Search PubMed⌕ Search

Biomedical subjects

N Tamura

Publications and source records attributed to N Tamura.

At least 55 records · Page 3Linked to original sources
PubMed2001

Effects of ticlopidine on von Willebrand factor-mediated shear-induced platelet activation and aggregation.

Ticlopidine, recently classified as a P2Y(12) ADP receptor blockade, is known to be an effective antiplatelet agent preventing arterial thrombosis, e.g., myocardial infarction or cerebral infarction, but the mechanism of the in vivo antithrombotic effects of ticlopidine is not fully understood. Blood was drawn from seven normal volunteers before and 7 days after consecutive intake of ticlopidine, and 1 day after oral intake of aspirin after 7 days of ticlopidine wash-out period. Effects of drug intake on shear-induced von Willebrand factor (vWF) binding to platelets, platelet activation evidenced by P-selectin surface expression and translocation of GP Ibalpha, and vWF-mediated platelet aggregation, were investigated by using an optically modified cone-plate viscometer and quantitative flow cytometry. The maximum extent of platelet aggregation occurring under a high shear rate of 10800 s(-1), presumably mediated by vWF, was not significantly influenced by either ticlopidine or aspirin. However, significant dissociation of once aggregated platelets occurred in samples obtained after ticlopidine intake (25.4 +/- 9.3%, P = 0.00030) and less significantly after aspirin intake (15.9 +/- 5.7%, P = 0.0013), while only insignificant and modest dissociation occurred in controls (6.3 +/- 4.4%, n.s.). Binding experiments also revealed that the shear-induced vWF binding to platelets was significantly inhibited by ticlopidine, and less significantly by aspirin, although other indicators of platelet activation, such as shear-induced P-selectin expression and GP Ibalpha translocation were not influenced by either ticlopidine or aspirin. We demonstrate here that platelet aggregation mediated by von Willebrand factor (vWF) under a high shear rate of 10800 s(-1) cannot be stabilized and that dissociation occurs readily when ADP receptor stimulation is blocked by continuous intake of ticlopidine, indicating that these effects on platelet thrombus formation may contribute to the in vivo antithrombotic effects of ticlopidine.

Adult↗
PubMed2001

Monoclonal antibody against brain natriuretic peptide and characterization of brain natriuretic peptide-transgenic mice.

OBJECTIVE: Brain natriuretic peptide (BNP) is a ventricular hormone with natriuretic, diuretic and vasodilatory actions. Acute infusion of BNP reduces cardiac pre- and after-load in healthy and diseased subjects, but its long-term therapeutic usefulness remains unclear. DESIGN: We prepared a monoclonal antibody specific to mouse BNP, and characterized transgenic mice overexpressing BNP in the liver (BNP-Tg mice) as a model of its chronic overproduction. METHODS: Radioimmunoassay and neutralization experiments using the monoclonal antibody, KY-mBNP-I, were performed in BNP-Tg mice in conjunction with examinations of blood pressure (BP) and other markers for body fluid homeostasis. RESULTS: We developed highly sensitive radioimmunoassay to mouse BNP. In BNP-Tg mice, the plasma BNP concentration increased more than 100-fold, while ventricular BNP concentration did not alter, suggesting that ventricular BNP production was not down-regulated in BNP-Tg mice. The BNP concentration in the kidneys was 10-fold higher than nontransgenic (nonTg) littermates, accompanied with marked reduction in the atrial natriuretic peptide (ANP) concentration, that may be due to binding of circulating BNP to the natriuretic peptide receptors. BNP-Tg mice showed significantly low arterial BP, and a bolus intraperitoneal administration of KYmBNP-I completely abolished enhanced cGMP excretion in the urine and significantly increased the systolic BP. CONCLUSION: These results suggested that biological actions of BNP last and reduce cardiac overload in its longterm overproduction in the transgenic mouse model.

Animals↗
PubMed2001

Establishment and characterization of a new cell line (SKS) from neuroendocrine small cell carcinoma of the uterine cervix and its chemosensitivity.

