Leaky intra-acinar arteries in rat lungs perfused with hydrogen peroxide

We have investigated the effects of H2O2 (150 or 300 microM) on the ultrastructure and permeability of the pulmonary endothelium in rat lungs perfused for 60 min with buffered Hanks' bovine serum albumin medium. In one group of experiments, we examined the effect of H2O2 on the uptake and transport of cationized ferritin (CF) by endothelial cells in intra-acinar arteries, alveolar capillaries, and interlobular veins. The influence of the oxidant on endothelial adsorptive endocytic processes was assessed by measuring the density of ferritin particles in luminal vesicles, multivesicular bodies, and basal lamina. In a second group of experiments, we examined the effects of H2O2 on the fine structure and permeability to electron-dense macromolecules of arterial, microvascular, and venous endothelium. For this purpose, at the end of the 60-min perfusion with H2O2, CF was perfused to identify leaky vessels. We found that H2O2 caused a dose-dependent inhibition of transcytosis of CF in all vascular segments. At the lower dose of H2O2, inhibition of transcytotic activity was not associated with structural injury to the vascular endothelium or with elevation of wet-to-dry ratios. At the higher oxidant dose, inhibition of transcytosis was associated with leaky arterial endothelium and elevation of wet-to-dry ratios (6.44 +/- 0.12 vs. 5.64 +/- 0.16, P less than 0.02). The effects of H2)2 were prevented by adding catalase to the perfusate. The selective loss of structural integrity and leakiness of the arterial endothelium were diminished but not completely abolished by perfusing the oxidant retrograde from the venous side.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of the annual flood-pulse on catch per unit effort,condition and reproduction of Clarias gariepinus from the upper Okavango Delta,Botswana

Catch per unit effort (CPUE), length, weight and maturity data for Clarias gariepinus were collected during monthly gillnet surveys in the upper Okavango Delta between 2001 and 2009 to investigate their relationship with the annual flood-pulse. CPUE, condition factor (K) and the proportion of ripe-running fish (P_RR) in the population followed a unimodal annual cycle that could be modelled using water temperature and flood-pulse hydrology. Increased CPUE during declining water levels was most likely a result of feeding migrations and aggregation behaviour. The observed increase in K during low floods in October and November preceded the increase in P_RR, which increased mainly with increasing temperature but appeared less dependent on flow. This study provided quantitative evidence that the biology of fish in the Okavango Delta is mainly dependent on the annual flood regime and, therefore, that conservation efforts should be focused on maintaining natural flow patterns in the face of climate change and potential water extraction schemes upstream.     

Proteolysis of very low density lipoprotein in perfused lung.

Perfusion of homologous 125I-labeled rat very low density lipoprotein through isolated rat lungs in the presence of heparin resulted in apoprotein proteolysis. At least the apoprotein C was degraded into two peptides smaller than 7500 daltons as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lung uptake of radioactivity was small and due mainly to the presence of the larger of the two peptides. The lung protease was not active against an 125-I-labeled albumin substrate and was not released into the medium by heparin.     

Traffic of Leukocytes and Cytokine Up-regulation in the Central Nervous System in a Murine Model of Neuroparacoccidioidomycosis

Background

Paracoccidioidomycosis is the most important systemic mycosis in South America. In the last decades, it was observed that central nervous system involvement is frequent, occurring in 12.5 % of the cases. The aim of this study was to report the early inflammatory changes associated with an experimental model of neuroparacoccidioidomycosis (NPCM).

Methods

C57BL/6 mice were infected by intracranial route with 10⁶ yeast cells of PB18 strain of Paracoccidioides brasiliensis. Leukocyte–endothelium interactions were assessed by intravital microscopy 1, 2, 4, and 8 weeks post-infection (p.i.). Chemokine/cytokine levels in the brain and histopathological changes were assessed 4 and 8 weeks p.i..

Results

Intravital microscopy analysis revealed a progressive increase in leukocyte recruitment in the vessels of pia mater with a peak 4 weeks p.i. The chemokine CXCL9 was increased at 4 and 8 weeks p.i., while CCL2, CCL3, and CCL5 were increased at 8 weeks p.i. Histopathological analysis revealed the infiltration of inflammatory cells and the development of progressive granulomatous meningoencephalitis. CCL3 levels correlated with clinical manifestations of disease, as measured by the SHIRPA battery.

Conclusions

The experimental model of NPCM showed increased leukocyte recruitment associated with increased expression of chemokines and nervous tissue inflammation which correlated with clinical manifestations of disease.     

