Gaps in the drug-free and methadone treatment program response to Hepatitis C

Drug treatment programs are sites of opportunity for the delivery of hepatitis C (HCV) prevention and care services to drug users. Using data collected from a random nationwide sample (N = 595) of drug treatment programs in the United States, this study compares the provision of HCV services by drug-free and methadone maintenance treatment programs (MMTPs). It then examines and compares perceived inadequacies in this service provision from the perspective of the managers in these two types of programs. Findings indicate that MMTPs are providing more HCV services to their patients, and that a greater proportion of MMTPs are dissatisfied with their current level of HCV service provision. Managers of drug-free programs would like to be offering patients more HCV education, while MMTP managers would like to be providing more HCV testing to their patients, and more support and care for patients who are HCV+.     

Integrating microarrays into disease-gene identification strategies

Positional cloning represents one of the most successful paradigm shifts in identifying the underlying patho-mechanisms in human disease. While traditional discovery tools focused on identifying defects at the tissue or cellular level, positional cloning identifies the damaged region of the genome as the preliminary step. While a large number of inherited single gene disorders have been mapped using this approach, a bottleneck still exists in combing through the genomic interval, often millions of nucleotides in length, to identify the nucleotide changes which result in a defective protein and subsequent disease. Along with the recent unravelling of the human genetic code, the development of massively parallel tools, such as microarrays, represent an equally important step forward in unraveling pathogenic genome dysfunctions. There are many emerging variants on microarray technology, such as expression arrays, exon arrays, array-based comparative genomic hybridization and sequencing arrays. Several of these platforms, if used properly, can accelerate the positional cloning process. The proper use of the platform is driven by knowledge of the underlying molecular defect being searched for and the operating characteristics of the array. The resultant insight forms the basis for improved molecular diagnostics and novel therapeutic targets.     

Targeted inactivation of αi2 or αi3 disrupts activation of the cardiac muscarinic K+ channel, IK+Ach, in intact cells

Cardiac muscarinic receptors activate an inwardly rectifying K⁺ channel, I_K^+Ach, via pertussis toxin (PT)-sensitive heterotrimeric G proteins (in heart G_i2, G_i3, or G_o). We have used embryonic stem cell (ES cell)-derived cardiocytes with targeted inactivations of specific PT-sensitive α subunits to determine which G proteins are required for receptor-mediated regulation of I_K^+Ach in intact cells. The muscarinic agonist carbachol increased I_K^+Ach activity in ES cell-derived cardiocytes from wild-type cells, in cells lacking α_o, and in cells lacking the PT-insensitive G protein α_q. In cells with targeted inactivation of α_i2 or α_i3, channel activation by both carbachol and adenosine was blocked. Carbachol-induced channel activation was restored in the α_i2- and α_i3-null cells by reexpressing the previously targeted gene and guanosine 5′-[γ-thio] triphosphate was able to fully activate I_K^+Ach in excised membranes patches from these mutants. In contrast, negative chronotropic responses to both carbachol and adenosine were preserved in cells lacking α_i2 or α_i3. Our results show that expression of two specific PT-sensitive α subunits (α_i2 and α_i3 but not α_o) is required for normal agonist-dependent activation of I_K^+Ach and suggest that both α_i2- and α_i3-containing heterotrimeric G proteins may be involved in the signaling process. Also the generation of negative chronotropic responses to muscarinic or adenosine receptor agonists do not require activation of I_K^+Ach or the expression of α_i2 or α_i3.     

Structure-based design of novel anticancer agents

Recently identified agents that interact with cytoskeletal elements such as tubulin include synthetic spiroketal pyrans (SPIKET) and monotetrahydrofuran compounds (COBRA compounds). SPIKET compounds target the spongistatin binding site of beta-tubulin and COBRA compounds target a unique binding cavity on alpha-tubulin. At nanomolar concentrations, the SPIKET compound SPIKET-P causes tubulin depolymerization and exhibits potent cytotoxic activity against cancer cells. COBRA-1 inhibits GTP-induced tubulin polymerization. Treatment of human breast cancer and brain tumor cells with COBRA-1 caused destruction of microtubule organization and apoptosis. Other studies have identified some promising protein tyrosine kinase inhibitors as anti-cancer agents. These include EGFR inhibitors such as the quinazoline derivative WHI-P97 and the leflunomide metabolite analog LFM-A12. Both LFM-A12 and WHI-P97 inhibit the in vitro invasiveness of EGFR positive human breast cancer cells at micromolar concentrations and induce apoptotic cell death. Dimethoxyquinazoline compounds WHI-P131 and WHI-P154 inhibit tyrosine kinase JAK3 in leukemia cells. Of particular interest is WHI-P131, which inhibits JAK3 but not JAK1, JAK2, SYK, BTK, LYN, or IRK at concentrations as high as 350 microM. Studies of BTK inhibitors showed that the leflunomide metabolite analog LFM-A13 inhibited BTK in leukemia and lymphoma cells. Consistent with the anti-apoptotic function of BTK, treatment of leukemic cells with LFM-A13 enhanced their sensitivity to chemotherapy-induced apoptosis.     

