Caracterização molecular de DNA de Delta papillomavirus bovino (BPV1, 2 e 13) em sarcoides equinos

Resumo:Sarcoides são tumores fibroblásticos, considerados os tumores de pele mais comuns em pele de equinos e que raramente apresentam regressão espontânea. Papilomavírus bovino (BPV) tipos 1 e 2 são relacionados com a patogenia do sarcoide e, provavelmente, o BPV tipo 13 (BPV13), recentemente descrito, também pode estar associado com a formação dessa lesão. Neste estudo, 20 amostras de lesões cutâneas, sendo 12 constituídas por tecidos frescos e 8 amostras de tecido fixado em formalina e embebido em parafina, provenientes de 15 cavalos foram utilizadas para a identificação do DNA de BPV. A análise histopatológica (HE) confirmou todas as lesões como sarcoide. Para a amplificação do DNA de papilomavírus (PV) foram realizadas três reações de PCR. Como triagem, os primers IFNR2/IDNT2 foram utilizados para amplificar um fragmento da ORF L1 do PV. O segundo par de primersutilizado é complementar a sequência dos genes E5 e L2 de BPVs 1, 2 e 13. O terceiro par de primers(FAP59/FAP64) utilizado tem o gene L1 como alvo. A primeira e a segunda PCRs permitiram amplificar produtos em todas as amostras avaliadas. Entretanto, na terceira reação, na qual foram utilizados os primers FAP, foi possível amplificar produtos com tamanho molecular esperado somente nas amostras constituídas por tecidos frescos. O sequenciamento de nucleotídeos e as análises filogenéticas realizadas nos fragmentos E5L2 resultaram na identificação de BPV1, 2 e 13 em 14 (70%), 2 (10%) e em 4 (20%) amostras de sarcoides, respectivamente. As amostras de sarcoides de um dos animais continha somente o DNA de BPV1. Entretanto, nas amostras provenientes do segundo cavalo foi possível identificar o DNA de três tipos de Deltapapillomavirus bovino (BPV1, 2 e 13) em lesões distintas. Este estudo ratifica a presença do DNA de BPV1, 2 e 13 em lesões de sarcoides em equinos, além de identificar três tipos de BPVs em um mesmo animal e descrever pela primeira vez no Brasil a presença de BPV1 e 2 nesse tipo de lesão.

A single-step purification of bothropstoxin-1

Bothrops venoms are complex mixtures of components with a wide range of biological activities. Among these substances, myotoxins have been investigated by several groups. Bothropstoxin-1 (Bthtx-1) is a phospholipase A2-like basic myotoxin from Bothrops jararacussu. The purification of this component involves two chromatographic steps. Although providing a pure material, the association of these two steps is time consuming and a single-step method using high performance chromatography media would be useful. In the present study, we describe a single-step purification method for Bthtx-1. Bothrops jararacussu venom was dissolved in 1 ml buffer. After centrifugation, the supernatant was injected into a Resource-S cation exchange column connected to an FPLC system and eluted with a linear salt gradient. The complete procedure took 20 min, representing a considerable time gain when compared to a previously described method (Homsi-Brandenburgo MI et al. (1988) Toxicon, 26: 615-627). Bthtx-1 purity and identity, assessed by SDS-PAGE and N-terminal sequencing, resulted in a single band with a molecular mass of about 14 kDa and the expected sequence of the first 5 residues, S-L-F-E-L. Although the amount of protein purified after each run is lower than in the previously described method, we believe that this method may be useful for small-scale purifications.

Structure and function of the selectin ligand PSGL-1

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric mucin-like 120-kDa glycoprotein on leukocyte surfaces that binds to P- and L-selectin and promotes cell adhesion in the inflammatory response. The extreme amino terminal extracellular domain of PSGL-1 is critical for these interactions, based on site-directed mutagenesis, blocking monoclonal antibodies, and biochemical analyses. The current hypothesis is that for high affinity interactions with P-selectin, PSGL-1 must contain O-glycans with a core-2 branched motif containing the sialyl Lewis x antigen (NeuAca2®3Galß1®4 1;Fuca1®3]GlcNAcß1®R). In addition, high affinity interactions require the co-expression of tyrosine sulfate on tyrosine residues near the critical O-glycan structure. This review addresses the biochemical evidence for this hypothesis and the evidence that PSGL-1 is an important in vivo ligand for cell adhesion.

