Effect of gamma irradiation on the behavioral properties of crotoxin

Crotoxin has been detoxified with gamma radiation in order to improve crotalic antiserum production. Nevertheless, present knowledge of the biological characteristics of irradiated crotoxin is insufficient to propose it as an immunizing agent. Crotoxin is known to increase the emotional state of rats and to decrease their exploratory behavior (Moreira EG, Nascimento N, Rosa GJM, Rogero JR and Vassilieff VS (1996) Brazilian Journal of Medical and Biological Research, 29: 629-632). Therefore, we decided 1) to evaluate the effects of crotoxin in the social interaction test, which has been widely used for the evaluation of anxiogenic drugs, and 2) to determine if irradiated crotoxin induces behavioral alterations similar to those of crotoxin in the social interaction, open-field and hole-board tests. Male Wistar rats (180-220 g) were used. Crotoxin (100, 250, and 500 µg/kg) was injected intraperitoneally 2 h before the social interaction test. Similarly, irradiated crotoxin (2000 Gy gamma radiation from a 60Co source) was administered at the doses of 100, 250, and 500 µg/kg for the hole-board test, and at the doses of 1000 and 2500 µg/kg for the open-field and social interaction tests. ANOVA complemented with the Dunnett test was used for statistical analysis (P<0.05). Crotoxin decreased the social interaction time (s) at the doses of 100, 250 and 500 µg/kg (means ± SEM) from 51.6 ± 4.4 to 32.6 ± 3.7, 28.0 ± 3.6 and 31.6 ± 4.4, respectively. Irradiated crotoxin did not induce behavioral alterations. These results indicate that 1) crotoxin may be an anxiogenic compound, and 2) in contrast to crotoxin, irradiated crotoxin was unable to induce behavioral alterations, which makes it a promising compound for the production of crotalic antiserum

Hyperalgesic effect induced by barbiturates, midazolam and ethanol: pharmacological evidence for GABA-A receptor involvement

The involvement of GABA-A receptors in the control of nociception was studied using the tail-flick test in rats. Non-hypnotic doses of the barbiturates phenobarbital (5-50 mg/kg), pentobarbital (17-33 mg/kg), and thiopental (7.5-30 mg/kg), of the benzodiazepine midazolam (10 mg/kg) or of ethanol (0.4-1.6 g/kg) administered by the systemic route reduced the latency for the tail-flick response, thus inducing a 'hyperalgesic' state in the animals. In contrast, non-convulsant doses of the GABA-A antagonist picrotoxin (0.12-1.0 mg/kg) administered systemically induced an increase in the latency for the tail-flick response, therefore characterizing an 'antinociceptive' state. Previous picrotoxin (0.12 mg/kg) treatment abolished the hyperalgesic state induced by effective doses of the barbiturates, midazolam or ethanol. Since phenobarbital, midazolam and ethanol reproduced the described hyperalgesic effect of GABA-A-specific agonists (muscimol, THIP), which is specifically antagonized by the GABA-A antagonist picrotoxin, our results suggest that GABA-A receptors are tonically involved in the modulation of nociception in the rat central nervous system

Pituitary-thyroid axis in short- and long-term experimental diabetes mellitus

Short-term experimental diabetes mellitus (DM) produces a significant decrease in serum thyroid hormones, a decreased or normal serum thyroid-stimulating hormone (TSH) and a reduction in hepatic and renal T4-5'-deiodination. However, little is known about the effects of chronic diabetes mellitus on the pituitary-thyroid axis function. We evaluated the changes induced by very short-term (6 days), short-term (15 days) and chronic (6 months) streptozotocin-induced diabetes mellitus in 3-month old female Dutch-Miranda rat serum T4, serum TSH and T4-5'-deiodinase activity in the thyroid and pituitary glands. Serum hormones were determined by specific radioimmunoassays. Iodothyronine-5'-deiodinase activities were assayed in the thyroid and pituitary microsomal fractions using 2 µM T4 as substrate. Mean serum T4 was significantly decreased from 3.3 to 2.0 µg/dl 6 days after diabetes mellitus induction, and from 2.2 to 1.5 µg/dl after 15 days of DM, with no significant changes in serum TSH, indicating a decreased pituitary TSH responsiveness to the diminished suppression by T4, even though pituitary T4-5'-deiodinase activity was unchanged. Thyroid T4-5'-deiodinase was unchanged after 6 days of diabetes mellitus, but was significantly increased from 20.6 to 37.0 pmol T3/mg protein after 15 days. Six months after diabetes mellitus induction, both serum T4 and thyroid T4-5'-deiodinase returned to normal ranges and serum TSH was unchanged, although pituitary T4-5'-deiodinase was now significantly decreased from 2.7 to 1.7 pmol T3/mg protein. These findings indicate that some kind of adaptation to chronic insulinopenia may occur at the thyroid level, but this does not seem to be true for the pituitary

