The effect of drugs which alter GABA-ergic function on cerebellar guanosine-3',5'-monophosphate content.

The actions of several classes of drugs, thought to be involved with gamma-amino-butyric acid (GABA) mechanisms, have been examined for effects on cerebellar cGMP content. Picrotoxin and TRH increased, while ethanol and diazepam decreased, cerebellar cGMP. Doses of gamma-hydroxybutyrate (GHB) and baclofen caused no significant effect at doses that caused behavioral changes. These cGMP actions were contrasted with those induced by the dopaminergic agents, apomorphine and haloperidol, which respectively, raised and lowered cerebellar cGMP. Apomorphine-induced increases in cGMP were blocked by haloperidol, but not by GHB or baclofen given eight min before sacrifice. However, baclofen given one hour before sacrifice caused effects similar to those of haloperidol. These results are discussed in terms of dopaminergic-GABAergic interactions.     

Peripheral hyaline blebs (podosomes) of macrophages

The plasmalemma and hyaline ectoplasm together constitute the sensory and motor organ of macrophages. The purpose of this study was to isolate this cell fraction in order to analyze it biochemically and functionally. Brief sonification of warmed rabbit lung macrophages caused release of heterodisperse hyaline blebs and filopodia, which were easily collected by differential centrifugation. Viewed in the electron microscope, these structures consisted of membrane-bounded sacs principally containing actin filaments. Some contained secondary lysosomes. They were enriched threefold over whole cell homogenates in specific adenylate cyclase activity and in trichloroacetic-acid-precipitable (125)I when derived from cells labeled with 125(I) by means of a lactoperoxidase-catalyzed reaction. These markers were found to have identical isopycnic densitites when macrophage homogenates were subjected to sedimentation in a focusing sucrose density gradient system, and these markers had densities distinct from those of other cytoplasmic organelles. These markers were therefore assumed to be associated with macrophage plasma membranes. The specific β- glucuronidase activity of the bleb fraction was similar to that of homogenates, but the blebs had considerably lower specific succinic dehydrogenase activity and RNA content, and DNA was undetectable. Electrophoresis of blebs solubilized in sodium dodecyl sulfate on polyacrylamide gels revealed polypeptides co-migrating with macrophage actin-binding protein, myosin, and actin; blebs also had EDTA-activated adenosine triphosphatase activity characteristic of myosin. The concentrations of actin-binding protein and myosin were higher in blebs than in cells or cytoplasmic extracts, whereas actin concentrations were similar (relative to extracts) or only slightly greater (than in cells). Blebs and intact cells had high lactate dehydrogenase activities in the presence but not the absence of Triton X-100. Blebs and cells oxidased 1-[(14)C]glucose, and the rate of glucose oxidation was increased substantially in the presence of latex beads. We conclude that intact sacs of plasmalemma encasing contractile proteins and cytoplasmic enzymes can be isolated from macrophages. They are enriched in myosin and actin-binding protein, indicating that the contractile apparatus is regulated in the cell periphery. These structures have the capacity to respond to environmental signals. We suggest the name "podosomes" for them because of their resemblance to macrophage pseudopodia. We propose that podosome formation results from rapid dissolution of the cortical gel when the membrane is in an actively extended configuration.     

Role of macrophage colony-stimulating factor in the differentiation and expansion of monocytes and dendritic cells from CD34+ progenitor cells

The present study focused on whether it is possible to expand monocytic cells from CD34⁺ progenitor cells by using macrophage colony-stimulating factor (M-CSF) in the absence and presence of mast cell growth factor (MGF) and IL-6. It was demonstrated that CD34⁺ cells differentiate without expansion to functional mature monocytic cells in the presence of M-CSF or combinations of M-CSF plus IL-6 and MGF. A different response pattern was observed for the number of clonogenic cells. The addition of IL-6 or both IL-6 and MGF to M-CSF containing cultures resulted in significant higher numbers of colony-forming unit-macrophage (CFU-M) as tested in clonogenic and³H-thymidine assays. Furthermore, M-CSF plus both IL-6 and MGF appeared to be the most potent combination to preserve the monocytic precursor in cell suspension culture assays. These results indicate that IL-6 and MGF in conjunction with M-CSF affect CD34⁺ cells especially at precursor level without distinct effect on the more mature stages. Secondly we studied whether M-CSF is only critical for the monocytic lineage or also affects dendritic cell (DC) development. Indeed, we were able to culture CD83⁺ DC from CD34⁺ progenitor cells in the presence of M-CSF in conjunction with TNF-α, IL-4, and MGF although their absolute number is almost threefold lower than the number of CD83⁺ cells yielded from GM-CSF plus TNF-α, IL-4, and MGF stimulated CD34⁺ cells.     

