Biomechanical and biochemical investigation of erythrocytes in late stage human leptospirosis.

Leptospirosis is a zoonotic disease caused by bacteria of the genus Leptospira, which can cause lipid changes in the erythrocyte membrane. Optical tweezers were used to characterize rheological changes in erythrocytes from patients with leptospirosis in the late stage. Biochemical methods were also used for quantification of plasma lipid, erythrocyte membrane lipid, and evaluation of liver function. Our data showed that the mean elastic constant of erythrocytes from patients with leptospirosis was around 67% higher than the control (healthy individuals), indicating that patient's erythrocytes were less elastic. In individuals with leptospirosis, several alterations in relation to control were observed in the plasma lipids, however, in the erythrocyte membrane, only phosphatidylcholine showed a significant difference compared to control, increasing around 41%. With respect to the evaluation of liver function of individuals with leptospirosis, there was a significant increase in levels of alanine transaminase (154%) and aspartate transaminase (150%), whereas albumin was 43.8% lower than control (P<0.01). The lecithin-cholesterol acyltransferase fractional activity was 3.6 times lower in individuals with leptospirosis than in the healthy individuals (P<0.01). The decrease of the erythrocyte elasticity may be related to the changes of erythrocyte membrane phospholipids composition caused by disturbances that occur during human leptospirosis, with phosphatidylcholine being a strong candidate in the erythrocyte rheological changes.

Biomechanical and biochemical investigation of erythrocytes in late stage human leptospirosis

Leptospirosis is a zoonotic disease caused by bacteria of the genus Leptospira, which can cause lipid changes in the erythrocyte membrane. Optical tweezers were used to characterize rheological changes in erythrocytes from patients with leptospirosis in the late stage. Biochemical methods were also used for quantification of plasma lipid, erythrocyte membrane lipid, and evaluation of liver function. Our data showed that the mean elastic constant of erythrocytes from patients with leptospirosis was around 67% higher than the control (healthy individuals), indicating that patient's erythrocytes were less elastic. In individuals with leptospirosis, several alterations in relation to control were observed in the plasma lipids, however, in the erythrocyte membrane, only phosphatidylcholine showed a significant difference compared to control, increasing around 41%. With respect to the evaluation of liver function of individuals with leptospirosis, there was a significant increase in levels of alanine transaminase (154%) and aspartate transaminase (150%), whereas albumin was 43.8% lower than control (P<0.01). The lecithin-cholesterol acyltransferase fractional activity was 3.6 times lower in individuals with leptospirosis than in the healthy individuals (P<0.01). The decrease of the erythrocyte elasticity may be related to the changes of erythrocyte membrane phospholipids composition caused by disturbances that occur during human leptospirosis, with phosphatidylcholine being a strong candidate in the erythrocyte rheological changes.

Antioxidant activity of anthocyanins from quixabeira (Sideroxylon obtusifolium) fruits / Atividade antioxidante de antocianinas de frutos de quixabeira (Sideroxylon obtusifolium)

ABSTRACT The fruits of Sideroxylon obtusifolium T.D. Penn. are small berries which have dark purple skin color at maturity due the anthocyanins presence. Therefore, the aim of this research was to evaluate the antioxidant activity of anthocyanins from mature fruits which were obtained at a semi-arid region of Paraiba/BR. In addition, the total anthocyanins content was measured by the pH differential method. The anthocyanins were purified by solid-phase extraction (SPE) using C18 Sep-Pak cartridges and submitted to antioxidant activity determined by scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH·). The butylated hydroxytoluene (BHT) was the positive control and the results were expressed as DPPH· scavenging activity (%). Anthocyanins content were higher in the husks (236.15 mg cyanidin-3-glucoside 100g-1 fw) than in the pulp (30.49 mg cyanidin-3-glucoside 100g-1 fw). The results showed that the potential free radical scavenging grew with the increase of concentration used and the reaction time. The ability of the anthocyanins in scavenging the free radical was statistically greater than the BHT one. The anthocyanins which were present in this fruit may supply substantial dietary source of antioxidant that may promote health and produce disease prevention effects or that could be applied in food industry as a good source of natural pigments.

