Acute exposure of cultured canine cerebral vascular smooth muscle cells to methanol (10-400 mM) results in concentration-dependent elevation of the concentration of intracellular free calcium ion ([Ca2+]i) as measured with the fluorescent indicator, fura-2, and digital imaging microscopy. The resting level of [Ca2+]i in the cerebral vascular smooth muscle cells was 89.3+/-5.3 nM. Exposure of these cells to 10 mM methanol for only 5 min resulted in significant elevation in [Ca2+]i (i.e., to 105.7+/-4.6) (p < 0.05). Methanol (10 mM) is a concentration found in the blood of victims demonstrating early CNS toxicity. Other, higher concentrations of methanol rapidly raised [Ca2+]i upwards of 60% over basal resting levels. These result suggest that methanol-induced cerebral vasospasm is a consequence of large rises in intracellular Ca2+. These events could play a crucial role in methanol-induced cerebral edema, brain hemorrhage, and cerebral and retinal infarcts, eventuating in severe deficits in brain blood flow and the known, subsequent CNS disturbances.

Animals↗

PubMedDate not supplied

Functional InsP3 receptors that may modulate excitation-contraction coupling in the heart.

The roles of the Ca2+-mobilising messenger inositol 1,4,5-trisphosphate (InsP3) in heart are unclear, although many hormones activate InsP3 production in cardiomyocytes and some of their inotropic, chronotropic and arrhythmogenic effects may be due to Ca2+ release mediated by InsP3 receptors (InsP3Rs) [1-3]. In the present study, we examined the expression and subcellular localisation of InsP3R isoforms, and investigated their potential role in modulating excitation-contraction coupling (EC coupling). Western, PCR and InsP3-binding analysis indicated that both atrial and ventricular myocytes expressed mainly type II InsP3Rs, with approximately sixfold higher levels of InsP3Rs in atrial cells. Co-immunostaining of atrial myocytes with antibodies against type II ryanodine receptors (RyRs) and type II InsP3Rs revealed that the latter were arranged in the subsarcolemmal space where they largely co-localised with the junctional RyRs. Stimulation of quiescent or electrically paced atrial myocytes with a membrane-permeant InsP3 ester, which enters cells and directly activates InsP3Rs, caused the appearance of spontaneous Ca2+-release events. In addition, in paced cells, the InsP3 ester evoked an increase in the amplitudes of action potential-evoked Ca2+ transients. These data indicate that atrial cardiomyocytes express functional InsP3Rs, and that these channels could modulate EC coupling.

Animals↗

PubMedDate not supplied

Anti-idiotypic antibody and recombinant antigen vaccines in colorectal cancer patients.

The colorectal carcinoma (CRC)-associated GA733 antigen (also known as CO17-1A, KS1-4, KSA or EpCAM) has been the target of a phase II/III randomized trial of passive immunotherapy with monoclonal antibody CO17-1A and phase I active immunotherapy trials with polyclonal anti-idiotypic antibodies mimicking the CO17-1A or GA733 epitope on the antigen. The CO17-1A antigen was molecularly cloned and the extracellular domain expressed in baculovirus (BV) GA733-2E. Whereas, anti-idiotypic antibody mimics a single epitope on the antigen, BV GA733-2E expresses multiple potentially immunogenic epitopes. In animals, the immunogenicity of BV GA733-2E in aluminum hydroxide was superior to that of anti-idiotype in the same adjuvant. Here, we compared the immunogenicity of anti-idiotypic antibody and GA733-2E antigen in CRC patients. These studies indicate that the antigen is superior to the anti-idiotype antibody in inducing humoral and cellular immunity in CRC patients.

Antibodies, Anti-Idiotypic↗

PubMedDate not supplied

Optimization of conditions for bacteriocin extraction in PEG/salt aqueous two-phase systems using statistical experimental designs.

The bacteriocin nisin was extracted in PEG/salt aqueous two-phase systems (ATPS) using the property that the systems can extract hydrophobic proteins. The concentrations of the phase-forming components, PEG 4000 and Na(2)SO(4), were optimized for nisin recovery by means of statistical experimental designs, and it was found that they strongly influenced nisin recovery. The optimal composition of ATPS was found to be 15.99% (w/w) PEG 4000 and 15.85% (w/w) Na(2)SO(4) (pH 2), and the optimal ATPS allowed an 11.60% increase of nisin recovery compared to the standard method of nisin assay.