A new cell line (SKS) established from ascites of a patient with neuroendocrine small cell carcinoma of the uterine cervix had a good tumorigenicity and caused marked peritoneal dissemination, and was also highly sensitive to gemcitabine in an in vitro chemosensitivity test. SKS cells were small round cells with a high nuclear/cytoplasmic (N/C) ratio and grew into colony-like aggregates, forming spherical aggregates of floating cells. The population doubling time was 44 h. The number of chromosomes ranged from 50 to 56. On examination of the ultrastructure, membrane-bound dense-core neurosecretory-type granules were observed in the cytoplasm. Neuron-specific enolase (NSE) was immunocytochemically positive in the cytoplasm, and 9.3 ng/ml of NSE was detected in the cell culture supernatant. Human papillomavirus was not detected. In the p53 gene, a 3-bp deletion, AAC (Asn), was detected at codon 131 in exon 5. SKS exhibited good tumorigenicity, and the tumor doubling time was 11 days. Intraperitoneal injection of the cells caused peritoneal dissemination, and marked ascites formation was observed. SKS was highly sensitive to gemcitabine, and the 50% growth inhibitory concentration was 30 nM. SKS cells are useful as a model of neuroendocrine small cell carcinoma of the cervix, and chemotherapy using gemcitabine may possibly be effective in this malignancy.

Adult↗
PubMed2001

Cyclin-dependent kinase inhibitor p27 is related to cell proliferation and prognosis in laryngeal squamous cell carcinomas.

An immunohistochemical study of both p27 and proliferating cell nuclear antigen (PCNA) was performed on 102 cases of laryngeal squamous cell carcinomas (LSCCs), and large variations in p27 expression were found among the tumours. Reduced expression of p27 was revealed in 54 (52.9 per cent) cases and correlated with tumour size, lymph node metastasis, and clinical stage, but did not correlate with age, sex, tumour site, or tumour grade. A significant positive correlation was found between the percentage of loss of p27 expression and the PCNA index (r = 0.844, p < 0.0001). Reduced expression of p27 was significantly correlated with both reducing disease-free and overall survival by univariate analysis. By multivariate analysis, reduced expression of p27, tumour grade, tumour size, lymph node metastasis as well as clinical stage were independent prognostic factors for overall survival of LSCCs. These findings indicate that reduced expression of p27 may be correlated with the malignant biological behaviour of LSCCs.

Aged↗
PubMed2001

Tricorn protease in bacteria: characterization of the enzyme from Streptomyces coelicolor.

Tricorn protease is believed to act downstream of the proteasome, or of other ATP-dependent proteases, cleaving the oligopeptides (mostly 6 to 12 residues) released by them into small peptides (2 to 4 residues), before an array of aminopeptidases finally converts them into free amino acids. Hitherto, the occurrence of Tricorn protease seemed to be limited to some archaea, but genes encoding Tricorn homologs have now been found in several bacterial genomes. Among them is Streptomyces coelicolor A3(2), which has, in fact, two Tricorn-like genes, ScC77.16c and ScE87.19. The proteins encoded by them (TRI-ScC77 and TRI-ScE87) are very similar in their PDZ and TSP domains, but rather divergent in their beta-propeller domains. We have expressed one of them, TRI-ScC77, in E. coil and characterized the recombinant protein structurally and functionally. TRI-ScC77 forms a homohexameric complex of approximately 700 kDa, both in E. coil and in S. coelicolor, with enzymatic properties very similar to the complex from the archaeon Thermoplasma acidophilum. The fact that Tricorn-like proteins exist not only in thermoacidophiles, but also in bacteria inhabiting radically different environments, rules out the possibility that Tricorn protease is an adaptive element that helps to meet the challenges of an extreme habitat.

Amino Acid Sequence↗
PubMed2001

Effect of labor on maternal dehydration, starvation, coagulation, and fibrinolysis.

BACKGROUND: Dehydration, starvation, and enhanced blood coagulability may occur during labor. METHODS: We studied 60 women who gave birth vaginally to a healthy singleton infant at term. Sampling of urine and blood specimens was performed on admission to the hospital with labor pains and just after birth. Paired samples were available from 50 women for urine analysis and 29 women for blood analysis. Total fluid intake (oral and intravenous) during labor was recorded. Changes in various parameters in the urine and blood in relation to the total fluid intake were analyzed. RESULTS: Osmolality and creatinine concentration in the urine, red blood cell count, hemoglobin concentration, hematocrit value, thrombin-antithrombin III complex, plasmin-alpha 2-plasmin inhibitor complex and D dimer significantly increased during labor. Ketone bodies were absent in the urine in 84% (42/50) of women on admission to the hospital whereas these were present in 74% (37/50) of women just after delivery (p < 0.01). The degree of these changes appeared to be smaller with an increase in fluid intake. CONCLUSION: Concentrated urine, hemoconcentration, starvation, and activation of the thrombogenic and fibrinolytic system occur in parturient women. Sufficient fluid intake during labor may ameliorate these unfavorable changes.

