Involvement of the Helicobacter pylori plasticity region and cag pathogenicity island genes in the development of gastroduodenal diseases.

Infection by Helicobacter pylori is associated with the development of several gastroduodenal diseases, including gastritis, peptic ulcer disease (gastric ulcers and duodenal ulcers), and gastric adenocarcinoma. Although a number of putative virulence factors have been reported for H. pylori, there are conflicting results regarding their association with specific H. pylori-related diseases. In this work, we investigated the presence of virB11 and cagT, located in the left half of the cag pathogenicity island (cagPAI), and the jhp917-jhp918 sequences, components of the dupA gene located in the plasticity zone of H. pylori, in Brazilian isolates of H. pylori. We also examined the association between these genes and H. pylori-related gastritis, peptic ulcer disease, and gastric and duodenal ulcers in an attempt to identify a gene marker for clinical outcomes related to infection by H. pylori. The cagT gene was associated with peptic ulcer disease and gastric ulcers, whereas the virB11 gene was detected in nearly all of the samples. The dupA gene was not associated with duodenal ulcers or any gastroduodenal disease here analyzed. These results suggest that cagT could be a useful prognostic marker for the development of peptic ulcer disease in the state of São Paulo, Brazil. They also indicate that cagT is associated with greater virulence and peptic ulceration, and that this gene is an essential component of the type IV secretion system of H. pylori.

Protective protein gene mutations in galactosialidosis.

Four different protective protein cDNA mutations, 146A-->G (Q49R), 193T-->C (W65R), 268-269TC-->CT (S90L), and 1184A-->G (Y395C), were identified in six Japanese galactosialidosis patients with various phenotypic manifestations, and another mutation, 746T-->A (Y249N), in a patient of French-German origin with an atypical clinical course. Y395C was a common mutation in four Japanese patients in infancy and childhood; two juvenile patients were compound heterozygotes of Y395C and another common mutation, SpDEx7, and the other two infants were compound heterozygotes of Y395C and mutant alleles other than SpDEx7. We confirmed these mutations in genomic DNA by direct-sequence analysis or restriction-site analysis. The mutant cDNA clones, transiently expressed in a transformed galactosialidosis cell line, did not restore the secondarily deficient beta-galactosidase or alpha-neuraminidase activity except for the Y249N mutation that expressed some carboxypeptidase activity and restored the two lysosomal enzyme activities. Pulse-chase analysis detected a small amount of the mature form, as well as the precursor, in the cells transfected with the Y249N cDNA. Only precursor proteins were detected, mature proteins not appearing for the other mutant cDNAs.

The static head method for determining the charge stoichiometry of coupled transport systems. Applications to the sodium-coupled D-glucose transporters of the renal proximal tubule.

The static head method for determining the charge stoichiometry (the number of moles of charge translocated per mole of substrate) of a coupled transport system is presented. The method involves establishing experimental conditions under which a membrane potential exactly balances the thermodynamic driving force of a known substrate gradient. The charge stoichiometry can then be calculated from thermodynamic principles. In contrast to the usual steady-state method for determining charge stoichiometry in cell suspensions and vesicle preparations, the static head method is applicable to systems which are not capable of maintaining a constant membrane potential over time. The charge stoichiometries of two renal sodium coupled D-glucose transporters previously identified in brush-border membrane vesicle preparations from the outer cortex (early proximal tubule) and outer medulla (late proximal tubule) are determined. The charge stoichiometries of these transporters are in good agreement with their sodium/glucose coupling ratios arguing against the possibility that glucose transport is coupled to ions other than sodium in these membranes.

Gentamicin inhibits Na+-dependent D-glucose transport in rabbit kidney brush-border membrane vesicles.

