Catabolism of Ap4A and Ap5A by rat brain synaptosomes

Adenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) and adenosine 5',5'''-P1,P5-pentaphosphate (Ap5A) are stored in and released from rat brain synaptic terminals. In the present study we investigated the hydrolysis of dinucleotides (Ap4A and Ap5A) in synaptosomes from the cerebral cortex of adult rats. Ap4A and Ap5A, but not Ap3A, were hydrolyzed at pH 7.5 in the presence of 20 mM Tris/HCl, 2.0 mM MgCl2, 10 mM glucose and 225 mM sucrose at 37oC. The disappearance of the substrates measured by FPLC on a mono-Q HR column was both time and protein dependent. Since synaptosome integrity was at least 90% at the end of the assay, hydrolysis probably occurred by the action of an ecto-enzyme. Extracellular actions of adenine dinucleotides at central nervous system terminate due to the existence of ecto-nucleotidases which specifically cleave these dinucleotides. These enzymes in association with an ATP diphosphohydrolase and a 5'-nucleotidase are able to promote the complete hydrolysis of dinucleotides to adenosine in the synaptic cleft.

Dialysis, time and death: comparisons of two consecutive decades among patients treated at the same Brazilian dialysis center

The survival of hemodialysis patients is likely to be influenced not only by well-known risk factors like age and comorbidity, but also by changes in dialysis technology and practices accumulated along time. We compared the survival curves, dialysis routines and some risk factors of two groups of patients admitted to a Brazilian maintenance hemodialysis program during two consecutive decades: March 1977 to December 1986 (group 1, N = 162) and January 1987 to June 1997 (group 2, N = 237). The median treatment time was 22 months (range 1-198). Survival curves were constructed using the Kaplan-Meier method and compared using the log-rank method. The Cox proportional hazard regression model was used to investigate the more important variables associated with outcome. The most important changes in dialysis routine and in patient care during the total period of observation were the progressive increase in the dose of dialysis delivered, the prohibition of potassium-free dialysate, the use of bicarbonate as a buffer and the upgrading of the dialysis equipment. There were no significant differences between the survival curves of the two groups. Survival rates at 1, 5 and 10 years were 84, 53 and 29%, respectively, for group 1 and 77, 42 and 21% for group 2. Patients in group 1 were younger (45.5 ± 15.2 vs 55.2 ± 15.9 years, P<0.001) and had a lower prevalence of diabetes (11.1 vs 27.4%, P<0.001) and of cardiovascular disease (9.3 vs 20.7%, P<0.001). According to the Cox multivariate model, only age (hazard ratio (HR) 1.04, confidence interval (CI) 1.03-1.05, P<0.001) and diabetes (HR 2.55, CI 1.82-3.58, P<0.001) were independent predictors of mortality for the whole group. Patients of group 2 had a lower prevalence of sudden death (19.1 vs 9.7%, P<0.001). After adjusting for age, diabetes and other mortality risk factors, the risk of death was 17% lower in group 2, although this difference was not statistically significant. We conclude that the negative effects of advanced age and of higher frequency of comorbidity on the survival of group 2 patients were probably offset by improvements in patient care and in the quality and dose of dialysis delivered, so that the survival curves did not undergo significant changes along time.

