No evidence of association between 118A>G OPRM1 polymorphism and heroin dependence in a large Bulgarian case-control sample

The μ-opioid receptor is the primary site of action of most opioids. The 118A>G (rs1799971) polymorphism in exon 1 of the μ-opioid receptor gene (OPRM1) leads to an Asn40Asp amino acid change that affects a putative N-glycosylation site. It has been widely investigated for association with alcohol and drug dependence and pain sensitivity, with mixed results. The aim of the current study was to examine whether this polymorphism was associated with heroin dependence in a large Bulgarian cohort of 1842 active users and 1451 population controls. SNP genotyping was done using Real-Time PCR TaqMan technology. Association analyses were conducted, separately for Roma and non-Roma participants. Our results suggest that there is no direct effect of 118A>G genotype on the risk for heroin dependence among active heroin users.     

Effects of the cyclin-dependent kinase inhibitor CYC202 (R-roscovitine) on the physiology of cultured human keratinocytes

CYC202 (R-roscovitine) is a potent cyclin-dependent kinase inhibitor, investigated as a potential anti-cancer agent. The knowledge of the action of this pharmacological agent on normal human cells is still limited. In this study, we have explored the effects of the cyclin-dependent kinase inhibitor CYC202 on normal human epidermal keratinocytes. The loss of cell viability induced by this compound was strongly dependent on the rate of keratinocyte proliferation. At slightly cytotoxic doses, CYC202 inhibited the proliferation of subconfluent keratinocytes in a dose-dependent manner, and at higher concentrations induction of early apoptosis was observed, evidenced by caspase-3 activation. The signal transduction pathways in subconfluent keratinocytes were altered, as CYC202 increased the phosphorylation of p38 MAP kinase. The activation of this kinase was confirmed by the increased phosphorylation of p38 MAPK substrate, the small heat shock protein HSP27. Prolonged inhibition of highly proliferative cells with CYC202 for 48 and 72 h altered the expression of epidermal differentiation markers. The use of the selective p38 kinase inhibitor PD169316 demonstrated that involucrin mRNA was upregulated by CYC202 via p38 MAPK pathway. These effects were strongly dependent on cell density and were observed only in highly proliferative keratinocytes. We concluded that CYC202 although highly potent against cancer cells inhibits also the proliferation and induces early apoptotic events in autocrine culture of normal human keratinocytes, activates p38 MAP kinase pathway and alters the expression of the epidermal differentiation markers. These results suggest that despite this potency against tumour cells, CYC202 must be used attentively in the clinical practice.     

Regulation of tyrosine phosphorylation during the CD40-mediated rescue of Ramos-BL B cells from BCR-triggered apoptosis

The regulation of tyrosine phosphorylation is essential for BCR-triggered cellular responses during the selection process in the germinal centres. We were interested in examining the temporal regulation of tyrosine phosphorylation following CD40 cross-linking of anti-IgM-triggered Ramos-BL B cells. CD40 co-stimulation of anti-IgM-treated Ramos-BL B cells rescued them from growth inhibition and apoptosis, even when anti-CD40 Abs were added up to 12 h after the cross-linking of the BCR. The initial up-regulation of tyrosine phosphorylation triggered by BCR cross-linking is followed by tyrosine dephosphorylation after 12 h of stimulation, coinciding with pro-caspase-3 processing and PARP cleavage. We find that CD40 co-stimulation rescues BCR-triggered Ramos-BL B cells only before the irreversible inhibition of tyrosine kinase activity after 12 h of BCR cross-linking and that this is coupled with up-regulation of tyrosine phosphorylation; thus demonstrating the importance of the late regulation of tyrosine phosphorylation for CD40-mediated rescue of Ramos-BL B cells from BCR-triggered G1 growth arrest and apoptosis.     

Inhibition of Intracellular Growth of Salmonella enterica Serovar Typhimurium in Tissue Culture by Antisense Peptide-Phosphorodiamidate Morpholino Oligomer