We studied the effect of gentamicin on Na+-dependent D-glucose transport into brush-border membrane vesicles isolated from rabbit kidney outer cortex (early proximal tubule) and outer medulla (late proximal tubule) in vitro. We found the same osmotically active space and nonspecific binding between control and gentamicin-treated brush-border membrane vesicles. There was no difference in the passive permeability properties between control and gentamicin-treated brush-border membrane vesicles. Kinetic analyses of D-glucose transport into 1 mM gentamicin-treated brush-border membrane vesicles demonstrated that gentamicin decreased Vmax in the outer cortical preparation, while it did not affect Vmax in the outer medullary preparation. With regard to Km, there was no effect of gentamicin in any vesicle preparation. When brush-border membrane vesicles were incubated with higher concentrations of gentamicin, Na+-dependent D-glucose transport was inhibited dose-dependently in both outer cortical and outer medullary preparations. Dixon plots yield inhibition constant Ki = 4 mM in the outer cortical preparation and Ki = 7 mM in the outer medullary preparation. These results indicate that the Na+-dependent D-glucose transport system in early proximal tubule is more vulnerable to gentamicin toxicity than that in late proximal tubule.

The mechanism of decreased Na+-dependent D-glucose transport in brush-border membrane vesicles from rabbit kidneys with experimental Fanconi syndrome.

In our previous paper (Yanase, M. et al. (1983) Biochim. Biophys. Acta 733, 95-101) we reported that the Na+-dependent D-glucose uptake into brush-border membrane vesicles is decreased in rabbits with experimental Fanconi syndrome (induced by anhydro-4-epitetracycline). In the present paper we investigate the mechanism underlying this decrease. D-Glucose is taken up into the osmotically active space in anhydro-4-epitetracycline-treated brush-border membrane vesicles and exhibits the same distribution volume and the same degree of nonspecific binding and trapping as in control brush-border membrane vesicles. The passive permeability properties of control and anhydro-4-epitetracycline-treated brush-border membrane vesicles are shown to be the same as measured by the time-dependence of L-glucose efflux from brush-border membrane vesicles. D-Glucose flux was measured by the equilibrium exchange procedure at constant external and internal Na+ concentrations and zero potential. Kinetic analyses of Na+-dependent D-glucose flux indicate that Vmax in anhydro-4-epitetracycline-treated brush-border membrane vesicles (79.3 +/- 7.6 nmol/min per mg protein) is significantly smaller than in control brush-border membrane vesicles (141.3 +/- 9.9 nmol/min per mg protein), while the Km values in the two cases are not different from each other (22.3 +/- 0.9 and 27.4 +/- 1.8 mM, respectively). These results suggest that Na+-dependent D-glucose carriers per se are affected by anhydro-4-epitetracycline, and that this disorder is an important underlying mechanism in the decreased Na+-dependent D-glucose uptake into anhydro-4-epitetracycline-treated brush-border membrane vesicles.

Immunological studies on the respiratory burst oxidase of pig blood neutrophils.

Recently, a flavin enzyme (pI 5.0), that is probably responsible for superoxide (O2-)-generated oxidase activity, was separated by isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) from neutrophil membranes in our laboratory [(1987) J. Biol. Chem. 262, 12316-12322]. In the present work, we performed immunological studies on this enzyme derived from pig blood neutrophils. The enzyme extract obtained on IEF-PAGE was injected into guinea pigs to raise antibodies. IgG antibody against the pI 5.0 protein inhibited maximally 54% of the O2- -generating activity of the membrane-solubilized oxidase, whereas the normal serum IgG was not inhibitory at all. Our results further confirmed that the enzyme (PI 5.0) is one of the component(s) of the O2- -generating system. The enzyme gave rise to a band corresponding to a major protein of 72 +/- 4 kDa on both non-denaturing and SDS-PAGE. Immunoblotting after SDS-PAGE demonstrated labelling of peptides of 70-72, 28-32 and 16-18 kDa.

Decrease in the fluidity of brush-border membrane vesicles induced by gentamicin. A spin-labeling study.