Renal effects of uroguanylin and guanylin in vivo

Uroguanylin and guanylin are newly discovered endogenous heat-stable peptides that bind to and activate a membrane bound guanylyl cyclase signaling receptor (termed guanylyl cyclase C; GC-C). These peptides are not only found in blood but are secreted into the lumen of the intestine and effect a net secretion of electrolytes (Na+, K+, Cl-, HCO3-) and fluid into the intestine via a cyclic guanosine-3',5'-monophosphate (cGMP) mechanism. GC-C is also the receptor for Escherichia coli heat-stable enterotoxin (STa) and activation by STa results in a diarrheal illness. Employing mouse renal in vivo models, we have demonstrated that uroguanylin, guanylin, and STa elicit natriuretic, kaliuretic, and diuretic effects. These biological responses are time- and dose-dependent. Maximum natriuretic and kaliuretic effects are observed within 30-40 min following infusion with pharmacological doses of the peptides in a sealed-urethra mouse model. Our mouse renal clearance model confirms these results and shows significant natriuresis following a constant infusion of uroguanylin for 30 min, while the glomerular filtration rate, plasma creatinine, urine osmolality, heart rate, and blood pressure remain constant. These data suggest the peptides act through tubular transport mechanisms. Consistent with a tubular mechanism, messenger RNA-differential display PCR of kidney RNA extracted from vehicle- and uroguanylin-treated mice show the message for the Na+/K+ ATPase g-subunit is down-regulated. Interestingly, GC-C knockout mice (Gucy2c -/-) also exhibit significant uroguanylin-induced natriuresis and kaliuresis in vivo, suggesting the presence of an alternate receptor signaling mechanism in the kidney. Thus, uroguanylin and guanylin seem to serve as intestinal and renal natriuretic peptide-hormones influencing salt and water transport in the kidney through GC-C dependent and independent pathways. Furthermore, our recent clinical probe study has revealed a 70-fold increase in levels of urinary uroguanylin in patients with congestive heart failure. In conclusion, our studies support the concept that uroguanylin and guanylin are endogenous effector peptides involved in regulating body salt and water homeostasis.

Molluscicidal activity of Punica granatum bark and Canna indica root

The molluscicidal activity of Punica granatum Linn. (Punicaceae) and Canna indica Linn. (Cannaceae) against the snail Lymnaea acuminata was studied. The molluscicidal activity of P. granatum bark and C. indica root was found to be both time and dose dependent. The toxicity of P. granatum bark was more pronounced than that of C. indica. The 24 h LC50 of the column-purified root of C. indica was 6.54 mg/l whereas that of the column-purified bark of P. granatum was 4.39 mg/l. The ethanol extract of P. granatum (24 h LC50: 22.42 mg/l) was more effective than the ethanol extract of C. indica (24 h LC50: 55.65 mg/l) in killing the test animals. P. granatum and C. indica may be used as potent molluscicides since the concentrations used to kill the snails were not toxic for the fish Colisa fasciatus, which shares the same habitat with the snail L. acuminata.

Nitric oxide, cholesterol oxides and endothelium-dependent vasodilation in plasma of patients with essential hypertension

The objective of the present study was to identify disturbances of nitric oxide radical (·NO) metabolism and the formation of cholesterol oxidation products in human essential hypertension. The concentrations of·NO derivatives (nitrite, nitrate, S-nitrosothiols and nitrotyrosine), water and lipid-soluble antioxidants and cholesterol oxides were measured in plasma of 11 patients with mild essential hypertension (H: 57.8 ± 9.7 years; blood pressure, 148.3 ± 24.8/90.8 ± 10.2 mmHg) and in 11 healthy subjects (N: 48.4 ± 7.0 years; blood pressure, 119.4 ± 9.4/75.0 ± 8.0 mmHg).Nitrite, nitrate and S-nitrosothiols were measured by chemiluminescence and nitrotyrosine was determined by ELISA. Antioxidants were determined by reverse-phase HPLC and cholesterol oxides by gas chromatography. Hypertensive patients had reduced endothelium-dependent vasodilation in response to reactive hyperemia (H: 9.3 and N: 15.1% increase of diameter 90 s after hyperemia), and lower levels of ascorbate (H: 29.2 ± 26.0, N: 54.2 ± 24.9 µM), urate (H: 108.5 ± 18.9, N: 156.4 ± 26.3 µM), ß-carotene (H: 1.1 ± 0.8, N: 2.5 ± 1.2 nmol/mg cholesterol), and lycopene (H: 0.4 ± 0.2, N: 0.7 ± 0.2 nmol/mg cholesterol), in plasma, compared to normotensive subjects. The content of 7-ketocholesterol, 5alpha-cholestane-3ß,5,6ß-triol and 5,6alpha-epoxy-5alpha-cholestan-3alpha-ol in LDL, and the concentration of endothelin-1 (H: 0.9 ± 0.2, N: 0.7 ± 0.1 ng/ml) in plasma were increased in hypertensive patients. No differences were found for ·NO derivatives between groups. These data suggest that an increase in cholesterol oxidation is associated with endothelium dysfunction in essential hypertension and oxidative stress, although ·NO metabolite levels in plasma are not modified in the presence of elevated cholesterol oxides.