Adolescent↗
PubMed2001

[Renal autotransplantation in a patient with acute renal infarction following surgery for a dissecting aneurysm].

A 58-year-old woman experienced a sudden onset of severe chest and back pain and thus visited our center in October 1999. Contrast-enhanced computed tomography (CT) revealed a Stanford type A acute aortic dissection. The CT also demonstrated a 50 mm ascending aorta and dissection from the ascending aorta via the abdominal aorta to the level of the left renal artery. The perioperative transesophageal echocardiogram showed an intimal tear in the ascending aorta without valvular abnormality. Therefore, we performed graft replacement of the ascending aorta. On the first postoperative day, she developed oliguria and showed a sudden rise in serum creatinine (Cr) and blood urea nitrogen (BUN) levels, necessitating hemodialysis. She required daily hemodialysis or hemofiltration for twenty days. Thereafter, renal function recovered and dialysis was no longer performed. However, on postoperative day 26, the patient complained of sudden lumber pain. Unheralded oliguria was associated with worsening renal function. A CT scan at this point revealed infarction of the left kidney. During surgery, the left kidney was excised for heterotopic autotransplantation. Extensive thrombosis within a true lumen of the left renal artery was revealed. Following removal of the thrombus and perfusion with heparinized cold saline, renal autotransplantation to a heterotopic site in the pelvis were performed. Although the patient required hemodialysis for five days, renal function recovered gradually. She was discharged five months later. In our experience, it appears that heterotopic renal autotransplantation by which normal arterial perfusion distal to the dissection is reestablished is a good therapeutic option for reperfusion of the ischemic kidney compromised by a progressive dissection of the thoracoabdominal aorta.

Aortic Dissection↗
PubMed2001

Brain natriuretic peptide appears to act locally as an antifibrotic factor in the heart.

In addition to cardiac myocyte hypertrophy, proliferation and increased extracellular matrix production of cardiac fibroblasts occur in response to cardiac overload. This remodeling of the cardiac interstitium is a major determinant of pathologic hypertrophy leading to ventricular dysfunction and heart failure. Atrial and brain natriuretic peptides (ANP and BNP) are cardiac hormones produced primarily by the atrium and ventricle, respectively. Plasma ANP and BNP concentrations are elevated in patients with hypertension, cardiac hypertrophy, and acute myocardial infarction, suggesting their pathophysiologic roles in these disorders. ANP and BNP exhibit diuretic, natriuretic, and vasodilatory activities via a guanylyl cyclase-coupled natriuretic peptide receptor subtype (guanylyl cyclase-A or GC-A). Here we report the generation of mice with targeted disruption of BNP (BNP-/- mice). We observed focal fibrotic lesions in ventricles from BNP-/- mice with a remarkable increase in ventricular mRNA expression of ANP, angiotensin converting enzyme (ACE), transforming growth factor (TGF)-beta3, and pro-alpha1(I) collagen [Col alpha1(I)], which are implicated in the generation and progression of ventricular fibrosis. Electron microscopic examination revealed supercontraction of sarcomeres and disorganized myofibrils in some ventricular myocytes from BNP-/- mice. No signs of cardiac hypertrophy and systemic hypertension were noted in BNP-/- mice. In response to acute cardiac pressure overload induced by aortic constriction, massive fibrotic lesions were found in all the BNP-/- mice examined, accompanied by further increase of mRNA expression of TGF-beta3 and Col alpha1(I). We postulate that BNP acts as a cardiocyte-derived antifibrotic factor in the ventricle.

Alleles↗
PubMed2001

[Expression of urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR) plays a role in destruction of joint tissue in rapidly destructive coxarthropathy (RDC)].