RESUMO Os frutos da Sideroxylon obtusifolium TD Penn., são bagas pequenas que, quando maduras, apresentam a coloração roxo-escuro devido à presença de antocianinas. Assim, o objetivo desta pesquisa foi avaliar a atividade antioxidante das antocianinas desses frutos maduros obtidos da região do semiárido da Paraíba/BR. Além disso, o teor de antocianinas total foi quantificado pelo método de pH diferencial. As antocianinas foram purificadas por extração em fase sólida (EFS) utilizando cartuchos Sep-Pak C18 e submetidas à avaliação da atividade antioxidante por meio da capacidade de sequestrar o radical DPPH·. Butil-hidroxi-tolueno (BHT) foi utilizado como controle positivo e os resultados foram expressos como percentual de sequestro de radical livre. Os resultados demonstraram que o teor de antocianinas presente nas cascas foi maior (236,15 mg de cianidina-3-glicosídeo 100g- 1, peso fresco) do que na polpa (30,49 mg de cianidina-3-glicosídeo 100g-1, peso fresco) e que o percentual de sequestro de radical livre aumentou com o aumento da concentração utilizada e o tempo de reação. A habilidade das antocianinas em sequestrar o radical livre foi estatisticamente superior à do BHT. As antocianinas presentes nesta fruta podem fornecer fonte dietética significativa de antioxidantes que podem promover a saúde e produzir efeitos de prevenção de doenças ou ser aplicada na indústria de alimentos como uma boa fonte de pigmentos naturais.

Selective cytotoxicity and apoptosis induction in glioma cell lines by 5-oxygenated-6,7-methylenedioxycoumarins from Pterocaulon species.

The coumarins 5-methoxy-6,7-methylenedioxycoumarin 1 5-(3-methyl-2-butenyloxy)-6,7-methylenedioxycoumarin 2 and 5-(2,3-dihydroxy-3-methylbutyloxy)-6,7-methylenedioxycoumarin 3 isolated from Pterocaulon species showed significant cytotoxicity against two glioma cells lines. Compound 1 presented IC(50) values of 34.6 µM and 31.6 µM against human (U138-MG) and rat (C6) glioma cells, respectively, and this compound was at least two times more potent than compounds 2 and 3. This result could be explained by the planar conformation adopted by 1 through a non-classical hydrogen bond between a hydrogen of the methoxy and the oxygen of the methylenedioxy groups. Another important finding was that the cytotoxic effect induced by 1 in glioma cells was not observed in organotypic cultures, indicating a selective cytotoxicity for tumor cells.

Parkinsonia aculeata aqueous extract fraction: biochemical studies in alloxan-induced diabetic rats.

The antidiabetic effect of Parkinsonia aculeata water soluble fraction (WSF) made of aerial parts of the plant (leaves and flowers) was investigated in alloxan diabetic rats. Its effect was compared with that of insulin (positive control). The physico-metabolic parameters measured were: body weight, food and liquid intake, urinary volume, hepatic glycogen, serum glucose, total cholesterol, HDL-cholesterol, triglycerides, urinary glucose and urea, and the weight of epididymal adipose tissue, liver, kidneys and the skeletal muscles (soleus and extensor digitorum longus). Oral administration of WSF (125 or 250 mg/kg) for 16 days exhibited a significant reduction in serum and urinary glucose, urinary urea, total cholesterol, HDL-cholesterol and triglycerides in alloxan diabetic rats. An improvement of hepatic glycogen, a decrease of liquid and food intake, and a significantly positive actions in the weight of skeletal muscles (soleus and extensor digitorum longus) and kidneys were also observed, but just diabetic group treated with WSF at a dose of 125 mg/kg showed significant reduction in urinary volume, body weight, an improvement of epididymal adipose tissue and a positive action in liver weight. The effects of WSF on the physico-metabolic parameters was comparable to those observed in diabetic insulin treated group. The results of this work suggest that P. aculeate may have new clinical significant choice in diabetes mellitus illness, and could explain the basis for its traditional use to manage diabetes-related complications by rural community of northeast of Brazil.

