Biomedical subjects

H Iwamoto

Publications and source records attributed to H Iwamoto.

At least 19 recordsLinked to original sources

PubMed2002

Near field optical microscopy in aqueous solution: implementation and characterization of a vibrating probe.

Near field optical microscopy (NSOM) is one of the possible solutions to circumvent the diffraction limit, but the control of the optical probe in solution has been a technical challenge for practical applications. Most recently, it has been shown that the pipette used in the scanning ion conductance microscope can be modified to form a high resolution near field optical probe. When combined with a novel distance modulation mechanism, a robust near field microscope can be constructed for operation in aqueous solution. In this paper, we present technical details of this design and a further characterization of the NSOM system for imaging in solution. Fundamental limitations of this approach in comparison to other systems are also discussed. Based on the current technology, it is concluded that better than 50 nm resolution should be achievable with this technique for fluorescence, as well as fluorescence resonance energy transfer, imaging of biological specimens.

Microscopy↗

PubMed2002

Comparison of the histological and histochemical properties of skeletal muscles between carbon dioxide and electrically stunned chickens.

1. Histological and histochemical profiles of Musculus pectoralis (PT, type IIB fibres), M. iliotibialis lateralis (ITL, types IIA + IIB fibres) and M. puboischiofemoralis pars medialis (PIF, type I fibres) were compared in carbon dioxide (37%, 70 s) and electrically (14 V, 5 s) stunned male chickens. 2. Muscle materials were taken at 0, 4 and 24 h from carcases dressed and cooled with ice-water mixture for 30 min. Glycogen and fat contents, and adenosine triphosphatase and reduced nicotinamide adenine dinucleotide dehydrogenase activities of fibres were measured. 3. In PT muscle at 0 h, gas stunned chickens showed many fibres with high glycogen content but those electrically stunned contained few such fibres. Fibres from gas stunned birds had lost almost all their glycogen after 24 h of cold storage. 4. In the ITL muscle of gas stunned chickens at 0 h residual glycogen was observed in type IIB fibres. In contrast, in the electrically stunned birds it was in type IIA, showing the different effects of the stunning methods. During cold storage, glycogen disappeared earlier in type IIB than IIA fibres. 5. In PIF muscle with fibres of low glycogen content, the gas stunned chickens maintained a good fibre structure for 4 h or more, but the electrically stunned had already lost intact fibre structure at 4 h. 6. These results indicated that the carbon dioxide stunning was a better method for chicken welfare and meat quality than electrical stunning.

Adenosine Triphosphatases↗

PubMed2002

Renin-producing serous cystoadenocarcinoma of the ovary: a case report.

BACKGROUND: Only a few renin-producing ovarian tumors have been reported, and most such ovarian tumors have been sex cord/stromal tumors. Renin-producing ovarian epithelial tumors are quite rare. CASE: A 46-year-old woman presented with hypertension and hypokalemia. Examinations of the patient revealed elevated plasma renin activity, hyperaldosteronism and a pelvic mass. Subsequently, a right ovarian tumor mass was resected. Microscopic observation of the tumor revealed a well-differentiated serous cystadenocarcinoma. Immediately after surgery, blood pressure, serum potassium, plasma renin activity and plasma aldosterone levels returned to normal ranges. RT-PCR analysis and immunohistochemical staining of this tumor indicated that it was producing renin. CONCLUSION: This is the first report of a renin-producing ovarian serous cystadenocarcinoma.

Aldosterone↗

PubMed2001

Characterization of the metal-substituted dipeptidyl peptidase III (rat liver).

