Biomedical subjects

Aurelian Radu

Publications and source records attributed to Aurelian Radu.

8 recordsLinked to original sources

PubMed2006

Long-term culture of human gingival keratinocyte progenitor cells by down-regulation of 14-3-3 sigma.

Human gingival keratinocytes in culture stop proliferating after a limited number of passages. This limitation is associated with a gradual depletion of the stem cell compartment of the cell population. Human skin keratinocytes have a three- to five-fold higher proliferation capacity under similar culture conditions, and previous studies indicated that stable down-regulation of the 14-3-3 sigma protein in these cultures prevents stem cell differentiation and generates immortal cell lines without the effects of tumorigenic transformation, e.g., genotypic alterations. In this report, we demonstrate the creation of an immortalized human gingival keratinocyte stem cell line by stable down-regulation of the 14-3-3 sigma protein. Keratinocyte cultures were generated from human subjects ranging from 17 to 92 years of age and retrovirally transduced with a 14-3-3 sigma antisense RNA expression construct. In contrast to the control cultures, which propagated for only 2-5 passages and 25-35 cell doublings, the 14-3-3 sigma-transduced cultures propagated for 11 passages and 110 cell doublings so far. The percentage of stem cells measured by clonal analysis, which gradually decreased in the control cultures, increased to a steady level of over 90% in the 14-3-3 sigma down-regulated culture. This gingival keratinocyte stem cell line and others, which can be generated using the same procedure, have the potential to be useful for studies on stem cell differentiation, for developing gene therapy procedures that target the gingival epithelium, as well as a stable platform for testing oral hygiene products and as potential material for preprosthetic surgery.

14-3-3 Proteins↗

PubMed2004

Paired-type homeodomain transcription factors are imported into the nucleus by karyopherin 13.

We report that the paired homeodomain transcription factor Pax6 is imported into the nucleus by the Karyopherin beta family member Karyopherin 13 (Kap13). Pax6 was identified as a potential cargo for Kap13 by a yeast two-hybrid screen. Direct binding of Pax6 to Kap13 was subsequently confirmed by in vitro assays with recombinant proteins, and binding in vivo was shown by coimmunoprecipitation. Ran-dependent import of Pax6 by Kap13 was shown to occur by using a digitonin-permeabilized cells assay. Kap13 binds to Pax6 via a nuclear localization sequence (NLS), which is located within a segment of 80 amino acid residues that includes the homeodomain. Kap13 showed reduced binding to Pax6 when either region located at each end of the homeodomain (208 to 214 and 261 to 267) was deleted. The paired-type homeodomain transcription factor family includes more than 20 members. All members contain a region similar to the NLS found in Pax6 and are therefore likely to be imported by Kap13. We confirmed this hypothesis for Pax3 and Crx, which bind to and are imported by Kap13.

Amino Acid Sequence↗

PubMed2003

PTEN induces cell cycle arrest by decreasing the level and nuclear localization of cyclin D1.

PTEN is a tumor suppressor frequently inactivated in brain, prostate, and uterine cancers that acts as a phosphatase on phosphatidylinositol-3,4,5-trisphosphate, antagonizing the activity of the phosphatidylinositol 3'-OH kinase. PTEN manifests its tumor suppressor function in most tumor cells by inducing G(1)-phase cell cycle arrest. To study the mechanism of cell cycle arrest, we established a tetracycline-inducible expression system for PTEN in cell lines lacking this gene. Expression of wild-type PTEN but not of mutant forms unable to dephosphorylate phosphoinositides reduced the expression of cyclin D1. Cyclin D1 reduction was accompanied by a marked decrease in endogenous retinoblastoma (Rb) protein phosphorylation on cyclin D/CDK4-specific sites, showing an early negative effect of PTEN on Rb inactivation. PTEN expression also prevented cyclin D1 from localizing to the nucleus during the G(1)- to S-phase cell cycle transition. The PTEN-induced localization defect and the cell growth arrest could be rescued by the expression of a nucleus-persistent mutant form of cyclin D1, indicating that an important effect of PTEN is at the level of nuclear availability of cyclin D1. Constitutively active Akt/PKB kinase counteracted the effect of PTEN on cyclin D1 translocation. The data are consistent with an oncogenesis model in which a lack of PTEN fuels the cell cycle by increasing the nuclear availability of cyclin D1 through the Akt/PKB pathway.

Active Transport, Cell Nucleus↗

PubMed2002

Oligonucleotide microarray data mining: search for age-dependent gene expression.

Information on gene expression in colon tumors versus normal human colon was recently generated by an oligonucleotide microarray study. We used the associated database to search for genes that display age-dependent variations in expression. Statistically significant evidence was obtained that such genes are present in both the tumor and normal tissue databases. Besides the analysis of all genes included in the database, three subsets of genes were analyzed separately: genes controlled by p53, and genes coding for ribosomal proteins and for nuclear-encoded mitochondrial proteins. Among the genes controlled by p53 some show an age-dependent change in expression in tumor tissues, in the sense compatible with an activation of p53 at higher age. A decreased expression of some ribosomal genes at advanced age was detected both in tumor and normal tissues. No significant age-dependent expression could be detected for genes encoding mitochondrial proteins.

