Thermal response in murine L929 cells lacking αB-crystallin expression and αB-crystallin expressing L929 transfectants

We investigated the role of B-crystallin expression in the development of thermotolerance in murine L929 cells. An initial heat-shock of 10 min at 45°C induced thermotolerance in these cells to a heat challenge at 45°C administered 24 h later. The thermotolerance ratio at 10^–1 isosurvival was 1.7. Expression of B-crystallin gene was not detected during the 24 h incubation at 37°C following heat shock by either northern or western blots. In contrast, inducible HSP70 synthesis was observed during this time period. Thus, this cell line provided an unique system in which to examine the effects of transfected B-crystallin on thermoresistance and thermotolerance. Cells stably transfected with B-crystallin under the control of an inducible promoter did not show a significant increase in the ability to develop thermotolerance. However, a stably transfected L929 clone expressing high levels of constitutive B-crystallin exhibited an approximately 50% increase in thermal resistance over parental and control cells. Though expression of B-crystallin is not requisite for the development of thermotolerance in L929 cells, overexpression of transfected B-crystallin can contribute to increased thermoresistance.     

14-3-3 Protein homologues play a central role in the fusicoccin signal transduction pathway

The plasma membrane located fusicoccin binding protein (FCBP) is an essential element in the fusicoccin (FC) signal transduction pathway. We obtained primary sequence information for the 31 kD subunit of the FCBP. These sequences showed that the FCBP is homologous to members of the 14-3-3 protein family. Both the 31 and 30 kD subunits cross-react with 14-3-3 antibodies. In native form the FCBP occurs as a dimer, but it is also part of a complex with higher molecular mass. The monomeric forms of the FCBP (the 30 and 31 kD subunits) do not have ³H-FC binding activity. We discuss how the FCBP, as a member of the 14-3-3 protein family, may be able to bind FC and how the FC-signal is transduced to the effector protein, the H⁺-ATPase.     

Liver and intestinal fatty acid binding proteins in control and TGFβ1 gene targeted deficient mice

The effect of transforming growth factor beta-1 (TGF1) expression on fatty acid binding proteins was examined in control and two strains of gene targeted TGF1-deficient mice. Homozygous TGF1-deficient 129 × CF-1, expressing multifocal inflammatory syndrome, had 25% less liver fatty acid binding protein (L-FABP) when compared to control mice. The decrease in L-FABP expression was not due to multifocal inflammatory syndrome since homozygous TGF1-deficient/immunodeficient C3H mice on a SLID background had 36% lower liver L-FABP than controls. This effect was developmentally related and specific to liver, but not the proximal intestine, where L-FABP is also expressed. Finally, the proximal intestine also expresses intestinal-FABP (1-FABP) which decreased 3-fold in the TGF1-deficient/immunodeficient C3H mice only. Thus, TGF1 appears to regulate the expression of L-FABP and I-FABP in the liver and the proximal intestine, respectively.Abbreviations L-FABP liver fatty acid binding protein - I-FABP intestinal fatty acid binding protein - TGF1 transforming growth factor beta-1 - TNF- tumor necrosis factor- - MIP- macrophage inflammatory protein- - PMSF phenylmethyl sulfonyl fluoride - PBS phosphate buffered saline     

Behavioral responses of three species of ground beetle (Coleoptera: Carabidae) to olfactory cues associated with prey and habitat

The responses ofPterostichus melanarius Illiger,Harpalus rufipes DeGeer, andNebria brevicollis Fabricius (Coleoptera: Carabidae) to olfactory cues of prey and habitat were studied in a four-arm continuous-airflow olfactometer. The process was semiautomated using time-lapse image analysis by microcomputer. The primary constituent of the aphid alarm pheromone, (E)--farnesene (EBF), was synthesized and tested for a kairomonal role in prey detection by carabids. In addition, individual beetles were exposed to odors from live aphids, live collembolans, and a crude extract of wheat. All three beetle species showed evidence of olfaction.P. melanarius responded to all the odors except collembolans,H. rufipes responded to EBF and wheat, andN. brevicollis to collembolans. The use of a defence allomone as a prey-finding kairomone by certain carabid species has implications for pest management. Manipulation of predator chemical ecology by the inclusion of behavior-modifying compounds in a crop spray mix with reduced amounts of insecticide may allow for efficient aphid control with less environmental contamination.     

