Expression of synaptotagmin and syntaxin associated with N-type calcium channels in small cell lung cancer

AbstractThe presence of synaptic proteins involved in excitation/secretion coupling was examined in ten small cell lung cancer lines. N-Type calcium channels (ω)-conotoxin receptors), synaptotagmin (p65) and syntaxin (HPC-1) were detected in eight. Co-immunoprecipitation experiments indicated that syntaxin can form a complex with synaptotagmin and calcium channels. The expression of synaptotagmin in small cell lung cancer may elicit an autoimmune response that reduces transmitter release at the nerve terminal

Binding of Ala-scanning analogs of ω-conotoxin MVIIC to N- and P/Q-type calcium channels

Abstractω-Conotoxin MVIIC binds to P/Q-type calcium channels with high affinity and N-type channels with low affinity. To reveal the residues essential for subtype selectivity, we synthesized Ala-scanning analogs of MVIIC. Binding assays using rat cerebellar P2 membranes suggested that Thr11, Tyr13 and Lys2 are essential for binding to both N- and P/Q-type channels, whereas Lys4 and Arg22 are important for binding to P/Q-type channels. These results suggest that MVIIC interacts with P/Q-type channels via a large surface, in good agreement with previous observations using chimeric analogs

Three Year Evaluation of Xpert MTB/RIF in a Low Prevalence Tuberculosis Setting

Objectives Xpert MTB/RIF (Cepheid) is a rapid molecular assay shown to be sensitive and specific for pulmonary tuberculosis (TB) diagnosis in highly endemic countries. We evaluated its diagnostic performance in a low TB prevalence setting, examined rifampicin resistance detection and quantitative capabilities predicting graded auramine microscopy and time to positivity (TTP) of culture. Methods Xpert MTB/RIF was used to test respiratory samples over a 3 year period. Samples underwent graded auramine microscopy, solid/ liquid culture, in-house IS6110 real-time PCR, and GenoType MTBDRplus (HAIN Lifescience) to determine rifampicin and/or isoniazid resistance. Results A total of 2103 Xpert MTB/RIF tests were performed. Compared to culture sensitivity was 95.8%, specificity 99.5%, positive predictive value (PPV) 82.1%, and negative predictive value (NPV) 99.9%. A positive correlation was found between auramine microscopy grade and Xpert MTB/RIF assay load. We found a clear reduction in the median TTP as Xpert MTB/RIF assay load increased. Rifampicin resistance was detected. Conclusions Xpert MTB/RIF was rapid and accurate in diagnosing pulmonary TB in a low prevalence area. Rapid results will influence infection prevention and control and treatment measures. The excellent NPV obtained suggests further work should be carried out to assess its role in replacing microscopy

Interaction of a synaptobrevin (VAMP)-syntaxin complex with presynaptic calcium channels

AbstractNerve terminal protein complexes implicated in exocytosis were examined by immuno-isolation from rat brain synaptosomes. Immunoprecipitation with anti-syntaxin or anti-VAMP antibodies revealed a syntaxin-SNAP25-VAMP-synaptotagmin complex. Anti-VAMP antibodies also trapped a distinct VAMP-synaptophysin complex. A similar fraction (about 70%) of N-type calcium channels ([125I]ω conotoxin GVIA receptors), was immunoprecipitated by either anti-syntaxin or anti-VAMP antibodies, but not by anti-synaptophysin antibodies (< 4%). The majority of N- but not L-type calcium channels ([3H]PN200-110 receptors), appear to be associated with a synaptic vesicle prefusion complex

Three Year Evaluation of Xpert MTB/RIF in a Low Prevalence Tuberculosis Setting

Objectives Xpert MTB/RIF (Cepheid) is a rapid molecular assay shown to be sensitive and specific for pulmonary tuberculosis (TB) diagnosis in highly endemic countries. We evaluated its diagnostic performance in a low TB prevalence setting, examined rifampicin resistance detection and quantitative capabilities predicting graded auramine microscopy and time to positivity (TTP) of culture. Methods Xpert MTB/RIF was used to test respiratory samples over a 3 year period. Samples underwent graded auramine microscopy, solid/ liquid culture, in-house IS6110 real-time PCR, and GenoType MTBDRplus (HAIN Lifescience) to determine rifampicin and/or isoniazid resistance. Results A total of 2103 Xpert MTB/RIF tests were performed. Compared to culture sensitivity was 95.8%, specificity 99.5%, positive predictive value (PPV) 82.1%, and negative predictive value (NPV) 99.9%. A positive correlation was found between auramine microscopy grade and Xpert MTB/RIF assay load. We found a clear reduction in the median TTP as Xpert MTB/RIF assay load increased. Rifampicin resistance was detected. Conclusions Xpert MTB/RIF was rapid and accurate in diagnosing pulmonary TB in a low prevalence area. Rapid results will influence infection prevention and control and treatment measures. The excellent NPV obtained suggests further work should be carried out to assess its role in replacing microscopy

