TGFβ signaling in lung epithelium regulates bleomycin-induced alveolar injury and fibroblast recruitment

The response of alveolar epithelial cells (AECs) to lung injury plays a central role in the pathogenesis of pulmonary fibrosis, but the mechanisms by which AECs regulate fibrotic processes are not well defined. We aimed to elucidate how transforming growth factor-β (TGFβ) signaling in lung epithelium impacts lung fibrosis in the intratracheal bleomycin model. Mice with selective deficiency of TGFβ receptor 2 (TGFβR2) in lung epithelium were generated and crossed to cell fate reporter mice that express β-galactosidase (β-gal) in cells of lung epithelial lineage. Mice were given intratracheal bleomycin (0.08 U), and the following parameters were assessed: AEC death by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay, inflammation by total and differential cell counts from bronchoalveolar lavage, fibrosis by scoring of trichrome-stained lung sections, and total lung collagen content. Mice with lung epithelial deficiency of TGFβR2 had improved AEC survival, despite greater lung inflammation, after bleomycin administration. At 3 wk after bleomycin administration, mice with epithelial TGFβR2 deficiency showed a significantly attenuated fibrotic response in the lungs, as determined by semiquantitatve scoring and total collagen content. The reduction in lung fibrosis in these mice was associated with a marked decrease in the lung fibroblast population, both total lung fibroblasts and epithelial-to-mesenchymal transition-derived (S100A4(+)/β-gal(+)) fibroblasts. Attenuation of TGFβ signaling in lung epithelium provides protection from bleomycin-induced fibrosis, indicating a critical role for the epithelium in transducing the profibrotic effects of this cytokine.     

Infection with Leishmania amazonensis upregulates purinergic receptor expression and induces host-cell susceptibility to UTP-mediated apoptosis

Nucleotides are released into the extracellular milieu from infected cells and cells at inflammatory sites. The extracellular nucleotides bind to specific purinergic (P2) receptors and thereby induce a variety of cellular responses including anti-parasitic effects. Here we investigated whether extracellular nucleotides affect leishmanial infection in macrophages, and found that UTP reduces strongly the parasite load in peritoneal macrophages. Ultrastructural analysis of infected cells revealed that UTP induced morphological damage in the intracellular parasites. Uridine nucleotides also induced dose-dependent apoptosis of macrophages and production of ROI and RNI only in infected macrophages. The intracellular calcium measurements of infected cells showed that the response to UTP, but not UDP, increased the sensitivity and amplitude of cytosolic Ca(2+) changes. Infection of macrophages with Leishmania upregulated the expression of P2Y(2) and P2Y(4) receptor mRNA. The data suggest indirectly that Leishmania amazonensis infection induces modulation and heteromerization of P2Y receptors on macrophages. Thus UTP modulates the host response against L. amazonensis infection. UTP and UTP homologues should therefore be considered as novel components of therapeutic strategies against cutaneous leishmaniasis.     

Evolution of the 5.8S nrDNA gene and internal transcribed spacers in Carapichea ipecacuanha (Rubiaceae) within a phylogeographic context

Nuclear ribosomal DNA (nrDNA) constitutes a multicopy gene family that is used widely to test evolutionary hypotheses across a broad range of organisms. It is presumed that, as a result of concerted evolution, tandem nrDNA repeats are homogeneous within species and different between species. We sampled 77 specimens of a disjunct species (Carapichea ipecacuanha) from throughout its three geographic ranges and obtained 266 nrDNA sequences, of which 26 were obtained by direct sequencing and 240 by cloning of PCR products. Complementary sequence analyses, which included analyses of secondary structure stability, the pattern of base substitutions, GC content, and the presence of conserved motifs, were used to characterize the internal transcribed spacer (ITS) region (ITS1-5.8S nrDNA-ITS2). Our results showed that concerted evolution of the ITS region was incomplete in C. ipecacuanha, particularly in the Atlantic range. In the highly polymorphic populations of the Atlantic range, intraindividual variation was observed and involved 56 functional paralogs and 15 pseudogenes from two highly divergent ribogroups. The Amazonian range (with 12 functional paralogs) and the Central-American range (with five functional paralogs) were genetically depauperate and exhibited no pseudogenes. In the two latter ranges, almost complete homogenization of the ITS sequences had occurred. We argue that it is important to consider past evolutionary history when making inferences about the efficiency with which concerted evolution homogenizes tandem nrDNA repeats a single sequence.     