Synthesis and biological activity of novel acridinylidene and benzylidene thiazolidinediones.

A novel set of acridinylidene thiazolidinediones and benzylidene thiazolidinediones was synthesized by nucleophilic addition of cyanoacrylates. Some of these compounds were evaluated for their glucose lowering capability and their effects on the triglyceride level in alloxan diabetic mice.

Elevated levels of erythrocyte-conjugated dienes indicate increased lipid peroxidation in schistosomiasis mansoni patients.

Schistosoma mansoni causes liver disease by inducing granulomatous inflammation. This favors formation of reactive oxygen species, including superoxide ions, hydrogen peroxide and hydroxyl radicals all of which may induce lipid peroxidation. We have evaluated lipid peroxidation in 18 patients with hepatosplenic schistosomiasis mansoni previously treated with oxamniquine followed by splenectomy, ligature of the left gastric vein and auto-implantation of spleen tissue, by measuring levels of erythrocyte-conjugated dienes and plasma malondialdehyde (MDA). Age-matched, healthy individuals (N = 18) formed the control group. Erythrocyte-conjugated dienes were extracted with dichloromethane/methanol and quantified by UV spectrophotometry, while plasma MDA was measured by reaction with thiobarbituric acid. Patient erythrocytes contained two times more conjugated dienes than control cells (584.5 +/- 67.8 vs 271.7 +/- 20.1 micromol/l, P < 0.001), whereas the increase in plasma MDA concentration (about 10%) was not statistically significant. These elevated conjugated dienes in patients infected by S. mansoni suggest increased lipid peroxidation in cell membranes, although this was not evident when a common marker of oxidative stress, plasma MDA, was measured. Nevertheless, these two markers of lipid peroxidation, circulating MDA and erythrocyte-conjugated dienes, correlated significantly in both patient (r = 0.62; P < 0.01) and control (r = 0.57; P < 0.05) groups. Our data show that patients with schistosomiasis have abnormal lipid peroxidation, with elevated erythrocyte-conjugated dienes implying dysfunctional cell membranes, and also imply that this may be attenuated by the redox capacity of antioxidant agents, which prevent accumulation of plasma MDA.

Elevated levels of erythrocyte-conjugated dienes indicate increased lipid peroxidation in schistosomiasis mansoni patients

Schistosoma mansoni causes liver disease by inducing granulomatous inflammation. This favors formation of reactive oxygen species, including superoxide ions, hydrogen peroxide and hydroxyl radicals all of which may induce lipid peroxidation. We have evaluated lipid peroxidation in 18 patients with hepatosplenic schistosomiasis mansoni previously treated with oxamniquine followed by splenectomy, ligature of the left gastric vein and auto-implantation of spleen tissue, by measuring levels of erythrocyte-conjugated dienes and plasma malondialdehyde (MDA). Age-matched, healthy individuals (N = 18) formed the control group. Erythrocyte-conjugated dienes were extracted with dichloromethane/methanol and quantified by UV spectrophotometry, while plasma MDA was measured by reaction with thiobarbituric acid. Patient erythrocytes contained two times more conjugated dienes than control cells (584.5 ± 67.8 vs 271.7 ± 20.1 æmol/l, P < 0.001), whereas the increase in plasma MDA concentration (about 10 percent) was not statistically significant. These elevated conjugated dienes in patients infected by S. mansoni suggest increased lipid peroxidation in cell membranes, although this was not evident when a common marker of oxidative stress, plasma MDA, was measured. Nevertheless, these two markers of lipid peroxidation, circulating MDA and erythrocyte-conjugated dienes, correlated significantly in both patient (r = 0.62; P < 0.01) and control (r = 0.57; P < 0.05) groups. Our data show that patients with schistosomiasis have abnormal lipid peroxidation, with elevated erythrocyte-conjugated dienes implying dysfunctional cell membranes, and also imply that this may be attenuated by the redox capacity of antioxidant agents, which prevent accumulation of plasma MDA.