Tacrolimus (FK506) reduces ischemia-induced hippocampal damage in rats: a 7- and 30-day study

The neuroprotective effect of the immunosuppressant agent FK506 was evaluated in rats after brain ischemia induced for 15 min in the 4-vessel occlusion model. In the first experimental series, single doses of 1.0, 3.0 or 6.0 mg FK506/kg were given intravenously (iv) immediately after ischemia. In the second series, FK506 (1.0 mg/kg) was given iv at the beginning of reperfusion, followed by doses applied intraperitoneally (ip) 6, 24, 48, and 72 h post-ischemia. The same protocol was used in the third series except that all 5 doses were given iv. Damage to the hippocampal field CA1 was assessed 7 or 30 days post-ischemia on three different stereotaxic planes along the septotemporal axis of the hippocampus. Ischemia caused marked neurodegeneration on all planes (P<0.001). FK506 failed to provide neuroprotection to CA1 both when applied iv as a single dose of 1.0, 3.0 or 6.0 mg/kg (experiment 1), and after five iv injections of 1.0 mg/kg (experiment 3). In contrast, the repeated administration of FK506 combining iv plus ip administration reduced CA1 cell death on all stereotaxic planes both 7 and 30 days post-ischemia (experiment 2; P<=0.01). Compared to vehicle alone, FK506 reduced rectal temperature in a dose-dependent manner (P<=0.05); however, this effect did not alter normothermia (37ºC). FK506 reduced ischemic brain damage, an effect sustained over time and apparently dependent on repeated doses and on delivery route. The present data extend previous findings on the rat 4-vessel occlusion model, further supporting the possible use of FK506 in the treatment of ischemic brain damage.

Curcumin induces human HT-29 colon adenocarcinoma cell apoptosis by activating p53 and regulating apoptosis-related protein expression

Curcumin, a major yellow pigment and active component of turmeric, has multiple anti-cancer properties. However, its molecular targets and mechanisms of action on human colon adenocarcinoma cells are unknown. In the present study, we examined the effects of curcumin on the proliferation of human colon adenocarcinoma HT-29 cells by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method and confirmed the curcumin-induced apoptosis by morphology and DNA ladder formation. At the same time, p53, phospho-p53 (Ser15), and other apoptosis-related proteins such as Bax, Bcl-2, Bcl-xL, pro-caspase-3, and pro-caspase-9 were determined by Western blot analysis. The colon adenocarcinoma cells were treated with curcumin (0-75 µM) for 0-24 h. We observed that p53 was highly expressed in HT-29 cells and curcumin could up-regulate the serine phosphorylation of p53 in a time- and concentration-dependent manner. An increase in expression of the pro-apoptotic factor Bax and a decrease in expression of the anti-apoptotic factor Bcl-2 were also observed in a time-dependent manner after exposure of 50 µM curcumin, while the expression of the anti-apoptotic factor Bcl-xL was unchanged. Curcumin could also down-regulate the expression of pro-caspase-3 and pro-caspase-9 in a time-dependent manner. These data suggest a possible underlying molecular mechanism whereby curcumin could induce the apoptosis signaling pathway in human HT-29 colon adenocarcinoma cells by p53 activation and by the regulation of apoptosis-related proteins. This property of curcumin suggests that it could have a possible therapeutic potential in colon adenocarcinoma patients.

Effects of gamma-radiation on cell growth, cycle arrest, death, and superoxide dismutase expression by DU 145 human prostate cancer cells

Gamma-irradiation (gamma-IR) is extensively used in the treatment of hormone-resistant prostate carcinoma. The objective of the present study was to investigate the effects of 60Co gamma-IR on the growth, cell cycle arrest and cell death of the human prostate cancer cell line DU 145. The viability of DU 145 cells was measured by the Trypan blue exclusion assay and the 3(4,5-dimethylthiazol-2-yl)-2,5,diphenyltetrazolium bromide test. Bromodeoxyuridine incorporation was used for the determination of cell proliferation. Cell cycle arrest and cell death were analyzed by flow cytometry. Superoxide dismutase (SOD), specifically CuZnSOD and MnSOD protein expression, after 10 Gy gamma-IR, was determined by Western immunoblotting analysis. gamma-IR treatment had a significant (P < 0.001) antiproliferative and cytotoxic effect on DU 145 cells. Both effects were time and dose dependent. Also, the dose of gamma-IR which inhibited DNA synthesis and cell proliferation by 50% was 9.7 Gy. Furthermore, gamma-IR induced cell cycle arrest in the G2/M phase and the percentage of cells in the G2/M phase was increased from 15% (control) to 49% (IR cells), with a nonsignificant induction of apoptosis. Treatment with 10 Gy gamma-IR for 24, 48, and 72 h stimulated CuZnSOD and MnSOD protein expression in a time-dependent manner, approximately by 3- to 3.5-fold. These data suggest that CuZnSOD and MnSOD enzymes may play an important role in the gamma-IR-induced changes in DU 145 cell growth, cell cycle arrest and cell death.

