Overexpression of P-glycoprotein in heat-and/or drug-resistant hepatoma variants

We have earlier isolated a glucocorticoid-resistant, dedifferentiated rat hepatoma variant, the clone 2, which exhibited deficient stress activation of the major stress-inducible heat-shock protein hsp68.Multidrug-resistant variants were isolated from clone 2 cells using increasing concentrations of colchicine. The induction deficiency of hsp68 was maintained in the colchicine-resistant clone 2 cells grown for several months in the presence of 1 g/ml colchicine (termed ashighly multidrug-resistant variant) indicating that this heat-shock protein is not involved in the multidrug resistance. No alteration of the protein synthesis pattern was observed except the strong increase of the P-glycoprotein, which correlated with high level of corresponding mRNA. Stableheat-resistant variants of clone 2 were also isolated, which showed increaseddrug resistance to several drugs, i.e. they becamemoderately multidrug-resistant. This moderate multidrug resistance of the heat-resistant variants was further increased by stepwise selection with colchicine (highly multidrug-resistant heat-resistant variants). The levels of P-glycoprotein mRNA and protein were elevated both in the heat-resistant, non drug selected, moderately drug-resistant and in heatresistant, colchicine selected, highly drug-resistant variants. Decreased retention of antitumor drugs was observed in all multidrug-resistant variants indicating that P-glycoprotein was functional. Verapamil increased doxorubicin retention and cytotoxicity significantly. Our results showing that severely stressed hepatoma cells overexpressed the multidrug resistance gene(s) raise the possibility that the P-glycoprotein may participate in protection against enviromental stress such as heat.Abbreviations hsp heat-shock protein - MDR multidrug resistance - P-gp P-glycoprotein     

Identification of members of the HSP30 small heat shock protein family and characterization of their developmental regulation in heat-shocked xenopus laevis embryos

In the present study we have characterized the synthesis of members of the HSP30 family during Xenopus laevis development using a polyclonal antipeptide antibody derived from the carboxyl end of HSP30C. Two-dimensional PAGE/immunoblot analysis was unable to detect any heat-inducible small HSPs in cleavage, blastula, gastrula, or neurula stage embryos. However, heat-inducible accumulation of a single protein was first detectable in early tailbud embryos with an additional 5 HSPs at the late tailbud stage and a total of 13 small HSPs at the early tadpole stage. In the Xenopus A6 kidney epithelial cell line, a total of eight heat-inducible small HSPs were detected by this antibody. Comparison of the pattern of protein synthesis in embryos and somatic cells revealed a number of common and unique heat inducible proteins in Xenopus embryos and cultured kidney epithelial cells. To specifically identify the protein product of the HSP30C gene, we made a chimeric gene construct with the Xenopus HSP30C coding sequence under the control of a constitutive promoter. This construct was microinjected into fertilized eggs and resulted in the premature and constitutive synthesis of the HSP30C protein in gastrula stage embryos. Through a series of mixing experiments, we were able to specifically identify the protein encoded by the HSP30C gene in embryos and somatic cells and to conclude that HSP30C synthesis was first heat-inducible at the early tailbud stage of development. The differential pattern of heat-inducible accumulation of members of the HSP30 family during Xenopus development suggests that these proteins may have distinct functions at specific embryonic stages during a stress response.     

细胞寒害的热力学熵理论(Ⅱ)──超熵产生作为寒害稳定性判据的理论研究

本文从物质和能量交换的角度,运用非平衡态热力学超熵产生理论,分析了寒害定态的稳定性,并建立了超熵产生判据．理论分析所得的结论与实验结果基本相符．     

用生物素化重组质粒探针检测产蛋下降综合症病毒核酸

用ＥＤＳ－７６标准毒株感染鸭胚后,提取病毒核酸,经ＰｓｔＩ酶切后,与质粒ｐＴ７／Ｔ３α－１９重组,并转化到大肠杆菌ＤＨ５α中,筛选出一个插入片段为３１８ｂｐ的重组质粒,并命名为ｐＴＥＺＰ３。用生物素标记该重组质粒后,分别对正常鸭胚尿囊液、各地分离的ＥＤＳ毒株和禽类易感的几种病毒进行核酸杂交试验,结果该探针对实验中收到的几种ＥＤＳ分离毒均产生阳性反应,而不与正常鸭胚尿囊液和其它几种禽类病毒交;该探针     