Dipeptidyl peptidase III (DPP III) (EC 3.4.14.4), which has a HELLGH-E (residues 450-455, 508) motif as the zinc binding site, is classified as a zinc metallopeptidase. The zinc dissociation constants of the wild type, Leu(453)-deleted, and E508D mutant of DPP III at pH 7.4 were 4.5 (+/-0.7) x 10(-13), 5.8 (+/-0.7) x 10(-12), and 3.2 (+/-0.9) x 10(-10) M, respectively. The recoveries of the enzyme activities by the addition of various metal ions to apo-DPP III were also measured, and Co(2+), Ni(2+), and Cu(2+) ions completely recovered the enzyme activities as did Zn(2+). The dissociation constants of Co(2+), Ni(2+), and Cu(2+) ions for apo-DPP III at pH 7.4 were 8.2 (+/-0.9) x 10(-13), 2.7 (+/-0.3) x 10(-12), and 1.1 (+/-0.1) x 10(-14) M, respectively. The shape of the absorption spectrum of Co(2+)-DPP III was very similar to that of Co(2+)-carboxypeptidase A or Co(2+)-thermolysin, in which the Co(2+) is bound to two histidyl nitrogens, a water molecule, and a glutamate residue. The absorption spectrum of Cu(2+)-DPP III is also very similar to that of Cu(2+)-thermolysin. The EPR spectrum and the EPR parameters of Cu(2+)-DPP III were very similar to those of Cu(2+)-thermolysin but slightly different from those of Cu(2+)-carboxypeptidase A. The five lines of the superfine structure in the perpendicular region of the EPR spectrum in Cu(2+)-DPP III suggest that nitrogen atoms should coordinate to the cupric ion in Cu(2+)-DPP III. All of these data suggest that the donor set and the coordination geometry of the metal ions in DPP III, which has the HExxxH motif as the metal binding site, are very similar to those of the metal ions in thermolysin, which has the HExxH motif.

Animals↗

PubMed2001

Ischemic preconditioning attenuates cardiac sympathetic nerve injury via ATP-sensitive potassium channels during myocardial ischemia.

BACKGROUND: During myocardial ischemia, massive norepinephrine (NE) is released from the cardiac sympathetic nerve terminals, reflecting the sympathetic nerve injury. A brief preceding ischemia can reduce infarct size; this is known as ischemic preconditioning (PC). The effect of PC on sympathetic nerves, however, including its underlying mechanisms in dog hearts, has remained unclear. Thus, this study was designed to elucidate whether the activation of ATP-sensitive potassium (K(ATP)) channels is involved in the mechanism of cardiac sympathetic nerve protection conferred by PC. METHODS AND RESULTS: Interstitial NE concentration was measured by the in situ cardiac microdialysis method in 45 anesthetized dogs. Five minutes of ischemia followed by 5 minutes of reperfusion was performed as PC. In the controls, the dialysate NE concentration (dNE) increased 15-fold after the 40-minute ischemia. PC decreased dNE at 40-minute ischemia by 59% (P<0.01), which was reversed by glibenclamide. A K(ATP) channel opener, nicorandil (25 microg. kg(-1). min(-1) IV), decreased dNE at 40 minutes of ischemia by 76% (P<0.01), which was also reversed by glibenclamide. During the PC procedure, no significant increase in dNE was detected, even with the uptake-1 inhibitor desipramine. CONCLUSIONS: Cardiac sympathetic nerve injury during myocardial ischemia was attenuated by PC via the activation of K(ATP) channels, but the trigger of the PC effect is unlikely to be NE release in dog hearts.

Adenosine Triphosphate↗

PubMed2001

Serum levels of HCV RNA and core protein before and after incubation at 37 degrees C for 24 h.

The kinetics of HCV during interferon (IFN) therapy have recently been described and the estimated virion half-life is an average of 2.7 h, suggesting that HCV infection is highly dynamic. The aim of this study was to evaluate serum levels of HCV-RNA and HCV core protein (HCV-Ag) before and after incubation at 37 degrees C for 24 h. We also evaluated the viral kinetics during IFN treatment by determining their serum levels at 0, 24 and 48 h, and day 8 after the start of treatment. The decay slope was calculated as the logarithm of the ratio of HCV-RNA levels at 0 and 24 h of incubation: log(virus load) 24 h-log(virus load) 0 h and the estimated half-life was also calculated. The decay slope was -1.66+/-0.75 (-4.12 to -0.18) (mean+/-S.D. (range)) and the estimated virion half-life was 6.2+/-6.9 h (1.8-39.3). The HCV-RNA level was rapidly decreased to 6.8+/-13.1% of the initial load after incubation independently of the serotype. In contrast, the HCV-Ag level after incubation for 24 h was 98.7+/-12.2% of the initial level. The synthesized naked HCV-RNA (equivalent to 10(7) copy/ml) was not detected after 1-min incubation. These data suggested that HCV virions are very unstable and collapsed rapidly and that HCV-RNA, existing outside of virions, is immediately degraded in serum, whereas HCV-Ag remains stable. IFN treatment caused a rapid decrease in the levels of both HCV-RNA and HCV-Ag. The HCV-RNA decay slope was -1.95+/-0.96 (range: -3.48 to -0.50) and was similar to that seen in the incubation study. Our result suggested the significance of measuring HCV-Ag during clinical management independently of HCV-RNA, especially because of its high stability.

