A novel SDS-stable dimer of a heterogeneous nuclear ribonucleoprotein at presynaptic terminals of squid neurons.

The presence of mRNAs in synaptic terminals and their regulated translation are important factors in neuronal communication and plasticity. Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes are involved in the translocation, stability, and subcellular localization of mRNA and the regulation of its translation. Defects in these processes and mutations in components of the hnRNP complexes have been related to the formation of cytoplasmic inclusion bodies and neurodegenerative diseases. Despite much data on mRNA localization and evidence for protein synthesis, as well as the presence of translation machinery, in axons and presynaptic terminals, the identity of RNA-binding proteins involved in RNA transport and function in presynaptic regions is lacking. We previously characterized a strongly basic RNA-binding protein (p65), member of the hnRNPA/B subfamily, in squid presynaptic terminals. Intriguingly, in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), p65 migrated as a 65-kDa protein, whereas members of the hnRNPA/B family typically have molecular masses ranging from 35 to 42kDa. In this report we present further biochemical and molecular characterization that shows endogenous p65 to be an SDS-stable dimer composed of ∼37-kDa hnRNPA/B-like subunits. We cloned and expressed a recombinant protein corresponding to squid hnRNPA/B-like protein and showed its propensity to aggregate and form SDS-stable dimers in vitro. Our data suggest that this unique hnRNPA/B-like protein co-localizes with synaptic vesicle protein 2 and RNA-binding protein ELAV and thus may serve as a link between local mRNA processing and presynaptic function and regulation.

Efeito do extrato aquoso e etanólico do angico preto sobre larvas de Rhipicephalus (Boophilus) microplus / The effect of aqueous and ethanolic extracts from the angico Preto about larvae of Rhipicephalus (Boophilus) microplus

Rhipicephalus (Boophilus) microplus representa um grande problema na bovinocultura e o uso de acaricidas é a medida de controle profilático e terapêutico mais comum contra esses ectoparasitos. Os principais problemas relacionados com essa prática dizem respeito ao desenvolvimento de linhagens resistentes de carrapatos. Assim, objetivou-se determinar o efeito de extratos da casca de Anadenanthera macrocarpa sobre as larvas de R. (B.) microplus, obtidas de um pool de ovos, acondicionadas em tubo de polietileno. De acordo com os resultados, o extrato aquoso na concentração de 8,26mg.mL-1 causou 85% de mortalidade nas primeiras 12 horas. Quanto ao extrato etanólico, observou-se maior mortalidade nas concentrações 12,5; 6,25 e 1,56mg.mL-1, em torno de 84%, percentuais semelhantes ao amitraz. Os controles negativos não apresentaram mortalidade durante o experimento. Assim, tanto o extrato aquoso como o extrato etanólico apresentaram efeito larvicida, embora o extrato etanólico tenha sido mais eficiente para a espécie, podendo ser uma alternativa no controle desse ectoparasito.

Rhipicephalus (Boophilus) microplus represents a major problem in cattle breeding and the use of acaricides is the most common prophylactic and therapeutic control measure against these ectoparasites. The main problems with this practice relate to the development of resistant strains of ticks. Thus, the objective was to determine the effect of bark extracts from Anadenanthera macrocarpa on the larvae of R.(B.) microplus, obtained from a pool of eggs packed in a polyethylene tube. According to the results, the aqueous extract at a concentration of 8.26mg.mL-1caused 85% mortality in the first 12 hours. As for the ethanolic extract, higher mortality of about 84%, a percentage similar to amitraz, was observed at concentrations of 12.5mg.mL-1, 6.25mg.mL-1 and 1.56mg.mL-1. The negative controls showed no mortality during the experiment. Thus, both the aqueous and ethanolic extracts showed larvicidal activity, although the ethanolic extract has been more efficient and could be an alternative to control this ectoparasite.

The combination of infrared and microwave radiation to quantify trace elements in organic samples by ICP OES.

