(-/-) mice were almost normal. Immunohistochemistry revealed the binding of rabbit IgG to the GBM in all mice injected with anti-GBM antibody. However, depositions of mouse IgG and complement to the glomeruli were not observed in CD40(-/-) mice, and deposition of fibrin was not observed in FcRgamma(-/-) or CD40(-/-) mice. These findings suggest that FcgammaR may initiate anti-GBM antibody-mediated renal disease. We conclude that FcgammaR rather than the complement system is critically involved in the development of type II hypersensitivity diseases.

Animals↗
PubMed2000

Molecular construction of a multidrug exporter system, AcrAB: molecular interaction between AcrA and AcrB, and cleavage of the N-terminal signal sequence of AcrA.

The AcrAB system of Escherichia coli is an intrinsic efflux protein with a broad substrate specificity. AcrA was thought to be localized in the periplasmic space, and to be linked to AcrB and TolC. The AcrAB-TolC system directly exports diverse substrates from the cell interior to the medium. In this study, we have determined the cellular localization of AcrA. By using the osmotic shock method, sucrose density gradient centrifugation, urea washing and Western blotting analysis, we reveal that AcrA is a peripheral inner membrane protein. A mutant plasmid encoding both the AcrA-TetBCt fusion protein and the AcrB-His fusion protein was constructed. Membrane vesicles prepared from cells expressing these fusion proteins were solubilized and AcrB-His was immunoprecipitated with an anti-polyhistidine antibody. After SDS-PAGE, Western blotting was performed with anti-TetBCt antiserum, resulting in the appearance of a 40 kDa band, indicating that AcrA co-precipitated with AcrB. Next we performed site-directed chemical labeling of Cys-introduced mutants of AcrA with [(14)C]N-ethylmaleimide. As judged from the labeling pattern and the molecular mass shift, the N-terminus of AcrA was removed and the mature protein is on the periplasmic surface. On the other hand, C25A mutants retained the N-terminal signal sequence on the cytoplasmic side of the membrane. We conclude that AcrA exists as a complex with AcrB on the periplasmic surface of the inner membrane after removal of the signal sequence.

Bacterial Proteins↗
PubMed2000

Early detection of haemobilia associated with percutaneous ethanol injection for hepatocellular carcinoma.

OBJECTIVE: Haemobilia often results from iatrogenic injury caused by therapeutic procedures. The objective of this study was to evaluate the efficacy of early diagnosis of haemobilia based on ultrasonography in patients with hepatocellular carcinoma undergoing percutaneous ethanol injection. PATIENTS AND METHODS: A combination retrospective and prospective study on the early detection of haemobilia caused by percutaneous ethanol injection was conducted on 365 patients in 1995-1996. The retrospective study reviewed the clinical, laboratory and imaging data of 172 patients who had undergone ethanol injection therapy in 1995. The results showed that ultrasonographic changes in the gallbladder, namely the rapid appearance of echogenic material in the gallbladder lumen, are a useful early sign of haemobilia. Based on the results of the retrospective study, a prospective study on the early detection of haemobilia was carried out in 1996. In the prospective study, percutaneous ethanol injection was halted as soon as haemobilia was detected. RESULTS: The incidence of haemobilia in the prospective group (3.6%) was not different from that in the retrospective group (4.7%). However, the mean duration between percutaneous ethanol injection and diagnosis of haemobilia was only 0.3 +/- 0.2 days in the prospective group, compared with 2.8 +/- 2.1 days in the retrospective group (P < 0.001), and the mean duration of jaundice in the prospective group (4.3 days) was significantly shorter than in the retrospective group (40.0 days) (P< 0.05). CONCLUSION: Early diagnosis of haemobilia based on ultrasonographic findings of the gallbladder lumen effectively reduces the severity of haemobilia-related complications due to immediate interruption of the interventional procedure.

Administration, Cutaneous↗
PubMed1999

Sensitization of cancer cells to DNA damage-induced cell death by specific cell cycle G2 checkpoint abrogation.