Journal Article↗

PubMed2001

X-ray diffraction evidence for the lack of stereospecific protein interactions in highly activated actomyosin complex.

The structure of actomyosin complex while hydrolyzing ATP was investigated by recording X-ray diffraction patterns from rabbit skeletal muscle fibers, in which exogenously introduced rabbit skeletal subfragment-1 (S1) was covalently cross-linked to the endogenous actin filaments in rigor by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Approximately two-thirds of the introduced S1 was cross-linked. The cross-linking procedure did not affect the profile of the S1-induced enhancement of the actin-based layer line reflections in rigor, indicating that the acto-S1 interactions remained highly stereospecific. In the presence of ATP, the MgATPase of the S1 was highly activated regardless of calcium levels, presumably because the availability of the stereospecific binding sites for both proteins was maximized by the cross-linking. However, the diffraction pattern in the presence of ATP was striking in that the intensity profile of the strong 1/5.9 nm(-1) layer lines was indistinguishable from that from bare actin filaments, despite the fact that the majority of the S1 was still associated with actin. The change of the intensity profiles upon addition of ATP was completely reversible. Model calculations showed that this result can be explained if the S1 is not only swinging around its pivoting point, but the pivoting point itself is also moving on the actin surface in a range of a few nanometers. The results suggest that the stereospecific binding sites, which have been considered important for actomyosin cycling, are paradoxically left unoccupied for most of the time in this highly activated actomyosin complex.

Actins↗

PubMed2001

Telomerase activity is repressed during differentiation along the hepatocytic and biliary epithelial lineages: verification on immortal cell lines from the same origin.

Recent investigations indicate that telomerase activity regulates the life span of cells by compensating for telomere shortening during DNA replication. In addition, as differentiation progresses, telomerase activity is reduced in several different cell lineages. These findings lend support to the theory that more immature cells have greater remaining proliferative capacity and longer life span. However, it has not been directly demonstrated that the differentiation along a hepatocytic or a bile ductal lineage is accompanied by reduction of telomerase activity. In this study, we present direct evidence that telomerase activity is reduced during hepatocytic and biliary epithelial differentiation by using our unique cell lines including a stem-like cell line, ETK-1. When hepatocytic differentiation was induced in ETK-1 by 5-azacytidine, telomerase activity decreased significantly. Similarly, when we compared the telomerase activity on SSP-25 and RBE cell lines from the same origin but representing different maturation stages of cholangiocarcinoma, more mature cells were found to possess significantly lower activity. These results indicate that the generally accepted relationship between telomerase activity and differentiation stage also applies in the hepatocytic and biliary epithelial lineages.

Azacitidine↗

PubMed2001

A selective ROCK inhibitor, Y27632, prevents dimethylnitrosamine-induced hepatic fibrosis in rats.

BACKGROUND: p160ROCK is a direct Rho target which mediates Rho-induced assembly of focal adhesions and stress fibers. We previously reported that Rho signaling pathways are involved in the activation of hepatic stellate cells (HSC) in vitro. The aim of the present study was to test the hypothesis that an inhibitor specific for p160ROCK (Y27632) could prevent experimental hepatic fibrosis induced by dimethylnitrosamine (DMN) in rats. METHODS: Y27632 was given orally at 30 mg/kg daily for 4 weeks after the first injection of DMN. The degree of fibrosis was evaluated by image analysis and also by measurements of collagen and hydroxyproline content in the liver. The expression of alpha-smooth muscle actin (alpha-SMA) in the liver and in the primary cultured HSC was also evaluated. Semi-quantitative RT-PCR was performed to evaluate the expression of type I collagen mRNA in the liver. RESULTS: Y27632 treatment significantly decreased the occurrence of DMN-induced hepatic fibrosis and reduced the collagen and hydroxyproline content and alpha-SMA expression in the liver. The expression of alpha-SMA in HSC was also suppressed in vitro. CONCLUSIONS: These findings indicate that inhibitors of the Rho-ROCK pathway might be useful therapeutically in hepatic fibrosis.

Actins↗

PubMed2001

Characterization of AC mode scanning ion-conductance microscopy.

