Lead in liver and kidney of exposed rats: Aging accumulation study

The concentration of lead in liver and kidneys of Wistar rats, fed with lead since fetal period in relation to their age and to a control group, was determined. A group of rats was exposed to lead acetate (n = 30) in drinking water and the other group was exposed to normal water (n = 20). Samples were collected from rats aging between 1 and 11 months and were analyzed by Energy Dispersive X-ray Fluorescence (EDXRF) without any chemical preparation. The EDXRF results were assessed by the PIXE (Proton Induced X-ray Emission) technique. The formaldehyde used to preserve the samples was also analyzed by ETAAS (Electro-Thermal Atomic Absorption Spectrometry) in order to verify if there was any loss of lead from the samples to the formaldehyde. We found that the loss was not significant (<2%).Concerning the mean values of the lead concentration measured in the contaminated soft tissues, in liver they range from 6 to 22 μg g^?1, and in kidneys from 44 to 79 μg g^?1. The control rats show, in general, values below the EDXRF detection limit (2 μg g^?1). The ratio kidney/liver ranges from 2 to 10 and is strongly positively correlated with the age of the animals. A Spearman correlation matrix to investigate the correlation between elemental concentrations and the dependence of these concentrations with age showed that there is a strong positive correlation with age for lead in the liver but not in the kidney. The correlation matrix showed also that the concentration of lead in these two soft tissues is not correlated. The lead accumulation in liver is made by different plateaus that strongly decrease with age. It was verified the existence of two levels of accumulation in kidney, not very highlighted, which might be indicative of a maximum accumulation level for lead in kidney.     

Electromyographic evaluation of upper limb muscles involved in armwrestling sport simulation during dynamic and static conditions

ObjectiveTo evaluate the electromyographic activity of the Pectoralis Major (PM), Biceps Brachii (BB), Pronator Teres (PT) and Flexor Carpi Ulnaris (FCU) muscles involved in simulated armwrestling.MethodsTen trained volunteers were selected to perform the armwrestling movement, during dynamic tests with 40% and 80% of maximum voluntary load (MVL) and static tests in the initial, intermediary and final positions. Electromyographic and force data were normalized for analyses.ResultsIn dynamic tests with 40% MVL, electric activity of the PT muscle was greater than FCU (p < 0.01) and BB (p < 0.05) muscles, and with 80% MVL, PM and PT muscles were the most active. In static tests, electric activity increased from the initial to final positions for the PM muscle (p < 0.05), while it decreased for the BB and PT muscles (p < 0.001 and p < 0.05, respectively). No significant changes were observed for force and no correlation was found with the simultaneous electric activity.ConclusionsIt can be concluded that the PM and FCU muscles participate as agonists in the simulated armwrestling whereas the BB and PT muscles seem to perform secondary functions. Electric activity showed to be dependent on the load and on the position of the upper limb, but not on the force produced during the movement.     

Expanding the genetic toolbox for Leptospira species by generation of fluorescent bacteria

Our knowledge of the genetics and molecular basis of the pathogenesis associated with Leptospira, in comparison to those of other bacterial species, is very limited. An improved understanding of pathogenic mechanisms requires reliable genetic tools for functional genetic analysis. Here, we report the expression of gfp and mRFP1 genes under the control of constitutive spirochetal promoters in both saprophytic and pathogenic Leptospira strains. We were able to reliably measure the fluorescence of Leptospira by fluorescence microscopy and a fluorometric microplate reader-based assay. We showed that the expression of the gfp gene had no significant effects on growth in vivo and pathogenicity in L. interrogans. We constructed an expression vector for L. biflexa that contains the lacI repressor, an inducible lac promoter, and gfp as the reporter, demonstrating that the lac system is functional in Leptospira. Green fluorescent protein (GFP) expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) in L. biflexa transformants harboring the expression vector. Finally, we showed that GFP can be used as a reporter to assess promoter activity in different environmental conditions. These results may facilitate further advances for studying the genetics of Leptospira spp.     

