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.

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.

Alterations of cell membrane composition during prolonged fasting in lizards.

Male lizards fasted for 35 days showed significant alterations in the lipid composition of membranes isolated from a variety of organs and tissues. Unesterified cholesterol was significantly increased in membranes from kidney, skeletal muscle, small intestine, testis and heart but not in those from lung, brain and liver; cholesterol/phospholipid molar ratio was also significantly increased in membranes from all organs except brain and liver. Membrane total phospholipids were reduced for all tissues except brain and liver, whilst significant reductions in the molar ratio of membrane phosphatidylcholine to sphingomyelin occurred in kidney, muscle, testis, intestine and liver. Similar alterations in lipid composition were found in plasma and significant correlations between plasma and membrane cholesterol/phospholipid and phosphatidylcholine/sphingomyelin molar ratios were found for those organs in which significant lipid changes occurred during fasting. Lizards fasted for 35 days and then refed a natural diet, such that they started to regain weight, had normal membrane lipid compositions. These observations suggest that during prolonged fasting certain tissues of the lizard are unable to maintain a normal membrane lipid composition and that abnormal plasma lipoproteins may be involved in this process.

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.

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.

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.

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.

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.

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.

Characterization and potential uses of rabbit polyclonal antibodies against human plasma lecithin-cholesterol acyltransferase.

Familial and secondary deficiency of plasma lecithin-cholesterol acyltransferase (LCAT) produce circulating lipoprotein particles with gross structural and compositional abnormalities; these have adverse effects on a variety of cellular functions. Factors affecting hepatic synthesis and secretion of this plasma enzyme are largely unknown but, potentially, some of them can be investigated with monospecific antibodies. In the present study, enzymically active LCAT was purified 40,000-fold from human plasma and then used to raise polyclonal antibodies in New Zealand White rabbits. Addition of this antiserum (1 microliter) to human plasma (25 microlitres) completely inhibited LCAT activity, although it was less effective against plasma from other species. The antibodies appeared to be monospecific to plasma LCAT. They gave a single precipitin arc by crossed immunoelectrophoresis, while immunodiffusion established that there was no cross-reactivity with several apolipoproteins or with serum albumin. Moreover, the antiserum was successfully used to detect LCAT in normal human plasma by Laurell rocket immunoelectrophoresis. By contrast, Western blotting of plasma proteins using whole LCAT antiserum was largely unsuccessful because of high background staining, although this could be substantially reduced by use of an IgG fraction. However, the whole antiserum readily immunoprecipitated LCAT secreted into the culture medium of HepG2 cells, a human hepatoblastoma cell line, pre-labelled with [35S]methionine, the [35S]-labelled LCAT appearing as a narrow 65-kDa protein band by electrophoresis and fluorography. We conclude that polyclonal antibodies may be an important tool to investigate the characteristics and underlying mechanisms of secondary LCAT deficiencies, including those associated with hepatic cirrhosis and schistosomiasis.

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.

[The prevalence, infection indices and feeding habits of the Triatominae captured in an area of epidemiological surveillance]. / Prevalência, índices de infecção e hábitos alimentares de triatomíneos capturados em uma área de vigilância epidemiológica.

Prevalence of triatomines and their infection by Trypanosoma cruzi, were studied during 1974-1981, in the Administrative Region of Campinas, São Paulo, Brazil. This investigation was continued (1982-1986), blood meal tests with bird, marsupial, rodent and human antisera having been added. Panstrongylus megistus, predominant in the region, whose winged instars are often found infected by T. cruzi in inhabited houses, displayed and alimentary eclecticism. From a sample of 7,785, 14.78 percent reacted positively with human antisera. A similar, but less marked behaviour was observed in Rhodnius neglectus. Triatoma sordida, the second most important species in the Region, was neither demonstrated to be feeding on humans nor to be infected by T. cruzi. Changes in their natural habitat probably caused sharp rises in T. arthurneivai populations in the human dwellings. The capture of three specimens of Microtriatoma borbai marks the first occurrence of this species in the State São Paulo. "Focus investigation" in areas in the surveillance phase can detect triatomines in the process of domiciliation.

[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.

[Urban schistosomiasis and social and epidemiological heterogeneity in Campinas, São Paulo, Brazil]. / A esquistossomose urbana e a heterogeneidade social e epidemiológica da população do município de Campinas, São Paulo, Brasil.

This study deals with the relationship between schistosomiasis and urbanization, particularly the urbanization process experienced by cities in dependent capitalist countries, with large groups of people in precarious living conditions. Six hundred and ten notified autochthonous cases in Campinas during the period from 1970 to 1990 are analyzed through notification files with regard to patients socio-economic conditions. Autochthonous cases of schistosomiasis were demonstrated as having a more heterogeneous geographical distribution within the urban area than could be inferred from previous studies that correlate the occurrence of transmission with precarious sanitary conditions.

[Aspects of "bug reporting" activity by the population in the control of Panstrongylus megistus in 12 countries of the northwestern region of São Paulo State, Brazil, 1974 to 1983]. / Aspectos da atividade de "notificação de barbeiros "pela população no controle de Panstrongylus megistus em 12 municípios da região noroeste do Estado de São Paulo, Brasil, 1974 a 1983.

The importance of "bug reporting", on the part of the local human population, to the epidemiological surveillance of Chagas' disease in S. Paulo State is demonstrated. The variations the number of "bug reports" from the local human population in the period from 1974 to 1983 relating to an area, infested by Panstrongylus megistus, in the northeast of S. Paulo State are studied. An increase in the number of "bug reporting" occurring in October, November and December was observed; however, no connection was discovered between domiciliary infestation rate and the "bug report" rate in this specific area.

[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).

[Congenital malaria in the county of Leme, SP]. / Malária congênita no município de Leme, SP.

Congenital malaria associated to Plasmodium vivax in a newborn baby (14 days old) is reported. The mother lived in a malaria-prone area, and came up to the county of Leme, São Paulo state, 40 days before baby's birth. Congenital malaria was classified through precocious symptoms, absence of malaria in the county and no history of blood transfusion or parenteral intervention of any sort. The case arises interest because of the necessity of malaria diagnoses suspicion on febrile newborns in non endemic areas, since the mother have lived or stayed in a malarious region.

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.

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.