OBJECTIVE: Proliferation of small vessels including capillaries in joint tissues is one of the significant histopathological features of rapidly destructive coxarthropathy (RDC). To examine the relationship between the angiogenesis and bone destruction, expression of urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR), which are necessary for angiogenesis was histochemically investigated. MATERIALS AND METHODS: Synovia and cartilage-bone tissues of femoral head were obtained from RDC patients at total hip replacement (THR). These joint tissues were fixed with 4% paraformaldehyde, and cartilage-bone tissues were decalcified, additionally. Immunohistochemical staining was performed on paraffin sections, using monoclonal antibodies against human uPA and uPAR. RESULT: There was a difference between expression of uPA and that of uPAR. Expression of uPA was observed in many types of cells such as osteoclasts, giant cells, fibroblast-like cells and macrophages, of which osteoclasts and giant cells were most prominent. On the other hand, expression of uPAR was detected mainly in fibroblast-like cells and macrophages and rarely seen in osteoclasts and giant cells. CONCLUSION: These results indicate that uPA-plasmin system contributes to bone destruction in RDC, and furthermore, that fibroblast-like cells and macrophages play an important role in activation of uPA-plasmin system.

Aged↗
PubMed2001

Soluble CD154 in rheumatoid arthritis: elevated plasma levels in cases with vasculitis.

OBJECTIVE: To determine the levels of soluble CD154 (sCD154) in the plasma of patients with rheumatoid arthritis (RA) and rheumatoid vasculitis (RV). and to examine the relationship between the levels of sCD154 in plasma and the clinical variables. METHODS: Levels of sCD154 were quantified in 39 plasma samples from patients with RA, including 9 patients who were also diagnosed with RV, and compared with those of 20 healthy subjects. An ELISA was established and specificity of the ELISA was tested by control ELISA using isotype-matched IgG and preabsorption assay. The titers of IgM and IgG rheumatoid factor (IgM-RF, IgG-RF) for each patient were determined simultaneously, and values of other laboratory variables were also determined. RESULTS: Levels of sCD 154 in plasma were higher in patients with RA than in the healthy subjects (p < 0.02). Compared with RA patients without vasculitis, patients with RV had significantly higher levels of sCD154 in their plasma (p < 0.001). Control ELISA and absorption assay of sCD154 indicated that our ELISA system was capable of measuring plasma sCD154 in RA patients. Levels of sCD154 in RA plasma correlated significantly with both IgM-RF and IgG-RF titers (r = 0.64 and 0.61, respectively, both p < 0.001). The levels of sCD154 decreased after commencement of treatment for vasculitis in cases with RV. CONCLUSION: We identified the presence of sCD154 in RA plasma, with especially high levels in cases with vasculitis. Correlation between sCD154 and RF titers indicates the CD154-CD40 pathway is likely related to pathogenic RF production.

Adult↗
PubMed2000

Treatment of acute stanford type B aortic dissection with a novel cylindrical balloon catheter in dogs.

BACKGROUND: Despite recent progress in medical and surgical treatment, acute type B aortic dissection still carries a high mortality rate. We have developed a novel cylindrical balloon catheter for less invasive treatment to block the entry of the dissection and induce thrombotic occlusion of the false lumen. The balloon has the shape of a sheet when deflated but a double-cylinder shape when inflated. Therefore, aortic blood flow is maintained through the cylindrical lumen during balloon inflation. METHODS AND RESULTS: Six beagle dogs underwent a left thoracotomy at the 6th intercostal space. An acute dissection of 4-cm length was created surgically on the descending aorta. The balloon catheter was inserted through the distal descending aorta and advanced to the entry site. The balloon catheter was inflated for 6 hours. The blood flow in the descending aorta and the position of the balloon was monitored by color Doppler echovasculography. Four dogs were killed humanely on the following day and 2 dogs 10 days after the surgery. The descending aorta was examined macroscopically and microscopically in all dogs. In all dogs, the false lumen was occluded by thrombi. Although no dog had clinical evidence of distal thromboembolism, 2 of the 4 dogs that were killed on the second postoperative day had fresh mural thrombi in the true lumen. CONCLUSIONS: The false lumen of the acute type B aortic dissection was effectively occluded by the novel cylindrical balloon catheter in the canine experimental model. The thrombus formation in the true lumen is the problem to be solved.

Acute Disease↗
PubMed2000

Novel method to enhance sternal healing after harvesting bilateral internal thoracic arteries with use of basic fibroblast growth factor.