Galectin-1, an alternative signal for T cell death, is increased in activated macrophages

Galectin-1 belongs to an evolutionarily conserved family of animal ß-galactoside-binding proteins, which exert their functions by crosslinking the oligosaccharides of specific glycoconjugate ligands. During the past decade, attempts to identify the functional role of galectin-1 suggested participation in the regulation of the immune response. Only in the last few years has the molecular mechanism involved in these properties been clearly elucidated, revealing a critical role for galectin-1 as an alternative signal in the generation of T cell death. In the present study we will discuss the latest advances in galectin research in the context of the regulation of the immune response, not only at the central level but also at the periphery. Moreover, we will review the purification, biochemical properties and functional significance of a novel galectin-1-like protein from activated rat macrophages, whose expression is differentially regulated according to the activation state of the cells. The novel role of a carbohydrate-binding protein in the regulation of apoptosis is providing a breakthrough in galectin research and extending the interface between immunology, glycobiology and clinical medicine.

Effects of aluminum sulfate on delta-aminolevulinate dehydratase from kidney, brain, and liver of adult mice

The purpose of the present study was to investigate the in vitro and in vivo effects of aluminum sulfate on delta-aminolevulinic acid dehydratase (ALA-D) activity from the brain, liver and kidney of adult mice (Swiss albine). In vitro experiments showed that the aluminum sulfate concentration needed to inhibit the enzyme activity was 1.0-5.0 mM (N = 3) in brain, 4.0-5.0 mM (N = 3) in liver and 0.0-5.0 mM (N = 3) in kidney. The in vivo experiments were performed on three groups for one month: 1) control animals (N = 8); 2) animals treated with 1 g% (34 mM) sodium citrate (N = 8) and 3) animals treated with 1 g% (34 mM) sodium citrate plus 3.3 g% (49.5 mM) aluminum sulfate (N = 8). Exposure to aluminum sulfate in drinking water inhibited ALA-D activity in kidney (23.3 ± 3.7%, mean ± SEM, P<0.05 compared to control), but enhanced it in liver (31.2 ± 15.0%, mean ± SEM, P<0.05). The concentrations of aluminum in the brain, liver and kidney of adult mice were determined by graphite furnace atomic absorption spectrometry. The aluminum concentrations increased significantly in the liver (527 ± 3.9%, mean ± SEM, P<0.05) and kidney (283 ± 1.7%, mean ± SEM, P<0.05) but did not change in the brain of aluminum-exposed mice. One of the most important and striking observations was the increase in hepatic aluminum concentration in the mice treated only with 1 g% sodium citrate (34 mM) (217 ± 1.5%, mean ± SEM, P<0.05 compared to control). These results show that aluminum interferes with delta-aminolevulinate dehydratase activity in vitro and in vivo. The accumulation of this element was in the order: liver > kidney > brain. Furthermore, aluminum had only inhibitory properties in vitro, while in vivo it inhibited or stimulated the enzyme depending on the organ studied.

Frequency of islet cell autoantibodies (IA-2 and GAD) in young Brazilian type 1 diabetes patients

Type 1 diabetes, as an autoimmune disease, presents several islet cell-specific autoantibodies such as islet cell antibody (ICA), anti-insulin, anti-glutamic acid decarboxylase (GAD) and the antibody (Ab) against tyrosine phosphatase (PTP)-like protein known as ICA-512 (IA-2). In order to determine the frequency of the anti-GAD and anti-IA-2 autoantibodies in Brazilian type 1 diabetes patients we studied 35 diabetes mellitus (DM) type 1 patients with recent-onset disease (£12 months) and 37 type 1 diabetes patients with long-duration diabetes (>12 months) who were compared to 12 children with normal fasting glucose. Anti-GAD65 and anti-IA-2 autoantibodies were detected with commercial immunoprecipitation assays. The frequency of positive results in recent-onset DM type 1 patients was 80.0% for GADAb, 62.9% for IA-2Ab and 82.9% for GADAb and/or IA-2Ab. The long-duration type 1 diabetes subjects presented frequencies of 54.1% for GADAb and IA-2Ab, and 67.5% for GAD and/or IA-2 antibodies. The control group showed no positive cases. Anti-GAD and IA-2 assays showed a high frequency of positivity in these Brazilian type 1 diabetes patients, who presented the same prevalence as a Caucasian population.

