Changes in endogenous tissue glutathione level in relation to murine ascites tumor growth and the anticancer activity of cisplatin

Changes in glutathione levels were determined in tissues of 11- to 12-week-old Swiss albino mice at different stages of Dalton's lymphoma tumor growth and following cisplatin (8 mg/kg body weight, ip) treatment for 24-96 h, keeping 4-5 animals in each experimental group. Glutathione levels increased in spleen of tumor-bearing compared to normal mice (9.95 ± 0.14 vs 7.86 ± 1.64 µmol/g wet weight, P<=0.05) but decreased in blood (0.64 ± 0.10 vs 0.85 ± 0.09 mg/ml) and testes (9.28 ± 0.15 vs 10.16 ± 0.28 µmol/g wet weight, P<=0.05). Dalton's lymphoma cells showed an increase in glutathione concentration (4.43 ± 0.26 µmol/g wet weight) as compared to splenocytes, their normal counterpart (3.62 ± 0.41 µmol/g wet weight). With the progression of tumor in mice, glutathione levels decreased significantly in testes (~10%) and bone marrow cells (~13%) while they increased in Dalton's lymphoma cells (28-46%) and spleen (15-27%). Glutathione levels in kidney, Dalton's lymphoma cells and bone marrow cells (8.50 ± 1.22, 4.43 ± 0.26 and 3.28 ± 0.17 µmol/g wet weight, respectively) decreased significantly (6.04 ± 0.42, 3.51 ± 0.32 and 2.17 ± 0.14 µmol/g wet weight, P<=0.05) after in vivo cisplatin treatment for 24 h. Along with a decrease in glutathione level, the glutathione-S-transferase (GST) activity also decreased by 60% in tumor cells after cisplatin treatment. The elevated drug uptake by the tumor cells under the conditions of reduced glutathione concentration and GST activity after treatment could be an important contributory factor to cisplatin's anticancer activity leading to tumor regression. Furthermore, lower doses of cisplatin in combination with buthionine sulfoximine (an inhibitor of glutathione synthesis) may be useful in cancer chemotherapy with decreased toxicity in the host.

The modulatory effect of estradiol benzoate on superoxide dismutase activity in the developing rat brain

The sensitivity of copper,zinc (CuZn)- and manganese (Mn)-superoxide dismutase (SOD) to exogenous estradiol benzoate (EB) was investigated in Wistar rats during postnatal brain development. Enzyme activities were measured in samples prepared from brains of rats of both sexes and various ages between 0 and 75 days, treated sc with 0.5 µg EB/100 g body weight in 0.1 ml olive oil/100 g body weight, 48 and 24 h before sacrifice. In females, EB treatment stimulated MnSOD activity on days 0 (66.1%), 8 (72.7%) and 15 (81.7%). In males, the stimulatory effect of EB on MnSOD activity on day 0 (113.6%) disappeared on day 8 and on days 15 and 45 it became inhibitory (40.3 and 30.5%, respectively). EB had no effect on the other age groups. The stimulatory effect of EB on CuZnSOD activity in newborn females (51.8%) changed to an inhibitory effect on day 8 (38.4%) and disappeared by day 45 when inhibition was detected again (48.7%). In males, the inhibitory effect on this enzyme was observed on days 0 (45.0%) and 15 (28.9%), and then disappeared until day 60 when a stimulatory effect was observed (38.4%). EB treatment had no effect on the other age groups. The sensitivity of MnSOD to estradiol differed significantly between sexes during the neonatal and prepubertal period, whereas it followed a similar pattern thereafter. The sensitivity of CuZnSOD to estradiol differed significantly between sexes during most of the study period. Regression analysis showed that the sensitivity of MnSOD to this estrogen tended to decrease similarly in both sexes, whereas the sensitivity of CuZnSOD showed a significantly different opposite tendency in female and male rats. These are the first reports indicating hormonal modulation of antioxidant enzyme activities related to the developmental process.