The effect of standing contrast and pattern adaptation on the human visually evoked potential

Human visually evoked potentials (VEPs) were recorded with sinusoidal gratings of low (0.5 c/deg), medium (4 c/deg) and high (16 c/deg) spatial frequency (SF). Simultaneous recording from Oz-A1, O2-A1 and Oz-O2 allowed the separation of the early and late VEP waves for most subjects. The effect of standing contrast and pattern adaptation on these waves was studied. At low SF both early and late waves were not affected by standing contrast or adaptation with motionless grating, while adaptation with a drifting grating reduced them. At medium and high SFs both principal negative waves. N1 and N2, were reduced by standing contrast and pattern adaptation. The amplitude of N2 saturated at low contrast level (0.1) and was reduced after adaptation regardless of the SF of test and adapting stimuli, while the amplitude of N1 did not saturate up to the contrast level of 0.3 and exhibited SF-selective adaptation. The adapted onset-VEP was similar to the offset-VEP. The data suggest that: the early wave is generated by multiple narrowly tuned SF-selective structures that are medially positioned while the generators of the late wave are not SF-selective and occupy a wider area centered laterally; the effects of standing contrast and pattern adaptation on VEPs, as well as the relationship of onset and offset VEPs, reflect the time-course of neural activity evoked by long-lasting stimuli.     

Inhibition of inactivation of single sodium channels by a site-directed antibody.

The effects of site-directed antibodies on single sodium channel currents in excised membrane patches from rat brain neurons have been examined. Of six antibodies directed against different intracellular domains of the sodium channel alpha subunit, only an antibody directed against a highly conserved intracellular segment between homologous transmembrane domains III and IV induced late single channel openings and prolonged single channel open times during depolarizing test pulses, resulting in nearly complete inhibition of sodium channel inactivation. The antibody effect was not observed if the membrane patches were depolarized to inactivate sodium channels before exposure to the antibody, indicating that the intracellular sequence recognized by the antibody is rendered inaccessible by inactivation. The results show that a conformational change involving the intracellular segment between domains III and IV of the alpha subunit of the sodium channel molecule is required for fast sodium channel inactivation and suggest that this segment may be the fast inactivation gate of the sodium channel.     

HMR1402, a potassium ATP channel blocker during hyperdynamic porcine endotoxemia: effects on hepato-splanchnic oxygen exchange and metabolism

Objective To assess the effects of the potassium ATP (KATP) channel blocker HMR1402 (HMR) on systemic and hepato-splanchnic hemodynamics, oxygen exchange and metabolism during hyperdynamic porcine endotoxemia.Design Prospective, randomized, controlled study with repeated measures.Setting Animal laboratory.Subjects Eighteen pigs allocated to receive endotoxin alone (control group, CON, n=10) or endotoxin and HMR (6 mg/kg h^–1, n=8).Interventions Anesthetized, mechanically ventilated, and instrumented pigs receiving continuous i.v. endotoxin were resuscitated with hetastarch to maintain mean arterial pressure (MAP) >60 mmHg. Twelve hours after starting the endotoxin infusion, they received HMR or its vehicle for another 12 h.Results HMR transiently increased MAP by about 15 mmHg, but this effect was only present during the first 1 h of infusion. The HMR decreased cardiac output due to a fall in heart rate, and thereby reduced liver blood flow. While liver O₂ delivery and uptake remained unchanged, HMR induced hyperlactatemia [from 1.5 (1.1; 2.0), 1.4 (1.2; 1.8), and 1.2 (0.8; 2.0) to 3.1 (1.4; 3.2), 3.2 (1.6; 6.5), and 3.0 (1.0; 5.5) mmol/l in the arterial, portal and hepatic venous samples, respectively] and further increased arterial [from 8 (3; 13) to 23 (11; 57); p<0.05], portal [from 9 (4; 14) to 23 (14; 39); p<0.05] and hepatic vein [from 7 (0; 15) to 30 (8; 174), p<0.05] lactate/pyruvate ratios indicating impaired cytosolic redox state.Conclusion The short-term beneficial hemodynamic effects of KATP channel blockers have to be weighted with the detrimental effect on mitochondrial respiration.P. Asfar and Z. Iványi equally contributed to this work