ChTX induces oscillatory contraction in guinea pig trachea: role of cyclooxygenase-2 and PGE2

We examined the possible role of cyclooxygenase (COX) in charybdotoxin (ChTX)-induced oscillatory contraction in guinea pig trachea. Involvement of prostaglandin E(2) (PGE(2)) in ChTX-induced oscillatory contraction was also investigated. ChTX (100 nM) induced oscillatory contraction in guinea pig trachea. The mean oscillatory frequency induced by ChTX was 10.7 +/- 0.8 counts/h. Maximum and minimum tensions within ChTX-induced oscillatory contractions were 68.4 +/- 1.8 and 14.3 +/- 1.7% compared with K(+) (72.7 mM) contractions. ChTX-induced oscillatory contraction was completely inhibited by indomethacin, a nonselective COX inhibitor. Valeryl salicylate, a selective COX-1 inhibitor, did not significantly inhibit this contraction, whereas N-(2-cyclohexyloxy-4-nitro-phenyl)-methanesulfonamide, a selective COX-2 inhibitor, abolished this contraction. Exogenously applied arachidonic acid enhanced ChTX-induced oscillatory contraction. SC-51322, a selective PGE receptor subtype EP(1) antagonist, significantly inhibited ChTX-induced oscillatory contraction. Exogenously applied PGE(2) induced only a slight phasic contraction in guinea pig trachea, but PGE(2) induced strong oscillatory contraction after pretreatment with indomethacin and ChTX. Moreover, ChTX time-dependently stimulated PGE(2) generation. These results suggest that ChTX specifically activates COX-2 and stimulates PGE(2) production and that ChTX-induced oscillatory contraction in guinea pig trachea is mediated by activation of EP(1) receptor.     

An in vitro time-kill assessment of linezolid and anaerobic bacteria

Linezolid is a novel oxazolidinone antibacterial agent active against staphylococci (including methicillin-resistant strains), enterococci (including vancomycin-resistant strains), streptococci (including penicillin-intermediate and -resistant Streptococcus pneumoniae), and other aerobic and facultative bacteria. The agent has also demonstrated activity against a broad spectrum of Gram-positive and Gram-negative anaerobic bacteria. Previous time-kill assessments have shown linezolid to be generally bacteriostatic against staphylococci and enterococci, and bactericidal against streptococci. In this study, an anaerobic glovebox technique was employed to conduct time-kill assessments for four strains of anaerobic Gram-positive, and seven strains of anaerobic Gram-negative bacteria. The time-kill experiment was performed using Anaerobe Broth medium. The drugs were tested at four-fold the minimum inhibitory concentration (MIC), or at the higher concentration of 8mg/L for linezolid, 2mg/L for clindamycin, and 8mg/L for metronidazole. Samples for viable count were taken at 0, 6, and 24h, and plated using the Bioscience International Autospiral DW. Exposure of samples to the aerobic environment during plating was held to less than 30min. Plates were counted after a 48h anaerobic incubation (37 degrees C). The species tested included Bacteroides fragilis (2), B. distasonis, B. thetaiotaomicron, Fusobacterium nucleatum, F. varium, Prevotella melaninogenica, Clostridium perfringens, Eubacterium lentum and Peptostreptococcus anaerobius (2). The activity of linezolid was compared to that of metronidazole and clindamycin, two standard anti-anaerobe agents. As expected, the control agents were very active in these assays. Metronidazole yielded log(10)CFU/mL reductions of 3.0 or greater for nine of ten strains; clindamycin yielded log(10)CFU/mL reductions of 2.0 or greater for six of 11 strains, and 3.0 or greater for three strains. Linezolid also produced significant in vitro killing in this model achieving log(10)CFU/mL reductions of 2.0 or greater for six of 11 strains, and 3.0 or greater for four strains. The profile of activity was similar to that of clindamycin indicating that additional developmental studies of linezolid with anaerobic bacteria are warranted.     

Signaling via immunoglobulin Fc receptors induces oligodendrocyte precursor cell differentiation

Dramatic changes in morphology and myelin protein expression take place during the differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes. Fyn tyrosine kinase was reported to play a central role in the differentiation process. Molecules that could induce Fyn signaling have not been studied. Such molecules are promising therapeutic targets in demyelinating diseases. We provide evidence that the common gamma chain of immunoglobulin Fc receptors (FcRgamma) is expressed in OPCs and has a role in triggering Fyn signaling. FcRgamma cross-linking by immunoglobulin G on OPCs promotes the activation of Fyn signaling and induces rapid morphological differentiation with upregulation of myelin basic protein (MBP) expression levels. Mice deficient in FcRgamma are hypomyelinated, and a significant reduction in MBP content is evident. Our findings indicate that the FcRgamma-Fyn-MBP cascade is pivotal during the differentiation of OPCs into myelinating oligodendrocytes, revealing an unexpected involvement of immunological molecules.     