Sample-decomposition methods using microwave radiation in closed systems have been commonly used in the analysis of inorganic constituents; however, these methods are limited to small amounts of organic samples. This work proposes the combined use of infrared radiation and microwave radiation (IR-MW) to increase the amount of organic samples digested. The determination of Al, Ca, Cu, Fe, K, Mg, Mn, Na, P and Zn in human-feed samples was accomplished by ICP OES. The results were in agreement with those obtained from conventional decomposition by microwave radiation (closed system). The results obtained using the proposed IR-MW system for standard reference material (whole milk powder, NIST 8435) were also compared. Agreements of 85-100% were obtained for Al, Ca, Cu, Fe, K, Mg, Na, P and Zn in the standard reference material. The IR-MW system is simple to implement and cheap because it uses commercially available infrared lamps and allows the use of infrared radiation in the microwave-digestion vessel. Additionally, it is possible to reach better precision in the analysis of the human-feed samples using the IR-MW system. The proposed method also allows total digestion of large sample amounts or samples rich in organic compounds can also be performed in the IR-MW system using small volumes of nitric acid.

The role of mitochondrial function in glutamate-dependent metabolism in neuronal cells.

Glutamate is an important neurotransmitter in neurons and glial cells and it is one of the keys to the neuron-glial interaction in the brain. Glutamate transmission is strongly dependent on calcium homeostasis and on mitochondrial function. In the present work we presented several aspects related to the role of mitochondria in glutamate signaling and in brain diseases. We focused on glutamateinduced calcium signaling and its relation to the organelle dysfunction with cell death processes. In addition, we have discussed how alterations in this pathway may lead or aggravate a variety of neurodegenerative diseases. We compiled information on how mitochondria can influence cell fate during glutamate stimulation and calcium signaling. These organelles play a pivotal role in neuron and glial exchange, in synaptic plasticity and several pathological conditions related to Aging, Alzheimer's, Parkinson's and Huntington's diseases. We have also presented autophagy as a mechanism activated during mitochondrial dysfunction which may function as a protective mechanism during injury. Furthermore, some new perspectives and approaches to treat these neurodegenerative diseases are offered and evaluated.

Alterations in calcium signaling and a decrease in Bcl-2 expression: possible correlation with apoptosis in aged striatum.

Aging is a multifaceted process associated with various functional and structural deficits that might be evolved in degenerative diseases. It has been shown that neurodegenerative disorders are associated with alterations in Ca(2+) homeostasis. Thus, in the present work, we have investigated Ca(2+) signaling and apoptosis in aged striatum. Our results show that glutamate and NMDA evoke a greater Ca(2+) rise in striatum slices from aged animals. However, this difference is not present when glutamate is tested in the absence of external Ca(2+). Immunostaining of glutamate receptors shows that only NMDA receptors (NR1) are increased in the striatum of aged rats. Increases in mitochondrial Ca(2+) content and in the reactive oxygen species levels were also observed in aged animals, which could be associated with tissue vulnerability. In addition, a decrease in the Bcl-2 protein expression and an enhancement in apoptosis were also present in aged striatum. Together the results indicate that, in aged animals, alterations in Ca(2+) handling coupled to an increase in ROS accumulation and a decrease in the prosurvival protein Bcl-2 may contribute to apoptosis induction and cell death in rat striatum.

Alterations in Calcium Signalingand a Decrease in Bcl-2 Expression: Possible Correlation With Apoptosisin Aged Striatum

Aging is a multifaceted process associated with various functional and structural deficits that might be evolved in degenerative diseases. It has been shown that neurodegenerative disorders are associated with alterations in Ca2+ homeostasis. Thus, in the present work, we have investigated Ca2+ signaling and apoptosis in aged striatum. Our results show that glutamate and NMDA evoke a greater Ca2+ rise in striatum slices from aged animals. However, this difference is not present when glutamate is tested in the absence of external Ca2+. Immunostaining of glutamate receptors shows that only NMDA receptors (NR1) are increased in the striatum of aged rats. Increases in mitochondrial Ca2+ content and in the reactive oxygen species levels were also observed in aged animals, which could be associated with tissue vulnerability. In addition, a decrease in the Bcl-2 protein expression and an enhancement in apoptosis were also present in aged striatum. Together the results indicate that, in aged animals, alterations in Ca2+ handling coupled to an increase in ROS accumulation and a decrease in the prosurvival protein Bcl-2 may contribute to apoptosis induction and cell death in rat striatum.

Bcl-x(L) inhibits Bax-induced alterations in mitochondrial respiration and calcium release.