Two types of phosphorodiamidate morpholino oligomers (PMOs) were tested for inhibition of growth of Salmonella enterica serovar Typhimurium. Both PMOs have the same 11-base sequence that is antisense to the region near the start codon of acpP, which is essential for lipid biosynthesis and viability. To the 3′ end of each is attached the membrane-penetrating peptide (RXR)₄XB (R, X, and B indicate arginine, 6-aminohexanoic acid, and β-alanine, respectively). One peptide-PMO (AcpP PPMO) has no charge on the PMO moiety. The second PPMO has three cations (piperazine) attached to the phosphorodiamidate linkages (3+Pip-AcpP PPMO). A scrambled-sequence PPMO (Scr PPMO) was synthesized for each type of PMO. The MICs of AcpP PPMO, 3+Pip-AcpP PPMO, and either one of the Scr PPMOs were 1.25 μM (7 μg/ml), 0.156 μM (0.94 μg/ml), and >160 μM (>900 μg/ml), respectively. 3+Pip-AcpP PPMO at 1.25 or 2.5 μM significantly reduced the growth rates of pure cultures, whereas AcpP PPMO or either Scr PPMO had no effect. However, the viable cell count was significantly reduced at either concentration of 3+Pip-AcpP PPMO or AcpP PPMO, but not with either Scr PPMO. In other experiments, macrophages were infected intracellularly with S. enterica and treated with 3 μM 3+Pip-AcpP PPMO. Intracellular bacteria were reduced >99% with 3+Pip-AcpP PPMO, whereas intracellular bacteria increased 3 orders of magnitude in untreated or Scr PPMO-treated cultures. We conclude that either AcpP PPMO or 3+Pip-AcpP PPMO inhibited growth of S. enterica in pure culture and that 3+Pip-AcpP PPMO reduced intracellular viability of S. enterica in macrophages.Salmonella is a food- and waterborne bacterial pathogen that causes a variety of diseases, including gastroenteritis and typhoid fever. It is an intracellular bacterium that invades, replicates, and grows inside epithelial cells and macrophages. Emerging antibiotic-resistant strains of Salmonella enterica serovar Typhi (27, 32) and Typhimurium (11, 19) have increased the need to discover new and effective antibiotics against resistant strains.Synthetic oligonucleotide mimetics are an experimental class of compounds that modulate expression of specific genes (12, 18, 21, 29, 30, 33). Some examples are peptide nucleic acids, phosphorothioate oligonucleotides, locked nucleic acids, 2′-O-methyloligoribonucleotides, and phosphorodiamidate morpholino oligonucleotides (PMOs). The specificity of action in bacteria is determined by complementary base pairing between the synthetic oligonucleotide and its target, which is usually, but not always, an RNA (12, 33).Antisense PMOs and peptide nucleic acids have been shown to inhibit growth in pure cultures of a few types of bacteria (16, 17, 22, 23, 26), including S. enterica (31). There are few, if any, reports of inhibition of intracellular bacterial growth using antisense oligomers.Antisense oligomers require assistance to gain entry into bacterial cells because of their relatively high molecular weights and polar characteristics. Recently, antisense oligomers have been conjugated to membrane-penetrating peptides, which are composed of repeating patterns of cationic and nonpolar residues. Peptide-oligomer conjugates are significantly more effective in inhibiting expression of their specific targets than their nonconjugated counterparts (13, 16). Apparently the membrane-penetrating peptide carries its cargo (the antisense oligomer) across the gram-negative outer membrane after which it traverses the plasma membrane by an unknown mechanism.Cationic charges were introduced into the linkages between the bases of PMO (B. Geller, B. Mellbye, D. Weller, J. Hassinger, and M. Reeves, submitted for publication). One type of cationic linkage included a side moiety of piperazine (Pip). It was hypothesized that cationic PMOs with Pip linkages would obviate the need for membrane-penetrating peptides. Although this was found not to be true, peptide conjugates of PMOs with Pip (Pip-PMOs) were significantly more potent than the peptide conjugate of the equivalent noncharged PMO. In the present report, a Pip-PMO-peptide conjugate is tested against S. enterica and compared to the equivalent noncharged PMO-peptide conjugate.Bacteria that grow within eukaryotic host cells pose a challenging situation for antisense antibiotics. Not only must the oligomer penetrate the bacterial cell, but it must first enter the eukaryotic cell and retain its structural integrity within the endosome. Cationic membrane-penetrating peptides are generally thought to enter eukaryotic cells by first binding to proteoglycans and then being internalized by endocytosis (1, 20, 28). Recently we reported that a conjugate of a PMO and the membrane-penetrating peptide (RXR)₄XB (X is 6-aminohexanoic acid, and B is β-alanine) was effective in delivering antisense PMOs into Escherichia coli (25). PMO conjugates of (RXR)₄XB have been shown previously to enter mammalian cells, where they can modulate gene expression (4, 7, 10, 24, 36). These results suggest that PMO-(RXR)₄XB conjugates have potential for use against intracellular bacteria.In this report, we test the hypothesis that peptide-PMOs (PPMOs) targeted to a specific, essential gene (acpP, which is required for synthesis of lipids), can inhibit intracellular growth of S. enterica.