Identification of nuclear import and export signals within Fli-1: roles of the nuclear import signals in Fli-1-dependent activation of megakaryocyte-specific promoters

The Ets factor Friend leukemia integration 1 (Fli-1) is an important regulator of megakaryocytic (Mk) differentiation. Here, we demonstrate two novel nuclear localization signals (NLSs) within Fli-1: one (NLS1) is located at the N terminus, and another (NLS2) is within the Ets domain. Nuclear accumulation of Fli-1 reflected the combined functional effects of the two discrete NLSs. Each NLS can independently direct nuclear transport of a carrier protein, with mutations within the NLSs affecting nuclear accumulation. NLS1 has a bipartite motif, whereas the NLS2 region contains a nonclassical NLS. Both NLSs bind importin alpha (IMPalpha) and IMPbeta, with NLS1 and NLS2 being predominantly recognized by IMPalpha and IMPbeta, respectively. Fli-1 also contains one nuclear export signal. Leptomycin B abolished its cytoplasmic accumulation, showing CRM1 dependency. We demonstrate that Ets domain binding to specific target DNA effectively blocks IMP binding, indicating that the targeted DNA binding plays a role in localizing Fli-1 to its destination and releasing IMPs for recycling back to the cytoplasm. Finally, by analyzing full-length Fli-1 carrying NLS1, NLS2, and combined NLS1-NLS2 mutations, we conclude that two functional NLSs exist in Fli-1 and that each NLS is sufficient to target Fli-1 to the nucleus for activation of Mk-specific genes.     

Postprenylation CAAX processing is required for proper localization of Ras but not Rho GTPases

The CAAX motif at the C terminus of most monomeric GTPases is required for membrane targeting because it signals for a series of three posttranslational modifications that include isoprenylation, endoproteolytic release of the C-terminal- AAX amino acids, and carboxyl methylation of the newly exposed isoprenylcysteine. The individual contributions of these modifications to protein trafficking and function are unknown. To address this issue, we performed a series of experiments with mouse embryonic fibroblasts (MEFs) lacking Rce1 (responsible for removal of the -AAX sequence) or Icmt (responsible for carboxyl methylation of the isoprenylcysteine). In MEFs lacking Rce1 or Icmt, farnesylated Ras proteins were mislocalized. In contrast, the intracellular localizations of geranylgeranylated Rho GTPases were not perturbed. Consistent with the latter finding, RhoGDI binding and actin remodeling were normal in Rce1- and Icmt-deficient cells. Swapping geranylgeranylation for farnesylation on Ras proteins or vice versa on Rho proteins reversed the differential sensitivities to Rce1 and Icmt deficiency. These results suggest that postprenylation CAAX processing is required for proper localization of farnesylated Ras but not geranygeranylated Rho proteins.     

具有蛋白酶及解旋酶活性的HCV-NS3重组蛋白的纯化及活性分析

为深入探讨HCV-NS3蛋白的酶动力学性质,制备了具有蛋白酶及解旋酶活性的HCV NS3重组蛋白.利用PCR扩增HCV非结构基因NS3,插入pPIC9,测序分析.携带NS3基因的重组质粒(pPIC9-NS3)转化毕氏酵母菌菌株GS115,甲醇诱导表达NS3蛋白.重组蛋白首先采用Hitrap chelating柱进行亲和分离,之后使用Mono S HR柱进一步纯化.对纯化后的NS3重组蛋白的酶活性进行分析,结果表明,获得的重组蛋白分别具有蛋白酶及解旋酶活性.本研究为深入探讨NS3编码酶的功能和开发抗病毒药物创造条件.     

Differential Stain for acid fast Bacteria and Spores

A modified method of staining acid-fast organisms is described. After staining with carbol-fuchsin as usual in the Ziehl-Neelson method, wash with water and while the slide is still wet cover with a saturated acetone solution of malachite green for three to five minutes. Wash and examine. The acid-fast organisms and spores are red in a green background. If the smear is thick and appears too dense, dry for three minutes and hold over the mouth of a bottle of ammonia until decolorized to suit. Upon exposure to the air the green returns. This can be prevented by keeping the smear alkaline, by the addition of sodium bicarbonate.

A second method is described for use with sputum in which acid-fast organisms are scarce. It permits the examination of thick smears and therefore increases the chances of finding tubercle organisms when few in number. Stain with carbol fuchsin as in the Ziehl-Neelson method. Decolorize with 30% phenol-disulfonic acid in water for a few seconds or until decolorized. Wash and examine at once. If color returns upon washing decolorize again. The tubercle organisms appear red in a colorless background.     

Cognitive ability is heritable and predicts the success of an alternative mating tactic

The ability to attract mates, acquire resources for reproduction, and successfully outcompete rivals for fertilizations may make demands on cognitive traits—the mechanisms by which an animal acquires, processes, stores and acts upon information from its environment. Consequently, cognitive traits potentially undergo sexual selection in some mating systems. We investigated the role of cognitive traits on the reproductive performance of male rose bitterling (Rhodeus ocellatus), a freshwater fish with a complex mating system and alternative mating tactics. We quantified the learning accuracy of males and females in a spatial learning task and scored them for learning accuracy. Males were subsequently allowed to play the roles of a guarder and a sneaker in competitive mating trials, with reproductive success measured using paternity analysis. We detected a significant interaction between male mating role and learning accuracy on reproductive success, with the best-performing males in maze trials showing greater reproductive success in a sneaker role than as a guarder. Using a cross-classified breeding design, learning accuracy was demonstrated to be heritable, with significant additive maternal and paternal effects. Our results imply that male cognitive traits may undergo intra-sexual selection.