2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) enhances antibody production and protein kinase activity in murine B cells

Treatment of murine spleen cells with 30 nM TCDD resulted in an approximately 3 fold increase in unstimulated antibody production after 3 days in culture. This response was not accompanied by increased cellular proliferation and may represent an effect of TCDD on B cell activation or differentiation. Since PMA is capable of activating B cells, presumably via PKC, we have compared the effects of PMA and TCDD on protein kinase activation and phosphorylation of endogenous proteins in a highly purified preparation of B cells. In contrast to a reduction of cytosolic PKC activity, the expected effect of PMA, TCDD caused an increase in basal kinase activity with no effect on PKC activity. Addition of either PMA or TCDD resulted in enhanced phosphorylation of a similar profile of proteins, including proteins of Mr 12.2, 14.6, 29.2, 52.3 and 62.7 KDa. Addition of TCDD also resulted in the increased phosphorylation of a protein of Mr 45.2, which was unaffected by PMA. Combined treatment with PMA and TCDD resulted in additive responses. The additive effects of PMA and TCDD suggest an interaction at the level of protein phosphorylation which is mediated by different kinases. Therefore, TCDD may be stimulating B cells via an early effect on an unidentified protein kinase.     

Rapid development of new protein biosensors utilizing peptides obtained via phage display

There is a consistent demand for new biosensors for the detection of protein targets, and a systematic method for the rapid development of new sensors is needed. Here we present a platform where short unstructured peptides that bind to a desired target are selected using M13 phage display. The selected peptides are then chemically synthesized and immobilized on gold, allowing for detection of the target using electrochemical techniques such as electrochemical impedance spectroscopy (EIS). A quartz crystal microbalance (QCM) is also used as a diagnostic tool during biosensor development. We demonstrate the utility of this approach by creating a novel peptide-based electrochemical biosensor for the enzyme alanine aminotransferase (ALT), a well-known biomarker of hepatotoxicity. Biopanning of the M13 phage display library over immobilized ALT, led to the rapid identification of a new peptide (ALT5-8) with an amino acid sequence of WHWRNPDFWYLK. Phage particles expressing this peptide exhibited nanomolar affinity for immobilized ALT (K_d,app = 85±20 nM). The newly identified ALT5-8 peptide was then chemically synthesized with a C-terminal cysteine for gold immobilization. The performance of the gold-immobilized peptides was studied with cyclic voltammetry (CV), QCM, and EIS. Using QCM, the sensitivity for ALT detection was 8.9±0.9 Hz/(µg/mL) and the limit of detection (LOD) was 60 ng/mL. Using EIS measurements, the sensitivity was 142±12 impedance percentage change %/(µg/mL) and the LOD was 92 ng/mL. In both cases, the LOD was below the typical concentration of ALT in human blood. Although both QCM and EIS produced similar LODs, EIS is preferable due to a larger linear dynamic range. Using QCM, the immobilized peptide exhibited a nanomolar dissociation constant for ALT (K_d = 20.1±0.6 nM). These results demonstrate a simple and rapid platform for developing and assessing the performance of sensitive, peptide-based biosensors for new protein targets.     

ConA激活小鼠胸腺T淋巴细胞增殖过程中c-myc与核骨架蛋白的结合

采用DNA-蛋白质体外吸附的方法研究伴刀豆球蛋白激活小鼠胸腺T淋巴细胞增殖过程中c-myc与核骨架蛋白的结合.实验结果显示,c-myc与核骨架蛋白的结合具有特异性,在淋巴细胞激活过程中c-myc与P₃₄/P₃₆核骨架蛋白及核纤层蛋白结合,并发生动态变化.     

Characterization of the Dihydropyridine Binding Sites of Rat Neocortical Synaptosomes and Microvessels

The dihydropyridine binding sites associated with rat neocortical synaptosomes and microvessels were compared using an in vitro [3H]PN 200-110 [(+)-[methyl-3H]-isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5- methoxycarbonylpyridine-3-carboxylate] binding assay. Saturation experiments yielded similar KD values (approximately 70 pM) and Bmax values (approximately 400 fmol/mg of protein) for the two membrane preparations. Interaction experiments with [3H]PN 200-110 and various calcium-modulating substances provided further evidence for the practically identical nature of the synaptosomal and microvascular dihydropyridine binding sites. These findings predict that lipophilic dihydropyridines, simultaneously occupying the two central binding sites, have the dual effect of altering neuronal function and local blood flow.     

An electroejaculator for the collection of semen from the domestic cat

An electroejaculator for the collection of cat semen and for the evaluation of electroejaculation protocols is described. The electroejaculator contains an adjustable signal generator and allows for the precise control and monitoring of the electrical stimulus to the animal. The electroejaculator incorporates controls for the selection of the frequency, potential and waveform of the electrical stimulus and controls for either manual or automatic delivery of stimuli of specified characteristics to the rectal probe. In the automatic mode, the operator may also preset the rate and duration of stimulus application and the interval between successive stimuli. The electroejaculator output to the probe is controlled with an on-off foot-switch which allows for the collection of semen from an anesthetized cat by one operator. Diagrams of the functional block, the component circuits of the electroejaculator, and the accessories which facilitate the collection of cat semen are provided.