Dissimilarities in auditory tuning in midwife toads of the genus Alytes (Amphibia: Anura)

The auditory sensitivity in three species of the anuran genus Alytes (Alytidae) was examined to determine patterns of intra‐ and interspecific variation, relating these measurements to behavioural preferences measured in previous studies and to the adaptive and evolutionary significance of this sensory function. The audiograms obtained with multi‐unit recordings in the torus semicircularis of 13 Alytes cisternasii, 10 Alytes obstetricans, and eight Alytes dickhilleni show two regions of enhanced sensitivity, between approximately 100–500 and 1200–2400 Hz, with minimum thresholds at approximately 40 and 45 dB SPL, respectively. The mean and range of the high‐frequency region differed among species, although the sensitivity, measured as minimum thresholds, was similar. The region of high‐frequency sensitivity was centred at approximately the frequency of the advertisement call in A. cisternasii but, in A. obstetricans and A. dickhilleni, was centred at frequencies higher than the conspecific calls. These results contrast with preferences for lower frequencies exhibited by Alytes in female phonotactic and in male evoked vocal responses. Such loose relationships between signals and receivers suggest that the divergence of the sound communication system in Alytes has implied environmental and phylogenetic factors in addition to sexual selection processes.     

Effects of maternal leptin treatment during lactation on the body weight and leptin resistance of adult offspring

We investigate whether leptin treatment to lactating rats affects food intake, body weight and leptin serum concentration and its anorectic effect on their adult offspring. Lactating rats were divided into 2 groups: Lep-single injected with recombinant rat leptin (8 microg/100 g of body weight, daily for the last 3 consecutive days of lactation) and control group (C) that received the same volume of saline. After weaning all pups had free access to the control diet, their body weight and food intake were monitored at each 4 days until 180 days of age, when they were tested for its food intake and response to either leptin (0.5 mg/kg body wt, ip) or saline vehicle. The offspring of the leptin-treated dams gained more weight and had higher food intake from day 37 onward (p<0.05), higher amount of retroperitoneal white adipose tissue (RPWAT) (37%, p<0.05) and higher leptin serum concentration (40%, p<0.05) at 180 days of age compared to control group. The food intake at 2, 4, 6 and 24 h was unaffected after acute injection of leptin in these animals, suggesting resistance to the anorectic effect of leptin. The maternal leptin treatment during lactation makes their adult offspring more susceptible to overweight with resistance to the anorectic effect of leptin.     

Detection of Mycobacterium leprae in nasal mucosa biopsies by the polymerase chain reaction

Several discoveries about leprosy indicate that Mycobacterium leprae transmission mainly occurs by inhalation, and the nose is major port of entry and exit. The present study evaluated the clinical application of PCR for detection of M. leprae DNA in nasal mucosa biopsies in untreated leprosy patients (52) and their contacts (99) from the State Reference Center in Sanitary Dermatology and Leprosy, Uberlandia, MG, Brazil. PCR detection of a 372-base pair DNA fragment from M. leprae was accomplished in 36 (69.2%) patients, from which 34 (91.9%) of them were multibacillaries. Furthermore, PCR was positive in 3 (16.7%) of 18 slit-skin smear negative, 4 (25.0%) of 16 skin lesion BI negative, 8 (33.3%) of 24 nasal mucosa BI negative patients, and 10 of 99 contacts (10.1%). The presence of bacilli in 10.1% of the contacts may potentially reflect an occult leprosy, and these patients must be accompanied, followed by a chemoprophylaxy treatment. Considering all PCR results against clinical and BI classification of patients and controls, we have found a sensitivity of 69.2%, a specificity of 89.9%, and an accuracy of 82.8%. It has been demonstrated here through PCR of nasal biopsies that the bacillus invades the mucosa, passing through the nasal inferior turbinate to reach peripheral blood. Therefore, the molecular investigation of invasive nasal biopsies by PCR tests has proven to be useful in defining patients of higher risk of transmission and risk-group contacts, which is an important step to reach the World Health Organization objective towards the elimination of leprosy as a public health problem.     

Identification of a trafficking motif involved in the stabilization and polarization of P2X receptors

Extracellular ATP-gated channels (P2X receptors) define the third major family of ionotropic receptors, and they are expressed widely in nerve cells, muscles, and endocrine and exocrine glands. P2X subunits have two membrane-spanning domains, and a receptor is thought to be formed by oligomerization of three subunits. We have identified a conserved motif in the cytoplasmic C termini of P2X subunits that is necessary for their surface expression; mutations in this motif result in a marked reduction of the receptors at the plasma membrane because of a rapid internalization. Transfer of the motif to a reporter protein (CD(4)) enhances the surface expression of the chimera, indicating that this motif is likely involved in the stabilization of P2X receptor at the cell surface. In neurons, mutated P2X(2) subunits showed reduced membrane expression and an altered axodendritic distribution. This motif is also present in intracellular regions of other membrane proteins, such as in the third intracellular loop of some G protein-coupled receptors, suggesting that it might be involve in their cellular stabilization and polarization.     