Maximal lactate steady-state independent of recovery period during intermittent protocol

Barbosa, LF, de Souza, MR, Corrêa Caritá, RA, Caputo, F, Denadai, BS, and Greco, CC. Maximal lactate steady-state independent of recovery period during intermittent protocol. J Strength Cond Res 25(12): 3385-3390, 2011-The purpose of this study was to analyze the effect of the measurement time for blood lactate concentration ([La]) determination on [La] (maximal lactate steady state [MLSS]) and workload (MLSS during intermittent protocols [MLSSwi]) at maximal lactate steady state determined using intermittent protocols. Nineteen trained male cyclists were divided into 2 groups, for the determination of MLSSwi using passive (VO(2)max = 58.1 ± 3.5 ml·kg·min; N = 9) or active recovery (VO(2)max = 60.3 ± 9.0 ml·kg·min; N = 10). They performed the following tests, in different days, on a cycle ergometer: (a) Incremental test until exhaustion to determine (VO(2)max and (b) 30-minute intermittent constant-workload tests (7 × 4 and 1 × 2 minutes, with 2-minute recovery) to determine MLSSwi and MLSS. Each group performed the intermittent tests with passive or active recovery. The MLSSwi was defined as the highest workload at which [La] increased by no more than 1 mmol·L between minutes 10 and 30 (T1) or minutes 14 and 44 (T2) of the protocol. The MLSS (Passive-T1: 5.89 ± 1.41 vs. T2: 5.61 ± 1.78 mmol·L) and MLSSwi (Passive-T1: 294.5 ± 31.8 vs. T2: 294.7 ± 32.2 W; Active-T1: 304.6 ± 23.0 vs. T2: 300.5 ± 23.9 W) were similar for both criteria. However, MLSS was lower in T2 (4.91 ± 1.91 mmol·L) when compared with in T1 (5.62 ± 1.83 mmol·L) using active recovery. We can conclude that the MLSSwi (passive and active conditions) was unchanged whether recovery periods were considered (T1) or not (T2) for the interpretation of [La] kinetics. In contrast, MLSS was lowered when considering the active recovery periods (T2). Thus, shorter intermittent protocols (i.e., T1) to determine MLSSwi may optimize time of the aerobic capacity evaluation of well-trained cyclists.     

A new steroidal saponin from Allium ampeloprasum var. porrum with antiinflammatory and gastroprotective effects

A new steroidal saponin was isolated from the bulbs of Allium ampeloprasum var. porrum. On the basis of chemical conversions and detailed analyses of ¹H and ¹³C NMR spectra including 2D NMR spectroscopic techniques, its structure was established as 3-[(O-β-d-glucopyranosyl-(1 → 3)-β-d-glucopyranosyl-(1 → 2)-O-[O-β-d-glucopyranosyl-(1 → 3)]-O-β-d-glucopyranosyl-(1 → 4)-β-d-galactopyranosyl)oxy]-2,6-dihydroxy-(2α,3β,5α,6β,25R)-spirostane. Results of the present study indicated that the steroidal saponin showed haemolytic effects in the in vitro assays and demonstrated antiinflammatory activity and gastroprotective property using in vivo models.     

Measurements on ground or sectioned otoliths: possibilities of bias

Back-calculation usually requires measurements of growth marks revealed on otoliths by specific preparations. The standardization of the grinding (or sectioning) plane is necessary, but difficult, especially along the antero-posterior axis. In order to show the importance of the grinding plane, tetracycline labelling of eel otoliths ( Anguilla anguilla L.) has been used. This marking has a calcio-traumatic effect on otoliths, which can be revealed with staining techniques. Unless the grinding plane is incorrect, the tetracycline labelling and the staining of the ground surface are then superposed.     

