New Approach for Inhibition of HIV Entry: Modifying CD4 Binding Sites by Thiolated Pyrimidine Derivatives.

Thiolated pyrimidine derivatives have been synthetized and their antiretroviral effect against human immunodeficiency virus type 1 (HIV-1IIIB) and HIV-1 chimeric pseudovirions have been quantitatively determined in cell-based viral infectivity assays including syncytium inhibition assay as well as a single-cycle viral infection assay on HeLaCD4-LTR/ß-gal cells. Pseudotype virions prepared bearing HIV-1 envelope preference for CCR5 coreceptor, CXCR4 coreceptor or for both, respectively, with a HIV-1 core containing luciferase reporter gene were able to infect susceptible cells but are replication defective so unable to replicate in the cells . Data indicate that thiolated pyrimidine derivatives inhibited effectively virally induced cell fusion in vitro as well as infectivity of primary HIV-1IIIB strain and HIV-1 pseudovirions using chemokine receptors CCR5 or CXCR4 or both for virus entry a dose dependent manner.

Myelotoxicity of carboplatin is increased in vivo in db/db mice, the animal model of obesity-associated diabetes mellitus.

Purpose: Some authors observed increased carboplatin-associated myelotoxicity in obese patients which was exclusively attributed to elevated AUC. To investigate the potential contribution of functional changes of cells primarily responsible for myelopoiesis, granulocyte-macrophage progenitors (CFU-GM) were studied in obesity-associated diabetes mellitus (DMT2).

Methods: The most frequently used animal model of human obesity with DMT2 is db/db mouse.

Thiolated pyrimidine nucleotides may interfere thiol groups concentrated at lipid rafts of HIV-1 infected cells.

Upon HIV infection, cells become activated and cell surface thiols are present in increased number. Earlier we demonstrated in vitro anti-HIV effect of thiolated pyrimidine nucleotide UD29, which interferes thiol function. To further analyse the redox processes required for HIV-1 entry and infection, toxicity assays were performed using HIV-1 infected monolayer HeLaCD4-LTR/ β-gal cells and suspension H9 T cells treated with several thiolated nucleotide derivatives of UD29.

A challenging epigenetic message: telomerase activity is associated with complex changes in lifestyle.

As an outcome of The 2009 Nobel Prize in Physiology or Medicine, a connection has been highlighted between the length of telomeres and epigenetic effects, such as intensive changes in lifestyle and nutrition as well as behavioural and psychological factors. In this review, the various elements of molecular, cell biological, nutritional and lifestyle changes are introduced and discussed.

In vitro and in vivo activity of 4-thio-uridylate against JY cells, a model for human acute lymphoid leukemia.

We have previously reported the in vitro anti-proliferative effect of 4-thio-uridylate (s(4)UMP) on OCM-1 uveal melanoma cells. Here, we assessed the efficacy of s(4)UMP on JY cells. Treatment of JY cells with s(4)UMP suppressed their colony forming activity and induced apoptosis; healthy human bone marrow granulocyte-macrophage progenitor cells were 14-fold less sensitive to the nucleotide.

[The 2009 Nobel Prize in Medicine and its surprising message: lifestyle is associated with telomerase activity].

The 2009 Nobel Prize in Physiology and Medicine was awarded to three scientists for their pioneer research on telomeres - and the enzyme that forms them - telomerase. Their work highlighted the considerable connection between the length of telomeres and intensive changes in lifestyle and nutrition (Ornish method) as well as behavioral and psychological factors. In this review the various elements of molecular, cell biological, nutritional and lifestyle changes are introduced and discussed.

4-Thio-uridylate (UD29) interferes with the function of protein -SH and inhibits HIV replication in vitro.

In this short communication, it is shown that 4-thio-uridylate (s(4)UMP, designated as UD29) inhibits glyceraldehyde 3-phosphate dehydrogenase (GAPDH), suggesting that the enol-form of the thiolated nucleotide may interfere with the function of the essential -SH group in the active center of the enzyme. Since HIV entry requires thiol/disulfide exchange processes, this activity prompted us to study the anti-HIV activity of the nucleotide. Indeed, UD29 inhibited the replication of HIV-1(IIIB) in the MT-4 cell line and HIV-1(Ada-M) in peripheral blood mononuclear cells (PBMC).

Bcl-2 antisense oligonucleotide inhibits the proliferation of childhood leukemia/lymphoma cells of the B-cell lineage.

An 18-mer phosphorothioate bcl-2 antisense oligonucleotide (ASO) inhibited colony formation of three B-cell leukemia/lymphoma cell lines in a dose dependent manner in the range of 0.125-0.5 micromol/l.

Supramolecular polymers based on the quadruplex formation of ditopic guanosine macromonomers in nonaqueous media.

The formation of supramolecular polymeric aggregates with a molecular mass of 100 kDa in a nonaqueous solution from a telechelic dimer of isopropylidene guanosine in the presence of K(+) ions is reported. The possible structure of macromonomers resulting from the development of G4 quartets was deduced from DOSY NMR, circular dichroism spectra, and dynamic light scattering measurements.

Effect of the extent of thiolation and introduction of phosphorothioate internucleotide linkages on the anti-HIV activity of Suligovir [(s4dU)35].