BACKGROUND: Poor healing of the sternum often limits the use of bilateral internal thoracic arteries (BITAs) in coronary bypass surgery, especially for diabetic patients. We have reported that basic fibroblast growth factor (bFGF) enhanced regeneration of the skull. This study was designed to evaluate the effects of topical use of bFGF on sternal healing after removing the BITAs. METHODS AND RESULTS: Forty-five Wistar rats were subjected to median sternotomy and were divided into 3 groups: 15 had the BITAs removed and had a bFGF sheet applied on the posterior table of the sternum (group A), 15 had just the BITAs removed (group B), and 15 had intact BITAs (group C). Five and 10 rats were euthanized 2 and 4 weeks after surgery, respectively, in all 3 groups. Peristernal blood flow, measured with use of a noncontact laser flowmeter, decreased after removal of the BITAs (P:<0.001). Four weeks after the surgery, PBF markedly increased only in group A (9.7+/-1.2, 6.5+/-0.6, and 8.2+/-0.5 mL x min(-1) x 100 g(-1) for groups A, B, and C, respectively; P:<0.01 by ANOVA). Four weeks after surgery, the following findings were obtained only in group A: (1) nearly completely healed sternum filled with regenerated bone tissue, (2) marked angiogenesis around the sternum, and (3) osteoblasts in an active form around the edge of the sternum. CONCLUSIONS: The results suggest that use of the bFGF sheet offset the sternal ischemia and accelerated sternal healing. This method may help to decrease sternal necrosis in high-risk patients or allow extended use of BITAs in coronary bypass surgery.

Administration, Topical↗
PubMed2000

Molecular mechanism of fibronectin gene activation by cyclic stretch in vascular smooth muscle cells.

Fibronectin plays an important role in vascular remodeling. A functional interaction between mechanical stimuli and locally produced vasoactive agents is suggested to be crucial for vascular remodeling. We examined the effect of mechanical stretch on fibronectin gene expression in vascular smooth muscle cells and the role of vascular angiotensin II in the regulation of the fibronectin gene in response to stretch. Cyclic stretch induced an increase in vascular fibronectin mRNA levels that was inhibited by actinomycin D and CV11974, an angiotensin II type 1 receptor antagonist; cycloheximide and PD123319, an angiotensin II type 2 receptor antagonist, did not affect the induction. In transfection experiments, fibronectin promoter activity was stimulated by stretch and inhibited by CV11974 but not by PD123319. DNA-protein binding experiments revealed that cyclic stretch enhanced nuclear binding to the AP-1 site, which was partially supershifted by antibody to c-Jun. Site-directed mutation of the AP-1 site significantly decreased the cyclic stretch-mediated activation of fibronectin promoter. Furthermore, antisense c-jun oligonucleotides decreased the stretch-induced stimulation of the fibronectin promoter activity and the mRNA expression. These results suggest that cyclic stretch stimulates vascular fibronectin gene expression mainly via the activation of AP-1 through the angiotensin II type 1 receptor.

Angiotensin II↗
PubMed2000

Enhanced expression and activation of Ca(2+)/calmodulin-dependent protein kinase IV in hepatocellular carcinoma.

BACKGROUND: Ca(2+)/calmodulin-dependent protein kinase IV (CaM-kinase IV) is a multifunctional protein kinase that is expressed abundantly in the central nervous system and, to a lesser degree, in nonneuronal tissues such as the liver. In the current study, the authors demonstrated the expression of CaM-kinase IV in hepatocytes from hepatocellular carcinoma (HCC) in both humans and rats. METHODS: Immunoblotting and immunohistochemical analysis were performed to confirm the expression of CaM-kinase IV and CaM-kinase kinase in HCC occurring in both humans and rats. The kinase activity of CaM-kinase IV in the lysate of each of these liver supernatant fluids was measured using a specific substrate (peptide gamma) for this enzyme before and after phosphorylation by exogenously added CaM-kinase kinase. RESULTS: Marked positive staining of HCC hepatocytes was found and the subcellular staining pattern mainly was cytosolic. One immunoreactive band with a molecular weight of 64 kilodaltons, which was identical to an isoform of rat cerebellum CaM-kinase IV, was demonstrated by immunoblotting. Ca(2+)/calmodulin-dependent CaM-kinase IV activity was high in HCC and showed almost no difference in activity in specimens with and without CaM-kinase kinase phosphorylation. CONCLUSIONS: CaM-kinase IV was found to be expressed in HCC and might have been involved in the development of HCC. CaM-kinase IV that was expressed in cancerous hepatocytes was phosphorylated mainly by CaM-kinase kinase that also was expressed in tumor cells.

Animals↗
PubMed2000

Cysteine-scanning mutagenesis of transmembrane segments 4 and 5 of the Tn10-encoded metal-tetracycline/H+ antiporter reveals a permeability barrier in the middle of a transmembrane water-filled channel.