Cystyl aminopeptidase activity in the plasma, viscera and brain of the snake Bothrops jararaca

The relationship between plasma osmolality and cystyl aminopeptidase was characterized in the snake Bothrops jararaca and comparisons were made with the emerging picture of this relationship in rats. The profile of cystyl aminopeptidase activity under basal conditions was determined in the soluble and membrane-bound forms in visceral organs and in the central nervous system in comparison with that of alanyl aminopeptidase. The regional localization of cystyl and alanyl aminopeptidase activities was studied in the central nervous system. The basal level of plasma cystyl aminopeptidase, four- to six-fold higher than in rats, suggests its importance to help regulate circulating levels of neurohypophysial peptides in B. jararaca snake. The osmotic sensitivity of this plasma enzyme, undetectable in male, but about three-fold higher in female snakes than in rats, reveals a sexual dimorphism. In marked contrast to those observed in rats, low levels of soluble and particulate forms in the kidney indicate that cystyl aminopeptidase plays a minor metabolizing role at this anatomical location in B. jararaca. Despite of the regional-specific divergence between the levels of rat and snake enzymes, the bilaterally symmetric pattern of the diencephalic distribution of alanyl aminopeptidase reflects functional homologies between these two distantly related species.     

Expression of Sara2 human gene in erythroid progenitors

A human homologue of Sar1, named Sara2, was shown to be preferentially expressed during erythropoiesis in a culture stimulated by EPO. Previous studies, in yeast, have shown that secretion-associated and Ras-related protein (Sar1p) plays an essential role in protein transport from the endoplasmic reticulum to the Golgi apparatus. Here, we report the molecular analysis of Sara2 in erythroid cell culture. A 1250 bp long cDNA, encoding a 198 amino-acid protein very similar to Sar1 proteins from other organisms, was obtained. Furthermore, we also report a functional study of Sara2 with Real-time quantitative PCR analysis, demonstrating that expression of Sara2 mRNA increases during the initial stages of erythroid differentiation with EPO and that a two-fold increase in expression occurs following the addition of hydroxyurea (HU). In K562 cells, Sara2 mRNA was observed to have a constant expression and the addition of HU also up-regulated the expression in these cells. Our results suggest that Sara2 is an important gene in processes involving proliferation and differentiation and could be valuable for understanding the vesicular transport system during erythropoiesis.     

A mannanase, ManA, of the polycentric anaerobic fungus Orpinomyces sp. strain PC-2 has carbohydrate binding and docking modules

The anaerobic fungus Orpinomyces sp. strain PC-2 produces a broad spectrum of glycoside hydrolases, most of which are components of a high molecular mass cellulosomal complex. Here we report about a cDNA (manA) having 1924 bp isolated from the fungus and found to encode a polypeptide of 579 amino acid residues. Analysis of the deduced sequence revealed that it had a mannanase catalytic module, a family 1 carbohydrate-binding module, and a noncatalytic docking module. The catalytic module was homologous to aerobic fungal mannanases belonging to family 5 glycoside hydrolases, but unrelated to the previously isolated mannanases (family 26) of the anaerobic fungus Piromyces. No mannanase activity could be detected in Escherichia coli harboring a manA-containing plasmid. The manA was expressed in Saccharomyces cerevisiae and ManA was secreted into the culture medium in multiple forms. The purified extracellular heterologous mannanase hydrolyzed several types of mannan but lacked activity against cellulose, chitin, or beta-glucan. The enzyme had high specific activity toward locust bean mannan and an extremely broad pH profile. It was stable for several hours at 50 degrees C, but was rapidly inactivated at 60 degrees C. The carbohydrate-binding module of the Man A produced separately in E. coli bound preferably to insoluble lignocellulosic substrates, suggesting that it might play an important role in the complex enzyme system of the fungus for lignocellulose degradation.