Modulation of endocytic traffic in polarized Madin-Darby canine kidney cells by the small GTPase RhoA

Efficient postendocytic membrane traffic in polarized epithelial cells is thought to be regulated in part by the actin cytoskeleton. RhoA modulates assemblies of actin in the cell, and it has been shown to regulate pinocytosis and phagocytosis; however, its effects on postendocytic traffic are largely unexplored. To this end, we expressed wild-type RhoA (RhoAWT), dominant active RhoA (RhoAV14), and dominant inactive RhoA (RhoAN19) in Madin-Darby canine kidney (MDCK) cells expressing the polymeric immunoglobulin receptor. RhoAV14 expression stimulated the rate of apical and basolateral endocytosis, whereas RhoAN19 expression decreased the rate from both membrane domains. Polarized basolateral recycling of transferrin was disrupted in RhoAV14-expressing cells as a result of increased ligand release at the apical pole of the cell. Degradation of basolaterally internalized epidermal growth factor was slowed in RhoAV14-expressing cells. Although apical recycling of immunoglobulin A (IgA) was largely unaffected in cells expressing RhoAV14, transcytosis of basolaterally internalized IgA was severely impaired. Morphological and biochemical analyses demonstrated that a large proportion of IgA internalized from the basolateral pole of RhoAV14-expressing cells remained within basolateral early endosomes and was slow to exit these compartments. RhoAN19 and RhoAWT expression had little effect on these postendocytic pathways. These results indicate that in polarized MDCK cells activated RhoA may modulate endocytosis from both membrane domains and postendocytic traffic at the basolateral pole of the cell.     

The mycetisms and their relevancy in medicine.

Poisonous mushrooms play an important role in public health, since the poisonings caused by their toxins have very high morbidity-mortality rates. No real data are available on this problem because diagnosis is often difficult. This work points out the important role of poisonous mushrooms. An analysis is included of the clinical features of the most important mycetisms, the mushrooms that produce them and the toxins that cause this pathology. A guide to aid clinical doctors in the diagnosis and treatment of mycetisms and recomendations to prevent these poisonings are provided.     

Characterization of a bacteriocin-like substance produced by a vaginal Lactobacillus salivarius strain

A novel bacteriocin-like substance produced by vaginal Lactobacillus salivarius subsp. salivarius CRL 1328 with activity against Enterococcus faecalis, Enterococcus faecium, and Neisseria gonorrhoeae was characterized. The highest level of production of this heat-resistant peptide or protein occurred during the late exponential phase. Its mode of action was shown to be bactericidal. L. salivarius subsp. salivarius CRL 1328 could be used for the design of a probiotic to prevent urogenital infections.     

Role of antioxidants in the survival of normal and vitiliginous avian melanocytes.

Mutant feather melanocytes from Barred Plymouth Rock (BPR) and White Leghorn (WL) chickens are currently being used as avian models of vitiligo. Feather melanocytes in BPR and WL chickens die prematurely in vivo due to low (50-66%) antioxidant glutathione and superoxide dismutase levels when compared to the wild type Jungle Fowl (JF) melanocytes. Excess superoxide anions, generated by xanthine:xanthine oxidase (X:XO), caused a 15-20% increase in mortality after 1 and 2 hrs. in all three genotypes of in vitro melanocytes as compared to control values that received no X:XO. Overall, the JF wild type melanocytes had the lowest mortality rate, WL melanocytes had the highest mortality rate and the BPR melanocytes had an intermediate mortality rate. Superoxide anion and hydroxyl radical production in the WL feather were double the production in the JF wild type feather. The production of reactive oxygen species in BPR was intermediate to the other two genotypes. In an effort to mimic the low antioxidant levels of the BPR and WL feathers in the JF feather, JF in vitro feather melanocytes were treated with buthionine sulfoximine (BSO), a glutathione synthesis inhibitor. With BSO added to the medium, the JF mortality rates increased by 20-25%, reaching the mortality levels of the mutant BPR melanocytes. The addition of iron to the JF melanocyte X:XO medium increased their mortality rate by 20%, probably via the Fenton reaction. Thus, antioxidants play an extremely important role in both the viability of normal avian melanocytes and the premature death of the vitiliginous avian melanocytes. A working hypothesis, supported in part by the current results, is that the premature death of the mutant melanocytes could be precipitated in the poorly vascularized feather by low antioxidant protection due to both low turnover of tissue fluids which contain SOD and to genetically determined low levels of internal antioxidant protection in these melanocytes. This same mechanistic hypothesis could apply as "a" cause of premature melanocyte cell death in human vitiligo wherein the vitiliginous melanocytes may have a genetic defect in their antioxidant protection system and blood flow to an area may be restricted.     