Neural expression of the Huntington's disease gene as a chordate evolutionary novelty

Huntington's disease is a progressive neuro-degenerative disorder in humans, which is scharacterized by onset of dementia, muscular ataxia, and death. Huntington's disease is caused by the expansion of the polyglutamine (polyQ) tract in the N-terminus of the HD protein (Huntingtin). CAG expansion is a dominant gain of function mutation that affects striated neurons in the brain (Cattaneo, 2003, News Physiol Sci 18:34). The evolutionary origins of the vertebrate Hd gene are not well understood. In order to address the evolutionary history of the Hd gene, we have cloned and characterized the expression of the Hd gene in two invertebrate deuterostomes, an echinoderm and an ascidian, and have examined the expression patterns in a phylogenetic context. Echinoderms are basal deuterostomes and ascidians are basal chordates; both are useful for understanding the origins of and evolutionary trends in genes important in vertebrates such as the Huntigton's disease gene. Expression of Hd RNA is detected at all stages of development in both the echinoderm and ascidian studied. In the echinoderm Heliocidaris erythrogramma, Hd is expressed in coelomic mesodermal tissue derivatives, but not in the central nervous system. In the ascidian Halocynthia roretzi expression is located in both mesoderm and nervous tissue. We suggest that the primitive deuterostome expression pattern is not neural. Thus, neural expression of the Hd gene in deuterostomes may be a novel feature of the chordate lineage, and the original role(s) of HD in deuterostomes may have been non-neural.     

Symmetric and asymmetric squarylium dyes as noncovalent protein labels: a study by fluorimetry and capillary electrophoresis

Noncovalent interactions between two squarylium dyes and various model proteins have been explored. NN127 and SQ-3 are symmetric and asymmetric squarylium dyes, respectively, the fluorescence emissions of which have been shown to be enhanced upon complexation with proteins such as bovine serum albumin (BSA), human serum albumin (HSA), beta-lactoglobulin A, and trypsinogen. Although these dyes are poorly soluble in aqueous solution, they can be dissolved first in methanol followed by dilution with aqueous buffer without precipitation, and are then suitable for use as fluorescent labels in protein determination studies. The nature of interactions between these dyes and proteins was studied using a variety of buffer systems, and it was found that electrostatic interactions are involved but not dominant. Dye/protein stoichiometries in the noncovalent complexes were found to be 1:1 for SQ-3, although various possible stoichiometries were found for NN127 depending upon pH and protein. Association constants on the order of 10(5) and 10(7) were found for noncovalent complexes of SQ-3 and NN127, respectively, with HSA, indicating stronger interactions of the symmetric dye with proteins. Finally, HSA complexes with NN127 were determined by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). In particular, NN127 shows promise as a reagent capable of fluorescently labeling analyte proteins for analysis by CE-LIF without itself being significantly fluorescent under the aqueous solution conditions studied herein.     

Comparative study of the N-terminal hydrophilic region in Streptomyces lividans and E. coli FtsY

Streptomyces lividans FtsY (SlFtsY) was cloned and overexpressed in Escherichia coli. Analysis of the amino acid (aa) sequence showed a concentration of hydrophilic aa's in the N-terminal half region of SlFtsY as observed in that of E. coli FtsY (EcFtsY). However, the length of the hydrophilic region was shorter in SlFtsY than in EcFtsY. Overexpression of SlFtsY in E. coli resulted in growth suppression as in the case of the overexpression of EcFtsY, while growth suppression as a result of the overexpression of the C-terminal half region of SlFtsY was limited. This result suggests that the N-terminal hydrophilic region of SlFtsY, regardless of its short length, would behave like its counterpart region of EcFtsY in E. coli.     

Functional uncoupling of T-cell receptor engagement and Lck activation in anergic human thymic CD4+ T cells

Human thymic CD1a-CD4+ T cells in the final stage of thymic maturation are susceptible to anergy induced by a superantigen, toxic shock syndrome toxin-1 (TSST-1). Thymic CD4+ T-cell blasts, established by stimulating human thymic CD1a-CD4+ T cells with TSST-1 in vitro, produce a low level of interleukin-2 after restimulation with TSST-1, whereas TSST-1-induced adult peripheral blood (APB) CD4+ T-cell blasts produce high levels of interleukin-2. The extent of tyrosine phosphorylation of the T-cell receptor zeta chain induced after restimulation with TSST-1 was 2-4-fold higher in APB CD4+ T-cell blasts than in thymic CD4+ T-cell blasts. The tyrosine kinase activity of Lck was low in both thymic and APB CD4+ T-cell blasts before restimulation with TSST-1. After restimulation, the Lck kinase activity increased in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Surprisingly, Lck was highly tyrosine-phosphorylated in both thymic and APB CD4+ T-cell blasts before restimulation with TSST-1. After restimulation, it was markedly dephosphorylated in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Lck from APB CD4+ T-cell blasts bound the peptide containing the phosphotyrosine at the negative regulatory site of Lck-505 indicating that the site of dephosphorylation in TSST-1-activated T-cell blasts is Tyr-505. Confocal microscopy demonstrated that colocalization of Lck and CD45 was induced after restimulation with TSST-1 in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Further, remarkable accumulation of Lck in the membrane raft was observed in restimulated APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. These data indicate that interaction between Lck and CD45 is suppressed physically in thymic CD4+ T-cell blasts and plays a critical role in sustaining an anergic state.