Comparison of the Polar S810i monitor and the ECG for the analysis of heart rate variability in the time and frequency domains

The aim of the present study was to compare heart rate variability (HRV) at rest and during exercise using a temporal series obtained with the Polar S810i monitor and a signal from a LYNX® signal conditioner (BIO EMG 1000 model) with a channel configured for the acquisition of ECG signals. Fifteen healthy subjects aged 20.9 ± 1.4 years were analyzed. The subjects remained at rest for 20 min and performed exercise for another 20 min with the workload selected to achieve 60% of submaximal heart rate. RR series were obtained for each individual with a Polar S810i instrument and with an ECG analyzed with a biological signal conditioner. The HRV indices (rMSSD, pNN50, LFnu, HFnu, and LF/HF) were calculated after signal processing and analysis. The unpaired Student t-test and intraclass correlation coefficient were used for data analysis. No statistically significant differences were observed when comparing the values analyzed by means of the two devices for HRV at rest and during exercise. The intraclass correlation coefficient demonstrated satisfactory correlation between the values obtained by the devices at rest (pNN50 = 0.994; rMSSD = 0.995; LFnu = 0.978; HFnu = 0.978; LF/HF = 0.982) and during exercise (pNN50 = 0.869; rMSSD = 0.929; LFnu = 0.973; HFnu = 0.973; LF/HF = 0.942). The calculation of HRV values by means of temporal series obtained from the Polar S810i instrument appears to be as reliable as those obtained by processing the ECG signal captured with a signal conditioner.

Growth inhibitory effect and Chk1-dependent signaling involved in G2/M arrest on human gastric cancer cells induced by diallyl disulfide

Diallyl disulfide (DADS) inhibits growth and induces cell cycle G2/M arrest in human gastric cancer MGC803 cells. In this study, 15 mg/L DADS exerted similar effects on growth and cell cycle arrest in human gastric cancer BGC823 cells. Due to the importance of cell cycle redistribution in DADS-mediated anti-carcinogenic effects, we investigated the role of checkpoint kinases (Chk1 and Chk2) during DADS-induced cell cycle arrest. We hypothesized that DADS could mediate G2/M phase arrest through either Chk1 or Chk2 signal transduction pathways. We demonstrated that DADS induced the accumulation of phosphorylated Chk1, but not of Chk2, and that DADS down-regulated Cdc25C and cyclin B1. The expression of mRNA and total protein for Chkl and Chk2 was unchanged. Chk1 is specifically phosphorylated by ATR (ATM-RAD3-related gene). Western blot analysis showed that phospho-ATR was activated by DADS. Taken together, these data suggest that cell cycle G2/M arrest, which was associated with accumulation of the phosphorylated forms of Chk1, but not of Chk2, was involved in the growth inhibition induced by DADS in the human gastric cancer cell line BGC823. Furthermore, the DADS-induced G2/M checkpoint response is mediated by Chk1 signaling through ATR/Chk1/Cdc25C/cyclin B1, and is independent of Chk2.