Suligovir is a 35-mer homo-oligonucleotide, containing exclusively 4-thio deoxyuridylate, proved to be a potent inhibitor of HIV entry. In this paper, we described the effect of extent of thiolation and the introduction of nuclease-resistant phosphorothioate linkages on the anti-HIV activity of Suligovir. We found that the decreased thiolated nucleotide content decreases the anti-HIV potency of the compound and the introduction of phosphorothioate linkages does not improve its antiviral activity.

Immunodetection of human telomerase reverse-transcriptase (hTERT) re-appraised: nucleolin and telomerase cross paths.

The involvement of telomerase in cellular immortalization and senescence has often been assessed by means of telomerase expression at the RNA level and quantification of telomerase activity by the telomeric repeat amplification protocol assay. However, these methods either neglected the existence of various telomerase splice variants, or ignored the nonconventional functions of telomerase independent of its ability to elongate and maintain telomere length. Immunodetection of telomerase is now being recognized as a necessary approach to precisely elucidate its roles in oncogenesis and senescence.

Apoptotic response of uveal melanoma cells upon treatment with chelidonine, sanguinarine and chelerythrine.

The benzophenanthridine alkaloids sanguinarine, chelerythrine and chelidonine were reported previously to provoke cell death in a variety of tumor cells suggesting their potential application as anticancer agents. Here we tested their effects on a primary human uveal melanoma cell line, OCM-1. Flow cytometric analysis of annexin V binding/PI exclusion and DNA fragmentation disclosed that all these alkaloids could induce apoptosis in OCM-1 cells.

Deoxy-adenosine-monophosphate (dAMP) di-n-butylester induces apoptosis by increasing the dATP level in HL-60 cells.

Deoxy-ATP is a potent inducer of apoptosis. We intended to synthesize a lipophilic dAMP derivative which, according to our working hypothesis penetrates into the cell, is converted to dAMP by intracellular esterases and to dATP by nucleotide kinases. We synthesized dAMP-di-n-butylester (DAB) and tested it.

Potent inhibition of HIV-1 entry by (s4dU)35.

We have previously reported the potent in vitro HIV-1 anti-reverse transcriptase activity of a 35-mer of 4-thio-deoxyuridylate [(s(4)dU)(35)]. In efforts to define its activity in a more physiological system, studies were carried out to determine the stage of viral infection that this compound mediates its anti-viral effect. Results of the studies reported herein show that (s(4)dU)(35) is nontoxic and is capable of inhibiting both single and multi-drug resistant HIV strains (IC(50): 0.

Inhibition of human telomerase by oligonucleotide chimeras, composed of an antisense moiety and a chemically modified homo-oligonucleotide.

Most tumor cells attain their immortality by reactivating telomerase. We report here the telomerase inhibitory potential of chimeric oligonucleotides composed of a 13mer antisense sequence targeting the telomerase RNA template region and a (s4dU)n moiety at its 3' or 5'-end. The increase of the thiolated chain length enhances the telomerase inhibitory potential, but decreases specificity, indicated by HIV reverse transcriptase inhibition.

A DNA uptake-stimulating protein increases the antiproliferative effect of c-myb antisense oligonucleotide on leukemic cells.

Proliferation of HL-60 and MOLT4 leukemia cells was inhibited by a c-myb antisense oligonucleotide (ASO) in the presence of a DNA uptake-stimulating protein (DNA uptake-stimulating factor, DUSF). The inhibitory effect was very mild in the absence of DUSF. Sense oligonucleotides or DUSF, alone or in combination, were found to be ineffective.

Effect of increasing thiolation of the polycytidylic acid strand of poly I:poly C on the alpha, beta and gamma interferon-inducing properties, antiviral and antiproliferative activities.

Double-stranded RNAs induce interferons and cause the development of antiviral and antiproliferative activities. Antiviral activity is related to the production of interferons and other proteins that stimulate various immunologic activities, which appear to contribute to their overall antiproliferative activity. The most active double-stranded RNA, polyI:polyC, was shown to be too toxic for therapeutic use.

Membrane topography of HLA I, HLA II, and ICAM-1 is affected by IFN-gamma in lipid rafts of uveal melanomas.

The lateral distribution and colocalization of HLA I, HLA-DR, and ICAM-1 proteins was studied for the first time in the plasma membrane of two human uveal melanoma cell lines, OCM-1 and OCM-3. Our fluorescence resonance energy transfer and confocal laser scanning microscopic experiments revealed that these molecules are mostly confined to the same membrane regions, where they form similar protein patterns (homo- and hetero-associates) to those found previously on other cell types of lymphoid as well as colorectal carcinoma origin. Confocal microscopic colocalization experiments with GM(1) gangliosides and the GPI-anchored CD59 molecules showed enrichment of HLA I, HLA-DR, and ICAM-1 molecules in specific membrane domains (lipid rafts) excluding the transferrin receptor.

Interleukin-2 family cytokines stimulate phosphorylation of the Pro-Ser-Pro motif of Stat5 transcription factors in human T cells: resistance to suppression of multiple serine kinase pathways.

Signal transducer and activator of transcription (Stat)5a and Stat5b are critical for normal immune function. Progression of T cells through G(1)-S phase of cell cycle requires T cell receptor (TCR)- and/or cytokine-inducible tyrosine phosphorylation of Stat5a/b. Stat5a/b may also, in a cell-dependent manner, be constitutively or cytokine-inducibly phosphorylated on a Pro-Ser-Pro (PSP) motif located within the transcriptional activation domain.