Dynamic modification of a mutant cytoplasmic cysteine residue modulates the conductance of the human 5-HT3A receptor

Structural models suggest that Arg(436) lies within five cytoplasmic portals of the 5-HT(3A) receptor. We tested both the accessibility of residue 436 and the influence of its charge on single channel conductance (gamma) by substituting Arg(436) with Cys and examining the effect of methanethiosulfonate (MTS) reagents on gamma. Inclusion of positively charged 2-aminoethyl-MTS (MTSEA) within the electrode solution reduced gamma of 5-HT(3A)(R436C) receptors in outside-out patches from 7.8 +/- 0.5 to 5.0 +/- 0.5 picosiemens (pS). To increase gamma, we substituted Arg(436) by Cys in the 5-HT(3A)(R432Q,R440A) mutant, yielding the 5-HT(3A)(QCA) construct with a gamma of 17.7 +/- 0.4 pS. Modification of 5-HT(3A)(QCA) receptors by MTSEA or 2-(trimethylammonium) ethyl-MTS reduced gamma to 8.7 +/- 0.5 and 6.7 +/- 0.4 pS, respectively, both significantly below that of channels exposed to nonpolar propyl-MTS. Extracellular MTSEA, but not 2-(trimethylammonium) ethyl-MTS, crossed the membrane and in so doing slowly (tau = 94 s) reduced gamma. MTSEA more rapidly (tau = 15 s) reduced the gamma of 5-HT(3A)(QCA) receptors in inside-out patches, an effect reversed by the reducing agent dithiothreitol. Cys(436) modification by negatively charged 2-carboxyethyl-MTS and 2-sulfonatoethyl-MTS increasedgamma to 23 +/- 1.0 and 26 +/- 0.7 pS, respectively. MTS reagents did not affect gamma values for 5-HT(3A)(QDA) constructs with Cys substituted for Lys(431) predicted to be outside the entrance to the portals. Collectively, the data demonstrate that the dynamic modification of the charge of a cytoplasmic residue regulates gamma, consistent with the existence of cytoplasmic portals that impose a rate-limiting barrier to ion conduction in Cys loop receptors.     

Acute beta-adrenergic overload produces myocyte damage through calcium leakage from the ryanodine receptor 2 but spares cardiac stem cells

A hyperadrenergic state is a seminal aspect of chronic heart failure. Also, "Takotsubo stress cardiomyopathy," is associated with increased plasma catecholamine levels. The mechanisms of myocyte damage secondary to excess catecholamine exposure as well as the consequence of this neurohumoral burst on cardiac stem cells (CSCs) are unknown. Cardiomyocytes and CSCs were exposed to high doses of isoproterenol (ISO), in vivo and in vitro. Male Wistar rats received a single injection of ISO (5 mg kg-1) and were sacrificed 1, 3, and 6 days later. In comparison with controls, LV function was impaired in rats 1 day after ISO and started to improve at 3 days. The fraction of dead myocytes peaked 1 day after ISO and decreased thereafter. ISO administration resulted in significant ryanodine receptor 2 (RyR2) hyperphosphorylation and RyR2-calstabin dissociation. JTV519, a RyR2 stabilizer, prevented the ISO-induced death of adult myocytes in vitro. In contrast, CSCs were resistant to the acute neurohumoral overload. Indeed, CSCs expressed a decreased and inverted complement of beta1/beta2-adrenoreceptors and absence of RyR2, which may explain their survival to ISO insult. Thus, a single injection of ISO causes diffuse myocyte death through Ca2+ leakage secondary to the acutely dysfunctional RyR2. CSCs are resistant to the noxious effects of an acute hyperadrenergic state and through their activation participate in the response to the ISO-induced myocardial injury. The latter could contribute to the ability of the myocardium to rapidly recover from acute hyperadrenergic damage.     