We devised two short peptides corresponding to amino acids 211-221 of human Cdc25C fused with a part of HIV1-TAT. These peptides inhibited hChk1 and Chk2/HuCds1 kinase activity in vitro and specifically abrogated the G2 checkpoint in vivo. These peptides sensitized p53-defective cancer cell lines to DNA-damaging agent to death without obvious cytotoxic effect on normal cells. Our results clearly indicate that the specific abrogation of the cell cycle G2 checkpoint is a feasible strategy for cancer therapy, and hChk1 and Chk2/HuCds1 are proper targets for that purpose.

Amino Acid Sequence↗
PubMed1999

NF-kappaB subunit p65 binds to 53BP2 and inhibits cell death induced by 53BP2.

Nuclear factor kappaB (NF-kappaB) is a transcription factor that controls the expression of many cellular and viral genes. The p65 (RelA) subunit plays a critical role as a transcriptional activator and recent observations have highlighted its role in the control of apoptosis. Here we report that 53BP2, a protein previously identified by interaction with wild type p53 and Bcl-2, also binds to p65 in a yeast two-hybrid system. This specific interaction was confirmed by pull-down assay in vitro and by a mammalian two-hybrid assay in vivo. We observed that full-length 53BP2 fused to GFP had a punctate distribution in cytoplasm, predominantly in perinuclear region whereas the N-terminal 53BP2 localized in cytoplasm and C-terminal 53BP2 localized in the nucleus. Furthermore, we found that overexpression of GFP-53BP2 induced apoptosis in transiently transfected cells. Neither the N-terminal nor the C-terminal of 53BP2 fused to GFP induced cell death. Interestingly, co-transfection with a p65 expression plasmid significantly inhibited 53BP2-induced cell death. The previous findings that 53BP2 bound to p53 and Bcl-2 together with our present observations suggest that 53BP2 may play a central role in the regulation of apoptosis and cell growth.

Animals↗
PubMed1999

Transmembrane remote conformational suppression of the Gly-332 mutation of the Tn10-encoded metal-tetracycline/H+ antiporter.

Gly-332 is a conformationally important residue of the Tn10-encoded metal-tetracycline/H+ antiporter (TetA(B)), which was found by random mutagenesis and confirmed by site-directed mutagenesis. A bulky side chain at position 332 is deleterious to the transport function. A spontaneous second-site suppressor revertant was isolated from G332S mutant and identified as the Ala-354-->Asp mutant. Gly-332 and Ala-354 are located on opposite ends of transmembrane segment XI. As judged from [14C]NEM binding to Cys mutants, the residue at position 354, which is originally exposed to water, was buried in the membrane by a G332S mutation through a remote conformational change of transmembrane segment XI. This effect is the same as that of a G62L mutation at position 30 through transmembrane segment II [Kimura, T., Sawai, T. and Yamaguchi, A. (1997) Biochemistry 36, 6941-6946]. Interestingly, the G332S mutation was also suppressed by the L30S mutation, and the G62L mutation was moderately suppressed by the A354D mutation. These results indicate the presence of a close conformational relationship between the flanking regions of the transmembrane segments II and XI.

Amino Acid Sequence↗
PubMed1999

Enhanced transport of anticancer agents and leukotriene C4 by the human canalicular multispecific organic anion transporter (cMOAT/MRP2).

We established stable human canalicular multispecific organic anion transporter (cMOAT/MRP2) cDNA transfectants, CHO/cMOAT from non-polarized Chinese hamster ovary (CHO)-K1 and LLC/cMOAT from polarized pig kidney epithelial LLC-PK1. Human cMOAT was mainly localized in the plasma membrane of CHO/cMOAT and in the apical membrane of LLC/cMOAT. The ATP-dependent uptake of leukotriene C4 (LTC4) into CHO/cMOAT membrane vesicles was enhanced compared with empty vector transfectants. Km values in CHO/cMOAT membrane vesicles were 0.24 microM for LTC4 and 175 microM for ATP. Drug sensitivity to vincristine and cisplatin in human cMOAT cDNA transfectants decreased, but not to etoposide. Cellular accumulation of vincristine and cisplatin in human cMOAT cDNA transfectants decreased, but not of etoposide. The uptake of LTC4 into CHO/cMOAT membrane vesicles was inhibited by exogenous administration of vincristine or cisplatin, but not that of etoposide. Moreover, this inhibition was more enhanced in the presence of glutathione. These consequences indicate that drug resistance to vincristine or cisplatin appears to be modulated by human cMOAT through transport of the agents, possibly in direct or indirect association with glutathione.