肾综合征出血热人体组织中病毒包涵体的性质及意义的进一步研究

应用免疫组化、原位分子杂交、电镜及免疫电镜等方法进一步对肾综合征出血热人体尸检组织中病毒包涵体（ＩＢ）的抗原、核酸性质和超微结构特点作了进一步观察。结果,在３９例中的２０例尸检病例组织中显示出病毒核蛋白抗原和血凝素抗抗原阳性的ＩＢ,其中包括１６例陕西尸检病例组织中的６例休克期和１例多尿期病例,１７例上海病例中的３例休克期和９例少尿期病例及３例江西病例中的１例休克期病例。ＩＢ主要分布在呼吸道和肺泡、肾远曲小管和集合管、胃肠道、腺垂体、扁桃体、胰腺、前列腺等组织的粘膜上皮和腺上皮细胞及肝细胞和睾丸生精上皮细胞胞浆中,阳性细胞形态基本正常。应用原位分子杂交,可在该组细胞小同时检测到病毒ＲＮＡ,多为胞浆内弥漫阳性,仅少数组织中显示出病毒ＲＮＡ阳性ＩＢ结构。电镜观察阳性组织细胞中出现由大量微丝微管及颗粒样结构组成的ＩＢ结构,其小的病毒颗粒状结构、内质网及纤维丝状结构呈病毒抗原阳性,上述结构位于高尔基体区。结果说明该病毒有感染上皮细胞的特性,对其宿主细胞的致细胞病变作用是极其温和的,且多表现在亚细胞水平。ＩＢ可能是病毒过量表达抗原的堆积或病毒复制部位,而微丝微管结构可能参与病毒的感染过程。     

The influence of polyalcohols and carbohydrates on the thermostability of alpha-amylase

The influence of polyhydric alcohols and carbohydrates on the thermostability, i.e., the heat inactivation kinetics, of Bacillus licheniformis alpha-amylase was studied in the temperature range 96 degrees to 130 degrees C. High concentrations (from 9 to 60 weight percent) of glycerol, sorbitol, mannitol, sucrose, or starch can markedly decrease the inactivation rate constant, k, and in the studied cases, this stabilizing effect grows stronger with increasing additive concentration. Statements about stabilization should, however, be specified carefully with respect to temperature, because E(A) is mostly altered likewise. For dissolved enzyme E(A) was almost always decreased in the presence of polyol or carbohydrate, whereas for immobilized enzyme it was augmented in each studied instance. The inactivation of dissolved enzyme can, in all the studied cases, be adequately described as a first-order process. Immobilized enzyme, however, shows biphasic then first-order inactivation kinetics, depending on the additive concentration and temperature.     

Temperature and chemical shocks induce chilling tolerance in germinating Cucumis sativus (cv. Poinsett 76) seeds

Roots of 24-h-old germinated cucumber ( Cucumis sativus cv. Poinsett 76) seeds were subjected to thermal and chemical stresses, equilibrated at 25°C for 2 h and chilled at 2.5°C for 96 h. The germinated seeds were then held at 25°C for 72 h after they were chilled and the elongation of the primary root was used as a measure of chilling tolerance. Control roots elongated from an initial length of 0.2 cm to a final length of 6.3 cm at the end of 72 h. while chilled roots elongated to a final length of only 0.4 to 0.6 cm. Exposure to 0.4 M ethanol for 4 h or to 40°C for 1 h induced substantial chilling tolerance and the roots had a final length of 4.1 and 3.1 cm. respectively. Exposure to 7.5°C for 3 h conferred less chilling tolerance (elongation to 1.4 cm). while exposure to other chemicals (i.e. aqueous solutions of Ca(NO₃)₂, mannitol. methanol and NaCl) produced less, though still significant increases in chilling tolerance. A more severe chilling treatment of 144 h at 2.5°C was required to consistently induce elevated rates of ion leakage. Only the heat and the ethanol shock treatments significantly reduced chilling-induced ion leakage. Inclusion of the protein synthesis inhibitor cycloheximide negated the protective effects of these shock treatments. It appears that de novo protein synthesis is required for induction of chilling tolerance by a variety of chemical and thermal shock treatments.     

Selection for higher yields and heat resistance in Pima cotton has caused genetically determined changes in stomatal conductances

Gas exchange analysis was used to characterize photosynthetic and stomatal responses to key environmental stimuli in five commercial lines of Pima cotton ( Gossypium barbadense L.) which represent a gradient of selection for higher yields and heat resistance, and a primitive, uncultivated G. barbadense. At constant light and vapor pressure deficit, stomatal conductance increased linearly with air temperature in the 23 to 36$C range in all five commercial lines, and conductance at each temperature increased as a function of selection. In contrast, photosynthetic rates had a low sensitivity to temperature in the 23 to 36$C range, particularly in the advanced lines. In a segregating F₂ population from a cross between the advanced line, Pima S-6, and the primitive cotton, B368, the slope of the stomatal response to temperature in each F₂ plant was positively correlated with the stomatal conductance measured at 40$C. An analysis of the frequency distribution of stomatal conductance in F₁ and F₂ progeny of the cross showed that the differences in stomatal conductance between lines were genetically determined. These data indicate that selection for higher yield and heat resistance in Pima cotton has caused genetically determined changes in stomatal properties. Characterization of the relationship between the altered stomatal properties and the attained increases in heat resistance and yields could make it possible to use these physiological traits as selection criteria in future breeding programs.