Castration induces changes in the cation‐dependent mannose‐6‐phosphate receptor in rat epididymis: Possible implications in secretion of lysosomal enzymes

It is believed that the mammalian epididymis participates in the maturation of the sperm due to its secretory activity. High concentrations of several secreted acid hydrolases are found in the epididymal lumen. Moreover, some of these enzymes are secreted by the epididymal epithelium in an androgen‐dependent fashion. In this study, we attempted to discern whether mannose‐6‐phosphate receptors (MPRs) regulate transport and secretion of lysosomal enzymes in the rat epididymis, and if these events are altered when the animals are subjected to hormonal manipulation. We observed that expression of cation‐dependent MPR (CD‐MPR) and cation‐independent MPR (CI‐MPR) increased significantly in caudal epididymis of castrated rats by immunoblot. This increase was corroborated by quantitation of MPRs, by binding assays. This change could be due to androgen deprivation, as a similar effect was observed after treatment with the anti‐androgenic drug flutamide. Furthermore, we observed that the CD‐MPR was redistributed to the apical area of the epithelium on castrated rats by immunohistochemistry, which is compatible with the redistribution of the receptors toward lighter fractions in a Percoll gradient. Consistent with a possible involvement of the CD‐MPR in the secretion, we observed an increase in pro‐cathepsin D levels in epididymal fluid after castration. We conclude that the CD‐MPR might be regulated by hormones and that this receptor might be involved in the secretion of specific enzymes into the rat epididymis. J. Cell. Biochem. 110: 1101–1110, 2010. Published 2010 Wiley‐Liss, Inc.     

In a rat model of night work, activity during the normal resting phase produces desynchrony in the hypothalamus

Internal synchrony among external cycles and internal oscillators allows adaptation of physiology to cyclic demands for homeostasis. Night work and shift work lead to a disrupted phase relationship between external time cues and internal rhythms, also losing internal coherence among oscillations. This process results in internal desynchrony (ID) in which behavioral, hormonal, and metabolic variables cycle out of phase. It is still not clear whether ID originates at a peripheral or at a central level. In order to determine the possible role of hypothalamic oscillators in ID, we explored with a rat model of "night work" daily rhythms of activity and clock gene expression in the hypothalamus. This study provides evidence that wakefulness and activity during the normal resting phase lead to a shift in the diurnal rhythms of c-Fos and induce a rhythm of PER1 in the arcuate and dorsomedial nucleus of the hypothalamus, both associated with metabolism and regulation of the sleep/wake cycle. Moreover, the number of orexin (ORX)-positive neurons and c-Fos in the perifornical area increased during the working period, suggesting a relevant switch of activity in this brain region induced by the scheduled activity; however, the colocalization of c-Fos in ORX-positive cells was not increased. In contrast, the suprachiasmatic nucleus and the paraventricular nucleus remained locked to the light/dark cycle, resulting in ID in the hypothalamus. Present data suggest that ID occurs already at the level of the first output projections from the SCN, relaying nuclei that transmit temporal signals to other brain areas and to the periphery.     

Durability of five native Argentine wood species of the genera <Emphasis Type="Italic">Prosopis</Emphasis> and <Emphasis Type="Italic">Acacia</Emphasis> decayed by rot fungi and its relationship with extractive content

The natural durability of four Argentinean species of Prosopis and one of Acacia was evaluated in laboratory tests, according to European standards, using three brown rot and one white rot fungi. These tests were complemented by assessing the wood chemical composition. All the species were from moderately slightly durable to very durable (classes 4–1), and in all cases the heartwood was the most resistant to fungal attack. Chemical extractives content (organic, aqueous, tannic and phenolic) was higher in the heartwood. However, species durability was not related to extractive contents nor with wood density. Instead, it is possible that extractives could contribute to natural durability in different ways, including the effects related to the antioxidant properties of some of them.     