A study of the suitability of gas chromatography-electron capture detection for the analysis of deoxynivalenol in cereals

A gas chromatographic-electron capture detection (GC-ECD) method for the analysis of deoxynivalenol (DON) in cereals was investigated. The sample was extracted with a mixture of acetonitrile-water and purified with a MycoSep #225 column. The silylation was performed with Tri-Sil-TBT reagent, followed by dilution with hexane and a washing step with buffer. By using Tri-Sil-TBT reagent no double peaks were observed for DON in the gas chromatograms, in comparison with two other silylation reagents TMSI and Tri-Sil-Z. The use of trichothecolone (TRI) as an internal standard for DON was studied in order to indicate possible problems in the derivatisation reaction. TRI proved to be a relatively good internal standard for DON in cereal samples, as well as 1,1-bis-(4-chlorophenyl)-2,2-dichloroethylene (DDE), which was used as a GC standard for ensuring the function of GC-ECD. During the study, a matrix effect was clearly observed between the cereal matrix-assisted calibration curve and the calibration curve prepared without cereal matrix. The results of spiked and reference material samples, quantified with the calibration curve prepared without and with matrix, demonstrated that the matrix affects the results. However, after recovery correction the results were comparable. The validation results demonstrated that the GC-ECD method for DON analysis in cereals is sufficiently reliable.     

Simultaneous determination of thioridazine and its S-oxidized and N-demethylated metabolites using high-performance liquid chromatography on radially compressed silica

A method for simultaneously quantifying thioridazine, northioridazine, thioridazine-2-sulfoxide, thioridazine-2-sulfone and thioridazine-5-oxide in serum and plasma is described. Following solvent extraction these compounds were separated by high-performance liquid chromatography on radially compressed silica gel and detected by UV absorbance at 254 nm. Chromatography time is less than 7 min. The relative retention of these compounds as a function of the methanol and methylamine content of the mobile phase is discussed. Practical limits of detection, based upon an assayed plasma or serum volume of 1 ml, were 20 ng/ml for thioridazine-5-oxide and 10 ng/ml for the other compounds. The coefficient of variation for all compounds was less than 13%. The method is compared with more conventional high-performance liquid chromatographic and gas chromatographic methodology.     

Fungal infections of micropropagated plants at weaning: a problem exemplified by downy mildews in Rubus and Rosa

Plant Cell, Tissue and Organ Culture (PCTOC) -     

CpG联合铝佐剂对HCV免疫原体液免疫作用的影响

目的:探讨胞苷酸鸟苷寡脱氧核苷酸(CpG ODN)联合铝佐剂对丙型肝炎病毒(HCV)重组免疫原的体液免疫作用。方法:采用高交叉HCV-HVR1和E1重组蛋白与CpG ODN、铝佐剂组合,免疫BALB/c小鼠后以ELISA、酶联免疫斑点测定、流式细胞术、免疫沉淀等方法检测相关体液免疫指标和免疫血清多抗的交叉反应性。结果:CpG联合铝佐剂激发了最高的特异性抗体滴度;佐剂通过提高抗体分泌细胞数量、增加脾脏中记忆B细胞数量、增加脾淋巴细胞IL-6、IL-10分泌浓度实现体液免疫增效;CpG则能提高免疫效率,联合铝佐剂时显著提高浆细胞数量;12份HCV阳性血清中有10份可与多抗HVR1 IgG发生免疫沉淀。结论:CpG和铝佐剂联合应用具有协同作用,多抗HVR1 IgG具有较好的交叉反应性。