Worker honeybee hemolymph lipid composition and synodic lunar cycle periodicities

The aim of the present investigation was to extend a previous study, showing a correlation of the variations of hemolymph carbohydrates with synodic lunar-like cycle and its circaseptan harmonics to worker honeybee hemolymph lipids. Hemolymph lipid concentrations of emerging worker imagos were analyzed in terms of one ideal synodic lunar cycle and processed by the cosinor method testing the null hypothesis versus the presence of 29.5-, 14.8- or 7.4-day periods in the data. A rhythmicity statistically compatible with a 29.5-day rhythm was observed for triacylglycerols and steroids as well as for body weight. A circadiseptan rhythm was determined for 1,3 diacylglycerols, while fatty acids and phospholipids exhibited a circaseptan rhythm. An agreement of peaks for triacylglycerols, steroids and body weight at the new moon, but not at the full moon, was noted with respect to trehalose and glucose circadiseptan rhythms. The latter moon-phase timing of peaks and nadirs, compared with that previously determined for trehalose and glucose, appeared to be identical to the circadiseptan rhythm and reciprocal for the circaseptan rhythms of 1,3 diacylglycerols. Reciprocal tendencies in circaseptans of trehalose and glucose on the one hand, and fatty acids and phospholipids on the other are indicated. The underlying causal nexus of these relationships is unknown

Animal models of anxiety: an ethological perspective

In the field of anxiety research, animal models are used as screening tools in the search for compounds with therapeutic potential and as simulations for research on mechanisms underlying emotional behaviour. However, a solely pharmacological approach to the validation of such tests has resulted in distinct problems with their applicability to systems other than those involving the benzodiazepine/GABAA receptor complex. In this context, recent developments in our understanding of mammalian defensive behaviour have not only prompted the development of new models but also attempts to refine existing ones. The present review focuses on the application of ethological techniques to one of the most widely used animal models of anxiety, the elevated plus-maze paradigm. This fresh approach to an established test has revealed a hitherto unrecognized multidimensionality to plus-maze behaviour and, as it yields comprehensive behavioural profiles, has many advantages over conventional methodology. This assertion is supported by reference to recent work on the effects of diverse manipulations including psychosocial stress, benzodiazepines, GABA receptor ligands, neurosteroids, 5-HT1A receptor ligands, and panicolytic/panicogenic agents. On the basis of this review, it is suggested that other models of anxiety may well benefit from greater attention to behavioural detail

Evolution of circadian organization in vertebrates

Circadian organization means the way in which the entire circadian system above the cellular level is put together physically and the principles and rules that determine the interactions among its component parts which produce overt rhythms of physiology and behavior. Understanding this organization and its evolution is of practical importance as well as of basic interest. The first major problem that we face is the difficulty of making sense of the apparently great diversity that we observe in circadian organization of diverse vertebrates. Some of this diversity falls neatly into place along phylogenetic lines leading to firm generalizations: i) in all vertebrates there is a "circadian axis" consisting of the retinas, the pineal gland and the suprachiasmatic nucleus (SCN), ii) in many non-mammalian vertebrates of all classes (but not in any mammals) the pineal gland is both a photoreceptor and a circadian oscillator, and iii) in all non-mammalian vertebrates (but not in any mammals) there are extraretinal (and extrapineal) circadian photoreceptors. An interesting explanation of some of these facts, especially the differences between mammals and other vertebrates, can be constructed on the assumption that early in their evolution mammals passed through a "nocturnal bottleneck". On the other hand, a good deal of the diversity among the circadian systems of vertebrates does not fall neatly into place along phylogenetic lines. In the present review we will consider how we might better understand such "phylogenetically incoherent" diversity and what sorts of new information may help to further our understanding of the evolution of circadian organization in vertebrates

