A 1-Mb Physical Map and PAC Contig of the Imprinted Domain in 11p15.5 That Contains TAPA1 and the BWSCR1/WT2 Region

We have constructed a 1-Mb contig in human chromosomal band 11p15.5, a region implicated in the etiology of several embryonal tumors, including Wilms tumor, and in Beckwith–Wiedemann syndrome. Cosmid, P1, PAC, and BAC clones were characterized byNotI/SalI digestion and hybridized to a variety of probes to generate a detailed physical map that extends from D11S517 to L23MRP. Included in the map are the CARS, NAP2, p57/KIP2, KVLQT1, ASCL2, TH, INS, IGF2, H19, and L23MRP genes as well as end probes isolated from PACs. The TAPA1 gene, whose protein product can transmit an antiproliferative signal, was also localized in the contig. However, Northern blot analysis demonstrated that its expression did not correlate with tumorigenicity in G401 Wilms tumor hybrids, suggesting that TAPA1 is not responsible for the tumor suppression associated with 11p15.5. Genomic clones were used as probes in FISH analysis to map the breakpoints from three Beckwith–Wiedemann syndrome patients and a rhabdoid tumor. Interestingly, each of the breakpoints disrupts the KVLQT1 gene, which is spread over a 400-kb region of the contig. Since 11p15.5 contains several genes with imprinted expression and one or more tumor suppressor genes, our contig and map provide a framework for characterizing this intriguing genetic environment.     

Microtubule cold stability in supporting cells of the gerbil auditory sensory epithelium: correlation with tubulin post-translational modifications

Sensory cells in the organ of Corti exhibit loose microtubule networks enriched in tyrosinated tubulin, whereas supporting cells have bundled microtubules containing post-translationally modified tubulin. The tubulin isoform distribution suggests that the microtubules in sensory cells are dynamic and those in supporting cells are stable. To test this, microtubule resistance to cold-induced depolymerization was examined by using immunocytochemical methods and antibodies to post-translationally modified tubulins. Microtubule labelling in cochleas perfused/immersed at room temperature was identical to that in previous studies of untreated cochleas. However, the microtubule patterns of perfused/immersed specimens were changed in cold-treated cochleas. Microtubules were no longer detected with antibodies to alpha- and tyrosinated tubulin in sensory cells from specimens exposed to cold, indicating their disassembly. Supporting cells in the same specimens showed almost total loss of detyrosinated and polyglutamylated tubulin in the middle and apical cochlear turns, and reduced labelling in the basal-most turn. Probing for alpha-, nontyrosinatable, acetylated and glycylated tubulin yielded decreased and sometimes patchy staining but these isoforms were observed even when detyrosinated and polyglutamylated tubulins were absent. The results indicate that sensory cells in the gerbil auditory sensory epithelium contain only cold-sensitive microtubules. In contrast, supporting cells possess a substantial subset of cold-stable microtubules, providing structural support to the vibratory sensory organ required for hearing.     

Self-antigen tetramers discriminate between myelin autoantibodies to native or denatured protein

The role of autoantibodies in the pathogenesis of multiple sclerosis (MS) and other demyelinating diseases is controversial, in part because widely used western blotting and ELISA methods either do not permit the detection of conformation-sensitive antibodies or do not distinguish them from conformation-independent antibodies. We developed a sensitive assay based on self-assembling radiolabeled tetramers that allows discrimination of antibodies against folded or denatured myelin oligodendrocyte glycoprotein (MOG) by selective unfolding of the antigen domain. The tetramer radioimmunoassay (RIA) was more sensitive for MOG autoantibody detection than other methodologies, including monomer-based RIA, ELISA or fluorescent-activated cell sorting (FACS). Autoantibodies from individuals with acute disseminated encephalomyelitis (ADEM) selectively bound the folded MOG tetramer, whereas sera from mice with experimental autoimmune encephalomyelitis induced with MOG peptide immunoprecipitated only the unfolded tetramer. MOG-specific autoantibodies were identified in a subset of ADEM but only rarely in adult-onset MS cases, indicating that MOG is a more prominent target antigen in ADEM than MS.     

Placental insufficiency results in temporal alterations in the renin angiotensin system in male hypertensive growth restricted offspring

Reduced uterine perfusion initiated in late gestation in the rat results in intrauterine growth restriction (IUGR) and development of hypertension by 4 wk of age. We hypothesize that the renin angiotensin system (RAS), a regulatory system important in the long-term control of blood pressure, may be programmed by placental insufficiency and may contribute to the etiology of IUGR hypertension. We previously reported that RAS blockade abolished hypertension in adult IUGR offspring; however, the mechanisms responsible for the early phase of hypertension are unresolved. Therefore, the purpose of this study was to examine RAS involvement in early programmed hypertension and to determine whether temporal changes in RAS expression are observed in IUGR offspring. Renal renin and angiotensinogen mRNA expression were significantly decreased at birth (80 and 60%, respectively); plasma and renal RAS did not differ in conjunction with hypertension (mean increase of 14 mmHg) in young IUGR offspring; however, hypertension (mean increase of 22 mmHg) in adult IUGR offspring was associated with marked increases in renal angiotensin-converting enzyme (ACE) activity (122%) and renal renin and angiotensinogen mRNA (7-fold and 7.4-fold, respectively), but no change in renal ANG II or angiotensin type 1 receptor. ACE inhibition (enalapril, 10 mg x kg(-1) x day(-1), administered from 2 to 4 wk of age) abolished hypertension in IUGR at 4 wk of age (decrease of 15 mmHg, respectively) with no significant depressor effect in control offspring. Therefore, temporal alterations in renal RAS are observed in IUGR offspring and may play a key role in the etiology of IUGR hypertension.     