In our previous paper (Horio et al., Biochim Biophys Acta 858: 153-160, 1986), we reported that the addition of gentamicin in vitro to rabbit renal brush-border membrane vesicles decreases the apparent Vmax of Na+-dependent D-glucose transport without affecting the apparent Km. In the present study, we investigated the effects of gentamicin on the physical state of spin-labeled rabbit renal brush-border membranes, using electron spin resonance spectrometry. Brush-border membrane vesicles were prepared from outer cortex (mainly contains early proximal tubule) and outer medulla (containing primarily late proximal tubule), and the gentamicin toxicities in both preparations were compared. Significant decreases were observed in the membrane fluidity of 5 mM gentamicin-treated brush-border membranes. The fluidity of outer cortical brush-border membranes was affected at both 25 degrees and 35 degrees, whereas that of outer medullary membranes was affected only at 35 degrees. Two different stearic acid spin labels revealed that gentamicin affected the fluidity only in the superficial region of the membranes. We also demonstrated that the gentamicin-induced decreases in Na+-dependent D-glucose transport and in the membrane fluidity were recovered by washing gentamicin-treated brush-border membranes. We suggest that gentamicin binds to the superficial region of brush-border membranes and inhibits Na+-dependent D-glucose transport across brush-border membranes through the decrease in the membrane fluidity.

Formation of mutagens by heating foods and model systems.

We have demonstrated that the pyrolysis products of amino acids and proteins in model systems are mutagenic. The mutagenic principles in the pyrolyzates of amino acids have been isolated and identified by Sugimura et al. We have isolated and identified amino-alpha-carbolines from pyrolyzed soybean globulin as mutagens. The yield of mutagens by the heating of food constituents is changed by the heating method. Effects of heating methods on the yield of amino-alpha-carbolines were studied in a series of experiments, and the results are shown in this paper. Additionally, it has been shown that by the heating of creatine-sugar mixtures imidazoquinoline or quinoxaline mutagens are formed.

Autocrine amplification of type I interferon gene expression mediated by interferon stimulated gene factor 3 (ISGF3).

Interferon regulatory factor (IRF)-1 and IRF-2 have been implicated for the virus-induced expression of the interferon-alpha and beta (type I IFN) genes. However, recent gene disruption studies in mice suggested the presence of other factor(s) interacting with overlapping promoter elements. In the present paper, we describe the characterization of a DNA binding factor which is strongly induced after virus infection and recognizes these promoter elements. After extensive purification, the factor was revealed to be identical to IFN-stimulated gene factor 3 (ISGF3), a transcription factor complex activated by IFN treatment. ISGF3 binds to the promoter element of IFN-beta, positive regulatory domain I (PRDI), with significantly higher affinity than IRF-1, 2, and mutational analysis of PRDI showed that the gene expression and binding of ISGF3, but not of IRF-1, 2, are highly correlated. Furthermore, our functional analysis involving a dominant negative inhibitor for ISGF3 activation and an anti-IFN neutralizing antibody clearly demonstrated the presence of a positive feedback path way for type I IFN genes mediated by ISGF3.

Effect of chronic renal failure on the level of albumin messenger RNA.

The effects of chronic renal failure on the level of albumin mRNA and the transcription rate of the albumin gene were studied in seven of eight nephrectomized rats. A paired feeding procedure was employed to eliminate a nutritional difference between sham-operated control and uremic rats. Total RNA was isolated from the livers of control and uremic rats fasted for 24 or 48 hours. The mRNA level was measured by RNA-cDNA dot blot hybridization, and the transcription rate was measured by the "run-on" transcription assay in isolated nuclei. The albumin mRNA levels in uremic rat livers were reduced to 75% at the 24-hour fasted state, and to 45% at 48-hour fasted state compared with those in the respective control groups. There was no difference in the level of beta-actin mRNA between these groups. However, there was no difference in the transcription rate of the albumin gene between control and uremic rats. Northern analysis showed that albumin mRNA isolated from uremic rat liver was identical in size with that from the control. These results suggested that a posttranscriptional process, involving the destabilization of cytoplasmic mRNA, is responsible for the uremia-induced repression of albumin synthesis.

Effects of chronic renal failure on the regulation of pyruvate kinase.

