Deposition of BaSO4 in the tight junctions of amphibian epithelia causes their opening; apical Ca2+ reverses this effect

Selective deposition of BaSO₄ in the tight junctions (TJs) of frog skins led to profound and reversible functional alterations of these structures, as revealed by changes of tissue conductance (G), clamping current (I), and fluxes of extracellular markers (sulfate (J^SO ₄) and sucrose (J^SUC)). Experiments were performed with nominally Ca²⁺ -free simple salt solutions on the apical side (usually KCl) and Na₂SO₄-Ringer on the inner side of skins. The deposition of BaSO₄ in the TJs was obtained by diffusion and/or migration through the paracellular path of Ba²⁺ from the apical solution and SO ₄ ^2– from the inner solution. A brief presence (2 to 6 min) of apical Ba²⁺ (Ba²⁺ pulse) is followed (i.e., when Ba²⁺ is removed from the apical fluid) by a large increase of G, I, J^SO ₄ and J^SUC, above pre-Ba²⁺ levels. These attain a steady state within 15 to 30 min (overshoot phase), characterizing a conspicuous increase of the paracellular permeability. During the overshoot phase, a second Ba²⁺ pulse blocks the paracellular route while apical Ba²⁺ is present, leading to a new and larger overshoot when the Ba²⁺ pulse is terminated. Addition of apical Ca²⁺ triggers the resealing of the TJs, resulting in a full recovery of G, I, J^SO ₄ and J^SUC. This Ca²⁺ -induced recovery persists when apical Ca²⁺ is removed. The presence of a normal Ca²⁺ concentration in the inner bathing Ringer does not induce the recovery process. Tissues remain viable after being submitted to the Ba²⁺ treatment and the subsequent overshoot. Experiments performed in the urinary bladder of Rana catesbeiana and skins and urinary bladders of Bufo marinus indicate that Ba²⁺ effect can also be elicited in these tissues. The above results seem to report general properties of the TJs. Incidentally, they warn about the use of Ba²⁺ as an ion channel blocker in epithelial membranes in association with SO ₄ ^2– -containing solutions on the contralateral side.This project was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (91/0293-7 to F.L.V., and 90/1788-1 to A.S.), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (410068/90-0 and 303633-85/BF to F.L.V.). J.A.C. received a doctoral fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/Fundação Universidade do Rio Grande. We thank Dr. Alice T. Ferreira for help in the measurements of free Ca²⁺ concentration.     

Conservation and alteration of HLA-B27-specific T cell epitopes on mouse cells. Implications for peptide-mediated alloreactivity.

Alloreactive CTL responses generate a great variety of clonal specificities. Such diversity may be related to recognition of multiple peptides constitutively bound to any given MHC alloantigen. Among human alloreactive CTL, only a fraction of the clones lyse mouse P815 cells expressing class I HLA proteins. In this study the fine specificity of HLA-B27 allorecognition on human or mouse cells by five human HLA-B27-specific CTL clones was comparatively analyzed. This was done to examine what degree of variation in epitope structure is compatible with recognition of HLA Ag on mouse cells. Nine site-specific HLA-B27 mutants were expressed on both human and mouse cells, after DNA-mediated gene transfer, to construct two analogous series of target cells. The reaction patterns of four of the five CTL clones with these cell panels were compatible with conservation of their corresponding epitopes upon expression of HLA-B27 on mouse cells. The reaction pattern of the fifth clone was different with either cell panel, indicating that its epitope was structurally altered on mouse cells. It also suggested a selectively increased expression of the determinant on these cells. The results suggest that most of the epitopes recognized by allospecific CTL clones reacting across species are either independent of any bound peptide or involve identical peptides from both cell types. However, some of these clones recognize alloantigen-bound peptides that are somewhat different in structure depending on the cell type, and may be expressed at the mouse cell surface in greater amounts. Such peptides could arise from related proteins in both species, and be polymorphic as a result of phylogenetic divergence.     

Physiographic and climatic events in the Chihuahuan Desert lead to the speciation and distinct demographic patterns of two sister Sceloporus lizards

The biogeographic history of the Chihuahuan Desert is known to be complex, and there is evidence of the effects of physiographic and climatic events in species diversification and demographic population changes in many taxa. Here, using DNA sequence data, we studied the influence of the physiographic and climatic events that occurred in the Chihuahuan Desert during the Pliocene–Pleistocene transition on the speciation and evolutionary history of the sister lizard species Sceloporus cyanostictus and S. gadsdeni. First, based on mtDNA and nDNA sequences, we estimated the divergence times of the sister species. Then, based on mtDNA sequences, we investigated the demographic history of both species within a phylogeographic framework. The divergence time was inferred to be 1.48 Mya, date that corresponds to the existence of a large lake in the Mapimian subprovince, between the current geographic locations of S. cyanostictus and S. gadsdeni. This lake could have acted as a barrier, leading to the speciation of both species. For the demographic history of the two species, we identified two distinct patterns: the population expansion of S. gadsdeni within the Last Glacial Maximum and the potential population decline of S. cyanostictus. Our results can be used as a guide for the study of other aspects that could be critical to developing conservation actions that ensure the survival of not only S. gadsdeni and S. cyanostictus, but also other co‐occurring lizard species.     