The importance of topography and climate on short-term revegetation of coal wastes in Spain

The initial colonization phase is the crucial start point for succession and therefore for restoration. However, little is known about abiotic factors that influence the early stages of revegetation dynamics on restored coal mines, particularly in a Mediterranean climate. This information is crucial for improving our ability to reclaim land despoiled by mining. Here, we characterized the short-term plant community development in the first 3 years after hydroseeding on a topographically diverse reclaimed open-pit coal mines in Spain. Topography influenced both community composition and diversity producing different trajectories between the three different aspects (north-facing, south-facing, flat). Hydroseeded species provided most of the initial vegetation cover, which brought about most of the compositional differences between these aspects. We also found that the changing climate through the growing season also influenced floristic composition and diversity. Summer drought reduced the cover of hydroseeded perennial species and allowed native species to colonize. These results emphasize the need to take topography into account when developing reclamation management plans in Mediterranean ecosystems, and an acknowledgement that climate might drive the succession in the desired direction, hence increasing success in restoring mining impacts.     

Expression and immunolocalization of ERG1 potassium channels in the rat kidney

Potassium (K⁺) channels participate in K⁺ secretion, K⁺ recycling, and cell volume regulation and help to maintain the resting potential in mammalian kidneys. Previously, we identified a set of voltage-gated K⁺ channels (Kv1) in the inner medullary collecting duct of the rat kidney. In the present work, we identified the voltage-gated K⁺ channel ether-à-go-go-related gene (ERG) in the rat kidney. mRNAs of ERG1a and its N-terminal splice-variant ERG1b were detected. Immunoblots of the cortex and medulla revealed two molecular mass proteins of 135 and 80 kDa, consistent in size with the nonglycosylated ERG1a and ERG1b isoforms, respectively. However, bands of 155 and 95 kDa, corresponding to mature glycosylated ERG1a and ERG1b, respectively, were also observed. In our immunohistochemical experiments, we could not differentiate the ERG1 isoforms because we used an antibody against a carboxy-terminal epitope. ERG1 was differentially localized in specific nephron segments: its localization was intracellular in the proximal tubule and medullary collecting ducts and in the apical membranes in the distal convoluted and connecting tubules. ERG1 was also abundant in glomerular arterioles and renal vessels. In summary, ERG1 displays a heterogeneous distribution in the rat kidney.     

Electrostatic interactions play a significant role in the affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase for Mn

Phosphoenolpyruvate (PEP) carboxykinases catalyse the reversible formation of oxaloacetate (OAA) and ATP (or GTP) from PEP, ADP (or GDP) and CO₂. They are activated by Mn²⁺, a metal ion that coordinates to the protein through the ?-amino group of a lysine residue, the N_?-2-imidazole of a histidine residue, and the carboxylate from an aspartic acid residue. Neutrality in the ?-amino group of Lys213 of Saccharomyces cerevisiae PEP carboxykinase is expected to be favoured by the vicinity of ionised Lys212. Glu272 and Glu284, located close to Lys212, should, in turn, electrostatically stabilise its positive charge and hence assist in keeping the ?-amino group of Lys213 in a neutral state. The mutations Glu272Gln, Glu284Gln, and Lys212Met increased the activation constant for Mn²⁺ in the main reaction of the enzyme up to seven-fold. The control mutation Lys213Gln increased this constant by ten-fold, as opposed to control mutation Lys212Arg, which did not affect the Mn²⁺ affinity of the enzyme. These observations indicate a role for Glu272, Glu284, and Lys212 in assisting Lys213 to properly bind Mn²⁺. In an unexpected result, the mutations Glu284Gln, Lys212Met and Lys213Gln changed the nucleotide-independent OAA decarboxylase activity of S. cerevisiae PEP carboxykinase into an ADP-requiring activity, implying an effect on the OAA binding characteristics of PEP carboxykinase.     