Antidepressant-like effect of lead in adult mice

It has been reported that lead can cause behavioral impairment by inhibiting the N-methyl-D-aspartate (NMDA) receptor complex. MK-801, a noncompetitive NMDA receptor antagonist, exhibits an antidepressant-like action in the forced swimming test. The purpose of the present study was to determine whether subacute lead exposure in adult male Swiss mice weighing 30-35 g causes an antidepressant-like action in a forced swimming test. Mice were injected intraperitoneally (ip) with 10 mg/kg lead acetate or saline daily for 7 consecutive days. Twenty-four hours after the last treatment, the saline and lead-treated mice received an injection of MK-801 (0.01 mg/kg, ip) or saline and were tested in forced swimming and in open-field tests. Immobility time was similarly reduced in the saline-MK-801, Pb-saline and Pb-MK-801 groups compared to the saline-saline group (mean ± SEM; 197.3 ± 18.5, 193.5 ± 15.8, 191.3 ± 12.3 and 264.0 ± 14.4 s, respectively; N = 9). These data indicate that lead may exert its effect on the forced swimming test by directly or indirectly inhibiting the NMDA receptor complex. Lead treatment caused no deficit in memory of habituation and did not affect locomotor activity in an open-field (N = 14). However, mice that received MK-801 after lead exhibited a deficit in habituation (22% reduction in rearing responses between session 3 and 1; N = 14) as compared to control (41% reduction in rearing responses; N = 15), further suggesting that lead may have affected the NMDA receptor activity. Forced-swim immobility in a basin in two daily consecutive sessions was also significantly decreased by lead exposure (mean ± SEM; day 1 = 10.6 ± 3.2, day 2 = 19.6 ± 3.6; N = 16) as compared to control (day 1 = 18.4 ± 3.8, day 2 = 34.0 ± 3.7; N = 17), whereas the number of crossings was not affected by lead treatment, further indicating a specific antidepressant-like action of lead.

Kallikrein-like amidase activity in renal ischemia and reperfusion

We assessed a kallikrein-like amidase activity probably related to the kallikrein-kinin system, as well as the participation of leukocyte infiltration in renal ischemia and reperfusion. Male C57BL/KSJmdb mice were subjected to 20 or 60 min of ischemia and to different periods of reperfusion. A control group consisted of sham-operated mice, under similar conditions, except for ischemia induction. Kallikrein-like amidase activity, Evans blue extravasation and myeloperoxidase activity were measured in kidney homogenates, previously perfused with 0.9% NaCl. Plasma creatinine concentration increased only in the 60-min ischemic group. After 20 min of ischemia and 1 or 24 h of reperfusion, no change in kallikrein-like amidase activity or Evans blue extravasation was observed. In the mice subjected to 20 min of ischemia, edema was evident at 1 h of reperfusion, but kidney water content returned to basal levels after 24 h of reperfusion. In the 60-min ischemic group, kallikrein-like amidase activity and Evans blue extravasation showed a similar significant increase along reperfusion time. Kallikrein-like amidase activity increased from 4 nmol PNA mg protein-1 min-1 in the basal condition to 15 nmol PNA mg protein-1 min-1 at 10 h of reperfusion. For dye extravasation the concentration measured was near 200 µg of Evans blue/g dry tissue in the basal condition and 1750 µg of Evans blue/g dry tissue at 10 h of reperfusion. No variation could be detected in the control group. A significant increase from 5 to 40 units of DAbs 655 nm g wet tissue-1 min-1 in the activity of the enzyme myeloperoxidase was observed in the 60-min ischemic group, when it was evaluated after 24 h of reperfusion. Histological analysis of the kidneys showed migration of polymorphonuclear leukocytes from the vascular bed to the interstitial tissue in the 60-min ischemic group after 24 h of reperfusion. We conclude that the duration of ischemia is critical for the development of damage during reperfusion and that the increase in renal cortex kallikrein-like amidase activity probably released from both the kidney and leukocytes may be responsible, at least in part, for the observed effects, probably through direct induction of increased vascular permeability.