Postprandial symptoms in dysmotility-like functional dyspepsia are not related to disturbances of gastric myoelectrical activity

Gastric dysrhythmias, such as tachy- or bradygastria, have been reported in patients with functional dyspepsia (FD), but their role in symptom production is uncertain. It is also not known whether gastric dysrhythmias in these patients can be elicited by physiological gastric distension with a meal. We investigated the relationships between symptoms after ingestion of different volumes of water following a test meal and gastric dysrhythmias in FD patients. Fourteen patients with dysmotility-like FD and 13 healthy volunteers underwent paired electrogastrography (EGG) studies. Fasted subjects ingested 150 ml of yoghurt with either 150 ml (low volume) or 300 ml (high volume) water in random order. Fasting and fed EGGs with monitoring of symptoms were performed in both studies. Ten FD patients (71.4%) reported upper abdominal discomfort and bloating after the low volume meal, but only one (7.1%) presented an abnormal EGG (dominant frequency in the 2-4-cpm range: 58%). Following the high volume meal, 7 patients (50%) had symptoms, but none had EGG abnormalities. No significant differences were found between FD patients and controls for any of the EGG variables, in any test. In FD patients with postprandial symptoms, the percentage of the EGG dominant frequency in the normal range (median, 84.6%; range, 76.0-100.0%) was similar (P > 0.20) to that in those without symptoms (88.5%; 75.0-100.0%). We conclude that disturbances of gastric myoelectrical activity are unlikely to play a role in the origin of postprandial upper abdominal discomfort and bloating in dysmotility-like FD.

Comparison of different monoclonal antibodies against immunosuppressive proteins of Ascaris suum

The extract of Ascaris suum suppresses the humoral and cellular immune responses to unrelated antigens in the mouse. In order to further characterize the suppressive components of A. suum, we produced specific monoclonal antibodies which can provide an important tool for the identification of these proteins. The A. suum immunosuppressive fractions isolated by gel filtration from an extract of adult worms were used to immunize BALB/c mice. Popliteal lymph node cells taken from the immunized animals were fused with SP2/O myeloma cells and the cloned hybrid cells obtained were screened to determine the specificity of secreted antibodies. Three monoclonal antibodies named MAIP-1, MAIP-2 and MAIP-3 were selected and were shown to react with different epitopes of high molecular weight proteins from the A. suum extract. All antibody molecules have kappa-type light chains but differ in heavy chain isotype. MAIP-1 is a mouse IgM, MAIP-2 is an IgA immunoglobulin and MAIP-3 is an IgG1 immunoglobulin and they recognize the antigen with affinity constants of 1.3 x 10(10) M-1, 7.1 x 10(9) M-1 and 3.8 x 10(7) M-1, respectively. The proteins recognized by these monoclonal antibodies (PAS-1, PAS-2 and PAS-3) were purified from the crude extract by affinity chromatography and injected with ovalbumin in BALB/c mice in order to determine their suppressive activity on heterologous antibody production. It was demonstrated that these three proteins are able to significantly suppress anti-ovalbumin antibody secretion, with PAS-1 being more efficient than the others.

Partial characterization and anticoagulant activity of a heterofucan from the brown seaweed Padina gymnospora

The brown algae Padina gymnospora contain different fucans. Powdered algae were submitted to proteolysis with the proteolytic enzyme maxataze. The first extract of the algae was constituted of polysaccharides contaminated with lipids, phenols, etc. Fractionation of the fucans with increasing concentrations of acetone produced fractions with different proportions of fucose, xylose, uronic acid, galactose, and sulfate. One of the fractions, precipitated with 50% acetone (v/v), contained an 18-kDa heterofucan (PF1), which was further purified by gel-permeation chromatography on Sephadex G-75 using 0.2 M acetic acid as eluent and characterized by agarose gel electrophoresis in 0.05 M 1,3 diaminopropane/acetate buffer at pH 9.0, methylation and nuclear magnetic resonance spectroscopy. Structural analysis indicates that this fucan has a central core consisting mainly of 3-ß-D-glucuronic acid 1-> or 4-ß-D-glucuronic acid 1 ->, substituted at C-2 with alpha-L-fucose or ß-D-xylose. Sulfate groups were only detected at C-3 of 4-alpha-L-fucose 1-> units. The anticoagulant activity of the PF1 (only 2.5-fold lesser than low molecular weight heparin) estimated by activated partial thromboplastin time was completely abolished upon desulfation by solvolysis in dimethyl sulfoxide, indicating that 3-O-sulfation at C-3 of 4-alpha-L-fucose 1-> units is responsible for the anticoagulant activity of the polymer.

