Differential patterns of expression of Eps15 and Eps15R during mouse embryogenesis

Eps15 and Eps15R are related tyrosine kinase substrates, which have been implicated in endocytosis and synaptic vesicle recycling. Through the protein:protein interaction abilities of their EH domains, they establish a complex network of interactions with several proteins, including Numb, a protein necessary for neuronal cell fate specification. We analyzed the expression of Eps15 and Eps15R during murine development, at the time of active neurogenesis. The most striking difference was at the level of subcellular localization, with Eps15 present in the cytosol and on the plasma membrane, while Eps15R exhibited mainly a nuclear localization. Interesting topographical differences also emerged. In the 12.5 days post coitum neuroepithelium, Eps15 was expressed in the ventricular zone, which contains proliferating neuroblasts, whereas Eps15R was found only in postmitotic neurons. Conversely, both proteins were expressed in sensory and cranial ganglia. At later times, the expression of Eps15 and Eps15R was widely maintained in neuronal structures. In other tissues, Eps15 was first seen in the liver primordium and at low levels in choroid plexus, lung, kidney and intestine; later on the expression was maintained at high levels in epithelia. Nuclear staining of Eps15R was present in kidney, intestine, lung and liver, as well as in heart and pancreas.     

Association between GM3 and CD4-Ick complex in human peripheral blood lymphocytes

The aim of this study was to further elucidate our previous observation on molecular interaction of GM3, CD4 and p56^lck in microdomains of human peripheral blood lymphocytes (PBL). We analyzed GM3 distribution by immunoelectron microscopy and the association between GM3 and CD4-p56^lck complex by scanning confocal microscopy and co-immunoprecipitation experiments. Scanning confocal microscopy analysis showed an uneven signal distribution of GM3 molecules over the surface of human lymphocytes. Nearly complete colocalization areas indicated that CD4 molecules were distributed in GM3-enriched plasma membrane domains. Co-immunoprecipitation experiments revealed that CD4 and p56^lck were immunoprecipitated by IgG anti-GM3, demonstrating that GM3 tightly binds to the CD4-p56^lck complex in human PBL. In order to verify whether GM3 association with CD4 molecules may depend on the presence of p56^lck, we analyzed this association in U937, a CD4+and p56^lck negative cell line. The immunoprecipitation with anti-GM3 revealed the presence of a 58[emsp4 ]kDa band immunostained with anti-CD4 Ab, suggesting that the GM3-CD4 interaction does not require its association with p56^lck. These findings support the view that GM3 enriched-domains may represent a functional multimolecular complex involved in signal transduction and cell activation.     

Nucleolar Organizing Regions, 18S and 5S rDNA in Astyanax Scabripinnis (Pisces, Characidae): Populations Distribution and Functional Diversity

Astyanax scabripinnis specimens from four distinct populations in Brazil were studied with respect to their karyotype macrostructure, nucleolar organizer regions, and 18S and 5S rRNA genes. The four populations showed a 2n = 50 chromosomes (3 M + 11 SM + 5 ST + 6 A pairs) and 1–2 B chromosomes. No chromosomal differentiations were observed between sexes. Although a karyotypic diversity has been characterized in this fish group, the populations now analyzed presented the same macrokaryotypic pattern. Chromosome mapping of 5S rDNA showed a total of eight sites located in four distinct chromosomal pairs, with no apparent differences among populations. A comparative study on 18S rDNA locations and Ag-NORs showed some secondary NOR sites that are not usually expressed in karyotypes and a probable differential NOR activity among populations. Correlations between these data, environmental conditions and B chromosomes are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inactivation of Mitochondrial Permeability Transition Pore by Octylguanidine and Octylamine

Mitochondrial permeability transition occurs through a Ca²⁺-dependent opening of atransmembrane pore, whose identity has been attributed to that of the adenine nucleotide translocase(ANT). In this work, we induced permeability transition by adding 0.5 M carboxyatractyloside.The process was evaluated analyzing Ca²⁺ efflux, a drop in transmembrane electric gradient,and swelling. We found that the amphiphyllic cations octylguanidine and octylamine, at theconcentration of 100 M, inhibited, almost completely, nonspecific membrane permeability.Hexylguanidine, hexylamine, as well as guanidine chloride and hydroxylamine failed to doso. The inhibition was reversed after the addition of 40 mM Li⁺, Na⁺ K⁺,Rb⁺, or Cs⁺; K⁺ wasthe most effective. We propose that the positive charge of the amines interact with negativecharges of membrane proteins, more likely the ADP/ATP carrier, while the alkyl chain penetratesinto the hydrophobic milieu of the inner membrane, fixing the reagent.     