Nisin↗

PubMedDate not supplied

A statistical-based approach to assessing the fidelity of combinatorial libraries encoded with electrophoric molecular tags. Development and application of tag decode-assisted single bead LC/MS analysis.

A statistical sampling protocol is described to assess the fidelity of libraries encoded with molecular tags. The methodology, termed library QA, is based on the combined application of tag decode analysis and single bead LC/MS. The physical existence of library compounds eluted from beads is established by comparing the molecular weight predicted by tag decode with empirical measurement. The goal of sampling is to provide information on overall library fidelity and an indication of the performance of individual library synthons. The minimal sampling size n for library QA is l0 x the largest synthon set. Data are reported as proportion (p) +/- lower and upper boundary (lb-ub) computed at the 95% confidence level (alpha = 0.05). As a practical demonstration, library QA was performed on a 25,200-member library of statine amides (size = 40 x 63 x 10). Sampling was conducted three times at n approximately 630 beads per run for a total of 1902 beads. The overall proportions found for the three runs were consistent with one another: p = 84.4%, lb-ub = 81.5-87.2%; p = 83.1%, lb-ub = 80.2-85.95; and p = 84.5%, lb-ub = 81.8-87.3%, suggesting the true value of p is close to 84% compound confirmation. The performance pi of individual synthons was also computed. Corroboration of QA data with biological screening results obtained from assaying the library against cathepsin D and plasmepsin II is discussed.

Journal Article↗

PubMedDate not supplied

Parallel electrosynthesis of 1,2-diamines.

Parallel reductive hydrocoupling of aldimines was performed to generate vicinal diamines using the spatially addressable electrolysis platform (SAEP). The stainless steel cathodes and sacrificial aluminum anodes were used in this electrosynthesis methodology. Introducing a large substituent on the imino nitrogen atom efficiently controls stereoselectivity of the electroreductive coupling process. This method can now be applied to the generation of libraries of 1,2-diamines, which further expands the techniques of parallel synthesis and combinatorial electrochemistry.

Combinatorial Chemistry Techniques↗

PubMedDate not supplied

Analysis of CARD15/NOD2 haplotypes fails to identify common variants associated with rheumatoid arthritis susceptibility.

OBJECTIVES: The CARD15/NOD2 gene product plays an important role in host response to bacterial lipopolysaccharides and bacterial muramyl dipeptide via activation of NF-kappaB in monocytes. Mutations in CARD15 are associated with Crohn's disease (CD), a chronic inflammatory bowel disease. In this study we sought to determine whether CD-associated mutations or any common variants of this gene might contribute to susceptibility to another chronic inflammatory disease, rheumatoid arthritis (RA). METHODS: We genotyped 376 Caucasian RA cases and 376 ethnically matched healthy controls for three CD-associated CARD15 mutations. We also genotyped these 752 individuals for 12 common CARD15 single nucleotide polymorphisms (SNPs), determined the linkage disequilibrium structure of the gene, and compared the frequencies of the common CARD15 haplotypes in the RA cases and controls. RESULTS: None of the CD-associated mutations or the CARD15 SNPs was associated with susceptibility to RA. We also found no significant difference in the frequencies of any of the common haplotypes of the CARD15 gene in RA patients and controls. Our haplotype analysis was consistent with earlier observations that all three CD-associated variants independently arose on the same ancestral haplotype. CONCLUSIONS: These data suggest that CARD15 variants are not associated with RA susceptibility.

Adolescent↗

PubMedDate not supplied

The effects of age on late components of the ERP and reaction time.

The effects of age on late component (N100, P200, N200, and P300) event-related potentials (ERP) and reaction time (RT) were investigated in 50 normal subjects, 18 to 70 years of age. A conventional auditory oddball paradigm was employed. An equal number of subjects, as well as an equal number of males and females, were examined in each decade. There were no significant associations found between ERP amplitude and age. A significant positive correlation was found between age and N200/P300 latency. There were no significant effects of age on RT in the overall group. However, females had slower RT and decreased P200 and N200 amplitude as compared with males. Moreover, males had significant correlations between N200 latency and RT, whereas females showed significant correlations between P300 amplitude and RT. ERP/RT effects should be further explicated between gender, as well as across age.

Adolescent↗