Stability of green fluorescent protein (GFP) in chlorine solutions of varying pH

Green fluorescent protein (GFP) is an excellent biosensor as a result of its ability to be easily monitored in a wide variety of applications. Enzymes and proteins have been used as biological indicators to evaluate the immediate efficacy of industrial procedures, such as blanching, pasteurization, and disinfection treatments, as well as to monitor the satisfactory preservation of a product subjected to disinfection or sterilization. The purpose of this work was to study GFP stability in chlorinated water for injection (WFI) and chlorinated buffered solutions at various pH ranges, with and without agitation, to evaluate the exposure time required for chlorine to decrease 90% of its fluorescence intensity (decimal reduction time, D-value, min, 25 degrees C). Fluorescence intensity (Ex/Emmax = 394/509 nm) was measured immediately after the addition of GFP (8.0-9.0 microg/mL) into buffered or unbuffered chlorine solutions with or without constant stirring. With solutions constantly stirred, GFP fluorescence decreased abruptly on contact with chlorine in concentrations greater than 150 ppm, with D-values between 1.3 min (147 ppm chlorine) and 1.7 min (183 ppm chlorine). In phosphate buffered chlorine solutions (pH = 7.15 +/- 0.08), GFP retained its structure between 52 and 94 ppm, but protein stability decreased 10-fold when exposed to 110 ppm chlorine. The recovery of GFP fluorescence intensity due to renaturation was observed between 30 and 100 ppm chlorine in WFI (final pH = 11.01 +/- 0.23) without stirring. Stirring enhanced the contact between GFP and chlorine throughout the assay and provided a more accurate D-value evaluation. GFP performed as a suitable fluorescent marker for monitoring disinfection effectiveness.     

P2X4 Activation Modulates Volume-sensitive Outwardly Rectifying Chloride Channels in Rat Hepatoma Cells

Volume-sensitive outwardly rectifying (VSOR) Cl⁻ channels are critical for the regulatory volume decrease (RVD) response triggered upon cell swelling. Recent evidence indicates that H₂O₂ plays an essential role in the activation of these channels and that H₂O₂ per se activates the channels under isotonic isovolumic conditions. However, a significant difference in the time course for current onset between H₂O₂-induced and hypotonicity-mediated VSOR Cl⁻ activation is observed. In several cell types, cell swelling induced by hypotonic challenges triggers the release of ATP to the extracellular medium, which in turn, activates purinergic receptors and modulates cell volume regulation. In this study, we have addressed the effect of purinergic receptor activation on H₂O₂-induced and hypotonicity-mediated VSOR Cl⁻ current activation. Here we show that rat hepatoma cells (HTC) exposed to a 33% hypotonic solution responded by rapidly activating VSOR Cl⁻ current and releasing ATP to the extracellular medium. In contrast, cells exposed to 200 μm H₂O₂ VSOR Cl⁻ current onset was significantly slower, and ATP release was not detected. In cells exposed to either 11% hypotonicity or 200 μm H₂O₂, exogenous addition of ATP in the presence of extracellular Ca²⁺ resulted in a decrease in the half-time for VSOR Cl⁻ current onset. Conversely, in cells that overexpress a dominant-negative mutant of the ionotropic receptor P2X4 challenged with a 33% hypotonic solution, the half-time for VSOR Cl⁻ current onset was significantly slowed down. Our results indicate that, at high hypotonic imbalances, swelling-induced ATP release activates the purinergic receptor P2X4, which in turn modulates the time course of VSOR Cl⁻ current onset in a extracellular Ca²⁺-dependent manner.     

Impact on growth and aflatoxin B1 accumulation by Kluyveromyces isolates at different water activity conditions

This study showed the impact on germination, mycelial growth and aflatoxin B₁ accumulation when interacting Aspergillus aflatoxigenic strains with Kluyveromyces isolates and the effect of water activity on this relationship. Isolates Y₁₄ and Y₁₆ reduced the percentage of germination of all Aspergillus strains and decrease germ tube elongation rate at majority of water activity assayed. Similarly they produced an increase of germination lag phase and lag phase of growth beside decreased growth rate of all Aspergillus strains. At water activities 0.994, 0.982, 0.955 and 0.937, no aflatoxins were produced in paired cultures with isolates Y_25, Y₂₂, Y₁₆, and Y₁₄, and Kluyveromyces isolates Y₁₄ and Y₁₆ impact both growth and aflatoxin accumulation at wide range of water activity.     