Multilocus and morphological analysis of south-eastern Iberian Wall lizards (Squamata,Podarcis)

The phylogenetic relationships among the wall lizards of the Podarcis hispanicus complex that inhabit the south-east (SE) of the Iberian Peninsula and other lineages of the complex remain unclear. In this study, four mitochondrial and two nuclear markers were used to study genetic relationships within this complex. The phylogenetic analyses based on mtDNA gene trees constructed with ML and BI, and a species tree using *BEAST support three divergent clades in this region: the Valencia, Galera and Albacete/Murcia lineages. These three lineages were also corroborated in species delimitation analyses based on mtDNA using bPTP, mPTP, GMYC, ABGD and BAPS. Bayesian inference species delimitation method (BPP) based on both nuclear data and a combined data set (mtDNA + nuclear) showed high posterior probabilities for these three SE lineages (≥0.94) and another Bayesian analysis (STACEY) based on combined data set recovered the same three groups in this region. Divergence time dating of the species tree provided an estimated divergence of the Galera lineage from the other SE group (Podarcis vaucheri, (Albacete/Murcia, Valencia)) at 12.48 Ma. During this period, the Betic–Rifian arc was isolated, which could have caused the isolation of the Galera form distributed to the south of the Betic Corridor. Although lizards from the Albacete/Murcia and Galera lineage are morphologically similar, they clearly represent distinct genetic lineages. The noteworthy separation of the Galera lineage enables us to conclude that this lineage must be considered as a new full species.     

High T cell epitope sharing between two HLA-B27 subtypes (B*2705 and B*2709) differentially associated to ankylosing spondylitis.

HLA-B*2705 is strongly associated with ankylosing spondylitis (AS) and reactive arthritis. In contrast, B*2709 has been reported to be more weakly or not associated to AS. These two molecules differ by a single amino acid change: aspartic acid in B*2705 or histidine in B*2709 at position 116. In this study, we analyzed the degree of T cell epitope sharing between the two subtypes. Ten allospecific T cell clones raised against B*2705, 10 clones raised against B*2703 but cross-reactive with B*2705, and 10 clones raised against B*2709 were examined for their capacity to lyse B*2705 and B*2709 target cells. The anti-B*2705 and anti-B*2703 CTL were peptide dependent as demonstrated by their failure to lyse TAP-deficient B*2705-T2 transfectant cells. Eight of the anti-B*2705 and five of the anti-B*2703 CTL clones lysed B*2709 targets. The degree of cross-reaction between B*2705 and B*2709 was donor dependent. In addition, the effect of the B*2709 mutation (D116H) on allorecognition was smaller than the effect of the other naturally occurring subtype change at this position, D116Y. These results demonstrate that B*2705 and B*2709 are the antigenically closest HLA-B27 subtypes. Because allospecific T cell recognition is peptide dependent, our results imply that the B*2705- and B*2709-bound peptide repertoires are largely overlapping. Thus, to the extent to which linkage of HLA-B27 with AS is related to the peptide-presenting properties of this molecule, our results would imply that peptides within a relatively small fraction of the HLA-B27-bound peptide repertoire influence susceptibility to this disease.     

Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis

1. Download : Download high-res image (226KB)
2. Download : Download full-size image

     

Control of the growth of Alicyclobacillus acidoterrestris in industrialized orange juice using rosemary essential oil and nisin

The use of rosemary essential oil (RO) and its combination with nisin (RO+N) in preventing the multiplication of Alicyclobacillus acidoterrestris in orange juice was evaluated. The minimum inhibitory and bactericidal concentrations (MIC and MBC) for RO were both 125 μg ml⁻¹ while RO+N displayed a synergistic effect. The use of RO and RO+N at concentrations of 1, 4 and 8× MIC in orange juice for 96 h was evaluated in terms of their sporicidal effectiveness. With regard to the action against A. acidoterrestris spores, RO at 8× MIC was sporostatic, whereas RO+N at 1× MIC was sporicidal. Morphological changes in the structure of the micro-organism after treatment were also observed by microscopy. Furthermore, flow cytometric analysis showed that most cells were damaged or killed after treatment. In general, the antioxidant activity after addition of RO+N decreased with time. The results demonstrate that using the combination of RO and nisin can prevent the A. acidoterrestris growth in orange juice.     

Cytological analysis of the effect of treatments visualizing the chromosome core on non-histone nuclear proteins

A silver-stainable chromatid core is visualized under the light microscope on human leukocyte chromosomes by treating fixed chromosomes with 2M NaCl.An aminoacid which is not part of histone composition, 3H-tryptophan, is used to label the synthesis and location of non-histone proteins and to study the effect on these proteins of treatment with 2M NaCl and 0.2N HCl to visualize the core. 2M NaCl is shown to remove from nuclei and chromosomes an appreciable fraction of acid proteins, as well as all those synthesized during the two hours preceding the metaphase. 3H-Try labelling seems to preferentially affect only one chromatid per chromosome. The silver-stainable halocs appearing around numerous nuclei after treatment with 2M NaCl and 0.2N HCl are found to be labelled by 3H-Try and not by 3H-TdR.