The Inhibition of Adenosine Kinase by α,ω-Di (Adenosin - N 6- YL) Alkanes1

Abstract

Members of a series of α,ω-di(adenosin- N ⁶-yl)alkanes, comprising two adenosine residues linked with alkyl bridges from 1 to 14 methylene units in length, were found to be inhibitors of rat liver and BHK cell adenosine kinase. The inhibition was competitive with respect to adenosine and non-competitive with respect to ATP. The corresponding α,ω-di(cytidin-N ⁴-yl) alkanes were not inhibitors and N ⁶-alkyladenosines inhibited only weakly.     

The PowerAtlas: a power and sample size atlas for microarray experimental design and research

Background  

Microarrays permit biologists to simultaneously measure the mRNA abundance of thousands of genes. An important issue facing investigators planning microarray experiments is how to estimate the sample size required for good statistical power. What is the projected sample size or number of replicate chips needed to address the multiple hypotheses with acceptable accuracy? Statistical methods exist for calculating power based upon a single hypothesis, using estimates of the variability in data from pilot studies. There is, however, a need for methods to estimate power and/or required sample sizes in situations where multiple hypotheses are being tested, such as in microarray experiments. In addition, investigators frequently do not have pilot data to estimate the sample sizes required for microarray studies.     

Gene expression profiling of single bovine embryos uncovers significant effects of in vitro maturation, fertilization and culture

In vitro production (IVP) has been shown to affect embryonic gene expression and often result in large offspring syndrome (LOS) in cattle and sheep. To dissect the effects of in vitro maturation, fertilization and culture on bovine embryos, we compared the expression profiles of single blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF); (2) in vivo maturation, fertilization and in vitro culture (IVD); and (3) in vivo maturation, fertilization and development (AI). To conduct expression profiling, total RNA was isolated from individual embryos, linearly amplified and hybridized to a custom bovine cDNA microarray containing approximately 6,300 unique genes. There were 306, 367, and 200 genes differentially expressed between the AI and IVD, IVF and IVD, and AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and IVD embryos, making these potential candidates for LOS. There were 60 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology category "RNA processing" was over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on the ability to transcribe maternal RNA stores. A culture effect on the expression of genes involved in translation was also observed by the comparison of AI with IVD embryos.     

Y;autosome translocations and mosaicism in the aetiology of 45,X maleness: assignment of fertility factor to distal Yq11

Summary Three 45,X males have been studied with Y-DNA probes by Southern blotting and in situ hybridization. Southern blotting studies with a panel of mapped Y-DNA probes showed that in all three individuals contiguous portions of the Y chromosome including all of the short arm, the centromere, and part of the euchromatic portion of the long arm were present. The breakpoint was different in each case. The individual with the largest portion (intervals 1–6) is a fertile male belonging to a family in which the translocation is inherited in four generations. The second adult patient, who has intervals 1–5, is an azoospermic, sterile male. These phenotypic findings suggest the existence of a gene involved in spermatogenesis in interval 6 in distal Yq11. The third case, a boy with penoscrotal hypospadias, has intervals 1–4B. In situ hybridization with the pseudoautosomal probe pDP230 and the Y chromosome specific probe pDP105 showed that Y-derived DNA was translocated onto the short arm of a chromosome 15, 14, and 14, respectively. One of the patients was a mosaic for the 14p+ translocation chromosome. Our data and those reported by others suggest the following conclusions based on molecular studies in eight 45,X males: The predominant aetiological factor is Y;autosome translocation observed in seven of the eight cases. As the remaining case was a low-grade mosaic involving a normal Y chromosome, the maleness in all cases was due to the effect of the testis determing factor, TDF. There is preferential involvement of the short arm of an acrocentric chromosome (five out of seven translocations) but other autosomal regions can also be involved. The reason why one of the derivative translocation chromosomes becomes lost may be that it has no centromere.     

Cat Heart Muscle In Vitro : IV. Inhibition of transport in quiescent muscles

Cellular concentrations, [K]_i, [Na]_i, and [Cl]_i, and cell water contents were measured in vitro at 27°C in cat papillary muscles. Measurements were made with and without ouabain at varying concentrations of K and ouabain, at pH 5.2 and 9.0, in absence of O₂, and in NaCl-free solution. Large losses of cell K and increases of cell Na occurred in presence of ouabain, at 2–3°C, and in K-free medium. The dependence of inhibition of cation transport by ouabain on external K concentration, studied at constant initial [K]_i, was consistent with a competition between K and ouabain localized to the external face of the membrane. In NaCl-free sucrose solution [K]_i remained at its physiological value and was not affected by exposure to ouabain or low temperature, except when Ca was also omitted. Ouabain inhibition persisted at pH 9.0 and in Ca-poor media. Cells swelled and lost K at pH 5.2, and residual ouabain effect was small. At pH 9.0, or in absence of O₂, or in Ca-poor solutions cells became permeable to mannitol. The ion movements observed after inhibition of active transport are compatible either with a passive K distribution and a primary inhibition of Na extrusion or with inhibition of a coupled active transport of both K and Na.     

GSK3 beta mediates acentromeric spindle stabilization by activated PKC zeta

Upon fertilization, the mammalian egg undergoes a precise series of signaling events that orchestrate its conversion into a zygote. Mouse eggs contain acentrosomal spindle poles when arrested at meiotic metaphase II. The meiotic spindle is thought to provide a scaffold that mediates spatial and temporal regulation of the signaling pathways orchestrating post-fertilization events. Many kinases have been found to be enriched at the MII meiotic spindle, such as Protein Kinase C (PKC), and are thought to have an important role in regulating signaling events initiated through fertilization. In this study phosphorylated PKCζ (p-PKCζ) and Glycogen Synthase Kinase 3β (GSK3β) were found to be enriched at both acentrosomal spindle poles and the kinetochore region. Phosphorylated PKCζ (p-PKCζ) was immunopurified from MII eggs and was found to co-localize with known microtubule stabilizing components found in somatic cells, including GSK3β and Partition deficit protein 6 (Par6). Both fluorescence resonance energy transfer (FRET) and immunofluorescence confirmed the existence and close association of these proteins with p-PKCζ at the meiotic spindle. When GSK3β is phosphorylated on ser9 its activity is inhibited and the spindle is stabilized. However, when GSK3β is dephosphorylated (on ser9) it becomes active and the spindle is destabilized. The mechanism by which p-PKCζ maintains spindle organization appears to be through GSK3β and suggests that p-PKCζ phosphorylates GSK3β on the ser9 position inactivating GSK3β and consequently maintaining spindle stability during meiotic metaphase arrest.