HSV-1 amplicon vectors are a highly efficient gene delivery system for skeletal muscle myoblasts and myotubes

Analysis of RyR1 structure function inmuscle cells is made difficult by the low (<5%) transfectionefficiencies of myoblasts or myotubes using calcium phosphate orcationic lipid techniques. We inserted the full-length 15.3-kb RyR1cDNA into a herpes simplex virus type 1 (HSV-1) amplicon vector,pHSVPrPUC between the ori/IE 4/5 promoter sequence and theHSV-1 DNA cleavage/packaging signal (pac). pHSVGN andpHSVGRyR1, two amplicons that expressed green fluorescent protein, wereused for fluorescence-activated cell sorter analysis of transductionefficiency. All amplicons were packaged into HSV-1 virusparticles using a helper virus-free packaging system and yielded10⁶ transducing vector units/ml. HSVRyR1, HSVGRyR1, andHSVGN virions efficiently transduced mouse myoblasts and myotubes,expressing the desired product in 70-90% of the cells atmultiplicity of infection 5. The transduced cells appeared healthy andRyR1 produced by this method was targeted properly and restoredskeletal excitation-contraction coupling in dyspedic myotubes. Themyotubes produced sufficient protein to allow single-channel analysesfrom as few as 10 100-mm dishes. In most cases this method couldpreclude the need for permanent transfectants for the study of RyR1structure function.

     

C terminus-mediated control of voltage and cAMP gating of hyperpolarization-activated cyclic nucleotide-gated channels

The hyperpolarization-activated cyclic nucleotide-gated (HCN) family of "pacemaker" channels includes 4 isoforms, the kinetics and cAMP-induced modulation of which differ quantitatively. Because HCN isoforms are highly homologous in the central region, but diverge more substantially in the N and C termini, we asked whether these latter regions could contribute to the determination of channel properties. To this aim, we analyzed activation/deactivation kinetics and the response to cAMP of heterologously expressed isoforms mHCN1 and rbHCN4 and verified that mHCN1 has much faster kinetics and lower cAMP sensitivity than rbHCN4. We then constructed rbHCN4 chimeras by replacing either the N or the C terminus, or both, with the analogous domains from mHCN1. We found that: 1) replacement of the N terminus (chimera N1-4) did not substantially modify either the kinetics or cAMP dependence of wild-type channels; 2) replacement of the C terminus, on the contrary, resulted in a chimeric channel (4-C1), the kinetics of which were strongly accelerated compared with rbHCN4, and that was fully insensitive to cAMP; 3) replacement of both N and C termini led to the same results as replacement of the C terminus alone. These results indicate that the C terminus of rbHCN4 contributes to the regulation of voltage- and cAMP-dependent channel gating, possibly through interaction with other intracellular regions not belonging to the N terminus.     

Notes on Scleroderma,Puff balls and Geastrum of Azad Jammu and Kashmir,Pakistan

Six species of mushrooms allied to the Family Sclerodermataceae, Lycoperdaceae and Geastraceae have been described for the first time from Azad Jammu and Kashmir. These are Scleroderma aurantium, Calvatia verrucosia sp. nov., Lycoperdon pedicellaton sp. nov. L. sphaericon sp. nov., L. echinulaton sp. nov., and Geastrum heptaplex sp. nov.     

Investigation of aryl halides as ketone bioisosteres: Refinement of potent and selective inhibitors of human cytochrome P450 19A1 (aromatase)

Bioisosteric replacement of cyclic ketone functionality with aryl halides was investigated on a centrally-flexible, five-component 1,2,3-triazole-containing pharmacophore, resulting in enhanced inhibition of aromatase (CYP450 19A1). Structure–activity data generated from both syn- and anti-aldol precursors provides significant insights into the requirements for enhanced potency, validating this novel ketone-to-aryl halide bioisostere hypothesis.     

Resequencing the susceptibility gene,ITGAM, identifies two functionally deleterious rare variants in systemic lupus erythematosus cases

Introduction

The majority of the genetic variance of systemic lupus erythematosus (SLE) remains unexplained by the common disease-common variant hypothesis. Rare variants, which are not detectable by genome-wide association studies because of their low frequencies, are predicted to explain part of this ”missing heritability.” However, recent studies identifying rare variants within known disease-susceptibility loci have failed to show genetic associations because of their extremely low frequencies, leading to the questioning of the contribution of rare variants to disease susceptibility. A common (minor allele frequency = 17.4% in cases) nonsynonymous coding variant rs1143679 (R77H) in ITGAM (CD11b), which forms half of the heterodimeric integrin receptor, complement receptor 3 (CR3), is robustly associated with SLE and has been shown to impair CR3-mediated phagocytosis.

Methods

We resequenced ITGAM in 73 SLE cases and identified two previously unidentified, case-specific nonsynonymous variants, F941V and G1145S. Both variants were genotyped in 2,107 and 949 additional SLE cases, respectively, to estimate their frequencies in a disease population. An in vitro model was used to assess the impact of F941V and G1145S, together with two nonsynonymous ITGAM polymorphisms, A858V (rs1143683) and M441T (rs11861251), on CR3-mediated phagocytosis. A paired two-tailed t test was used to compare the phagocytic capabilities of each variant with that of wild-type CR3.

Results

Both rare variants, F941V and G1145S, significantly impair CR3-mediated phagocytosis in an in vitro model (61% reduction, P = 0.006; 26% reduction, P = 0.0232). However, neither of the common variants, M441T and A858V, had an effect on phagocytosis. Neither rare variant was observed again in the genotyping of additional SLE cases, suggesting that there frequencies are extremely low.

Conclusions

Our results add further evidence to the functional importance of ITGAM in SLE pathogenesis through impaired phagocytosis. Additionally, this study provides a new example of the identification of rare variants in common-allele-associated loci, which, because of their extremely low frequencies, are not statistically associated. However, the demonstration of their functional effects adds support to their contribution to disease risk, and questions the current notion of dismissing the contribution of very rare variants on purely statistical analyses.     

Analysis of a genome-wide association study-linked locus (CCR6) in Asian rheumatoid arthritis

A genome-wide association study in Japan identified the C-C chemokine receptor type 6 gene (CCR6) as associated with rheumatoid arthritis (RA). This finding has not been validated in other Asian populations. A case-control study involving 996 subjects, comprising 440 controls and 556 RA patients, was done to determine their anticyclic citrullinated peptide (anti-CCP) antibody status and CCR6 polymorphism (rs3093024) genotype. Three hundred eighty-seven patients were anti-CCP positive and 153 anti-CCP negative. Logistic regression showed that allele A was likely to increase the risk of developing RA among females via a recessive model (odds ratio [OR]=1.55, 95% confidence interval [CI]=1.01, 2.39), whereas the risk effect appeared to be reduced among males via an additive model (OR=0.60, 95% CI=0.42, 0.85). Considering only subjects who are anti-CCP positive, allele A increased RA risk among females via a recessive model (OR=1.68, 95% CI=1.07, 2.64) but decreased the risk among males via an additive model (OR=0.59, 95% CI=0.39, 0.89). We showed that CCR6 polymorphism was a risk factor among females but a protective factor among males. Functional studies are warranted to unravel the pathophysiological relevance of the gene variant and other linked variants with RA.