Ultrastructural Study of the Filum Terminate and Caudal Ampulla of the Spinal Cord of Amphioxus (Branchiostoma lanceolatum Pallas)

The filum terminale and caudal ampulla of amphioxus were studied by electron microscopy. The filum terminale consists of ependymal cells whose cilia are directed caudally. Remarkably, nerve fibres course through the filum terminale and caudal ampulla and end on the basal lamina forming neuro-connective structures. Moreover, these nerve boutons are divisible into several classes according to their vesicle content. Boutons containing large dense-cored vesicles are very similar in appearance to the neurosecretory terminals found in the caudal spinal cord of some vertebrates. These observations on nerve fibres suggest that a primitive neurosecretory system similar to the fish urophysis is present in the amphioxus.     

Preliminary studies of the 2D crystallization of Omp1 of Serratia marcescens: observation by atomic force microscopy in native membranes environment and reconstituted in proteolipid sheets

In this work the porin Omp1 of Serratia marcescens was expressed in a porin deficient mutant (Escherichia coli UH302) and its functionality studied following the accumulation of ciprofloxacin in bacteria. The protein was extracted, purified and reconstituted in proteoliposomes of different composition (lipopolysaccharide (LPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)). Maximum extraction of the detergent was achieved applying different steps of dialysis and centrifugation. Proteolipid sheets with different composition were spread onto mica and observed by atomic force microscopy. Two-dimensional crystal of Omp1 was not observed in any case due to low resolution achieved. Judging from the images features POPC is the most suitable phospholipid to enhance 2D lattice formation for Omp1.     

Overexpression of human DNA polymerase mu (Pol mu) in a Burkitt's lymphoma cell line affects the somatic hypermutation rate

DNA polymerase μ (Pol μ) is a DNA-dependent DNA polymerase closely related to terminal deoxynucleotidyl transferase (TdT), and prone to induce template/primer misalignments and misincorporation. In addition to a proposed general role in non-homologous end joining of double-strand breaks, its mutagenic potential and preferential expression in secondary lymphoid tissues support a role in somatic hypermutation (SHM) of immunoglobulin genes. Here, we show that human Pol μ protein is expressed in the nucleus of centroblasts obtained from human tonsils, forming a characteristic foci pattern resembling that of other DNA repair proteins in response to DNA damage. Overexpression of human Pol μ in Ramos cells, in which the SHM process is constitutive, augmented the somatic mutations specifically at the variable (V) region of the immunoglobulin genes. The nature of the mutations introduced, mostly base substitutions, supports the contribution of Pol μ to mutation of G and C residues during SHM. In vitro analysis of Pol μ misincorporation on specific templates, that mimic DNA repair intermediates and correspond to mutational hotspots, indicated that many of the mutations observed in vivo can be explained by the capacity of Pol μ to induce transient template/primer misalignments.     

Tagging quantitative trait loci for maturity-corrected late blight resistance in tetraploid potato with PCR-based candidate gene markers

Late blight caused by the oomycete Phytophthora infestans is the economically most important and destructive disease in potato cultivation. Quantitative resistance to late blight available in tetraploid cultivars is correlated with late maturity in temperate climates, which is an undesirable characteristic. A total of 30 DNA-based markers known to be linked to loci for pathogen resistance in diploid potato were selected and tested as polymerase chain reaction-based markers for linkage with quantitative trait loci (QTL) for late blight resistance and plant maturity in two half-sib families of tetraploid potatoes. Most markers originated from within or were physically closely linked to candidate genes for quantitative resistance factors. The families were repeatedly evaluated in the field for quantitative resistance to late blight and maturity. Resistance was corrected for the maturity effect. Nine of eleven different map segments tagged by the markers harbored QTL affecting maturity-corrected resistance. Interactions were found between unlinked resistance QTL, providing testable strategies for marker-assisted selection in tetraploid potato. Based on the linkage observed between QTL for resistance and plant maturity and based on the genetic interactions observed between candidate genes tagging resistance QTL, we discuss models for the molecular basis of quantitative resistance and maturity.     

Clofibric and ethacrynic acids prevent experimental pyelonephritis by Escherichia coli in mice

Interfering Escherichia coli attachment to the urinary tract, using P-fimbriation inhibitors, can prevent pyelonephritis. Clofibric and ethacrynic acids are organic compounds structurally related, but with different pharmacological uses. These agents are potentially active in the urinary tract due to its elimination in an unaltered form by the renal route. This study described a pyelonephritogenic E. coli strain, grown in the presence of sub-inhibitory concentrations of clofibric or ethacrynic acids (0.1 and 1 mM, respectively), which exhibits inhibition of P1 erythrocytes agglutination and a drastic decrease in fimbriation, using electron microscopy and quantitative analyses of superficial proteins (decrease to a 17-25% in comparison with the control). In vivo assays were performed using ascending urinary tract infection in mice. The treatment with therapeutic doses of the drugs, administered 2 days before the bacterial challenge and daily until the end of the experiment (22 days), abolished renal infection after 7-10 days of drug exposure. Within this period clofibric acid did not produce adverse effects on the renal parenchyma. However, ethacrynic acid caused pyelitis and tubular cellular desquamation. These results suggested that clofibric acid might be useful in the short-term prophylaxis of urinary tract infection.