Improvement of Transfection Efficiency of Epithelioma Papulosum Cyprini Carp Cells by Modification of Cell Cycle and Use of an Optimal Promoter

Several methods to improve transfection of epithelioma papulosum cyprini (EPC) carp cells have been tested and are reported here. By modifying the cell cycle state of EPC cell monolayers and selecting the best promoter for the plasmid to be transfected, we increased transfection efficiency from 12.8% to 55.1% and decreased the coefficient of variation among different experiments from 54.1% to 11.8%. Thus 2- to 3-fold higher transfection efficiencies were obtained when the EPC monolayers were treated with colchicine or thymidine before transfection. In addition, the plasmids pMOKgal and its shorter derivative pMVC1.4gal, both containing 218 bp of additional sequences upstream of the cytomegalovirus promoter contained in plasmid pCMV, consistently produced higher transfection efficiencies than pCMV. Combination of the two methods resulted in an improvement of both efficiency and reproducibility. These results should facilitate transfection of EPC cells to use as a model to obtain transgenics, to conduct quantitative transfected-cell fusion assays, to improve DNA-immersion-vaccination methods, or to obtain infectious cDNA from fish RNA viruses.     

Time Course of Brain Neuronal Degeneration and Heat Shock Protein (72) Expression Following Neck Tourniquet-Induced Cerebral Ischemia in the Rat

1. The present study was designed to examine the regional expression of HSP72/73 protein after a 7.5-min period of cerebral ischemia and to compare the distribution of HSP neurons with the localization of irreversible neuronal degeneration as analyzed by silver impregnation technique.2. During 6–24 hr after cerebral ischemia clear-cut neuronal argyrophilia developed in several brain regions including the hippocampal hilus, nucleus reticularis thalami, and colliculi inferiores. With the exception of the hippocampal hilus, the structures which showed silver impregnability were HSP72 negative at 6–24 hr.3. Despite the clear HSP72 expression seen in hippocampal CA1 neurons, a significant loss of these neurons was seen at 7 days after ischemia.4. These data show that in some structures the presence of HSP72 is indicative of higher resistance of these neurons to ischemia-induced degeneration, however, the process of delayed neuronal degeneration appears to be independent of the accelerated synthesis of HSP72 seen during the early period of reflow.     

Silver electrode as a sensor for determination of zinc in cell cultivation medium.

Use of the silver electrode as a sensor for the monitoring of zinc in cell growth medium is described. Zinc at silver electrodes provides specific voltammetric signal, which is affected by solution components. Signals of zinc ions in phosphate buffer solutions with and without cell growth medium were compared. Common DMEM cell culture medium was used for the cultivation of a cell line of v-myb-transformed chicken monoblasts and its variants expressing v-jun and c-jun in a zinc-dependent manner. Electrochemical results showed zinc concentrations in the medium coincide very well with the jun expression. With respect to the low toxicity of silver for eukaryotic cells, silver electrodes represent promising tools for the determination of zinc concentrations in vivo without the potential risk of a cell culture damage.     

A new method for fish leucocyte counting and partial differentiation by flow cytometry

A simple and rapid method for analysis of fish blood cells is presented. Carp (Cyprinus carpio) blood was diluted 200 times with Hanks' solution containing 1 microg/ml of DiOC6(3) which is a fluorescent, lipophilic dye. After staining for 10 min, the blood cells were measured by a flow cytometer (FACS). Several blood cell populations were identified by different FL-1 (green fluorescence), FSC (forward scatter), and SSC (side scatter) properties. FL-1 v. SSC or FSC v. SSC dot-plot of stained blood cells displayed five separate cell populations: erythrocytes: a mixture of thrombocytes plus lymphocytes; monocytes; neutrophils; and basophils. The number of each type of blood cell counted by the FACS was in good agreement with those counted microscopically.     