Selenium deficiency and hypothyroidism

Three female children presented with different clinical symptoms that could be related to impaired thyroid function. They underwent an accurate pediatric-endocrinologic diagnosis. Laboratory tests revealed no pathological findings, except latent hypothyroidism and selenium deficiency. Hypothyroidism was diagnosed by elevated basal TSH and by a pathological iv-TRH-stimulation test. After treating the children with sodium selenite orally for 4 wk, their metabolism had returned to normal and we saw a marked improvement of all clinical symptoms. For the first time, we have been able to describe hypothyroidism caused exclusively by selenium deficiency, the pathophysiology of which may be expressed as a malfunction of human 5′-deiodinases.     

The Arabidopsis thaliana PPX/PP4 phosphatases: molecular cloning and structural organization of the genes and immunolocalization of the proteins to plastids

The PPX/PP4 Ser/Thr protein phosphatases belong to the type 2A phosphatase subfamily and are present in most eukaryotic organisms. We have previously isolated two closely related DNAs encoding PPX isoforms (PPX-1 and PPX-2) of Arabidopsis thaliana. Here we report the molecular cloning of the genes encoding these proteins. The genes PPX-1 and PPX-2 are composed of eight exons and seven introns located at equivalent positions related to the coding sequences. Whereas the intron-exon organization of the PPX genes is completely different from that of the PP2A-3/PP2A-4 A. thaliana family, specific intron-exon boundaries are conserved among PPX genes from distantly related organisms. Based on GUS expression, both PPX genes show the same spatial and temporal pattern of expression: they are expressed in all the organs and tissues analyzed, and from the earliest stage of development. When PPX proteins were localized to the root in semi-thin methacrylate sections by immunofluorescence, staining was predominantly confined to small organelles, shown to be plastids by co-localization of PPX and ferredoxin. Interestingly, only some ferredoxin-positive plastids were also PPX-positive, and PPX staining was consistently brighter in the epidermis. The localization was confirmed with immunogold and electron microscopy. Our results suggest that, despite its strong sequence conservation, PPX in plants functions differently than in animals.     

PCR for Detection of Shigella spp. in Mayonnaise

The use of PCR to amplify a specific virA gene fragment serves as a highly specific and sensitive method to detect virulent bacteria of the genus Shigella and enteroinvasive Escherichia coli. Amplification of a 215-bp DNA band was obtained by using isolated genomic DNA of Shigella, individual cells of Shigella dysenteriae, and mayonnaise contaminated with S. dysenteriae. Moreover, a multiplex PCR with specific (virA) and bacterium-restricted (16S ribosomal DNA) primers generated an amplification product of approximately 755 bp for all bacteria tested and an additional 215-bp product for Shigella and enteroinvasive E. coli.     

Mitochondrial Membrane Potential and DNA Stainability in Human Sperm Cells: A Flow Cytometry Analysis with Implications for Male Infertility

Sperm cells from control donors of proven fertility and men from barren couples were studied by conventional procedures, i.e., light microscopy as well as flow cytometry. Light microscopy analysis of semen included the measurement of spermatozoa concentration, morphology, and motility. All the men from barren couples were asthenozoospermic at the conventional analysis of semen samples. Flow cytometry was applied to study two important parameters of sperm cells: mitochondrial membrane potential (MMP) assessed by the cationic dye JC-1 and DNA stainability with propidium iodide (PI). JC-1 staining was more reliable than the classical procedure used for this purpose, i.e., rhodamine 123 (Rh123) staining, and allowed us to show a positive correlation between MMP and spermatozoa motility. Regarding DNA analysis, a higher relative percentage of immature spermatozoa, showing a high accessibility of DNA to the intercalating PI fluorochrome, was found in men from barren couples compared to donors of proven fertility. The relative percentage of immature spermatozoa was significantly higher in semen from oligoasthenozoospermic subjects. Moreover, a positive correlation was found between immature spermatozoa, as evaluated by PI staining, and cells with depolarized mitochondria, as evaluated by JC-1 staining, suggesting that spermatozoa defective for nuclear maturity could be functionally defective cells. No correlation between immature spermatozoa determined by FCM and immature spermatozoa determined by light microscopy was found, suggesting that these two techniques assess sperm cell maturity at different levels.