Hypoglycemic activity of polysaccharide fractions containing ß-glucans from extracts of Rhynchelytrum repens (Willd.) C.E. Hubb., Poaceae

ß-Glucans are soluble fibers with physiological functions, such as interference with absorption of sugars and reduction of serum lipid levels. The objective of the present study was to analyze the distribution of ß-glucans in different tissues of the African grass species Rhynchelytrum repens and also to evaluate their hypoglycemic activity. Leaf blades, sheaths, stems, and young leaves of R. repens were submitted to extraction with 4 M KOH. Analysis of the fractions revealed the presence of arabinose, glucose, xylose, and traces of rhamnose and galactose. The presence of ß-glucan in these fractions was confirmed by hydrolyzing the polymers with endo-ß-glucanase from Bacillus subtilis, followed by HPLC analysis of the characteristic oligosaccharides produced. The 4 M KOH fractions from different tissues were subjected to gel permeation chromatography on Sepharose 4B, with separation of polysaccharides with different degrees of polymerization, the highest molecular mass (above 2000 kDa) being found in young leaves. The molecular mass of the leaf blade polymers was similar (250 kDa) to that of maize coleoptile ß-glucan used for comparison. The 4 M KOH fraction injected into rats with streptozotocin-induced diabetes showed hypoglycemic activity, reducing blood sugar to normal levels for approximately 24 h. This performance was better than that obtained with pure ß-glucan from barley, which decreased blood sugar levels for about 4 h. These results suggest that the activity of ß-glucans from R. repens is responsible for the use of this plant extract as a hypoglycemic drug in folk medicine.

Some physico-chemical parameters that influence proteinase K resistance and the infectivity of PrP Sc after high pressure treatment

Crude brain homogenates of terminally diseased hamsters infected with the 263 K strain of scrapie (PrP Sc) were heated and/or pressurized at 800 MPa at 60ºC for different times (a few seconds or 5, 30, 120 min) in phosphate-buffered saline (PBS) of different pH and concentration. Prion proteins were analyzed on immunoblots for their proteinase K (PK) resistance, and in hamster bioassays for their infectivity. Samples pressurized under initially neutral conditions and containing native PrP Sc were negative on immunoblots after PK treatment, and a 6-7 log reduction of infectious units per gram was found when the samples were pressurized in PBS of pH 7.4 for 2 h. A pressure-induced change in the protein conformation of native PrP Sc may lead to less PK resistant and less infectious prions. However, opposite results were obtained after pressurizing native infectious prions at slightly acidic pH and in PBS of higher concentration. In this case an extensive fraction of native PrP Sc remained PK resistant after pressure treatment, indicating a protective effect possibly due to induced aggregation of prion proteins in such buffers.

Linking immunity and hematopoiesis by bone marrow T cell activity

Two different levels of control for bone marrow hematopoiesis are believed to exist. On the one hand, normal blood cell distribution is believed to be maintained in healthy subjects by an "innate" hematopoietic activity, i.e., a basal intrinsic bone marrow activity. On the other hand, an "adaptive" hematopoietic state develops in response to stress-induced stimulation. This adaptive hematopoiesis targets specific lineage amplification depending on the nature of the stimuli. Unexpectedly, recent data have shown that what we call "normal hematopoiesis" is a stress-induced state maintained by activated bone marrow CD4+ T cells. This T cell population includes a large number of recently stimulated cells in normal mice whose priming requires the presence of the cognate antigens. In the absence of CD4+ T cells or their cognate antigens, hematopoiesis is maintained at low levels. In this review, we summarize current knowledge on T cell biology, which could explain how CD4+ T cells can help hematopoiesis, how they are primed in mice that were not intentionally immunized, and what maintains them activated in the bone marrow.