Cytoplasmatic domain of Na,K-ATPase α-subunit is responsible for the aggregation of the enzyme in proteoliposomes

We studied the thermal dependence of amide I′ infrared absorption and fluorescence emission of Trp residues in the Na,K-ATPase of rabbit kidney. We studied the whole enzyme solubilized with detergent, the whole enzyme reconstituted in proteoliposomes and the protein fraction that remained in the lipid membrane after the trypsin digestion of the proteoliposomes. Cooperative unfolding and aggregation with increasing temperature were observed in the whole protein, whether solubilized or reconstituted, but not in the fraction remaining after trypsinization. The protein influenced the physical state of the lipid, decreasing the temperature of the gel to liquid-crystalline phase transition and the degree of cooperativity. This study provides new information for the understanding of the processes controlling the association mechanisms that are important for enzyme function in natural membranes.     

Perspectives on Neonatal Hypoxia/Ischemia-Induced Edema Formation

Neonatal hypoxia/ischemia (HI) is the most common cause of developmental neurological, cognitive and behavioral deficits in children, with hyperoxia (HHI) treatment being a clinical therapy for newborn resuscitation. Although cerebral edema is a common outcome after HI, the mechanisms leading to excessive fluid accumulation in the brain are poorly understood. Given the rigid nature of the bone-encased brain matter, knowledge of edema formation in the brain as a consequence of any injury, as well as the importance of water clearance mechanisms and water and ion homeostasis is important to our understanding of its detrimental effects. Knowledge of the pathological process underlying the appearance of dysfunctional outcomes after development of cerebral edema after neonatal HI in the developing brain and the molecular events triggered will allow a rational assessment of HHI therapy for neonatal HI and determine whether this treatment is beneficial or harmful to the developing infant.     

Genotyping of Mycobacterium tuberculosis isolates from a low-endemic setting in northwestern state of Paraná in Southern Brazil

The purpose of this study was to provide information about the genetic diversity and prevalent genotype of Mycobacterium tuberculosis in a low-endemic setting in northwestern state of Paraná in Southern Brazil. We employed spoligotyping and mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) techniques to genotype M. tuberculos isisolates from patients with pulmonary tuberculosis (TB). The 93 isolates analyzed by spoligotyping were divided into 36 different patterns, 30 of which were described in the SITVIT database. Latin American and Mediterranean, Haarlem and T families were responsible for 26.9%, 17.2% and 11.8% of TB cases, respectively. From the 84 isolates analyzed by MIRU-VNTR, 58 shared a unique pattern and the remaining 26 belonged to nine clusters. The MIRU loci 40, 23, 10 and 16 were the most discriminatory. A combination of MIRU-VNTR and spoligotyping resulted in 85.7% discriminatory power (Hunter-Gaston index = 0.995). Thus, combining spoligotyping and MIRU-VNTR typing proved to be most useful for epidemiological study in this low-endemic setting in Southern Brazil. The current study demonstrated that there is significant diversity in circulating strains in the city of Maringá and the surrounding regions, with no single genotype of M. tuberculosis predominating.     

Prevalence of Bartonella henselae and Bartonella clarridgeiae in cats in the south of Brazil: a molecular study

Bartonella spp are the causative agent of cat scratch disease in humans. Cats are the natural reservoir of these bacteria and may infect humans through scratches, bites or fleas. Blood samples from 47 cats aged up to 12 months were collected for this study. All animals were lodged in municipal animal shelters in the Vale do Sinos region, Rio Grande do Sul, Brazil. Bartonella spp were detected by genus-specific polymerase chain reaction (PCR) and when the PCR was positive, the species were determined by DNA sequencing. A Giemsa-stained blood smear was also examined for the presence of intraerythrocytic elements suggestive of Bartonella spp infection. Phylogenetic analysis was also performed for all positive samples. Using molecular detection methods, Bartonella spp were detected in 17.02% (8/47) of the samples. In seven out of eight samples confirmed to be positive for Bartonella spp, blood smear examination revealed the presence of intraerythrocytic elements suggestive of Bartonella spp. Phylogenetic analysis characterized positive samples as Bartonella henselae (5) or Bartonella clarridgeiae (3). To the best of our knowledge, this is the first molecular study demonstrating the presence of Bartonella spp in cats from the Southern Region of Brazil.