Pyrazole inhibitors of HMG-CoA reductase: an attempt to dramatically reduce synthetic complexity through minimal analog re-design

An extraordinarily potent and hepatoselective class of HMG-CoA reductase inhibitors containing a pyrazole core was recently reported; however, its development was hampered by a long and difficult synthetic route. We attempted to circumvent this obstacle by preparing closely related analogs wherein the key dihydroxyheptanoic acid sidechain was tethered to the pyrazole core via an oxygen linker ('oxypyrazoles'). This minor change reduced the total number of synthetic steps from 14 to 7. Although the resulting analogs maintained much of the in vitro and cell activity of the pyrazoles, inferior in vivo activity precluded further development. Caco-2 cell permeability data suggest that enhanced cellular efflux of the oxypyrazoles relative to the pyrazoles may be responsible for the poor in vivo activity.     

Phencyclidine (PCP) injected in the nucleus accumbens increases extracellular dopamine and serotonin as measured by microdialysis

Phencyclidine (PCP; 20 micrograms in 0.5 microliter) was tested by local brain injection for neurochemical effects in the nucleus accumbens and striatum of rats. Changes in dopamine turnover could not be detected in postmortem tissue assays. In contrast, extracellular levels of dopamine significantly increased as measured by microdialysis in freely moving animals. PCP also increased extracellular levels of serotonin and decreased 3,4-dihydroxyphenylacetic acid (DOPAC), but did not change homovanillic acid (HVA) or 5-hydroxyindoleacetic acid (5HIAA). Microdialysis suggests that PCP acts in some dopamine terminal regions to increase extracellular dopamine and serotonin.     

Evaluation of portable point-of-care CD4 counter with high sensitivity for detecting patients eligible for antiretroviral therapy

Background

Accurate, inexpensive point-of-care CD4+ T cell testing technologies are needed that can deliver CD4+ T cell results at lower level health centers or community outreach voluntary counseling and testing. We sought to evaluate a point-of-care CD4+ T cell counter, the Pima CD4 Test System, a portable, battery-operated bench-top instrument that is designed to use finger stick blood samples suitable for field use in conjunction with rapid HIV testing.

Methods

Duplicate measurements were performed on both capillary and venous samples using Pima CD4 analyzers, compared to the BD FACSCalibur (reference method). The mean bias was estimated by paired Student''s t-test. Bland Altman plots were used to assess agreement.

Results

206 participants were enrolled with a median CD4 count of 396 (range; 18–1500). The finger stick PIMA had a mean bias of −66.3 cells/µL (95%CI −83.4−49.2, P<0.001) compared to the FACSCalibur; the bias was smaller at lower CD4 counts (0–250 cells/µL) with a mean bias of −10.8 (95%CI −27.3−+5.6, P = 0.198), and much greater at higher CD4 cell counts (>500 cells/µL) with a mean bias of −120.6 (95%CI −162.8, −78.4, P<0.001). The sensitivity (95%CI) of the Pima CD4 analyzer was 96.3% (79.1–99.8%) for a <250 cells/ul cut-off with a negative predictive value of 99.2% (95.1–99.9%).

Conclusions

The Pima CD4 finger stick test is an easy-to-use, portable, relatively fast device to test CD4+ T cell counts in the field. Issues of negatively-biased CD4 cell counts especially at higher absolute numbers will limit its utility for longitudinal immunologic response to ART. The high sensitivity and negative predictive value of the test makes it an attractive option for field use to identify patients eligible for ART, thus potentially reducing delays in linkage to care and ART initiation.     

Action of nicotine and analogs on acetylcholine receptors having mutations of transmitter-binding site residue αG153

A primary target for nicotine is the acetylcholine receptor channel (AChR). Some of the ability of nicotine to activate differentially AChR subtypes has been traced to a transmitter-binding site amino acid that is glycine in lower affinity and lysine in higher affinity AChRs. We studied the effects of mutations of this residue (αG153) in neuromuscular AChRs activated by nicotine and eight other agonists including nornicotine and anabasine. All of the mutations increased the unliganded gating equilibrium constant. The affinity of the resting receptor (K_d) and the net binding energy from the agonist for gating (ΔG_B) were estimated by cross-concentration fitting of single-channel currents. In all but one of the agonist/mutant combinations there was a moderate decrease in K_d and essentially no change in ΔG_B. The exceptional case was nicotine plus lysine, which showed a large, >8,000-fold decrease in K_d but no change in ΔG_B. The extraordinary specificity of this combination leads us to speculate that AChRs with a lysine at position αG153 may be exposed to a nicotine-like compound in vivo.     

Clastogen-induced chromosomal breakage as a marker for first trimester prenatal diagnosis of Fanconi anemia

Summary Using cultured trophoblast cells obtained by chorionic villus biopsy, we diagnosed Fanconi anemia (FA) in two pregnancies and excluded it in eight pregnancies at risk for the syndrome. Baseline chromosomal breakage and breakage induced by diepoxybutane (DEB) were analyzed. Increased breakage was used as a marker for the syndrome. Our results were unambiguous and provide a reliable method for prenatal detection of FA in the first trimester of pregnancy.     

Acetylcholine receptor gating at extracellular transmembrane domain interface: the cys-loop and M2-M3 linker

Acetylcholine receptor channel gating is a propagated conformational cascade that links changes in structure and function at the transmitter binding sites in the extracellular domain (ECD) with those at a "gate" in the transmembrane domain (TMD). We used Phi-value analysis to probe the relative timing of the gating motions of alpha-subunit residues located near the ECD-TMD interface. Mutation of four of the seven amino acids in the M2-M3 linker (which connects the pore-lining M2 helix with the M3 helix), including three of the four residues in the core of the linker, changed the diliganded gating equilibrium constant (K(eq)) by up to 10,000-fold (P272 > I274 > A270 > G275). The average Phi-value for the whole linker was approximately 0.64. One interpretation of this result is that the gating motions of the M2-M3 linker are approximately synchronous with those of much of M2 (approximately 0.64), but occur after those of the transmitter binding site region (approximately 0.93) and loops 2 and 7 (approximately 0.77). We also examined mutants of six cys-loop residues (V132, T133, H134, F135, P136, and F137). Mutation of V132, H134, and F135 changed K(eq) by 2800-, 10-, and 18-fold, respectively, and with an average Phi-value of 0.74, similar to those of other cys-loop residues. Even though V132 and I274 are close, the energetic coupling between I and V mutants of these positions was small (< or =0.51 kcal mol(-1)). The M2-M3 linker appears to be the key moving part that couples gating motions at the base of the ECD with those in TMD. These interactions are distributed along an approximately 16-A border and involve about a dozen residues.