Calcium handling by vascular myocytes in hypertension

Calcium ions (Ca2+) trigger the contraction of vascular myocytes and the level of free intracellular Ca2+ within the myocyte is precisely regulated by sequestration and extrusion mechanisms. Extensive evidence indicates that a defect in the regulation of intracellular Ca2+ plays a role in the augmented vascular reactivity characteristic of clinical and experimental hypertension. For example, arteries from spontaneously hypertensive rats (SHR) have an increased contractile sensitivity to extracellular Ca2+ and intracellular Ca2+ levels are elevated in aortic smooth muscle cells of SHR. We hypothesize that these changes are due to an increase in membrane Ca2+ channel density and possibly function in vascular myocytes from hypertensive animals. Several observations using various experimental approaches support this hypothesis: 1) the contractile activity in response to depolarizing stimuli is increased in arteries from hypertensive animals demonstrating increased voltage-dependent Ca2+ channel activity in hypertension; 2) Ca2+ channel agonists such as Bay K 8644 produce contractions in isolated arterial segments from hypertensive rats and minimal contraction in those from normotensive rats; 3) intracellular Ca2+ concentration is abnormally increased in vascular myocytes from hypertensive animals following treatment with Ca2+ channel agonists and depolarizing interventions, and 4) using the voltage-clamp technique, the inward Ca2+ current in arterial myocytes from hypertensive rats is nearly twice as large as that from myocytes of normotensive rats. We suggest that an alteration in Ca2+ channel function and/or an increase in Ca2+ channel density, resulting from increased channel synthesis or reduced turnover, underlies the increased vascular reactivity characteristic of hypertension

Control of the phosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in the immature rat hippocampus by glutamate and calcium ions: possible key factor in astrocytic plasticity

The present review describes recent research on the regulation by glutamate and Ca2+ of the phosphorylation state of the intermediate filament protein of the astrocytic cytoskeleton, glial fibrillary acidic protein (GFAP), in immature hippocampal slices. The results of this research are discussed against a background of modern knowledge of the functional importance of astrocytes in the brain and of the structure and dynamic properties of intermediate filament proteins. Astrocytes are now recognized as partners with neurons in many aspects of brain function with important roles in neural plasticity. Site-specific phosphorylation of intermediate filament proteins, including GFAP, has been shown to regulate the dynamic equilibrium between the polymerized and depolymerized state of the filaments and to play a fundamental role in mitosis. Glutamate was found to increase the phosphorylation state of GFAP in hippocampal slices from rats in the post-natal age range of 12-16 days in a reaction that was dependent on external Ca2+. The lack of external Ca2+ in the absence of glutamate also increased GFAP phosphorylation to the same extent. These effects of glutamate and Ca2+ were absent in adult hippocampal slices, where the phosphorylation of GFAP was completely Ca2+-dependent. Studies using specific agonists of glutamate receptors showed that the glutamate response was mediated by a G protein-linked group II metabotropic glutamate receptor (mGluR). Since group II mGluRs do not act by liberating Ca2+ from internal stores, it is proposed that activation of the receptor by glutamate inhibits Ca2+ entry into the astrocytes and consequently down-regulates a Ca2+-dependent dephosphorylation cascade regulating the phosphorylation state of GFAP. The functional significance of these results may be related to the narrow developmental window when the glutamate response is present. In the rat brain this window corresponds to the period of massive synaptogenesis during which astrocytes are known to proliferate. Possibly, glutamate liberated from developing synapses during this period may signal an increase in the phosphorylation

Genomic characterization of Brazilian hepatitis C virus genotypes 1a and 1b

Parts of 5' non-coding (5' NC) and of E1 envelope regions of the hepatitis C virus (HCV) genome were amplified from sera of 26 Brazilian anti-HCV antibody-positive patients using the reverse transcription-polymerase chain reaction (RT-PCR). Fourteen samples were PCR positive with primers from the 5' NC region and 8 of them were also positive with primers from the E1 region. A genomic segment of 176 bp from the E1 region of 7 isolates was directly sequenced from PCR products. The sequences were compared with those of HCV strains isolated in other countries and the Brazilian isolates were classified by phylogenetic analysis into genotypes 1a and 1b. This could have a clinical importance since it has been shown that individuals infected with type 1 viruses are less likely to respond to treatment with interferon than individuals infected with types 2 and 3 viruses. Two quasispecies isolated from the same patient with an interval of 13 months differed by two base substitutions (1.1%). The sequence of another isolate presented a three-nucleotide deletion at codon 329

Assessment of the degree of contamination of rat germ cell preparations using specific cDNA probes