Validated HPLC method for determination of LASSBio-581, a new heterocyclic N-phenylpiperazine derivative, in rat plasma.

A rapid, simple and accurate high performance liquid chromatography (HPLC) method was developed and validated for the determination of LASSBio-581 (1-[1-(4-chloro-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-4-phenyl-piperazine) in rat plasma using ketoconazole as internal standard. Plasma samples were deproteinized with methanol. A good chromatographic separation was achieved using a reversed phase C18 column. Mobile phase consisting of sodium dihydrogen phosphate monohydrate (pH 4.5, 0.02 M) and methanol mixture (35:65, v/v) was used at a flow rate of 1.0 ml/min. The eluate was monitored using a UV detector at 248 nm. The retention times of LASSBio-581 and the internal standard were approximately 3.8 and 5.6 min, respectively. The calibration curves were linear over the concentration range of 0.25-8.0 microg/ml with correlation coefficients >0.99. The limit of quantitation was 0.25 microg/ml. The accuracy of the method was >90%. The intra-day relative standard deviation (R.S.D.) ranged from 6.15 to 10.52% at 0.4 microg/ml, 7.44 to 13.81% at 1.5 microg/ml and 6.10 to 13.94% at 6.0 microg/ml. The inter-day R.S.D. were 9.54, 8.42 and 8.25% at 0.4, 1.5 and 6.0 microg/ml, respectively. No interference from endogenous substances or metabolites were observed. The method has been used to measure plasma concentrations of LASSBio-581 in pharmacokinetic studies in rats.

[Triatoma infestans in area under entomological surveillance for Chagas' disease in São Paulo State, Brazil]. / Triatoma infestans em área sob vigilância entomológica para doença de Chagas, Estado de São Paulo, Brasil.

In response to notification of the capture of a winged triatomine bug by local inhabitants, an epidemiological investigation was investigated in the rural area of the Municipality of Paulínia, São Paulo State (Brazil). This led to the collection of 109 specimens of Triatoma infestans from peridomiciliary breeding sites. Local conditions favored colonization by triatomine bugs: many abandoned outbuildings were inhabited by pigeons and sparrows, which meant abundant feed for these bugs. Thus, surveillance is mandatory as long as conditions for triatomine breeding persist. Nevertheless, entomological and serological indicators ruled out immediate concern over vectorial transmission of Chagas' disease in this municipality. All domiciliary units of the region were sprayed with residual-effect insecticides and the triatomine breeding sites were destroyed by the use of mechanical control of bird nests. The importance of reporting the occurrence of triatomine bugs, particularly Triatoma infestans, in this region, is emphasized.

Microwave assisted synthesis of N-arylphthalamic acids with hyperlipidemic activity.

A series of substituted N-arylphthalamic acids 3a-i has been synthesized by the reaction of phthalic anhydride 1 and aryl- or heterocyclic amines 2a-i, in the absence of solvents, in a domestic microwave oven. The formation of nine N-arylphthalamic acids was accomplished in 1-3 min giving excellent yields for compounds 3a-g, but moderate yield of compounds 3h and 3i, respectively. Compounds 3h and 3i are new. Interestingly, N-arylphthalamic acids 3a-i induced hyperlipidemia in Swiss white mice and also increased animals' body weight.

Ofloxacin/beta-cyclodextrin complexation.