In vitro modulation of alkaline phosphatase activity of Saccharomyces cerevisiae grown in low or high phosphate medium

Our objective was to characterize the modulation of the activity of Saccharomyces cerevisiae alkaline phosphatases (ALPs) by classic inhibitors of ALP activity, cholesterol and steroid hormones, in order to identify catalytic similarities between yeast and mammalian ALPs. S. cerevisiae expresses two ALPs, coded for by the PHO8 and PHO13 genes. The product of the PHO8 gene is repressible by Pi in the medium. ALP activity from yeast (grown in low or high phosphate medium) homogenates was determined with p-nitrophenylphosphate as substrate, pH 10.4 (lPiALP or hPiALP, respectively). Activation of hPiALP was observed with 5 mM L-amino acids (L-homoarginine _ 186%, L-leucine _ 155% and L-phenylalanine - 168%) and with 1 mM levamisole (122%; percentage values, in comparison to control, of recovered activity). EDTA (5 mM) and vanadate (1 mM) distinctly inhibited hPiALP (2 and 20%, respectively). L-homoarginine (5 mM) had a lower activating effect on lPiALP (166%) and was the strongest hPiALP activator. Corticosterone (5 mM) inhibited hPiALP to 90%, but no effect was observed in low phosphate medium. Cholesterol, ß-estradiol and progesterone also had different effects on lPiALP and hPiALP. A concentration-dependent activation of lPiALP minus hPiALP was evident with all three compounds, most especially with ß-estradiol and cholesterol. These results do not allow us to identify similarities of the behavior of S. cerevisiae ALPs and any of the mammalian ALPs but allow us to raise the hypothesis of differential regulation of S. cerevisiae ALPs by L-homoarginine, ß-estradiol and cholesterol and of using these compounds to discriminate between S. cerevisiae lPiALP and hPiALP.

Cytotoxicity and antitumoral activity of dichloromethane extract and its fractions from Pothomorphe umbellata

The cytotoxicity of the dichloromethane crude extract (DCE), obtained from the aerial parts of Pothomorphe umbellata (L.) Miq (Piperaceae), was evaluated against nine human cancer cell lines (MCF-7, NCI-ADR/RES, OVCAR-3, PC-3, HT-29, NCI-H460, 786-O, UACC-62, K-562). The DCE presented antiproliferative activity with good potency against all cell lines at low concentrations (between 4.0 and 9.5 µg/mL) and with selectivity (1.55 µg/mL) for the leukemia cell line (K-652). DCE (100, 200, 300 and 400 mg/kg, ip) was also evaluated in the Ehrlich ascites tumor model. Both the survival number and the life span of the animals that died increased by at least 45 and 50%, respectively (8 animals per group), demonstrating P. umbellata extract potential anticancer activity. The results of the in vivo antitumor activity prompted the fractionation of the crude extract. The crude extract was submitted to dry column chromatography with dichloromethane-methanol (99:1). The column effluent fractions were extracted with methanol, dried under vacuum yielding fractions FR1 (less polar), FR2 (medium polarity), and FR3 (polar), which were analyzed for their growth inhibition or cytotoxic properties by a 48-h sulforhodamine B cell viability assay by measuring the total protein content. FR1 demonstrated high potency and cytotoxicity, a result compatible with the high toxicity of oxalic acid; FR2, containing 4-nerolidylcathecol, presented the lowest cytotoxic activity compared to the other two fractions but with selectivity for prostate cancer cell line; FR3, containing a mixture of steroids described in the literature as possessing various biological activities, also presented potent anticancer in vitro activity. These results suggest that P. umbellata DCE in vivo antitumor activity may be a consequence of the activity of different active principles.