Apoptosis is a natural cell elimination process involved in a number of physiological and pathological events. This process can be regulated by members of the Bcl-2 family. Bax, a pro-apoptotic member of this family, accelerates cell death, while the pro-survival member, Bcl-x(L), can antagonize the pro-apoptotic function of Bax to promote cell survival. In the present study, we have evaluated the effect of Bcl-x(L) on Bax-induced alterations in mitochondrial respiration and calcium release. We found that in primary cultured astrocytes, recombinant Bcl-x(L) is able to antagonize Bax-induced decrease in mitochondrial respiration and increase in mitochondrial calcium release. In addition, we found that Bcl-x(L) can lower the calcium store in the endoplasmic reticulum, thus limiting potential calcium flux induced by apoptosis. This regulation of calcium flux by Bcl-x(L) may represent an important mechanism by which this protein promotes cell survival.

Increase in bax expression and apoptosis are associated in Huntington's disease progression.

Huntington's disease (HD) is a hereditary dominant neurodegenerative disorder and the progression of the disease may be associated with apoptosis and altered expression of apoptotic proteins. The aim of this study was to investigate gene expression of bax and bcl-2 in tissues from R6/1 transgenic (TGN) mice of different ages (3, 6 and 9 months). The mRNA expression was investigated and related to apoptotic cells measured by TUNEL. Results showed a significant and progressive increase in bax levels in the cortex of TGN (from 10 to 33%) when compared to control (CT) (8 to 20%) mice with 3, 6 and 9-month-old. The increase in bax was correlated with the elevation in the number of apoptotic nuclei, especially in the cortex of 6 (10%) and 9 (18%)-month-old mice. Increase in bax expression might be related to an apoptotic induction which contributes to the HD progression.

Prevalence of IgG diphtheria antitoxin in blood donors in Rio de Janeiro.

The lack of information on the immunity of adults in Brazil against diphtheria prompted us to analyse sera from 234 blood donors aged 18-61 years (30.3% females and 69.7% males). IgG diphtheria antitoxin levels determined by means of an ELISA, validated by toxin neutralization test in Vero cells, showed that 30.7% (95% CI 25.0-37.1) of the population was fully protected (>or=1 IU/ml). The highest percentage of subjects fully protected was in the 31-40 years age group. Most of the subjects with uncertain or no protection (<1 IU/ml) were found in the 18-30 years age group (43.8%, OR 2.18, P=0.01). Antitoxin levels were not influenced by the increase in age. Males were more protected than females (80.5%, OR 0.44, P=0.01). The prevalence of 30% of individuals fully protected against diphtheria in blood donors in Rio de Janeiro supports the fact that immunity to diphtheria among healthy Brazilian adults is inadequate. To avoid diphtheria epidemics in the future the immunity among adults should be raised in the coming years.

Effect of unilateral nephrectomy on renal function of diabetic rats.

Glomerular alterations of experimental diabetes mellitus are observed in animals submitted to a reduction in renal mass, suggesting that some mechanisms responsible for the progression of renal disease are common. The aim of this study was to investigate the effect of nephrectomy on the renal function and morphology of diabetic rats. Male Wistar rats were divided into 4 groups: control (C), n=8; diabetic (DM), n=8; non-diabetic nephrectomized (Nx), n=8; (DMNx), n=9. DM was induced by streptozotocin (65 mg/Kg), and animals were treated with insulin. After 12 weeks, the glomerular filtration rate (GFR), renal plasma flow (RPF) and mean arterial pressure (MAP) were evaluated in unanaesthetized animals. Glomerular volume (GV), glomerular sclerosis index (GSI), mesangial volume density (Vvmes) and glomerular capillary surface density (Svcap) were also evaluated. Results show that kidney weight increased in Nx groups, being higher in DMNx. GFR was higher in Nx groups as was RPF, being higher in DMNx. RVR was lower in Nx groups, especially in DMNx. MAP was not different among the groups. RPF and GFR showed a high correlation for the DMNx group (r=0.95, p=0.02). The DMNx group showed a correlation between RVR and GFR (r=-0.96, p=0.005). The GV increased in Nx groups, and the GSI was higher in DMNx. Vvmes and Svcap increased in DMNx group. In summary, Nx groups developed similar degrees of glomerular hypertrophy, but only DMNx showed an increased value for GSI. The present data suggest that the acceleration of glomerular lesions in DMNx animals was more closely associated to hemodynamic adaptations than to glomerular hypertrophy.

Flow-injection spectrophotometric determination of ascorbic acid in pharmaceutical products with the Prussian Blue reaction.