Specific α- and β-Tubulin Isotypes Optimize the Functions of Sensory Cilia in Caenorhabditis elegans

Primary cilia have essential roles in transducing signals in eukaryotes. At their core is the ciliary axoneme, a microtubule-based structure that defines cilium morphology and provides a substrate for intraflagellar transport. However, the extent to which axonemal microtubules are specialized for sensory cilium function is unknown. In the nematode Caenorhabditis elegans, primary cilia are present at the dendritic ends of most sensory neurons, where they provide a specialized environment for the transduction of particular stimuli. Here, we find that three tubulin isotypes—the α-tubulins TBA-6 and TBA-9 and the β-tubulin TBB-4—are specifically expressed in overlapping sets of C. elegans sensory neurons and localize to the sensory cilia of these cells. Although cilia still form in mutants lacking tba-6, tba-9, and tbb-4, ciliary function is often compromised: these mutants exhibit a variety of sensory deficits as well as the mislocalization of signaling components. In at least one case, that of the CEM cephalic sensory neurons, cilium architecture is disrupted in mutants lacking specific ciliary tubulins. While there is likely to be some functional redundancy among C. elegans tubulin genes, our results indicate that specific tubulins optimize the functional properties of C. elegans sensory cilia.THE fitness of all organisms depends on an ability to appropriately sense and respond to the environment. At the cellular level, many specific architectures have evolved to optimize these sensory functions. Prominent among these is the sensory cilium, a tubulin-based cytoplasmic extension that interrogates the extracellular environment in many biological contexts (Davenport and Yoder 2005; Berbari et al. 2009). Cilia are important for the transduction of a broad range of visual, auditory, mechanical, thermal, and chemical stimuli. They also function during development to receive a variety of signals, both chemical and mechanical, that regulate proliferation and differentiation (Goetz and Anderson 2010). Indeed, the disruption of cilium assembly and function can give rise to a spectrum of human diseases collectively known as ciliopathies (Berbari et al. 2009; Lancaster and Gleeson 2009). These disorders, which include autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD), Bardet–Biedl syndrome, Meckel–Gruber syndrome, and Joubert syndrome, are associated with a variety of pathogenic conditions including polycystic kidneys and neurological impairments.At the core of all cilia and flagella is the microtubule axoneme. This characteristic structural element comprises nine doublet outer microtubules that may surround a central pair, the presence of which often indicates a motile cilium/flagellum. Like all microtubule-based structures, ciliary axonemes are built of heterodimers of α- and β-tubulins, highly conserved small GTP-binding proteins. The recruitment of other cilium components, including signal transduction machinery, requires a conserved assembly and maintenance process called intraflagellar transport (IFT) (Blacque et al. 2008; Pedersen and Rosenbaum 2008). IFT employs two major complexes that transport ciliary cargo bidirectionally by traveling along the axonemal microtubules. Loss of individual IFT components can cause a broad spectrum of defects in the assembly, maintenance, and function of cilia.Important insights into cilium structure and function have come from studies of genetically tractable organisms, particularly the green alga Chlamydomonas and the nematode Caenorhabditis elegans (Bae and Barr 2008; Pedersen and Rosenbaum 2008). In C. elegans, sensory cilia are found exclusively at the dendritic ends of sensory neurons. These cilia constitute a highly specialized sensory environment characterized by localized sensory receptors and specific signaling components. Cilium morphology is quite distinctive in many of these cells and likely contributes to their functional specialization (Ward et al. 1975). Recent progress has shed light on the mechanisms that confer this specialization onto more general pan-ciliary pathways (Evans et al. 2006; Mukhopadhyay et al. 2007; Jauregui et al. 2008; Mukhopadhyay et al. 2008; Silverman and Leroux 2009).The genomes