Recent reports showing a decrease in sperm count in men have brought new concerns about male infertility. Animal models have been widely used to provide some relevant information about the human male gamete, and extrapolations are made to men and to the clinical context. The present study assesses one of the methods used for separation of germ cells of the adult rat testis, namely centrifugal elutriation followed by density gradients (Percoll®). This method was chosen since it presents the best results for cell purity in separating germ cells from the rat testis. A comparison between continuous and discontinuous Percoll® gradients was performed in order to identify the best type of gradient to separate the cells. Maximal cell purity was obtained for spermatocytes (81 ± 8.2%, mean ± SEM) and spermatids (84 ± 2.6%) using centrifugal elutriation followed by continuous Percoll® gradients. A significant difference in purity was observed between elongating spermatids harvested from continuous Percoll® gradients and from discontinuous gradients. Molecular analysis was used to assess cell contamination by employing specific probes, namely transition protein 2 (TP2), mitochondrial cytochrome C oxidase II (COX II), and sulfated glycoprotein 1 (SGP1). Molecular analysis of the samples demonstrated that morphological criteria are efficient in characterizing the main composition of the cell suspension, but are not reliable for identifying minimal contamination from other cells. Reliable cell purity data should be established using molecular analysis

Synergistic effect of glucose and prolactin on GLUT2 expression in cultured neonatal rat islets

We studied the synergistic effect of glucose and prolactin (PRL) on insulin secretion and GLUT2 expression in cultured neonatal rat islets. After 7 days in culture, basal insulin secretion (2.8 mM glucose) was similar in control and PRL-treated islets (1.84 ± 0.06% and 2.08 ± 0.07% of the islet insulin content, respectively). At 5.6 and 22 mM glucose, insulin secretion was significantly higher in PRL-treated than in control islets, achieving 1.38 ± 0.15% and 3.09 ± 0.21% of the islet insulin content in control and 2.43 ± 0.16% and 4.31 ± 0.24% of the islet insulin content in PRL-treated islets, respectively. The expression of the glucose transporter GLUT2 in B-cell membranes was dose-dependently increased by exposure of the islet to increasing glucose concentrations. This effect was potentiated in islets cultured for 7 days in the presence of 2 µg/ml PRL. At 5.6 and 10 mM glucose, the increase in GLUT2 expression in PRL-treated islets was 75% and 150% higher than that registered in the respective control. The data presented here indicate that insulin secretion, induced by different concentrations of glucose, correlates well with the expression of the B-cell-specific glucose transporter GLUT2 in pancreatic islets

Mini-Mu insertions in the tetracycline resistance determinant from Proteus mirabilis

The inducible tetracycline resistance determinant isolated from Proteus mirabilis cloned into the plasmid pACYC177 was mutagenized by insertion of a mini-Mu-lac phage in order to define the regions in the cloned sequences encoding the structural and regulatory proteins. Three different types of mutants were obtained: one lost the resistance phenotype and became Lac+; another expressed the resistance at lower levels and constitutively; the third was still dependent on induction but showed a lower minimal inhibitory concentration. The mutant phenotypes and the locations of the insertions indicate that the determinant is composed of a repressor gene and a structural gene which are not transcribed divergently as are other known tetracycline determinants isolated from Gram-negative bacteria

Diclofenac plasma protein binding: PK-PD modelling in cardiac patients submitted to cardiopulmonary bypass

Twenty-four surgical patients of both sexes without cardiac, hepatic, renal or endocrine dysfunctions were divided into two groups: 10 cardiac surgical patients submitted to myocardial revascularization and cardiopulmonary bypass (CPB), 3 females and 7 males aged 65 ± 11 years, 74 ± 16 kg body weight, 166 ± 9 cm height and 1.80 ± 0.21 m2 body surface area (BSA), and control, 14 surgical patients not submitted to CPB, 11 female and 3 males aged 41 ± 14 years, 66 ± 14 kg body weight, 159 ± 9 cm height and 1.65 ± 0.16 m2 BSA (mean ± SD). Sodium diclofenac (1 mg/kg, im Voltaren 75® twice a day) was administered to patients in the Recovery Unit 48 h after surgery. Venous blood samples were collected during a period of 0-12 h and analgesia was measured by the visual analogue scale (VAS) during the same period. Plasma diclofenac levels were measured by high performance liquid chromatography. A two-compartment open model was applied to obtain the plasma decay curve and to estimate kinetic parameters. Plasma diclofenac protein binding decreased whereas free plasma diclofenac levels were increased five-fold in CPB patients. Data obtained for analgesia reported as the maximum effect (EMAX) were: 25% VAS (CPB) vs 10% VAS (control), P<0.05, median measured by the visual analogue scale where 100% is equivalent to the highest level of pain. To correlate the effect versus plasma diclofenac levels, the EMAX sigmoid model was applied. A prolongation of the mean residence time for maximum effect (MRTEMAX) was observed without any change in lag-time in CPB in spite of the reduced analgesia reported for these patients, during the time-dose interval. In conclusion, the extent of plasma diclofenac protein binding was influenced by CPB with clinically relevant kinetic-dynamic consequences