Kinetics of retroviral production from the amphotropic ΨCRIP murine producer cell line

Rapidly expanding development and practice of gene therapy requires the availability of large quantities of high titer retroviral supernatants. One way to achieve high retroviral titers is through improved understanding of the kinetics of retroviral production and decay, and the subsequent development of improved cell culture methods. In the present study we investigated the effects of different operational modes on the retroviral production of the NIH 3T3 fibroblast derived amphotropic murine retroviral producing cell line pMFG/CRIP. Semi-continuous culture (exchange of 50% of medium volume daily) was found to promote cell growth and enhance retroviral production. The rapid medium exchange resulted in significantly larger amounts of high titer supernatants and an extended production phase as compared to the batch control cultures. The specific viral productivity of the pMFG/CRIP cells was in the range of 10 to 40 infectious viruses produced per thousand producer cells per day. The CV-1 African Green Monkey kidney cell line was used as the infection target. Lowering the serum level form 20% to 10% improved retroviral production slightly. However, at lower serum levels (1%, 5% and 10% (v/v)) growth of the producer cell line, and thus retroviral production, was directly proportional to the serum level. The half-life of the virus at 37°C was found to be 5.5 hours. Promoting the growth of producer cell lines can improve retroviral vectors titers and viral production. High cell density systems that allow for rapid cell growth and waste product removal are likely to be used to generate high-titer retroviral supernatants.     

Development of insect resistance in tomato plants expressing the δ-endotoxin gene ofBacillus thuringiensis subsp.tenebrionis

A crystal -endotoxin gene ofBacillus thuringiensis subsp.tenebrionis (B.t.t.) encoding a coleopteran insect-specific toxin was used to construct a chimeric gene which expressed the toxin in plant cells. Via anAgrobacterium tumefaciens binary vector system, the toxin gene was transferred into tomato cells. From leaf disks recombinant plants were regenerated. Hybridization experiments demonstrated that these plants synthesized toxin-specific mRNA of the expected size. Transgenic tomato plants with the chimericB.t.t. toxin gene contained a 74 kDa protein which cross-reacted with toxin antibodies. The expression caused a significant insecticidal activity of the transgenic tomato plants against Colorado potato beetle larvae.     

Epstein─Barr病毒膜抗原在中国仓鼠卵巢（CHO）细胞中的表达

将切去３’端穿膜序列的ＥＢ病毒膜抗原（ＭＡ）基因,插入ｐＳＶ２－ｄｈｆｒ质粒的ＳＶ４０早期启动子下游,构建了真核表达载体ｐＳＶ２－ｄｈｆｒＧＰＴＲ,使两个ＳＶ４０早期启动子分别调控ＭＡ和二氢叶酸还原酶（ｄｈｆｒ）基因。将该重组质粒转化ＣＨＯ－ｄｈｆｒ细胞,在选择培养基中筛选阳性克隆,用氨甲喋呤加压扩增,建立了表达ＥＢＶ－ＭＡ的克隆细胞系。ｗｅｓｔｅｒｎｂｌｏｔ分析证明,所表达的蛋白的分子量大约为３４０ｋｄ和２２０ｋｄ。经过细Ｓｅｐｈａｒｏｓｅ２Ｂ琼脂糖凝胶层析初步纯化的抗原与福氏佐剂混合免疫小鼠,２周后小鼠血清中出现明显的ｇｐ３４０／２２０特异性抗体,表明切去嵌膜区结构的ＥＢＶ－ＭＡ基因在ＣＨＯ细胞中的表达产物具有同天然膜抗原相似的分子量大小、糖基化程度、免疫特异性和免疫原性,可望成为ＥＢ病毒人用基因工程亚单位疫苗。