Association between dental-oral health in young adults and salivary glutathione, lipid peroxidation and sialic acid levels and carbonic anhydrase activity

The aim of the present study was to evaluate the relationship between salivary oxidative stress and dental-oral health. Healthy young adults, matched for gender and age, with (N = 21, 10 men, mean age: 20.3 ± 1 years) and without (N = 16, 8 men, mean age: 21.2 ± 1.8 years) caries were included in this study. The World Health Organization (WHO) caries diagnostic criteria were used for determining the decayed, missing, filled teeth (DMFT) index. The oral hygiene and gingival status were assessed using the simplified oral hygiene index and gingival index, respectively. Unstimulated salivary total protein, glutathione (GSH), lipid peroxidation and total sialic acid levels, carbonic anhydrase activity, and salivary buffering capacity were determined by standard methods. Furthermore, salivary pH was measured with pH paper and salivary flow rate was calculated. Simplified oral hygiene index and gingival index were not significantly different between groups but DMFT scores were significant (P < 0.01). Only, GSH values were significantly different (P < 0.05) between groups (2.2 and 1.6 mg/g protein in young adults without caries and with caries, respectively). There was a significant negative correlation between DMFT and GSH (r = -0.391; P < 0.05; Pearson's correlation coefficient). Our results suggest that there is an association between caries history and salivary GSH levels.

Modulation of peritoneal macrophage activity by the saturation state of the fatty acid moiety of phosphatidylcholine

To determine the effects of saturated and unsaturated fatty acids in phosphatidylcholine (PC) on macrophage activity, peritoneal lavage cells were cultured in the presence of phosphatidylcholine rich in saturated or unsaturated fatty acids (sat PC and unsat PC, respectively), both used at concentrations of 32 and 64 µM. The treatment of peritoneal macrophages with 64 µM unsat PC increased the production of hydrogen peroxide by 48.3% compared to control (148.3 ± 16.3 vs 100.0 ± 1.8%, N = 15), and both doses of unsat PC increased adhesion capacity by nearly 50%. Moreover, 64 µM unsat PC decreased neutral red uptake by lysosomes by 32.5% compared to the untreated group (67.5 ± 6.8 vs 100.0 ± 5.5%, N = 15), while both 32 and 64 µM unsat PC decreased the production of lipopolysaccharide-elicited nitric oxide by 30.4% (13.5 ± 2.6 vs 19.4 ± 2.5 µM) and 46.4% (10.4 ± 3.1 vs 19.4 ± 2.5 µM), respectively. Unsat PC did not affect anion production in non-stimulated cells or phagocytosis of unopsonized zymosan particles. A different result pattern was obtained for macrophages treated with sat PC. Phorbol 12-miristate 13-acetate-elicited superoxide production and neutral red uptake were decreased by nearly 25% by 32 and 64 µM sat PC, respectively. Sat PC did not affect nitric oxide or hydrogen peroxide production, adhesion capacity or zymosan phagocytosis. Thus, PC modifies macrophage activity, but this effect depends on cell activation state, fatty acid saturation and esterification to PC molecule and PC concentration. Taken together, these results indicate that the fatty acid moiety of PC modulates macrophage activity and, consequently, is likely to affect immune system regulation in vivo.

Trailing end-point phenotype antibiotic-sensitive strains of Candida albicans produce different amounts of aspartyl peptidases

Candida albicans is an opportunistic fungal pathogen that causes severe systemic infections in immunosuppressed individuals. C. albicans resistance to antifungal drugs is a severe problem in patients receiving prolonged therapy. Moreover, trailing yeast growth, which is defined as a resistant MIC after 48 h of incubation with triazole antifungal agents but a susceptible MIC after 24 h, has been noted in tests of antifungal susceptibility against some C. albicans isolates. In this context, we recently noticed this phenomenon in our routine susceptibility tests with fluconazole/itraconazole and C. albicans clinical isolates. In the present study, we investigated the production of cell-associated and secreted aspartyl peptidases (Saps) in six trailing clinical isolates of C. albicans, since this class of hydrolytic enzymes is a well-known virulence factor expressed by this fungal pathogen. Sap2, which is the best-studied member of the Sap family, was detected by flow cytometry on the cell surface of yeasts and as a 43-kDa polypeptide in the culture supernatant, as demonstrated by Western blotting assay using an anti-Sap1-3 polyclonal antibody. Released aspartyl peptidase activity was measured with BSA hydrolysis and inhibited by pepstatin A, showing distinct amounts of proteolytic activity ranging from 5.7 (strain 44B) to 133.2 (strain 11) arbitrary units. Taken together, our results showed that trailing clinical isolates of C. albicans produced different amounts of both cellular and secreted aspartyl-type peptidases, suggesting that this phenotypic feature did not generate a regular pattern regarding the expression of Sap.