Ofloxacin (OFX) is a fluorquinolone characterized by photochemical instability. With the goal to improve its photostability in aqueous solutions, the complexation of ofloxacin with beta-cyclodextrin was investigated. The complexes showed a water solubility enhancement of approximately 2.6 times; nevertheless, the photodegradation of ofloxacin was not reduced. The complexes obtained were characterized by thermal and 1H nuclear magnetic resonance (NMR) analysis, which revealed an interaction between ofloxacin and beta-cyclodextrin. The last analysis indicated that only partial inclusion of the N-methylpiperazinyl moiety occurred, which can explain the fact that photostabilization was not improved. This partial inclusion phenomenon could be explained also by computer-aided molecular modeling.

Synthesis and hypolipidemic activity of N-phthalimidomethyl tetra-O-acyl-alpha-D-mannopyranosides.

A facile synthesis of anomerically pure phthalimidomethyl 2,3,4,6-tetra-O-acetyl- and phthalimidomethyl 2,3-di-O-acetyl-4,6-di-O-benzoyl-alpha-D-mannopyranosides (6 and 9b) starting from N-hydroxymethylphthalimide and tri-O-acetyl-D-glucal is described. Compounds 3, 6, 8, 9a and 9b have been tested for their hypolipidemic activity in mice. All these compound showed significant reduction of plasma cholesterol and triglyceride levels. Compound 9b has been found to possess the highest activity.

[Dengue fever: a post-epidemic sero-epidemiological survey in an urban area setting at a northwestern county of São Paulo State, Brazil]. / Dengue: inquérito sorológico pós-epidêmico em zona urbana do Estado de São Paulo (Brasil).

OBJECTIVE: The objective of this study was to evaluate the real size of the epidemics registered in the urban area of the county of Santa Bárbara D'Oeste, SP, Brazil, from April to June, 1995. The measurement of the epidemiological validity of the official surveillance system criteria and its positive predicted value were adopted as specific goals. METHODS: A sero-epidemiological survey was carried out over a sample of 1,113 sera from citizens of Santa Barbara D'Oeste, through a systematic random sampling of houses, five months after the end of the epidemics. Infection rates were compared with the infestation indexes by Aedes aegipty and the notified cases amongst the county sections. The importance of submitting patients with clinical suspicion of dengue to laboratory tests was discussed. RESULTS AND DISCUSSION: It was found that infection rates by dengue virus varied in the same direction and proportion as the presence of Aedes aegipty larvae reported by the "Breteau Index", as well as the number of cases reported by the official notifiable diseases surveillance system during the epidemics. A prevalence of 630 by 100 thousand inhabitants was found, a 15-fold rate when compared to the laboratory positive sera from cases detected by the surveillance system during the epidemics. A retrospective comparison with the surveillance reports, using serological results as a gold standard, also showed that the majority of dengue specific serum-positive individuals were not detected during the epidemics, otherwise cases that did not present serological reaction were notified exhibiting a low positive predictive value of clinical diagnosis (15,6).

An evaluation of the marmoset Callithrix jacchus (sagüi) as an experimental model for the dyslipoproteinemia of human Schistosomiasis mansoni.