Modeling H3 histone N-terminal tail and linker DNA interactions

Molecular dynamics computer simulations were performed for the 25-residue N-terminal tail of the H3 histone protein in the proximity of a DNA segment of 10 base pairs (bp), representing a model for the linker DNA in chromatin. Several least biased configurations were used as initial configurations. The secondary structure content of the protein was increased by the presence of DNA close to it, but the locations of the secondary motifs were different for different initial orientations of the DNA grooves with respect to the protein. As a common feature to all simulations, the electrostatic attraction between negatively charged DNA and positively charged protein was screened by the water solvent and counterbalanced by the intrinsic compaction of the protein due to hydrophobic effects. The protein secondary structure limited the covering of DNA by the protein to 4-5 bp. The degree of compaction and charge density of the bound protein suggests a possible role of H3 tail in a nonspecific bending and plasticity of the linker DNA when the protein is located in the crowded dense chromatin.     

The effects of paradoxical sleep deprivation on estrous cycles of the female rats

The present purpose was to examine how sleep deprivation affects the estrous cycle of the female rat. Paradoxical sleep-deprived (PSD) adult female Wistar rats were compared to home-cage control (CTRL) on their estrous cyclicity. Forty-four PSD and forty-four CTRL female rats were distributed into 4 subgroups of 11 animals each according to the phase of estrous cycle and were subjected to sleep deprivation for 96 h by the multiple platform technique. After PSD period, vaginal estrous cycles were taken for an additional 9 days. Animals that were submitted to PSD in diestrus phase (PSD-diestrus) had their estrous cycles disrupted during the recovery period by showing a constant diestrus during the first week. As for hormone alterations, progesterone concentrations were statistically higher in PSD-diestrus compared to respective phase control and to PSD-proestrus and PSD-estrus rats while CTRL-metestrus had higher levels than CTRL-proestrus and estrus groups. Testosterone was significantly decreased in PSD-estrus in relation to PSD-proestrus and PSD-diestrus groups and was lower in CTRL-diestrus rats than in home-cage rats in proestrus. In addition, PSD-diestrus phase exhibited higher concentrations of corticosterone and lower estrogen than the respective control rats. These data indicate that PSD may modulate the ovarian hormone release through alterations in hormonal-neurochemical mechanisms.     

Time-Series Evolution of Toxic Organisms and Related Environmental Factors in a Brackish Ecosystem of the Mediterranean Sea

In the framework of the EU Project STRATEGY, a short-term study was carried out in the Marinello ecosystem, a small brackish area located on the Tyrrhenian coast of Sicily (Italy). The investigation was aimed at understanding the dynamics of phytoplankton toxic blooms in relation to other planktonic species and environmental conditions. The study started on 10 March 2003, in coincidence with the first detection of Alexandrium minutum, a dinoflagellate known as a producer of Paralyzing Shellfish Toxins (PST) and lasted until 4 June 2003, when the bloom collapsed. The specific identity of A. minutum was confirmed on field mixed samples, through the use of species-specific PCR-primers targeting the 5.8S rDNA-ITS regions. Water samples and phytoplankton net hauls were taken approximately at 10 days intervals in the Verde Pond, one of the five basins of the Marinello ecosystem, in order to evaluate the incidence of toxic and non-toxic dinoflagellate species over the whole planktonic community. The evolution of the main environmental and trophic parameters (temperature, salinity, dissolved oxygen, POC, C/N, DIN, PO₄–P) was simultaneously investigated. Alexandrium blooms were mostly characterized by A. minutum (max. 6 × 10⁵ cells l⁻¹ on April 11) and Alexandrium tamarense as an associated species (max. 2.5 × 10⁴ cells l⁻¹ on March 25). During the bloom, dinoflagellates or small flagellates dominated over the other taxa, with a minimum incidence of diatoms. The load of dissolved inorganic nitrogen was maximum in the pre-bloom phase (29 μM on March 19), after which it decreased sharply. An oxygen supersaturation event was registered in coincidence with the A. minutum bloom. The amounts of POC ranged between 266 and 658 μg l⁻¹ showing a discontinuous temporal trend. A recent introduction of A. minutum into the Verde Pond is suggested on the basis of the absence of this species in past years.