A scanning ion-conductance microscope (SICM) with a vibrating probe has been recently developed (vSICM). In this system, the amplitude of the AC ionic current is detected by using a lock-in amplifier locked to the vibration frequency of the probe. Such a scheme allows for a better control of the tip position because the AC ionic current is more sensitive to the probe-surface distance than the DC ionic current used previously. In this paper, we demonstrate the utility of this technique to the imaging of topographically rough specimens and high-resolution imaging over selected small areas. We also show that it is possible to record the DC ionic current simultaneously during the scan, which can reveal additional information not apparent in the images obtained with the AC ionic current.

Electric Conductivity↗

PubMed2001

S-myotrophin promotes the hypertrophy of myotube as insulin-like growth factor-I does.

We have reported previously that a novel muscle cell growth factor, having a structure of a peptide with sugar chains, was successfully purified from porcine skeletal muscle. It was named s-myotrophin. To determine the role of s-myotrophin in skeletal muscle growth, the effect of s-myotrophin on primary cultured chick skeletal muscle cells (composed almost totally of multinucleated myotubes) was investigated by comparing s-myotrophin with Insulin-like growth factor-I (IGF-I). Both s-myotrophin and IGF-I significantly increased creatine kinase activity of the cultures; both substances gave similar responses. Intracellar protein content was also increased by the addition of these factors. The content of myosin and actin in s-myotrophin treated culture in the differentiation medium was significantly higher than that of the control (unstimulated). The content of those proteins in IGF-I treated culture was also higher than that of control, but the differences were not statistically significant. Immunoblot analysis confirmed that the amounts of myosin and actin in the myocytes were greatly increased by s-myotrophin stimulation and also by IGF-I stimulation. Morphological observations using an anti-desmin antibody staining procedure demonstrated that the size of both s-myotrophin and IGF-I treated myotubes was appreciably larger than that of control myotubes. These results suggest that s-myotrophin is a potent mediator of skeletal muscle cell hypertrophy thorough the accumulations of muscle structural proteins.

Actins↗

PubMed2001

Pirfenidone inhibits dimethylnitrosamine-induced hepatic fibrosis in rats.

1. In the present study, we investigated the preventive effects of pirfenidone (PFD), an antifibrotic agent, on experimental hepatic fibrosis induced by dimethylnitrosamine (DMN) in rats. 2. Treatment with DMN caused a significant decrease in bodyweight and liver weight. Oral PFD (500 mg/kg daily for 4 weeks) essentially prevented this DMN-induced loss in bodyweight and tended to suppress the loss in liver weight. There were no significant differences in liver weight and serum L-alanine aminotransferase levels between PFD-treated and -untreated groups. Pirfenidone has no major side effects in vivo. 3. Pirfenidone suppressed the induction of hepatic fibrosis determined by histological evaluation and reduced hepatic hydroxyproline levels. Expression of mRNA for type I collagen and transforming growth factor-beta in the liver was also suppressed by PFD treatment. 4. Because hepatic stellate cells (HSC) are the major cellular source of extracellular matrix in hepatic fibrosis, we examined the effects of PFD on type I collagen production in vitro using rat primary HSC cultures. Pirfenidone inhibited collagen production in HSC culture in a dose-dependent manner. 5. These results demonstrate that the inhibitory effects of PFD against hepatic fibrosis may be due, at least in part, to blockade of collagen production by HSC and suggest that PFD may be potentially useful in the prevention of the development of hepatic fibrosis.

Animals↗

PubMed2001

Kinetics of the hepatitis C virus during interferon therapy as a marker of therapeutic response.

BACKGROUND: The viral load and subtype of hepatitis C virus (HCV) are predictors of the efficacy of interferon (IFN) therapy. The kinetics of HCV during IFN therapy have been described recently, suggesting that HCV infection is highly dynamic. These observations have raised the issue as to whether early monitoring of the viral load can help guide IFN therapy. METHODS: We measured HCV-RNA levels at 0, 24 and 48 h after the start of IFN-alpha treatment (10 MU daily for 2 weeks and then three times weekly for 22 weeks) or IFN-beta treatment (6 MU daily for 6 weeks). Then we analyzed the relationship between HCV kinetics and therapeutic response using stepwise multivariate logistic regression analysis. RESULTS: The exponential decay slope of the viral load during the first 24 h, not the first 48 h or the next 24 h, was a predictor of viral eradication at 6 months after completion of the treatment (sustained response; P = 0.0023). This decay slope was not affected by the HCV serotype or the type of IFN used. Initial viral load and HCV serotype were also predictors, as reported previously (P < 0.0001 and P = 0.0347, respectively). We also proposed a model using a prognostic index that predicted a sustained response with more than 80% sensitivity, specificity and efficacy in an independent and external group of patients. CONCLUSION: This study demonstrated that the exponential decay slope of the viral load during the first 24 h was an important predictor of the response to IFN therapy as well as the initial viral load and HCV serotype. The model may also be useful for the clinical management of IFN therapy.