Cysteine-scanning mutants as to putative transmembrane segments 4 and 5 and the flanking regions of Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) were constructed. All mutants were normally expressed. Among the 57 mutants (L99C to I155C), nine conserved arginine-, aspartate-, and glycine-replaced ones exhibited greatly reduced tetracycline resistance and almost no transport activity, and five conserved glycine- and proline-replaced mutants exhibited greatly reduced tetracycline transport activity in inverted membrane vesicles despite their high or moderate drug resistance. All other cysteine-scanning mutants retained normal drug resistance and normal tetracycline transport activity except for the L142C and I143C mutants. The transmembrane (TM) regions TM4 and TM5 were determined to comprise 20 amino acid residues, Leu-99 to Ile-118, and 17 amino acid residues, Ala-136 to Ala-152, respectively, on the basis of N-[(14)C]ethylmaleimide ([(14)C]NEM) reactivity. The NEM reactivity patterns of the TM4 and TM5 mutants were quite different from each other. TM4 could be divided into two halves, that is, a NEM nonreactive periplasmic half and a periodically reactive cytoplasmic half, indicating that TM4 is tilted toward a water-filled transmembrane channel and that only its cytoplasmic half faces the channel. On the other hand, NEM-reactive mutations were observed periodically (every two residues) along the whole length of TM5. A permeability barrier for a membrane-impermeable sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, was present in the middle of TM5 between Leu-142 and Gly-145, whereas all the NEM-reactive mutants as to TM4 were not accessible to 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, indicating that the channel-facing side of TM4 is located inside the permeability barrier. Tetracycline protected the G141C mutant from the NEM binding, whereas the other mutants in TM4 and TM5 were not protected by tetracycline.

Amino Acid Sequence↗
PubMed2000

Cysteine-scanning mutagenesis around transmembrane segments 1 and 11 and their flanking loop regions of Tn10-encoded metal-Tetracycline/H+ antiporter.

Putative transmembrane helices (TM) 1 and 11 in the metal-tetracycline/H(+) antiporter are predicted to be close to each other on the basis of disulfide cross-linking experiments of the double-cysteine mutants in the periplasmic loop regions (Kubo, Y., Konishi, S., Kawabe, T., Nada, S., and Yamaguchi, A. (2000) J. Biol. Chem. 275, 5270-5274). In this study, each amino acid from Asn-2 to Gly-44 in the putative TM1 and loop1-2 regions or that from Ser-328 to Gly-366 in TM11 and its flanking regions was individually replaced with cysteine. With respect to the TM1 region, 10 mutants, from T5C to L14C, were all not reactive with N-ethylmaleimide (NEM), and from D15C to I22C, NEM-reactive and non-reactive mutations periodically appeared every two residues. Three mutants, M23C to V25C, were all NEM-reactive, but the degree of the latter two mutants was very low. Seven mutants, from L26C to E32C, were all highly reactive with NEM. Therefore, the region of TM1 is composed of the 21 amino acid residues from Thr-5 to Val-25. It is a partially amphiphilic helix, that is, the N-terminal (cytoplasmic) half is embedded in the hydrophobic interior, and the C-terminal (periplasmic) half faces a water-filled channel. With respect to TM11, nine mutants, from S328C to G336C, and six mutants, from L361C to G366C, were all reactive with NEM. On the other hand, out of the 24 mutants, from L337C to S360C, 17 were not reactive with NEM, and the 7 NEM-reactive mutants were scattered, indicating that this region is a transmembrane segment. The 7 residues from Val-347 to Phe-353 including Pro-350 formed a central hydrophobic core, and the 7 NEM-reactive mutations were periodically distributed in its flanking regions, indicating that both ends of TM11 face a water-filled channel. Ala-354 is located at about 1/3 of the length from the periplasmic end of TM11. Disulfide cross-linking experiments on double-cysteine mutants having the combination of A354C and a cysteine-scanning mutation in the loop1-2 region indicated that loop1-2 is very flexible and close to the periplasmic end of TM11. Tetracycline prevented the cross-linking formation between the periplasmic ends of TM1 and TM11; however, it did not affect the cross-linking between loop1-2 and TM11, indicating that the substrate-induced conformational change involves a shift in the relative locations of TM1 and TM11.

Amino Acid Sequence↗