Adenosine Triphosphate↗
PubMed1999

Decreased alpha-adrenergic response in the intestinal microcirculation after "two-hit" hemorrhage/resuscitation and bacteremia.

BACKGROUND: The two-hit theory of multiple organ dysfunction syndrome proposes that an initial insult primes the host for an altered response to subsequent stimuli. We have previously documented enhanced dilator tone in the small intestine after a two-hit insult; however, the effects on vasoconstrictor function are unknown. We postulated that prior hemorrhage and resuscitation followed by bacteremia would alter microvascular responsiveness to alpha-adrenergic stimulation. METHODS: Male Sprague-Dawley rats underwent fixed-volume hemorrhage with resuscitation (H/R) or sham procedure (Sham). At 24 or 72 h, in vivo videomicroscopy of the small intestine was performed (inflow A1 and premucosal A3 arterioles). Constrictor function was assessed by topical application of norepinephrine (NE; 10(-8)-10(-6) M) before and 1 h after intravenous Escherichia coli or saline. RESULTS: Sham, 24 or 72 h H/R, and E. coli alone produced no significant changes in A1 or A3 response to NE. Sequential H/R + E. coli resulted in decreased constrictor response in both A1 (72 h H/R + E. coli-38% from baseline vs Sham - 54%, P < 0.05) and A3 arterioles (-8% vs -51%, P < 0.05) at high doses of NE (10(-6) M). CONCLUSIONS: Prior H/R primes the intestinal microvasculature for an altered response during a subsequent stress and these effects persist for up to 72 h following H/R. Sequential insults in this two-hit model caused marked hyporesponsiveness to NE. These alterations in control of microvascular tone might contribute to the hemodynamic compromise of sepsis, impair mucosal blood flow, and contribute to the development of MODS.

Adrenergic alpha-Agonists↗
PubMed1999

In vitro sonographic evaluation of common bile duct stones and fragments with a high-frequency microprobe.

PURPOSE: This study was conducted to evaluate the sonographic characteristics of common bile duct (CBD) stones and stone fragments scanned in vitro with a high-frequency (20-MHz) microprobe. METHODS: We sonographically examined 4 whole CBD stones (1 cholesterol stone with radiant cross section, 1 cholesterol stone with lamellar cross section, 1 black pigment stone, and 1 brown pigment stone), 44 fragments of a cholesterol stone (diameter range, 1.1-3.0 mm; mean diameter, 2.0 mm), and 75 fragments of a black pigment stone (diameter range, 1.1-3.0 mm; mean diameter, 2.0 mm). The chemical composition of all stones was analyzed by infrared absorption spectroscopy. All stones and fragments were placed in a plastic box filled with physiologic saline solution for scanning with a 20-MHz microprobe and a 5-MHz probe. Echogenic foci and acoustic shadows revealed at each frequency were compared. RESULTS: At 5 MHz, the 4 CBD stones showed different sonographic characteristics. The cholesterol stone with radiant cross section showed an echogenic focus but no definite acoustic shadow, whereas the cholesterol stone with lamellar cross section showed an echogenic line, several echogenic spots, and a definite acoustic shadow. The black pigment stone showed an echogenic line with a vague acoustic shadow, and the brown pigment stone showed echogenicity of the whole stone and a definite acoustic shadow. At 20 MHz, the 4 stones showed an echogenic line with a definite acoustic shadow. All fragments appeared as echogenic foci at 20 and 5 MHz. Seventy-seven percent (34) and 5% (2) of 44 cholesterol stone fragments cast a definite acoustic shadow at 20 and 5 MHz, respectively. Sixty-nine percent (52) and 15% (11) of 75 black pigment stone fragments cast a definite acoustic shadow at 20 and 5 MHz, respectively. Among fragments 1.5 mm in diameter and smaller, only 45% (5 of 11) from a cholesterol stone and 30% (6 of 20) from a black pigment stone showed a definite acoustic shadow at 20 MHz. CONCLUSIONS: High-frequency (20-MHz) sonography demonstrates a definite acoustic shadow more frequently among CBD stones and fragments 1.6 mm in diameter or larger than does low-frequency (5-MHz) sonography, but it does not discriminate the chemical composition.