Antiviral Agents↗

PubMed2001

Prevalence of hepatitis B or C virus infections in patients with non-Hodgkin's lymphoma.

BACKGROUND: Hepatitis C virus (HCV) and hepatitis B virus (HBV) are not only hepatotropic, but also lymphotropic viruses. Recently, some reports suggested that these viruses may participate in the development of malignant lymphoproliferative disorders. METHODS: We investigated the prevalence of HCV or HBV infection in 348 patients with non-Hodgkin's lymphoma (NHL). We also compared these prevalences with those in blood donors as a control group representing the general population in our area (n= 1,513,358). Next, we evaluated the clinical and pathologic characteristics of HCV- or HBV-infected NHL cases. Non-Hodgkin's lymphoma was classified according to the Working Formulation classification. RESULTS: Thirty-seven cases (14.9%) were found to be infected with HCV or HBV; of these, 20 (8.1%) were infected with HCV, and 17 (6.9%) with HBV. In male NHL patients, the rate of HCV infection was significantly higher than in an age- and sex-matched population in the same area (P < 0.001, Mantel-Haenszel test). The rate of HBV infection also tended to be higher in the population (P = 0.0551). In contrast, in female NHL patients, the rate of HCV or HBV infection was not higher than in the general population. In HCV-infected cases, 15 cases (75%) had B-cell NHL and 16 cases (80%) were classified as being in the intermediate grade; B-cell NHL comprised 83% of all NHL cases. In HBV-infected NHL cases, 11 (65%) were of B-cell type and 10 (58%) were classified as being in the intermediate grade. CONCLUSIONS: The high prevalence of HCV or HBV infections in our study population provides epidemiologic evidence suggesting that HCV and HBV infections may be involved in the development of a subgroup of NHL in males. Our investigation also revealed that both HCV- and HBV-infected NHL patients showed certain similarities in clinical and pathologic manifestations.

Adult↗

PubMed2001

Changes of cytokines in direct endotoxin adsorption treatment on postoperative multiple organ failure.

Multiple organ failure (MOF) is a serious condition that involves simultaneous or consecutive functional failure of several important organs. Furthermore, sepsis is known to play an important role in the occurrence of MOF. Hemoadsorption therapy with the endotoxin adsorption column containing polymyxin B immobilized fibers by direct hemoperfusion (PMX-DHP) is reportedly effective in the treatment of septic shock. This study examined the changes induced on cytokines upon PMX-DHP treatment in 25 patients who underwent emergency abdominal surgery and were immediately started on a postoperative regimen of continuous hemodiafiltration (CHDF) and PMX-DHP. Postoperative MOF was observed in these patients with a mean APACHE II SCORE of 25.5. Eighty percent of patients survived for more than 1 month. We were able to reduce the necessary dose of dopamine in 85.7% of patients because hemodynamic stability improved after administration of PMX-DHP. Interleukin 6 blood levels did not change significantly before or after PMX-DHP treatment in either the surviving or nonsurviving patients. Blood interleukin 1 receptor antagonist levels decreased in both groups. Intercellular adhesion molecular-1, NOx, and thrombomodulin did not change significantly during the course of treatment in either group. Decreased blood levels of PAI-1 levels were found in the surviving patients whereas increased levels of PAI-1 were found in the non-surviving patients. In conclusion, PMX-DHP treatment may be limited clinically in its ability to remove inflammatory cytokines and humoral mediators. However, PMX-DHP treatment is useful for hemodynamic stabilization, which prevents development of MOF.

APACHE↗

PubMed2001

Activation of retinoic X receptor and peroxisome proliferator-activated receptor-gamma inhibits nitric oxide and tumor necrosis factor-alpha production in rat Kupffer cells.