The effects of chronic renal failure on the enzyme activity of pyruvate kinase and the mRNA level of this enzyme were studied in 7 out of 8 nephrectomized rats. The mRNA level was measured by RNA-DNA dot blot hybridization, using cloned pyruvate kinase cDNA as hybridized probe. Neither the activity of M1-type pyruvate kinase nor the level of this enzyme in rat gastrocnemius muscle was affected by chronic renal failure, whereas L-type pyruvate kinase enzyme activity in uremic rat liver was lower than that in control at both fasted and refed states. The levels of L-type pyruvate kinase mRNA were not different between two groups at the fasted state. Induction of L-type pyruvate kinase mRNA after high carbohydrate diet refeeding was suppressed proportionally to the severity of chronic renal failure, which was expressed by the serum creatinine concentrations (r = -.876, P less than .005). These results indicate that the suppression of L-type pyruvate kinase activity in uremia was partly reflected by the decreased accumulation of this enzyme mRNA. There was a significantly negative correlation between L-type pyruvate kinase mRNA levels and plasma glucagon/insulin ratios (r = -.719, P less than .05). Hyperglucagonemia in uremia might play a major role in this suppression.

GAPDH knockdown rescues mesencephalic dopaminergic neurons from MPP+ -induced apoptosis.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) has a number of diverse functions apart from glycolytic function. We explored the possible involvement of GAPDH in 1-methyl-4-phenylpyridinium (MPP+)-induced death of mesencephalic dopaminergic neurons (MDNs) in culture. MPP+ (10 and 20 microM, 24 h) exposure selectively decreased the survival of tyrosine hydroxylase positive (TH+) MDNs, which manifested apoptotic features including shrinkage of the cell body, chromatin condensation and nuclear fragmentation. Two types of GAPDH antisense oligonucleotides almost completely rescued MDNs from MPP+ toxicity. GAPDH was strongly expressed in apoptotic TH+ neurons, and MPP+ exposure significantly increased the percentage of TH+ neurons in which GAPDH is over-expressed. Confocal microscopic analysis demonstrated the nuclear accumulation of GAPDH in neurons undergoing MPP+-induced apoptosis. These results suggest that MPP+ causes apoptosis of MDNs, concomitant with the over-expression and nuclear accumulation of GAPDH.

Aclacinomycin, an anti-leukemic anthracycline, impairs human neutrophil functions.

The effects of aclacinomycin, an anti-leukemic anthracycline, on human neutrophil functions were investigated. The release of superoxide (O2-) in neutrophils stimulated by opsonized zymosan, myristate, or phorbol myristate acetate was inhibited by aclacinomycin in a dose- and time-dependent manner. The phagocytosis of yeast particles and oil droplets, and membrane potential changes stimulated by phorbol myristate acetate were also inhibited by aclacinomycin. On the other hand, the O2(-)-producing enzyme (NADPH oxidase) in the particulate fraction prepared from myristate-stimulated neutrophils was not affected by aclacinomycin. When high concentrations of aclacinomycin (10-100 micrograms/ml) were employed, significant inhibition of O2- release, phagocytosis, and membrane potential changes was observed within 5 min. Phagocytic activity was also inhibited when neutrophils were preincubated for 13 h at 37 degrees C with a low concentration (40 ng/ml) of aclacinomycin, which could be obtained by intravenous administration of 20 mg aclacinomycin. Myristate-induced O2- release was not impaired by cytosine arabinoside (2-800 micrograms/ml), vincristine (0.1-100 micrograms/ml), adriamycin (25-100 micrograms/ml), or daunomycin (5-75 micrograms/ml) when the cells were preincubated with these drugs for 5 min at 37 degrees C. These findings suggest that aclacinomycin inhibits the respiratory burst by impairing the activating system of NADPH oxidase and phagocytic activity.

Increased plasma levels of IL-1beta, IL-6, IL-8, IL-10 and TNF-alpha in patients moderately or severely envenomed by Tityus serrulatus scorpion sting.

Scorpion envenomation is a common medical problem in many countries and an important cause of morbidity and mortality, especially among children. The plasma levels of pro-inflammatory (IL-1beta, IL-6, IL-8 and TNF-alpha) and anti-inflammatory (IL-10) cytokines were measured in individuals stung by Tityus serrulatus (Ts) scorpions. According to clinical manifestations patients were classified, as defined by the Brazilian Ministry of Health, as having mild (n=15, mean age=42.2 years), moderate (n=8, mean age=26 years) or severe (n=4, mean age=14 years) envenomation. Blood samples were taken immediately (T1) and 6h (T2) after admission to the hospital. Eighteen age-matched healthy volunteers were used as control. TNF-alpha, IL-1beta, IL-6 and IL-8 levels were significantly increased in moderate and severe cases and the levels of these cytokines were positively correlated with the severity of envenomation, as evaluated by clinical profile and plasma venom concentration. IL-10 levels were increased in severe and moderate cases and reduced in mild cases. The results reported in the present study suggest that the physiopathological manifestation of Ts envenomation may be mediated, at least in part, by cytokines, and that the early treatment after scorpion sting with drugs that inhibit cytokine production, such as glucocorticoids, may have a potential beneficial effect, ameliorating the severity of the clinical manifestations observed, particularly in severe and moderate cases.