Human infection with the parasite Schistosoma mansoni is a relatively common occurrence in regions of South America and is associated with liver dysfunction and dyslipoproteinemia. Specifically, the activity of plasma lecithin:cholesterol acyltransferase (LCAT) activity is reduced, the concentration of plasma cholesterol esters falls, phospholipid concentrations are elevated and erythrocyte membranes become cholesterol enriched. Previous studies have utilized rodents (rats and mice) as experimental models to study the dyslipoproteinemia induced by S. mansoni infection. However, the plasma lipoprotein profiles in these animals is very different from humans and infection is not accompanied by decreases in LCAT activity or cholesterol enrichment of their erythrocyte membranes. Here we have evaluated the suitability of the marmoset Callithrix jacchus (sagüi) which is small and readily available in Brazil, as a potential animal model for the study of the dyslipoproteinemia of S. mansoni infections. The plasma lipoprotein compositions and distributions in sagüi, unlike rats or mice, approximate those of man with the LDL representing a major lipoprotein species. The molecular species of phospholipids, cholesterol esters and triglycerides present in sagüi plasma are also very similar to man, whereas those of rats and mice favor the longer chain more unsaturated species, Sagüi, like rodents, can be successfully infected with S. mansoni and after 60 days, this results in a 50% reduction in plasma LCAT activity, an 11% reduction in plasma cholesterol esters, an absolute increase of 46% in plasma phospholipids and an 18% increase in the cholesterol content of erythrocyte membranes. These changes are qualitatively and quantitatively very similar to those previously reported following human infections. Based upon these changes, and the observation that the plasma lipoprotein profile of sagüi and human is similar, we conclude that C. jacchus (sagüi) is an appropriate animal model for the study of dyslipoproteinemia associated with S. mansoni infections.

Immunofluorescence test on Schistosoma mansoni worm paraffin sections (IgM-IFT) for the study of schistosomiasis transmission in Campinas, São Paulo, Brazil.

The detection of IgM antibodies for Schistosoma mansoni using gut-associated antigens (IgM-IFT) was compared to the parasitological Kato-Katz method for study of the transmission of schistosomiasis in an urban area in Campinas. About 400 schoolchildren whose ages ranged from 6 to 18 years, were observed for a period of two years. Blood samples on filter paper and fecal samples were collected, at intervals of six months. Serological (IgM-IFT) prevalence rates of 1.2%, 4.3%, 3.6%, 2.9% and 3.4% were obtained in five surveys carried out. S. mansoni eggs were detected in only one child out of the 225 children (0.4%) who were submitted to the Kato-Katz method (three slides for each fecal sample) in the 1st survey. Sixty eight children who submitted five blood samples, one for each survey, were found IFT negative throughout the study. No child was found to be IFT positive in all five surveys, and only four children showed IFT positive results in at least four surveys. Seroconversion from IFT negative to positive was observed from the 1st to the 2nd survey in six children, from the 2nd to the 3rd survey in three children, from the 3rd to the 4th survey in four children, and from the 4th to the 5th survey in two cases. However, confirmation of S. mansoni infection using the fecal examination was not possible in any of the cases. Also, in most of them the IFT result oscillated from negative to positive and vice versa. Our data implied that recent transmission of schistosomiasis in the study area was not possible.

Inhibition of tumor growth and polyamine uptake by tetracyclic amidines bearing a putrescine moiety.

Tetracyclic amidines (tetrahydroquino[4.3-b][l]benzazepine: compound 11 and tetrahydrobenzo[k]naphthyridine: compound 12) bearing a putrescine moiety possess significant DNA-binding affinity. We report here that these compounds and their a and b isomers inhibit tumor cell growth and putrescine uptake in 3LL carcinoma cells in vitro. Moreover, compound 11 reduced by 50% the accumulation of putrescine in intestinal brush border membrane vesicles. In CHO-MG, a cell line deficient for the specific polyamine uptake system, the cytotoxicity of these compounds was significantly reduced compared to the CHO wild cell line. The IC50 for CHO-MG was significantly higher than for CHO, demonstrating that the polyamine transport system increased the efficacy of these compounds. The efficacy of compounds 11 and 12 might therefore be related to their ability to interact with DNA as well as their structural analogy with polyamines. Moreover, we clearly show that DFMO enhances the efficacy of these tetracyclic amidines in vivo. Potential mechanisms include: a) lower intracellular polyamine levels reduces polyamine DNA-stabilizing functions, increasing accessibility for DNA-binding drugs; b) DFMO enhances the polyamine uptake system in tumor cells, increasing the entry of tetracyclic amidines bearing a putrescine moiety as well as their accessibility to final DNA-binding sites. The fact that natural polyamine uptake is reduced by the same compounds constitutes an additive mechanism for antitumoral efficiency.