Adult↗
PubMed1999

Immunological diagnosis of Werner syndrome by down-regulated and truncated gene products.

Although immunological methods are widely used to diagnose various infectious diseases, they have rarely been employed to detect genetic diseases. In this study, we have established an immunoblot analysis system for the diagnosis of Werner syndrome (WS), a recessive genetic disorder causing premature aging and an enhanced risk of rare cancers. The method uses an immunoblot technique with specific monoclonal antibodies to WS gene product, and B-lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus; these cell lines express an increased level of normal WS gene product DNA helicase. The method clearly distinguishes normal from patient LCLs containing any of the mutation types found so far in Japan, primarily because of the drastically reduced levels of mutated gene products, and secondarily because of the truncated product sizes. A comparison of this immunological diagnosis with the symptom-based clinical diagnosis has narrowed down the criteria of symptoms essential for WS diagnosis. This procedure is compatible with, and has some advantage over, the genetic method, because WS patients can be diagnosed without determining the mutated gene sequences. The method exemplified in WS may also be applied to detect some other genetic diseases.

Adult↗
PubMed1999

Washout of small stones in the bile duct by saline infusion using a side-holed balloon catheter in patients undergoing endoscopic papillary balloon dilation.

BACKGROUND: Complete bile duct clearance of stones should be achieved in patients managed with endoscopic papillary balloon dilation. However, complete retrieval of small stones or tiny fragments sometimes proves difficult using conventional devices. METHODS: We attempted the removal of fine stone fragments by saline infusion using a specially designed retrieval balloon catheter with a blind tip and a side hole located proximal to the balloon in 14 patients. RESULTS: Using this technique, bile duct clearance was achieved in a single attempt in 13 of 14 patients; 2 attempts were required in 1 patient. Cholecystitis occurred in 2 patients with gallstones in situ after the procedure. CONCLUSIONS: Saline washout using a side-holed retrieval balloon catheter effectively cleans stones and stone fragments from the bile duct in patients treated with endoscopic papillary balloon dilation.

Aged↗
PubMed1999

Comparison of controlled and Glisson's pedicle transections of hepatic hilum occlusion for hepatic resection.

BACKGROUND: For hepatic resection, intraoperative bleeding is reduced by clamping the afferent blood flow. Selective clamping at the hepatic hilum can be accomplished using the standard controlled method or Glisson's pedicle transection method. The safety and efficacy of these two methods have not previously been compared. STUDY DESIGN: The intraoperative findings and complications were retrospectively reviewed in 90 patients who underwent major hepatectomy with selective inflow clamping between 1988 and 1997. RESULTS: Blood loss and operative time did not differ between the two groups. Bile leakage was observed in 3 of 43 patients (7.0%) in the standard controlled method group and 11 of 47 patients (23.4%) in the Glisson's pedicle transection method group (p = 0.031). In the Glisson's pedicle transection method group, bile leakage occurred more frequently in patients who underwent left lobectomy than in those who underwent right lobectomy (p = 0.023). CONCLUSIONS: There were no differences in intraoperative findings and postoperative complications analyzed between the standard controlled method and Glisson's pedicle transection method for major hepatectomy except for the rate of bile leakage. In particular, attention must be paid to the bile duct injuries when the Glisson's pedicle is dissected.

Bile↗
PubMed1999

SHIP recruitment attenuates Fc gamma RIIB-induced B cell apoptosis.

Fc gammaRIIB is an inhibitory receptor that terminates activation signals initiated by antigen cross-linking of the BCR through the recruitment of SHIP. Fc gammaRIIB can also signal independently of BCR coligation to directly mediate an apoptotic response, requiring only an intact transmembrane domain. Failure to recruit SHIP, either by deletion of SHIP or mutation of Fc gammaRIIB, results in enhanced Fc gammaRIIB-triggered apoptosis. Thus, in the germinal center, where ICs are retained by FDCs, Fc gammaRIIB may be an active determinant in the negative selection of B cells whose BCRs have reduced affinity for antigen as a result of somatic hypermutation. Selection of B cells may represent the sum of opposing signals generated by the interaction of ICs with the BCR and Fc gammaRIIB through pathways modulated by SHIP.

Agammaglobulinaemia Tyrosine Kinase↗