Influence of different sanitizers on food contaminant bacteria: effect of exposure temperature, contact time, and product concentration

The efficiency of four Sanitizers - peracetic acid, chlorhexidine, quaternary ammonium, and organic acids - was tested in this work using different bacteria recognized as a problem to meat industry, Salmonella sp., S. aureus, E. coli and L. monocytogenes. The effects of sanitizer concentration (0.2, 0.5, 0.6, 1.0, 1.1 and 1.4%), at different temperatures (10 and 45 °C) and contact time (2, 10, 15, 18 and 25 minutes) were evaluated. Tests in an industrial plant were also carried out considering previously obtained results. In a general way, peracetic acid presented higher efficiencies using low concentration (0.2%) and contact time (2 minutes) at 10 °C. The tests performed in industrial scale showed that peracetic acid presented a good performance in concentration and contact time lower than that suggested by the suppliers. The use of chlorhexidine and quaternary ammonium led to reasonable results at the indicated conditions, and organic acids were ineffective under concentration and contact time higher than those indicated by the suppliers in relation to Staphylococcus aureus. The results, in general, show that the choice for the most adequate sanitizer depends on the microorganism contaminant, the time available for sanitizer application, and also on the process cost.

Influence of distillation time and sample mass on sulfur dioxide analysis in passion fruit juice through Monier-Williams method

AbstractThis study aimed to evaluate the effect of the distillation time and the sample mass on the total SO2 content in integral passion fruit juice (Passiflora sp). For the SO2 analysis, a modified version of the Monier-Williams method was used. In this experiment, the distillation time and the sample mass were reduced to half of the values proposed in the original method. The analyses were performed in triplicate for each distilling time x sample mass binomial, making a total of 12 tests, which were performed on the same day. The significance of the effects of the different distillation times and sample mass were evaluated by applying one-factor analysis of variance (ANOVA). For a 95% confidence limit, it was found that the proposed amendments to the distillation time, sample mass, and the interaction between distilling time x sample mass were not significant (p > 0.05) in determining the SO2 content in passion fruit juice. In view of the results that were obtained it was concluded that for integral passion fruit juice it was possible to reduce the distillation time and the sample mass in determining the SO2 content by the Monier-Williams method without affecting the result.

Efficacy of euphorbia hirta latex as plant derived molluscicides against freshwater snails

The toxic effect of binary and tertiary combinations of Euphorbia hirta Linn latex powder with other plant molluscicidal compounds, were evaluated against the freshwater snails Lymnaea (Radix) acuminata and Indoplanorbis exustus in pond. These combinations showed significant time and dose dependent effect against both the snails. These compounds at higher doses were also lethal to freshwater fish Channa punctatus (Bloch) (Channidae {Ophicephalidae}), which shares the habitat with these snails, but the LC90 (24h) doses of snails have no apparent killing properties in fish populations when treated in mixed population of snails and fish.

Effect of exposure to sublethal concentrations of sodium cyanide on the carbohydrate metabolism of the Indian Major Carp Labeo rohita (Hamilton, 1822)

Experiments were designed to study in-vivo effects of sodium cyanide on biochemical endpoints in the freshwater fish Labeo rohita. Fish were exposed to two sublethal concentrations (0.106 and 0.064mg/L) for a period of 15 days. Levels of glycogen, pyruvate, lactate and the enzymatic activities of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G6PDH), phosphorylase, alkaline phosphatase (ALP), acid phosphatase (AcP) were assessed in different tissues (liver, muscle and gills). Result indicated a steady decrease in glycogen, pyruvate, SDH, ALP and AcP activity with a concomitant increase in the lactate, phosphorylase, LDH and G6PD activity in all selected tissues. The alterations in all the above biochemical parameters were significantly (p<0.05) time and dose dependent. In all the above parameters, liver pointing out the intensity of cyanide intoxication compare to muscle and gills. Study revealed change in the metabolic energy by means of altered metabolic profile of the fish. Further, these observations indicated that even sublethal concentrations of sodium cyanide might not be fully devoid of deleterious influence on metabolism in L. rohita.

Inverse convection problem of simultaneous estimation of two boundary heat fluxes in parallel plate channels

This paper deals with the use of the conjugate gradient method of function estimation for the simultaneous identification of two unknown boundary heat fluxes in parallel plate channels. The fluid flow is assumed to be laminar and hydrodynamically developed. Temperature measurements taken inside the channel are used in the inverse analysis. The accuracy of the present solution approach is examined by using simulated measurements containing random errors, for strict cases involving functional forms with discontinuities and sharp-corners for the unknown functions. Three different types of inverse problems are addressed in the paper, involving the estimation of: (i) Spatially dependent heat fluxes; (ii) Time-dependent heat fluxes; and (iii) Time and spatially dependent heat fluxes.