Activators of peroxisome proliferator-activated receptor gamma (PPAR gamma), which forms a heterodimer with retinoic X receptor (RXR), inhibit the production of certain inflammatory mediators. To clarify the role of the PPAR gamma:RXR signaling pathway in Kupffer cells, we studied the effect of an RXR agonist and PPARgamma agonist on LPS-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production. An RXR-specific agonist, Ro47-5944, and a PPAR gamma-specific agonist, AD4833 (pioglitazone hydrochloride), each inhibited LPS-induced NO and TNF-alpha production. The combined treatment of Ro47-5944 and AD4833 resulted in enhanced inhibition, and suppressed the mRNA levels of NO and TNF-alpha. PPAR gamma:RXR activation did not affect the level of LPS-induced phosphorylation of c-jun N-terminal kinase and p38 mitogen-activated protein kinase. PPAR gamma:RXR activation also did not affect nuclear factor kappa B (NF-kappa B) nuclear translocation nor NF-kappa B and activator protein 1 (AP-1) activation in the electrophoretic mobility-shift assay. Finally, PPAR gamma:RXR activation suppressed the LPS-induced promoter activity of the NF-kappa B-luciferase reporter gene in RAW 264.7 cells. These data imply that PPARgamma:RXR activation suppresses LPS-induced NO and TNF-alpha production in Kupffer cells, and that this inhibition occurred at the transcriptional level. Although no consensus PPAR gamma:RXR-responsive element in the promoter regions of the inducible isoform of nitric oxide synthase (iNOS) and TNF-alpha genes was found, PPAR gamma:RXR may interfere with NF-kappa B and AP-1 transcriptional activity. Our data also suggest a potential therapeutic approach for moderating hepatic injury such as endotoxin shock in which Kupffer cell activation has been implicated.

Animals↗

PubMed2001

Scanning electron microscopic observation of the architecture of collagen fibres in chicken M. iliotibialis lateralis.

1. The collagen architecture of M. iliotibialis lateralis in chicken was observed under the scanning electron microscope after muscle maceration in NaOH. 2. Immunohistochemical methods showed Type I and III collagens to be distributed over both perimysium and endomysium. 3. Thick perimysium around secondary myofibre fasciculi was composed of many large longitudinal collagen bundles and a few small circumferential bundles. In contrast, thin perimysium around primary myofibre fasciculi showed mainly circumferential bundles. 4. Endomysium had a honeycomb-like structure and consisted of a fine collagen mesh, its main fibre striation being circumferential. 5. It is suggested that functional demand differs between thick perimysium and thin endomysium.

Animals↗

PubMed2001

A 12-amino-acid segment, present in type s2 but not type s1 Helicobacter pylori VacA proteins, abolishes cytotoxin activity and alters membrane channel formation.

Helicobacter pylori, a gram-negative bacterium associated with gastritis, peptic ulceration, and gastric adenocarcinoma in humans, secretes a protein toxin, VacA, that causes vacuolar degeneration of epithelial cells. Several different families of H. pylori vacA alleles can be distinguished based on sequence diversity in the "middle" region (i.e., m1 and m2) and in the 5' end of the gene (i.e., s1 and s2). Type s2 VacA toxins contain a 12-amino-acid amino-terminal hydrophilic segment, which is absent from type s1 toxins. To examine the functional properties of VacA toxins containing this 12-amino-acid segment, we analyzed a wild-type s1/m1 VacA and a chimeric s2/m1 VacA protein. Purified s1/m1 VacA from H. pylori strain 60190 induced vacuolation in HeLa and Vero cells, whereas the chimeric s2/m1 toxin (in which the s1 sequence of VacA from strain 60190 was replaced with the s2 sequence from strain Tx30a) lacked detectable cytotoxic activity. Type s1/m1 VacA from strain 60190 formed membrane channels in a planar lipid bilayer assay at a significantly higher rate than did s2/m1 VacA. However, membrane channels formed by type s1 VacA and type s2 VacA proteins exhibited similar anion selectivities (permeability ratio, P(Cl)/P(Na) = 5). When an equimolar mixture of the chimeric s2/m1 toxin and the wild-type s1/m1 toxin was added to HeLa cells, the chimeric toxin completely inhibited the activity of the s1/m1 toxin. Thus, the s2/m1 toxin exhibited a dominant-negative phenotype similar to that of a previously described mutant toxin, VacA-(Delta6-27). Immunoprecipitation experiments indicated that both s2/m1 VacA and VacA-(Delta6-27) could physically interact with a c-myc epitope-tagged s1/m1 VacA, which suggests that the dominant-negative phenotype results from the formation of heterooligomeric VacA complexes with defective functional activity. Despite detectable differences in the channel-forming activities and cytotoxic properties of type s1 and type s2 VacA proteins, the conservation of type s2 sequences in many H. pylori isolates suggests that type s2 VacA proteins retain an important biological activity.

Animals↗