Reaction of diphenyl diselenide with hydrogen peroxide and inhibition of delta-aminolevulinate dehydratase from rat liver and cucumber leaves

The interaction of the product of H2O2 and (PhSe)2 with delta-aminolevulinate dehydratase (delta-ALA-D) from mammals and plants was investigated. (PhSe)2 inhibited rat hepatic delta-ALA-D with an IC50 of 10 µM but not the enzyme from cucumber leaves. The reaction of (PhSe)2 with H2O2 for 1 h increased the inhibitory potency of the original compound and the IC50 for animal delta-ALA-D inhibition was decreased from 10 to 2 µM. delta-ALA-D from cucumber leaves was also inhibited by the products of reaction of (PhSe)2 with H2O2 with an IC50 of 4 µM. The major product of reaction of (PhSe)2 with H2O2 was identified as seleninic acid and produced an intermediate with a lambdamax at 265 nm after reaction with t-BuSH. These results suggest that the interaction of (PhSe)2 with mammal delta-ALA-D requires the presence of cysteinyl residues in close proximity. Two cysteine residues in spatial proximity have been recently described for the mammalian enzyme. Analysis of the primary structure of plant delta-ALA-D did not reveal an analogous site. In contrast to (PhSe)2, seleninic acid, as a result of the higher electrophilic nature of its selenium atom, may react with additional cysteinyl residue(s) in mammalian delta-ALA-D and also with cysteinyl residues from cucumber leaves located at a site distinct from that found at the B and A sites in mammals. Although the interaction of organochalcogens with H2O2 may have some antioxidant properties, the formation of seleninic acid as a product of this reaction may increase the toxicity of organic chalcogens such as (PhSe)2.

Absence-like seizures in adult rats following pilocarpine-induced status epilepticus early in life

Administration of pilocarpine causes epilepsy in rats if status epilepticus (SE) is induced at an early age. To determine in detail the electrophysiological patterns of the epileptogenic activity in these animals, 46 Wistar rats, 7-17 days old, were subjected to SE induced by pilocarpine and electro-oscillograms from the cortex, hippocampus, amygdala, thalamus and hypothalamus, as well as head, rostrum and vibrissa, eye, ear and forelimb movements, were recorded 120 days later. Six control animals of the same age range did not show any signs of epilepsy. In all the rats subjected to SE, iterative spike-wave complexes (8.1 ± 0.5 Hz in frequency, 18.9 ± 9.1 s in duration) were recorded from the frontal cortex during absence fits. However, similar spike-wave discharges were always found also in the hippocampus and, less frequently, in the amygdala and in thalamic nuclei. Repetitive or single spikes were also detected in these same central structures. Clonic movements and single jerks were recorded from all the rats, either concomitantly with or independently of the spike-wave complexes and spikes. We conclude that rats made epileptic with pilocarpine develop absence seizures also occurring during paradoxical sleep, showing the characteristic spike-wave bursts in neocortical areas and also in the hippocampus. This is in contrast to the well-accepted statement that one of the main characteristics of absence-like fits in the rat is that spike-wave discharges are never recorded from the hippocampal fields.

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.

The compulsive-like aspect of the head dipping emission in rats with chronic electrolytic lesion in the area of the median raphe nucleus

Head dipping (HD) is a behavioral pattern considered to have a risk assessment or an exploratory role and is used as a complementary parameter to evaluate anxiety in experimental animals. Since rats with electrolytic lesion in the area of the median raphe nucleus displayed high frequencies of HD in a previous study, the present investigation was undertaken to confirm this observation and to determine its anxiety-related origin. HD episodes were counted in adult male Wistar rats (270-350 g) with electrolytic lesion (N = 11) and sham-lesioned controls (N = 12). When HD was measured for 60 min on an elevated open platform, lesioned rats emitted 13 times more HD than controls (264.7 ± 93.3 vs 20.3 ± 7.6 episodes), with the difference being statistically significant (P < 0.05). HD counts during 10-min sessions held 7, 14, 21, 27, and 63 days after lesion showed significantly higher means (range: 28.14 ± 5.38 to 62.85 ± 9.48) compared to sham-lesioned controls (range: 7.37 ± 1.13 to 8.5 ± 1.45). Normal rats stepped down into their home cages when the vertical distance between them and the cage was short (16 cm), and the step-down latencies increased with increasing depths (36.7 ± 7.92 to 185.87 ± 35.44 s). Lesioned rats showed a similar behavior when facing the shortest depth, but had a significantly increased number (23.28 ± 2.35 episodes) and latency (300 ± 0.00 s) of HD compared to normal rats (9.25 ± 1.37 episodes and 185.87 ± 35.44 s) when facing the greatest depth (30 cm). This suggests that HD may be a depth-measuring behavior related to risk assessment.