A lot of modern analytical strategies for exploiting chemistries have been developed by using flow-injection analysis. However, even after 20 years of flow-injection evolution, there still are new quantitative procedures being established using old qualitative assays. The formation of Prussian Blue is a classical test to detect Fe(2+) using hexacyanoferrate(III) as a precipitating reagent. This reaction was evaluated for spectrophotometric determination of ascorbic acid employing Fe(3+) and hexacyanoferrate(III) as chromogenic reagents. An excess of the complexing anion avoids the formation of precipitate and forms a deep blue solution when Fe(3+) is reduced to Fe(2+) by ascorbic acid. The maximum absorbance of the colored complex occurs at 700 nm and the molar absorptivity is 3.0 x 10(4) 1 mol(-1) cm(-1). Under flow-injection conditions the Prussian Blue reaction was employed with an intermittent flow of an oxalate alkaline solution for removing the colored product adsorbed on tube and flow-cell walls. Reference solutions containing 5.0 x 10(-6)-1.0 x 10(-4) M of ascorbic acid were employed to obtain the analytical curve (r = 0.9999). For all solutions the relative standard deviation was lower than 1.0% (n=10). Results obtained for ascorbic acid determination in pharmaceutical products (Cewin, Redoxon and Cebion) are in good agreement with those obtained by using a flow-injection procedure involving the reaction between triiodide and ascorbic acid. The sampling frequency is 140 h(-1) and only 430 microl of reagents is consumed in each determination.

Urinary tract infection in men with AIDS.

OBJECTIVE--To investigate whether bacteriuria and, specifically, symptomatic urinary tract infection (UTI) occur with increased frequency in men with HIV infection. METHODS--In this cross-sectional study we investigated three groups of men, aged from 18 to 50 years. Group A was composed of patients with a diagnosis of AIDS; Group B, of patients without HIV infection, and group C of patients with asymptomatic HIV infection. Patients with any known predisposing factor for UTI were excluded from the study. A clean-catch midstream urine sample was collected from each patient on the first day of hospital admission (groups A and B) or during a visit to the outpatient clinic (group C). Bacteriuria was diagnosed when > or = 100,000 colony forming units/ml, urine were grown. RESULTS--There were 415 patients, 151 in group A, 170 in group B and 94 in group C. Bacteriuria was significantly more frequently in group A (20 cases, 13.3%) than in groups B (3 cases, 1.8%, p = 0.00007) and C (3 cases, 3.2%, p = 0.009). Ten cases of bacteriuria in group A (6.6%) were symptomatic while no case of symptomatic UTI was seen in groups B (p = 0.0004) and C (p = 0.008). The frequency of UTI in homosexual men with AIDS (7 cases, 6.7%) was not significantly different from that observed in men with AIDS who denied homosexuality (3 cases, 6.5%). E coli was the predominant pathogen associated with UTI. Although adequate response to a two-week course of antibiotics was observed in most cases, an in-hospital mortality rate of 20% was found among AIDS patients with symptomatic UTI. CONCLUSIONS--In the present study, the frequency of bacteriuria and symptomatic UTI was found to be increased in men with AIDS. E coli was the predominant pathogen in these cases. These data suggest that symptomatic UTI may represent a relevant cause of morbidity for men with AIDS.

Glomerular disease and human immunodeficiency virus infection in Brazil.

Clinically overt glomerular disease was detected in 6 (1.1%) of 543 patients with HIV infection followed at a Brazilian National Referral Center for AIDS. In 4 cases, glomerulosclerosis was present (focal and segmental in 3, diffuse and global in 1) and rapid progression to terminal renal failure was observed 1-10 months after clinical presentation. The other 2 patients died with normal renal function, and autopsy studies suggested the diagnosis of minimal change disease. Clinically overt glomerular disease was significantly more common among Black patients, whether all the cases with glomerulopathy (p < 0.001) or just the cases with glomerular sclerosis were considered (p = 0.011). Autopsy study of renal fragments from patients without clinical evidence of glomerular disease was additionally performed and revealed the presence of focal and segmental glomerulosclerosis in 3 cases (7.5%). We concluded that a glomerulopathy with clinicopathological features which match the definition of HIV nephropathy can be found among Brazilian patients with HIV infection. Accordingly to what has been described in American series, Brazilian Black patients seem to be at increased risk of the development of that nephropathy.