Molecular Longitudinal Tracking of Mycobacterium abscessus spp. during Chronic Infection of the Human Lung

<div><p>The <i>Mycobacterium abscessus</i> complex is an emerging cause of chronic pulmonary infection in patients with underlying lung disease. The <i>M. abscessus</i> complex is regarded as an environmental pathogen but its molecular adaptation to the human lung during long-term infection is poorly understood. Here we carried out a longitudinal molecular epidemiological analysis of 178 <i>M. abscessus</i> spp. isolates obtained from 10 cystic fibrosis (CF) and 2 non CF patients over a 13 year period. Multi-locus sequence and molecular typing analysis revealed that 11 of 12 patients were persistently colonized with the same genotype during the course of the infection while replacement of a <i>M. abscessus sensu stricto</i> strain with a <i>Mycobacterium massiliense</i> strain was observed for a single patient. Of note, several patients including a pair of siblings were colonized with closely-related strains consistent with intra-familial transmission or a common infection reservoir. In general, a switch from smooth to rough colony morphology was observed during the course of long-term infection, which in some cases correlated with an increasing severity of clinical symptoms. To examine evolution during long-term infection of the CF lung we compared the genome sequences of 6 sequential isolates of <i>Mycobacterium bolletii</i> obtained from a single patient over an 11 year period, revealing a heterogeneous clonal infecting population with mutations in regulators controlling the expression of virulence factors and complex lipids. Taken together, these data provide new insights into the epidemiology of <i>M. abscessus</i> spp. during long-term infection of the CF lung, and the molecular transition from saprophytic organism to human pathogen.</p></div

Gβγ and the C Terminus of SNAP-25 Are Necessary for Long-Term Depression of Transmitter Release

Short-term presynaptic inhibition mediated by G protein-coupled receptors involves a direct interaction between G proteins and the vesicle release machinery. Recent studies implicate the C terminus of the vesicle-associated protein SNAP-25 as a molecular binding target of Gβγ that transiently reduces vesicular release. However, it is not known whether SNAP-25 is a target for molecular modifications expressing long-term changes in transmitter release probability.This study utilized two-photon laser scanning microscopy for real-time imaging of action potential-evoked [Ca(2+)] increases, in single Schaffer collateral presynaptic release sites in in vitro hippocampal slices, plus simultaneous recording of Schaffer collateral-evoked synaptic potentials. We used electroporation to infuse small peptides through CA3 cell bodies into presynaptic Schaffer collateral terminals to selectively study the presynaptic effect of scavenging the G-protein Gβγ. We demonstrate here that the C terminus of SNAP-25 is necessary for expression of LTD, but not long-term potentiation (LTP), of synaptic strength. Using type A botulinum toxin (BoNT/A) to enzymatically cleave the 9 amino acid C-terminus of SNAP-25 eliminated the ability of low frequency synaptic stimulation to induce LTD, but not LTP, even if release probability was restored to pre-BoNT/A levels by elevating extracellular [Ca(2+)]. Presynaptic electroporation infusion of the 14-amino acid C-terminus of SNAP-25 (Ct-SNAP-25), to scavenge Gβγ, reduced both the transient presynaptic inhibition produced by the group II metabotropic glutamate receptor stimulation, and LTD. Furthermore, presynaptic infusion of mSIRK, a second, structurally distinct Gβγ scavenging peptide, also blocked the induction of LTD. While Gβγ binds directly to and inhibit voltage-dependent Ca(2+) channels, imaging of presynaptic [Ca(2+)] with Mg-Green revealed that low-frequency stimulation only transiently reduced presynaptic Ca(2+) influx, an effect not altered by infusion of Ct-SNAP-25.The C-terminus of SNAP-25, which links synaptotagmin I to the SNARE complex, is a binding target for Gβγ necessary for both transient transmitter-mediated presynaptic inhibition, and the induction of presynaptic LTD