of many eukaryotes harbor multiple α- and β-tubulin genes. Two hypotheses, which are not mutually exclusive, have been proposed to account for these paralogs (Cleveland 1987; Wade 2007). At one extreme, different tubulin isotypes might be functionally redundant, such that their minor coding differences are largely irrelevant. According to this model, multiple genes allow the maintenance of a stable pool of available monomers and dimers. The small amount of sequence variation within the α- and β-tubulin families supports this idea, as do studies of functionally redundant mitotic tubulins in C. elegans (Ellis et al. 2004; Lu et al. 2004; Phillips et al. 2004; Lu and Mains 2005). The alternative hypothesis proposes that specific structures, e.g., ciliary axonemes or axonal microtubules, rely on tubulins optimized for specific roles. Support for this idea has come from studies of cultured mammalian neurons (Joshi and Cleveland 1989), Drosophila (Hutchens et al. 1997; Raff et al. 1997), and human tubulins (Vent et al. 2005; Jaglin et al. 2009). In Drosophila, studies of motile sperm flagella have revealed that the sperm-specific β2 tubulin isoform builds not only the specialized motile axoneme but also all other tubulin-based structures (Kemphues et al. 1982). However, sequences both within and outside the axoneme motif in the C-terminal tail of this tubulin isoform are required for the flagellar axoneme, and other closely related β-tubulins cannot support this role (Fuller et al. 1987; Raff et al. 1997; Popodi et al. 2008). Genetic interactions have provided evidence that β2 tubulin heterodimerizes with the α-tubulin 84B (Hays et al. 1989), which also possesses specific functional properties not provided by structurally similar α-tubulins (Hutchens et al. 1997). In C. elegans, a specific role for tubulin isoforms has been described in the six touch receptor neurons. These nonciliated cells harbor unusual 15-filament microtubules composed of dimers of the α-tubulin MEC-12 and the β-tubulin MEC-7. The loss of mec-7 or mec-12, the expression of which is largely restricted to these cells, results in the conversion of 15-filament microtubules to the standard 11-microfilament variety and a commensurate loss of light-touch response (Savage et al. 1989; Fukushige et al. 1999; Bounoutas et al. 2009). Thus experimental support exists for both of these opposing views, and it seems likely that the role of specific tubulin isoforms in regulating microtubule structure and function differs according to cell and organelle type.The C. elegans genome encodes nine α- and six β-tubulin genes (Gogonea et al. 1999). Some of these genes, particularly tba-1, tba-2, tbb-1, and tbb-2, are expressed broadly during embryogenesis and function redundantly in spindle assembly and positioning (Ellis et al. 2004; Lu et al. 2004; Phillips et al. 2004; Lu and Mains 2005). tba-1 and tbb-2 have also been recently shown to be important for axon outgrowth and synaptogenesis (Baran et al. 2010). Several others, including mec-7, mec-12, and the β-tubulin ben-1, have been identified through genetic screens for particular phenotypes, such as touch insensitivity or benzimidazole resistance (Driscoll et al. 1989; Savage et al. 1989; Fukushige et al. 1999). However, the extent to which specific tubulin isoforms are required for structural and functional diversity in the C. elegans nervous system remains unknown. Here, taking advantage of several existing genome-wide data sets, we identify the α-tubulins TBA-6 and TBA-9 and the β-tubulin TBB-4 as strong candidates for tubulins that have roles in sensory cilia. We find that each of these genes are expressed in characteristic, partially overlapping, sets of sensory neurons, where their products localize to ciliary axonemes. While the loss of any one (or all three) of these genes does not abolish ciliogenesis, tubulin mutants exhibit significant defects in the localization of cilium proteins and in some cilium-dependent behavioral responses. Together, our results indicate that specific α- and β-tubulin isoforms are important, although not essential, for the efficient assembly and function of specific classes of C. elegans sensory cilia. Sensory cilia throughout the animal kingdom may therefore employ specific tubulin isoforms to optimize their function.     