Effect of oligochitosan-coated silver nanoparticles (OCAgNPs) on the growth and reproduction of three species Phytophthora in vitro

Phytophthora diseases cause billions of dollars annually in damage to crops. Nanotechnology provides various metallic nanoparticles exhibiting a strong activity against microbial pathogens. Silver nanoparticles-based products are outstanding samples, which has been produced in large scale and performed well with a high activity against several fungal pathogens. Our previous study indicated oligochitosan-coated silver nanoparticles (OCAgNPs) which were prepared from 3,4 dihydroxyphenyl acetic acid – conjugated oligochitosan and silver nitrate salt performed an enhancement in antibacterial ability at a very low concentration. Objective of this study was to determine the effect of the OCAgNPs on growth and reproduction of Phytophthora capsici, P. nicotianae and P. colocasiae in vitro. The study shown that strong inhibition of mycelial growth, sporangium production, zoospore release and zoospore germination of P. capsici, P. nicotianae and P. colocasiae occurred when exposed to at 9 ppm of OCAgNP. The results demonstrated a great potential of OCAgNPs for controlling growth of Phytophthora.     

武汉东湖中浮游植物与鲢鱼相互作用的量的评价

以东湖郭郑湖为研究对象,将其生态系统中在质和量方面占重要地位的浮游植物-鲢作为亚系统,研究它们之间的相互作用。比较了夏季两个月中鲢的生长和让其充分摄食后的生长,发现最初20天没有差异,20天后两者开始出现差别。将两者的相互作用模型化,计算的结果是,浮游植物被鲢摄食后密度下降,而鲢得不到充足的饵料,导致生长缓慢。观察此期间的氮平衡,浮游植物所生产的氮几乎全部被鲢利用,但浮游植物所需要的78%的氮又是鲢提供的。这说明在湖区整个生态系统中,此相互作用系统在量的方面是很重要的。     

鲤鱼不同品种（系）红血球抗原特异性的初步研究

用血球凝集和血清吸收试验的方法,对若干个鲤鱼品种及鲤鱼人工雌核发育系的红血球中的某些同种异型抗原纯合性进行了初步研究。①以抗雌核发育红鲤抗血清(abs／R)为试剂,三个雌核发育系(R8305,R8413,RMGF1)的RBC反应值分别为1521—2360,1521-2028,853—1521,普通红鲤、镜鲤、白鲢的反应值在80—782。②abs／8305,与abs／8413经靶细胞相互吸收后的残留活性显著降低(均为320),而以白鲢RBC吸收后抗血清活性不变。作者认为这部分抗原在鲤鱼种以下的品种和品系中具有特异性,可考虑将其作为鱼类品系鉴定的一种手段。     

用环境毒物对草鱼组织ATPase的影响作为生态毒理学指标的初步研究

不同季节捕获的草鱼在实验室(16±4℃)驯化3—7天,其鳃ATPase活力在显著性水平为0.05时,不同批鱼无显著差异。离体实验条件下,汞、三氯联苯、直链烷基苯磺酸钠对草鱼组织ATPase的半抑制浓度分别为1.8ppM(肾),1.84ppM(鳃),3.9ppM(鳃);Hg~(2 )对肾的作用大于对鳃的作用。草鱼组织ATPase可作为一项指标用于毒理学研究。     

Molecular and functional diversity of non-histone protein fraction NHCP1 from hamster Kirkman-Robbins hepatoma and liver

Non-histone protein fraction NHCP1 of micrococcal nuclease-sensitive and nuclease-resistant chromatin from Kirkman-Robbins hepatoma and hamster liver was studied by two-dimensional electrophoresis followed by Coomassie and silver staining and by microcomplement fixation technique in the presence of antibodies elicited against NHCP1 of both tissues. Apart from many common spots several tissue specific components associated with either nuclease-sensitive or nuclease-resistant chromatin were found. The presence of tissue specific components among NHCP1 from hepatoma and liver was confirmed by immunological analysis. It was stated that these components are exclusively localized in nuclease-resistant part of chromatin from neoplastic and normal tissues thus suggesting their structural function.