Adult↗

PubMed2002

Karyopherin beta 2B participates in mRNA export from the nucleus.

Transport of macromolecules between the cell nucleus and cytoplasm occurs through the nuclear pores and is mediated by soluble carriers known as karyopherins (Kaps), transportins, importins, or exportins. We report that Kap beta2B (transportin-2) forms complexes with the mRNA export factor TAP in the presence of RanGTP, as shown by coimmunoprecipitation from HeLa cells. The interaction strictly depends on the presence of RanGTP. In digitonin-permeabilized cells, Kap beta2B mediates TAP-GFP export from the nuclei in the presence of RanGTP. A TAP mutant that does not coimmunoprecipitate with Kap beta2B is also not exported by Kap beta2B. In the permeabilized cells assay, TAP is also exported independently of Kap beta2B by direct interaction with nucleoporins, in agreement with previous reports. The export rate is, however, significantly lower than the Kap beta2B-mediated pathway. Both Kap beta2B and TAP are present and enriched in the poly(A)(+) RNA complexes isolated from HeLa cell nuclear lysates. Poly(A)(+) RNA strongly accumulates in the nuclei of HeLa cells treated with Kap beta2B short interfering RNA, indicating that Kap beta2B is involved in the export of at least a large proportion of the mRNA species. The export of beta-actin and GAPDH mRNA is also inhibited, whereas 28S RNA is not affected. The data support the conclusion that Kap beta2B participates directly in the export of a large proportion of cellular mRNAs, and TAP connects Kap beta2B to the mRNAs to be exported.

Active Transport, Cell Nucleus↗

PubMed2002

An approach to increased polyplex gene delivery by peptides selected from a phage display library.

Phage display libraries were screened for peptides to be incorporated in nonviral gene delivery vehicles. Cells in culture were incubated with heptamer random peptide libraries displayed on M13 bacteriophages in three to five copies per phage. Surface-adherent phages were removed or inactivated and the cells were fractionated in a nuclear pellet and supernatant. Bacteriophages from each of the two fractions were amplified and reincubated with the cells. Three successive rounds of selection were performed. Eighteen sequenced clones revealed 14 different sequences. Two sequences were homologous to segments of the HIV gp120 protein. For three sequences, the corresponding synthetic peptides were generated and attached via avidin-biotin to polylysine-condensed plasmid DNA containing a reporter gene. The addition of the peptides led to 8-14 times increase in the expression of the reporter.

Amino Acid Sequence↗

PubMed2002

Age-associated reduction of nuclear protein import in human fibroblasts.

Age-dependent decreases in the protein concentrations of the nucleocytoplasmic transport factors karyopherin alpha2, CAS, and RanBP1 were found by comparing fibroblast cultures obtained from young, mature, and old human donors. Karyopherin beta1 levels do not change with age and present very little variation among donors. The decrease in the concentration of transport factors is accompanied by a reduction in the protein import rate in fibroblasts from old donors, as detected by a change in the intracellular localization of a test transport substrate that shuttles between the cytoplasm and the nucleus. Measurements of concentrations of the same import factors in organs and tissues of old mice revealed a decrease of CAS in kidney, lung, and spleen. The import reduction in old age is expected to lead to impaired activity of proteins whose functions depend on timely import into the nuclei.

Active Transport, Cell Nucleus↗

PubMedDate not supplied

DNA condensation by high-affinity interaction with avidin.

Avidin, the basic biotin-binding glycoprotein from chicken egg white, is known to interact with DNA, whereas streptavidin, its neutral non-glycosylated bacterial analog, does not. In the present study we investigated the DNA-binding properties of avidin. Its affinity for DNA in the presence and absence of biotin was compared with that of other positively charged molecules, namely the protein lysozyme, the cationic polymers polylysine and polyarginine and an avidin derivative with higher isoelectric point (pI approximately 11) in which most of the lysine residues were converted to homoarginines. Gel-shift assays, transmission electron microscopy and dynamic light scattering experiments demonstrated an unexpectedly strong interaction between avidin and DNA. The most pronounced gel-shift retardation occurred with the avidin-biotin complex, followed by avidin alone and then guanidylated avidin. Furthermore, ultrastructural and light-scattering studies showed that avidin assembles on the DNA molecule in an organized manner. The assembly leads to the formation of nanoparticles that are about 50-100 nm in size (DNA approximately 5 kb) and have a rod-like or toroidal shape. In these particles the DNA is highly condensed and one avidin is bound to each 18 +/- 4 DNA base pairs. The complexes are very stable even at high dilution ([DNA] =10 pM) and are not disrupted in the presence of buffers or salt (up to 200 mM NaCl). The other positively charged molecules also condense DNA and form particles with a globular shape. However, in this case, these particles disassemble by dilution or in the presence of low salt concentration. The results indicate that the interaction of avidin with DNA may also occur under physiological conditions, further enhanced by the presence of biotin. This DNA-binding property of avidin may thus shed light on a potentially new physiological role for the protein in its natural environment.

Avidin↗