Synthesis and antiviral activity of azoles obtained from carbohydrates

Herein we describe the synthesis of 1,2,4-triazolyl-3-thione;1,3,4-oxadiazole, and imidazo[2,1-b]thiazole derivatives from carbohydrates. The antiviral activity of these compounds was tested against Dengue and Junin virus (the etiological agent of Argentine hemorrhagic fever). The 3-(p-bromobenzoyl)-5-(1,2-O-isopropylidene-3-O-methyl-alpha-d-xylofuranos-5-ulos-5-yl)imidazo[2,1-b]thiazole was able to inhibit the replication of both viruses in Vero cells at concentration significantly lower than the CC(50).     

CULTURING,ULTRASTRUCTURE AND COLONY FORMATION IN PECTODICTYON CUBICUM (CHLOROPHYCEAE,CHLOROCOCCALES) 12

Pectodictyon cubicum Taft collected in California develops typical eight-celled, cuboidal colonies only in alkaline media. Vegetative cell ultrastructure is similar to that in other genera of the Chlorococcales, except for an extensive endoplasmic reticulum system paralleling the plasmalemma (termed a paramural ER). Autosporogenesis proceeds inside the parent cell wall by three mitotic divisions producing eight nuclei in two tiers of four. Cleavage furrows following paths delineated by long ER segments and indistinct microtubules bisect and then radically separate the protoplast into eight pyramidal units. Walls comprised of a thick, presumably cellulose layer and a thinner trilaminar layer (not acetolysis resistant) form as cells become rounded, except where touching. A gelatinous matrix is produced around and between cells with concentration at the three sites of prior cell contact 90 degrees apart. Pulsed production of mucilage in three solid strands forces cells to separate, and the expanding colony becomes a hollow cube with a cell at each corner.     

Activation of fermentation by salts in Debaryomyces hansenii

The presence of 1.0 M KCl or NaCl during growth of Debaryomyces hansenii results in increased ethanol production. An additional increase of fermentation was observed when the salts were also present during incubation under nongrowing conditions. Extracts of cells grown in the presence of salt showed increased alcohol dehydrogenase and phosphofructokinase activities, indicating that these enzymes are responsible for the increased fermentation capacity. This is confirmed by measurements of the glycolytic intermediates. The increased fermentation capacity of the cells grown with salts seems to enable them to cope with the additional energy required for uptake and/or efflux of cations.     

Novel cellular mechanism that mediates the collecting duct formation during postnatal renal development

We have previously demonstrated that kidney embryonic structures are present in rats, and are still developing until postnatal Day 20. Consequently, at postnatal Day 10, the rat renal papilla contains newly formed collecting duct (CD) cells and others in a more mature stage. Performing primary cultures, combined with immunocytochemical and time-lapse analysis, we investigate the cellular mechanisms that mediate the postnatal CD formation. CD cells acquired a greater degree of differentiation, as we observed that they gradually lose the ability to bind BSL-I lectin, and acquire the capacity to bind Dolichos biflorus. Because CD cells retain the same behavior in culture than in vivo, and by using DBA and BSL-I as markers of cellular lineage besides specific markers of epithelial/mesenchymal phenotype, the experimental results strongly suggest the existence of mesenchymal cell insertion into the epithelial CD sheet. We propose such a mechanism as an alternative strategy for CD growing and development.     

A new rapid fluorogenic method for measuring bacteriocin activity

We describe a novel bacteriocin screening assay based on fluorescence emitted by berberine following its influx into compromised cells. This technique showed agreement with the conventional well-diffusion method, and results can be obtained within one hour. This assay could facilitate the rapid identification of bacteriocinogenic bacterial isolates.     

Ketoconazole and miconazole alter potassium homeostasis in Saccharomyces cerevisiae

The effects of ketoconazole and miconazole uptake on K(+) transport and the internal pH of Saccharomyces cerevisiae were studied. The uptake of both drugs was very fast, linear with concentration and not dependent on glucose, indicating entrance by diffusion and concentrating inside. Low (5.0μM) to intermediate concentrations (40μM) of both drugs produced a glucose-dependent K(+) efflux; higher ones also produced a small influx of protons, probably through a K(+)/H(+) exchanger, resulting in a decrease of the internal pH of the cells and the efflux of material absorbing at 260nm and phosphate. The cell membrane was not permeabilized. The K(+) efflux with miconazole was dependent directly on the medium pH. This efflux results in an increased membrane potential, responsible for an increased Ca(2+) uptake and other effects. These effects were not observed with two triazolic antifungals. A decrease of the Zeta (ζ) potential was observed at low concentrations of miconazole. Although the main effect of these antifungals is the inhibition of ergosterol synthesis, K(+) efflux is an important additional effect to be considered in their therapeutic use. Under certain conditions, the use of single mutants of several transporters involved in the movements of K(+) allowed to identify the participation of several antiporters in the efflux of the cation.