Cataract development induced by repeated oral dosing with FK506 (tacrolimus) in adult rats.

FK506 (tacrolimus), a potent immunosuppressant, is used for inhibiting allograft rejection in the organ transplantation field. In a preclinical toxicity study in rats, FK506 induced various toxicities, including renal and pancreatic injuries. One of these toxic findings was cataract, and we have found that cataract appeared in rats dosed orally with FK506 for 13 weeks and more. Therefore, to better elucidate the onset mechanism of FK506-induced cataract, we measured biochemical parameters, such as sorbitol, Na,K-ATPase and glutathione in the lens of rats. Rats were dosed with FK506 in oral daily doses of 0.2, 1 or 5 mg/kg for 13 weeks, the lowest dose of which approximated the expected clinical dosage. Cataract developed in the 5-mg/kg/day group, with an incidence of 25%, whereas no cataract formation was observed in the 0.2- or 1-mg/kg/day groups. Five mg/kg/day led an increase of sorbitol and a decrease of reduced type glutathione, but did not affect Na,K-ATPase activity of the lens. FK506 is known to have diabetogenicity mediated through pancreatic injury, which appears as vacuolation of islet cell in rats. Five mg/kg/day of FK506 induced an elevation of blood glucose associated with glucose intolerance, and decrease of both basal insulin level and insulin content in the pancreas, and the changes were in parallel with the cataract development in the present study. On the other hand, diabetic parameters did not change in the 0.2- or 1-mg/kg/day groups. These observation suggest that diabetes developed in the rats dosed with 5 mg/kg/day of FK506. Coadministration of a novel aldose reductase inhibitor, Zenarestat, at an oral dose of 50 mg/kg/day resulted in a reduction of incidence of the FK506-induced cataract and a decrease of sorbitol levels in the lens when compared to that in the lens of rats dosed with 5 mg/kg/day of FK506. These results suggest that FK506-induced cataract in rats is due to an accumulation of sorbitol in the lens, secondary to the diabetogenic effect of FK506. FK506 treatment at the doses of 0.2 and 1 mg/kg/day neither affected parameters indicative of diabetes nor induced cataract in rats, suggesting that the cataract would not develop with FK506 if diabetic parameters were kept under control.

Mutagenicity and co-mutagenicity of catechol on Salmonella.

Catechol was not mutagenic for Salmonella typhimurium TA98, TA100 or TA1537 in the presence or absence of S9 mix. At the lower level of S9 in the Ames method, the mutagenic activity of benzo[a]pyrene decreased with the increased addition of catechol. When catechol was added to the pre-incubation mixture at a higher concentration than in the conventional Ames method, the mutagenic activity of benzo[a]pyrene increased with the increased addition of catechol. Catechol is believed to be a co-mutagen for benzo[a]pyrene in the presence of a sufficient amount of S9 in the incubation mixture.

Relation between chemical constituents of tobacco and mutagenic activity of cigarette smoke condensate.

Mutagenic activities of cigarette smoke condensate were assayed in the presence of S-9 Mix using Salmonella typhimurium TA 98. The results were examined in relation to chemical data of tobacco leaves. Among the nitrogenous constituents examined, the contents of total nitrogen and protein nitrogen and the soluble nitrogenous fraction were positively and significantly related to an increase in mutagenic activity of the smoke condensate, whereas nicotine and nitrate were not important in contributing to mutagenic potency of such condensates. The age of tobacco leaves influenced the mutagenic potency of the condensate, which was lowest in leaves from the lower stalk position and increased with ascending leaf position on the stalk. Smoke condensate from tobacco with higher sugar content resulted in lower mutagenic activity. The present results, together with the previous study on the mutagenicity of the amino acid pyrolyzates, suggest that potent mutagens in cigarette smoke condensate are nitrogen-containing compounds, which may be formed from proteins and amino acids during the burning of a cigarette.