Detection of Leishmania infantum in animals and their ectoparasites by conventional PCR and real time PCR

Visceral leishmaniosis (VL) is a parasitic disease caused by Leishmania infantum, which is primarily transmitted by phlebotomine sandflies. However, there has been much speculation on the role of other arthropods in the transmission of VL. Thus, the aim of this study was to assess the presence of L. infantum in cats, dogs and their ectoparasites in a VL-endemic area in northeastern Brazil. DNA was extracted from blood samples and ectoparasites, tested by conventional PCR (cPCR) and quantitative real time PCR (qPCR) targeting the L. infantum kinetoplast DNA. A total of 280 blood samples (from five cats and 275 dogs) and 117 ectoparasites from dogs were collected. Animals were apparently healthy and not previously tested by serological or molecular diagnostic methods. Overall, 213 (76.1 %) animals and 51 (43.6 %) ectoparasites were positive to L. infantum, with mean parasite loads of 795.2, 31.9 and 9.1 fg in dogs, cats and ectoparasites, respectively. Concerning the positivity between dogs and their ectoparasites, 32 (15.3 %) positive dogs were parasitized by positive ectoparasites. The overall concordance between the PCR protocols used was 59.2 %, with qPCR being more efficient than cPCR; 34.1 % of all positive samples were exclusively positive by qPCR. The high number of positive animals and ectoparasites also indicates that they could serve as sentinels or indicators of the circulation of L. infantum in risk areas.     

Complex Patterns of Chromosome 11 Aberrations in Myeloid Malignancies Target CBL,MLL, DDB1 and LMO2

Exome sequencing of primary tumors identifies complex somatic mutation patterns. Assignment of relevance of individual somatic mutations is difficult and poses the next challenge for interpretation of next generation sequencing data. Here we present an approach how exome sequencing in combination with SNP microarray data may identify targets of chromosomal aberrations in myeloid malignancies. The rationale of this approach is that hotspots of chromosomal aberrations might also harbor point mutations in the target genes of deletions, gains or uniparental disomies (UPDs). Chromosome 11 is a frequent target of lesions in myeloid malignancies. Therefore, we studied chromosome 11 in a total of 813 samples from 773 individual patients with different myeloid malignancies by SNP microarrays and complemented the data with exome sequencing in selected cases exhibiting chromosome 11 defects. We found gains, losses and UPDs of chromosome 11 in 52 of the 813 samples (6.4%). Chromosome 11q UPDs frequently associated with mutations of CBL. In one patient the 11qUPD amplified somatic mutations in both CBL and the DNA repair gene DDB1. A duplication within MLL exon 3 was detected in another patient with 11qUPD. We identified several common deleted regions (CDR) on chromosome 11. One of the CDRs associated with de novo acute myeloid leukemia (P=0.013). One patient with a deletion at the LMO2 locus harbored an additional point mutation on the other allele indicating that LMO2 might be a tumor suppressor frequently targeted by 11p deletions. Our chromosome-centered analysis indicates that chromosome 11 contains a number of tumor suppressor genes and that the role of this chromosome in myeloid malignancies is more complex than previously recognized.     

On the Dynamical Behavior of the Cysteine Dioxygenase‐l‐Cysteine Complex in the Presence of Free Dioxygen and l‐Cysteine

In this work, viable models of cysteine dioxygenase (CDO) and its complex with l ‐cysteine dianion were built for the first time, under strict adherence to the crystal structure from X‐ray diffraction studies, for all atom molecular dynamics (MD). Based on the CHARMM36 FF, the active site, featuring an octahedral dummy Fe(II) model, allowed us observing water exchange, which would have escaped attention with the more popular bonded models. Free dioxygen (O₂) and l ‐cysteine, added at the active site, could be observed being expelled toward the solvating medium under Random Accelerated Molecular Dynamics (RAMD) along major and minor pathways. Correspondingly, free dioxygen (O₂), added to the solvating medium, could be observed to follow the same above pathways in getting to the active site under unbiased MD. For the bulky l ‐cysteine, 600 ns of trajectory were insufficient for protein penetration, and the molecule was stuck at the protein borders. These models pave the way to free energy studies of ligand associations, devised to better clarify how this cardinal enzyme behaves in human metabolism.     

On Dioxygen and Substrate Access to Soluble Methane Monooxygenases: An all‐Atom Molecular Dynamics Investigation in Water Solution

In a preliminary exploration of the dummy model for diiron proteins, random‐acceleration molecular dynamics (RAMD) revealed that a pure four‐helix bundle structure, like hemerythrin, constitutes an efficient cage for dioxygen (O₂), which can only leave from defined, albeit very broad, gates. However, this well ordered structure does not constitute an archetype on which to compare O₂ permeation of other diiron proteins, like the complex of soluble methane monooxygenase hydroxylase with the regulatory protein (sMMOH‐MMOB). The reason is that with this complex, unlike hemerythrin, the four helices of the four‐helix bundle are heavily bent, and RAMD showed that most traps for O₂ lie outside them. It was also observed that, in spite of a nearly identical van der Waals radius for O₂ and the natural substrate CH₄, the latter behaves under RAMD as a bulkier molecule than O₂, requiring a higher external force to be brought out of sMMOH‐MMOB along trajectories of viable length. All that determined with sMMOH‐MMOB multiple gates and multiple pathways to each of them through several binding pockets, for both O₂ and CH₄. Of the two equally preferred pathways for O₂, at right angle with one another, one proved to be in accordance with the Xe‐atom mapping for sMMOH. In contrast, none of the pathways identified for CH₄ proved to be in accordance with such mapping, CH₄ looking for more open avenues instead.