Common STAT3 variants are not associated with obesity or insulin resistance in female twins

In animal models, STAT3 action in the hypothalamus and liver appears essential for normal body weight and glucose homeostasis in response to insulin. We hypothesized that variation in the STAT3 gene may be associated with body fat and/or insulin resistance in the general population. Five tagging SNPs spanning the STAT3 gene, rs8074524, rs2293152, rs2306580, rs6503695, and rs7211777 were genotyped in 2776 white female twins (mean age, 47.4+/-12.5 yrs) from the St Thomas' United Kingdom Adult Twin Registry (Twins UK). Minor allele frequencies were as follows: rs8074524 (0.19), rs2293152 (0.37), rs2306580 (0.06), rs6503695 (0.35), and rs7211777 (0.34). The minor allele of rs2293152 was associated with higher homeostasis model assessment index of insulin resistance (p=0.013) in the full cohort and confirmed in sib-transmission/disequilibrium test (TDT): (p=0.015; n=60). However, there were no associations with fasting serum insulin or glucose or with obesity variables. Although defective STAT3 action results in obesity and insulin resistance in animal models, we failed to establish any indicative associations with common SNPs in this human study.     

Immunodominant Francisella tularensis antigens identified using proteome microarray

Stimulation of protective immune responses against intracellular pathogens is difficult to achieve using non-replicating vaccines. BALB/c mice immunized by intramuscular injection with killed Francisella tularensis (live vaccine strain) adjuvanted with preformed immune stimulating complexes admixed with CpG, were protected when systemically challenged with a highly virulent strain of F. tularensis (Schu S4). Serum from immunized mice was used to probe a whole proteome microarray in order to identify immunodominant antigens. Eleven out of the top 12 immunodominant antigens have been previously described as immunoreactive in F. tularensis. However, 31 previously unreported immunoreactive antigens were revealed using this approach. Twenty four (50%) of the ORFs on the immunodominant hit list belonged to the category of surface or membrane associated proteins compared to only 22% of the entire proteome. There were eight hypothetical protein hits and eight hits from proteins associated with different aspects of metabolism. The chip also allowed us to readily determine the IgG subclass bias, towards individual or multiple antigens, in protected and unprotected animals. These data give insight into the protective immune response and have potentially important implications for the rational design of non-living vaccines for tularemia and other intracellular pathogens.     

A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice

No single animal model for severe acute respiratory syndrome (SARS) reproduces all aspects of the human disease. Young inbred mice support SARS-coronavirus (SARS-CoV) replication in the respiratory tract and are available in sufficient numbers for statistical evaluation. They are relatively inexpensive and easily accessible, but their use in SARS research is limited because they do not develop illness following infection. Older (12- to 14-mo-old) BALB/c mice develop clinical illness and pneumonitis, but they can be hard to procure, and immune senescence complicates pathogenesis studies. We adapted the SARS-CoV (Urbani strain) by serial passage in the respiratory tract of young BALB/c mice. Fifteen passages resulted in a virus (MA15) that is lethal for mice following intranasal inoculation. Lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. Abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. These observations suggest that mice infected with MA15 die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. The MA15 virus has six coding mutations associated with adaptation and increased virulence; when introduced into a recombinant SARS-CoV, these mutations result in a highly virulent and lethal virus (rMA15), duplicating the phenotype of the biologically derived MA15 virus. Intranasal inoculation with MA15 reproduces many aspects of disease seen in severe human cases of SARS. The availability of the MA15 virus will enhance the use of the mouse model for SARS because infection with MA15 causes morbidity, mortality, and pulmonary pathology. This virus will be of value as a stringent challenge in evaluation of the efficacy of vaccines and antivirals.     

Personalized medicine and proteomics: lessons from non-small cell lung cancer

Personalized medicine allows the selection of treatments best suited to an individual patient and disease phenotype. To implement personalized medicine, effective tests predictive of response to treatment or susceptibility to adverse events are needed, and to develop a personalized medicine test, both high quality samples and reliable data are required. We review key features of state-of-the-art proteomic profiling and introduce further analytic developments to build a proteomic toolkit for use in personalized medicine approaches. The combination of novel analytical approaches in proteomic data generation, alignment and comparison permit translation of identified biomarkers into practical assays. We further propose an expanded statistical analysis to understand the sources of variability between individuals in terms of both protein expression and clinical variables and utilize this understanding in a predictive test.