Evaluation of tubulointerstitial injury by Doppler ultrasonography in glomerular diseases.

AIMS: While Doppler ultrasonography is used commonly in various renal diseases, its clinical value in diagnosis of renal parenchymal diseases, especially glomerular diseases, remains controversial. We investigated whether Doppler ultrasonography in glomerular diseases could discriminate tubulointerstitial lesions, which correlated closely with long-term prognosis for renal function. METHODS: Sixty patients with primary or secondary glomerular diseases were examined by Doppler ultrasonography immediately before renal biopsy. The resistive index was calculated, as was the atrophic index (a newly proposed parameter defined as renal sinus length/renal length). These were compared with histologic changes in biopsy specimens. RESULTS: Receiver operator characteristic analysis showed a resistive index of 0.65 to be the optimal for discriminating tubulointerstitial changes with specificity of 100% and sensitivity of 57.1%. Tubulointerstitial injury scores were significantly higher in patients with resistive indices exceeding 0.65 than in patients with a lower value. An atrophic index of 0.70 was also shown to be optimal with specificity 100% and sensitivity 61.9%. In combination, the 2 indices showed improved sensitivity; when the patients were divided into groups where both resistive and atrophic indices were normal (respectively < or = 0.65 and < or = 0.70) or where either or both were high, sensitivity rose to 85.7%, while specificity remained 94.4%. CONCLUSIONS: In combination, the resistive and atrophic indices discriminated tubulointerstitial injury in glomerular diseases with high specificity and sensitivity.

Prognosis of chronic glomerulonephritis in adult patients estimated on the basis of the Markov process.

The prognosis of chronic glomerulonephritis based on renal function was assessed using a statistical technique of the Markov process, where the absorbing state was assumed to be an uremic state, 194 adult patients with different types of disease were subjected to study. The 15 min value obtained in the intravenous PSP excretion test was divided into five states; SI (greater than 34%, normal), SII(25-34), SIII(15-24), SIV(5-14) and SV (greater than 5, uremic). The rates of SV with time were calculated with respect to several clinical characteristics. The prognosis of the patients with hypertension, distinct proteinuria and hematuria, or cellular cylindruria appeared to be relatively poor. The estimated number of years from each state to SV were also calculated. The results were similar to those already reported and gave us more exact information about the prognosis.

Comparative effects of gentamicin and netilmicin on Na(+)-dependent d-glucose transport in rabbit renal brush-border membrane vesicles.

The effects of gentamicin and netilmicin administration on Na(+)-dependent d-glucose transport in rabbit renal brush-border membrane vesicles (BBMV) were investigated. BBMV were isolated from rabbit kidney outer cortex (early proximal tubule) and outer medulla (late proximal tubule) 60 min after iv drug administration. Pharmacokinetics and renal accumulation of gentamicin and netilmicin were also studied. We found decreased affinity in the Na(+)-dependent d-glucose transport system after gentamicin administration only in the outer cortical preparations and not in the outer medullary ones. Gentamicin administration did not affect the membrane permeability or d-glucose distribution space of BBMV. Netilmicin had no effect on Na(+)-dependent d-glucose transport. The V(max) values of Na(+)-dependent d-glucose transport were not affected by either gentamicin or netilmicin administration in both outer cortical and outer medullary preparations. Thus, gentamicin was shown to have stronger effects on Na(+)-dependent d-glucose transport than did netilmicin. This result is consistent with the rank of nephrotoxicity for gentamicin and netilmicin. Furthermore, the Na(+)-dependent d-glucose transport system in the early proximal tubule seems to be more vulnerable to gentamicin toxicity than that in the late proximal tubule. Pharmacokinetic parameters and renal accumulation of gentamicin and netilmicin were not significantly different from each other. Thus, the difference in transport inhibition by gentamicin and netilmicin cannot be accounted for by using pharmacokinetic factors of these two drugs.