Requirement for proliferating cell nuclear antigen expression during stages of the Chinese hamster ovary cell cycle

Proliferating cell nuclear antigen (PCNA/cyclin) is a nuclear protein that can stimulate purified DNA polymerase delta in vitro, and its synthesis correlates with the proliferation rate of cells. We have attempted to determine whether synthesis of PCNA/cyclin in Chinese hamster ovary cells is necessary to regulate entry into S phase. We have measured cellular PCNA/cyclin concentration of the mRNA or protein throughout the cell cycle. Cells were separated by centrifugal elutriation into populations enriched for G-1, S, and G-2/M phases. Quantitative Northern hybridization analysis was performed on RNA isolated from each cell population by using a cDNA clone of PCNA/cyclin as a probe. Results demonstrated that although intact PCNA/cyclin mRNA is present during all phases of the cell cycle, an induction of about 3-fold occurs during S phase. Two-parameter staining for PCNA/cyclin and DNA, and analysis by flow cytometry, confirmed that the quantity of PCNA/cyclin protein in the cells increases severalfold in G-1 or early S phase but generally is invariant in S and G-2/M phases. This cell cycle dependence of PCNA/cyclin expression suggests that the observed synthesis is a prerequisite for initiation of DNA replication. Introduction of an antisense oligonucleotide complementary to the PCNA/cyclin mRNA to inhibit PCNA/cyclin synthesis effectively prevented entry of G-1 phase cells into S phase. A complementary sense oligonucleotide used as a control did not have an inhibitory effect. This result suggests that a threshold concentration of PCNA/cyclin is necessary for entry into S phase.     

Identification of 3- and 4-phosphorylated phosphoinositides and inositol phosphates in stomatal guard cells

Components of the polyphosphoinositide signalling pathway have been identified in stomatal guard cells of Commelina communis L., one of the few plant systems shown unequivocally to be capable of responding to release of inositol 1,4,5-trisphosphate in the cytoplasm by increase in cytoplasmic Ca²⁺. 'Isolated' epidermal strips of C. communis (in which all cells other than guard cells have been killed by treatment at low pH) were radiolabelled with myo -[2n-³H]inositol or [³²P]orthophosphate for 17–18 h. The phosphoinositides and inositol phosphates were extracted. Phosphoinositides were deacylated and the head groups resolved by HPLC. The water-soluble products generated by mild periodate cleavage of HPLC-purified, deacylated lipid fractions were examined. The resulting biochemical analysis led to the identification of: PtdIns, PtdIns3 P , PtdIns4 P , PtdIns(3,4) P ₂ and PtdIns(4,5) P ₂. Thex inositol phosphates were resolved by HPLC. Preliminary analysis of HPLC-purified putative inositol phosphate fractions resulted in the identification of each inositol phosphate class, that is, Ins P , Ins P ₂, Ins P ₃, Ins P ₄, Ins P ₅ and Ins_P6. Many of these inositol phosphates occurred in different isomeric forms. The presence of 3-phosphorylated phosphoinositides suggests that they may have a role in signalling in stomatal guard cells.     

Interaction of endothelial cells with a laminin A chain peptide (SIKVAV) in vitro and induction of angiogenic behavior in vivo.

Endothelial cells are known to bind to laminin, and two peptides derived from the laminin A (CTFALRGDNP) and B1 (CDPGYIGSR) chains block the capillary-like tube formation on a laminin-rich basement membrane matrix, Matrigel. In the present study, we have used various in vitro and in vivo assays to investigate the angiogenic-biologic effects of a third active site in the laminin A chain, CSRARKQAASIKVAVSADR (designated PA22-2) on endothelial cells. The SIKVAV-containing peptide was as active as the YIGSR-containing peptide for endothelial cell attachment but was less active than either the RGD-containing peptide or intact laminin. Endothelial cells seeded on this peptide appeared fibroblastic with many extended processes, unlike the normal cobblestone morphology observed on tissue culture plastic. In addition, in contrast to normal tube formation on Matrigel, short irregular structures formed, some of which penetrated the matrix and sprouting was more apparent. Analysis of endothelial cell conditioned media of cells cultured in the presence of this peptide indicated degradation of the Matrigel and zymograms demonstrated active collagenase IV (gelatinase) at 68 and 62 Kd. A murine in vivo angiogenesis assay and the chick yolk sac/chorioallantoic membrane assays with the peptide demonstrated increased endothelial cell mobilization, capillary branching, and vessel formation. These data suggest that the -SIKVAV-site may play an important role in initiating branching and formation of new capillaries from the parent vessels, a behavior that is observed in vivo in response to tumor growth or in the normal vascular response to injury.     

Potential pathophysiological role of microRNA 193b-5p in human placentae from pregnancies complicated by preeclampsia and intrauterine growth restriction

Molecular Biology Reports - Preeclampsia (PE) and intrauterine growth restriction (IUGR) are pregnancy complications resulting from abnormal placental development. MicroRNAs can regulate placental...     

Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON—bisLNA—with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson–Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.