Ventricular performance and Na+-K+ ATPase activity are reduced early and late after myocardial infarction in rats

Myocardial infarction leads to compensatory ventricular remodeling. Disturbances in myocardial contractility depend on the active transport of Ca2+ and Na+, which are regulated by Na+-K+ ATPase. Inappropriate regulation of Na+-K+ ATPase activity leads to excessive loss of K+ and gain of Na+ by the cell. We determined the participation of Na+-K+ ATPase in ventricular performance early and late after myocardial infarction. Wistar rats (8-10 per group) underwent left coronary artery ligation (infarcted, Inf) or sham-operation (Sham). Ventricular performance was measured at 3 and 30 days after surgery using the Langendorff technique. Left ventricular systolic pressure was obtained under different ventricular diastolic pressures and increased extracellular Ca2+ concentrations (Ca2+e) and after low and high ouabain concentrations. The baseline coronary perfusion pressure increased 3 days after myocardial infarction and normalized by 30 days (Sham 3 = 88 ± 6; Inf 3 = 130 ± 9; Inf 30 = 92 ± 7 mmHg; P < 0.05). The inotropic response to Ca2+e and ouabain was reduced at 3 and 30 days after myocardial infarction (Ca2+ = 1.25 mM; Sham 3 = 70 ± 3; Inf 3 = 45 ± 2; Inf 30 = 29 ± 3 mmHg; P < 0.05), while the Frank-Starling mechanism was preserved. At 3 and 30 days after myocardial infarction, ventricular Na+-K+ ATPase activity and contractility were reduced. This Na+-K+ ATPase hypoactivity may modify the Na+, K+ and Ca2+ transport across the sarcolemma resulting in ventricular dysfunction.

In vitro larvicidal activity of geraniol and citronellal against Contracaecum sp (Nematoda: Anisakidae)

Human infection with fish parasites can result from the ingestion of incompletely cooked or raw fish, giving origin to parasitic diseases such as anisakiasis, caused by parasites of the Anisakidae family. The present study assessed the in vitro larvicidal effect of two monoterpene compounds, geraniol and citronellal, against Contracaecum sp (Nematoda: Anisakidae). Four hundred live larvae of Contracaecum sp obtained from "traíra" fish (Hoplias malabaricus, Bloch, 1974) were analyzed on 40 Petri dishes (10 larvae each) with the compounds to be tested. The final concentrations tested for each compound were 250, 125, 62.5, and 31.2 µg/mL and the evaluation was carried out at five different times (2, 4, 8, 24, and 48 h). The larvicidal action of geraniol and citronellal was statistically superior (P < 0.005) to the control (1% ethanol) at concentrations of 250 and 31.2 µg/mL (geraniol) and 250, 125, and 62.5 μg/mL (citronellal). However, no larvicidal activity was observed at concentrations of 125 and 62.5 µg/mL for geraniol and 31.2 µg/mL for citronellal. When the larvicidal action of geraniol was compared to that of citronellal, the former was found to be statistically superior (P < 0.05) to the latter at concentrations of 250 and 31.2 μg/mL. On the other hand, citronellal was statistically superior (P < 0.005) to geraniol at concentrations of 125 and 62.5 μg/mL. The larval mortality rate in terms of time (hours) was higher for geraniol with the passing of time at the 250 μg/mL concentration. At this concentration (in 48 h) the best larvicidal effect was observed with 90% lethality. The larvae were considered to be dead using no motility and loss of structural integrity as parameters. The data indicate that natural terpene compounds should be more explored for antihelminthic activity and can be useful for other studies about anisakiasis treatment.