Sequence analysis of the growth hormone gene of the South American catfish Rhamdia quelen

Rhamdia quelen is an important Neotropical catfish species for fisheries and aquaculture in southern Brazil, where it is called Jandia. Like other native Brazilian species of economic importance, R. quelen genetics needs more attention for animal breeding programs. The growth hormone gene is known to be linked to a number of molecular markers and quantitative trait loci. We sequenced the coding region of the growth hormone gene with the primer walking technique. As in other Siluriformes, the R. quelen growth hormone gene has four introns and five exons, in a 1465-bp coding region. The tertiary structure of the encoded protein was predicted by bioinformatics; it has four α-helix structures connected by loops, which form a compressed complex maintained by two disulfide bridges.     

All three isoforms of the voltage-dependent anion channel (VDAC1, VDAC2, and VDAC3) are present in mitochondria from bovine, rabbit, and rat brain

All three isoforms of the voltage-dependent anion channel (VDAC) were detected by immunoblot analysis of mitochondria isolated from rat, rabbit, and bovine brain. All three isoforms were associated with mitochondria after fractionation of rat brain extracts on sucrose density gradients. No VDAC isoforms were detected in non-mitochondrial fractions. Relative levels of the mRNAs coding the VDAC isoforms in rat, rabbit, and bovine brain were determined by RT-PCR. In all three species, the mRNA for VDAC2 was predominant. Relative to the mRNA for VDAC3, mRNAs for both VDAC1 and VDAC2 were more highly expressed in bovine brain than in rat brain. These results are consistent with the possibility that differences in relative expression of VDAC isoforms may be a factor in determining the species-dependent ratio of Type A:Type B hexokinase binding sites on brain mitochondria.     

Genetic damage in exfoliated cells from oral mucosa of individuals exposed to X-rays during panoramic dental radiographies

The genotoxic effects of X-ray emitted during dental panoramic radiography were evaluated in exfoliated cells from oral epithelium through a differentiated protocol of the micronucleus test. Thirty-one healthy individuals agreed to participate in this study and were submitted to this procedure for diagnosis purpose after being requested by the dentist. All of them answered a questionnaire before the examination. Cells were obtained from both sides of the cheek by gentle scrapping with a cervical brush, immediately before the exposure and after 10 days. Cytological preparations were stained according to Feulgen-Rossenbeck reaction and analyzed under light and laser scanning confocal microscopies. Micronuclei, nuclear projections (buds and broken eggs) and degenerative nuclear alterations (condensed chromatin, karyolysis and karyorrhexis) were scored. The frequencies of micronuclei, karyolysis and pycnosis were similar before and after exposure (P > 0.90), whereas the condensation of the chromatin and the karyorrhexis increased significantly after exposure (P < 0.0001). In contrast, both bud and broken egg frequencies were significantly higher before the examination (P < 0.005), suggesting that these structures are associated to the normal epithelium differentiation. The results suggest that the X-ray exposure during panoramic dental radiography induces a cytotoxic effect by increasing apoptosis. We also believe that the score of other nuclear alterations in addition to the micronucleus improves the sensitivity of genotoxic effects detection.     

Comparative trajectories of active and S195A inactive trypsin upon binding to serpins

Serpins inhibit proteinases through a complicated multistep mechanism. The precise nature of these steps and the order by which they occur are still debated. We compared the fate of active and S195A inactive rat trypsin upon binding to alpha(1)-antitrypsin and P(1)-Arg-antichymotrypsin using stopped-flow kinetics with fluorescence resonance energy transfer detection and time-resolved fluorescence resonance energy transfer. We show that inhibition of active trypsin by these serpins leads to two irreversible complexes, one being compatible with the full insertion of the serpin-reactive site loop but not the other one. Binding of inactive trypsin to serpins triggers a large multistep reversible rearrangement leading to the migration of the proteinase to an intermediate position. Binding of inactive trypsin, unlike that of active trypsin, does not perturb the rhodamine fluorescence at position 150 on the helix F of the serpin. Thus, inactive proteinases do not migrate past helix F and do not trigger full serpin loop insertion.     

Effect of Chronic Central Administration of Glucagon-Like Peptide-1 (7–36) Amide on Food Consumption and Body Weight in Normal and Obese Rats

Glucagon-like peptide (7–36) amide (GLP-1) acutely inhibits food and water consumption in rats after intrace-rebroventricular (icv) administration. To assess the potential for desensitization of these effects, we investigated the effects of chronic icv administration of GLP-1 on food consumption and body weight in Sprague-Dawley (SD) rats and Zucker (fa/fa) obese rats. In vitro functional densensitization of the GLP-1 receptor was not observed after overnight exposure of Rin m5F insulinoma cells to GLP-1 at concentrations up to 10 nM. Administration of GLP-1 to SD rats (30 ug icv twice a day for 6 days) resulted in significant reductions in 24-hour food consumption each day (25 ±1%). Continuous icv infusion of GLP-1 for 7 and 14 days significantly inhibited cumulative food consumption and reduced body weight in SD rats. In the genetically obese Zucker rat, chronic dosing with GLP-1 (30 ug icv) once a day for 6 days caused significant reductions in food consumption each day and a reduction in body weight. These results indicate that the GLP-1 pathways in the central nervous system controlling food consumption do not desensitize after chronic exposure to GLP-1 and suggest that agonists of the central GLP-1 receptor may be effective agents for the treatment of obesity.