A role for histone-like protein H1 (H-NS) in the regulation of hemolysin expression by Serratia marcescens

The histone-like protein H1 (H-NS) is an abundant structural component of the bacterial nucleoid and influences many cellular processes including recombination, transcription and transposition. Mutations in the hns gene encoding H-NS are highly pleiotropic, affecting the expression of many unrelated genes. We have studied the role of H-NS on the regulation of hemolysin gene expression in Serratia marcescens. The Escherichia coli hns mutant carrying S. marcescens hemolysin genes on a plasmid constructed by ligation of the 3.2-kb HindIII-SacI fragment of pR02 into pBluescriptIIKS, showed a high level of expression of this hemolytic factor. To determine the osmoregulation of wild-type and hns defective mutants the cells were grown to mid-logarithmic phase in LB medium with 0.06 or 0.3 M NaCl containing ampicillin and kanamycin, whereas to analyze the effect of pH on hemolysin expression, the cells were grown to late-logarithmic phase in LB medium buffered with 0.1 M Tris-HCl, pH 4.5 to 8.0. To assay growth phase-related hemolysin production, bacterial cells were grown in LB medium supplemented with ampicillin and kanamycin. The expression of S. marcescens hemolysin genes in wild-type E. coli and in an hns-defective derivative at different pH and during different growth phases indicated that, in the absence of H-NS, the expression of hemolysin did not vary with pH changes or growth phases. Furthermore, the data suggest that H-NS may play an important role in the regulation of hemolysin expression in S. marcescens and its effect may be due to changes in DNA topology influencing transcription and thus the amount of hemolysin expression. Implications for the mechanism by which H-NS influences gene expression are discussed.

Activation of a P2Y4-like purinoceptor triggers an increase in cytosolic [Ca2+] in the red blood cells of the lizard Ameiva ameiva (Squamata, Teiidae)

An increasing number of pathophysiological roles for purinoceptors are emerging, some of which have therapeutic potential. Erythrocytes are an important source of purines, which can be released under physiological and physiopathological conditions, acting on purinergic receptors associated with the same cell or with neighboring cells. Few studies have been conducted on lizards, and have been limited to ATP agonist itself. We have previously shown that the red blood cells (RBCs) of the lizard Ameiva ameiva store Ca2+ in the endoplasmic reticulum (ER) and that the purinergic agonist ATP triggers a rapid and transient increase of [Ca2+]c by mobilization of the cation from internal stores. We also reported the ability of the second messenger IP3 to discharge the ER calcium pool of the ER. Here we characterize the purinoceptor present in the cytoplasmic membrane of the RBCs of the lizard Ameiva ameiva by the selective use of ATP analogues and pyrimidine nucleotides. The nucleotides UTP, UDP, GTP, and ATPgammaS triggered a dose-dependent response, while interestingly 2MeSATP, 2ClATP, alpha, ß-ATP, and ADP failed to do so in a 1- to 200-µm con- centration. The EC50 obtained for the compounds tested was 41.77 µM for UTP, 48.11 µM for GTP, 53.11 µM for UDP, and 30.78 µM for ATPgammaS. The present data indicate that the receptor within the RBCs of Ameiva ameiva is a P2Y4-like receptor due to its pharmacological similarity to the mammalian P2Y4 receptor.

Perspectives of digestive pest control with proteinase inhibitors that mainly affect the trypsin-like activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)

The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4ºC and the supernatants were used in enzymatic assays at 30ºC, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis) was 1.14 ± 0.15 absorbance variation min-1 mg protein-1, at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis) was 0.217 ± 0.02 mmol p-nitroaniline min-1 mg protein-1. The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50ºC, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.