U(L)31 and U(L)34 proteins of herpes simplex virus type 1 form a complex that accumulates at the nuclear rim and is required for envelopment of nucleocapsids

The herpes simplex virus type 1 (HSV-1) U(L)34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the U(L)31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with U(L)34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing U(L)31 protein fused to glutathione-S-transferase (U(L)31-GST) and U(L)34 protein fused to GST (U(L)34-GST) were demonstrated to specifically recognize the U(L)31 and U(L)34 proteins of approximately 34,000 and 30,000 Da, respectively. The U(L)31 and U(L)34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. U(L)34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of U(L)31 protein at the nuclear rim required the presence of U(L)34 protein, inasmuch as cells infected with a U(L)34 null mutant virus contained U(L)31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of U(L)34 protein exclusively at the nuclear rim required the presence of the U(L)31 gene product, inasmuch as U(L)34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a U(L)31 null virus. When transiently expressed in the absence of other viral factors, U(L)31 protein localized diffusely in the nucleoplasm, whereas U(L)34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the U(L)31 and U(L)34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the U(S)3-encoded protein kinase, previously shown to phosphorylate the U(L)34 gene product, U(L)31 and U(L)34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, U(S)3 kinase is required for even distribution of U(L)31 and U(L)34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the U(L)31 and U(L)34 deletion mutants, these data strongly suggest that the U(L)31 and U(L)34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment at the inner nuclear membrane.     

两种过路黄的核型研究

对国产报春花科珍珠菜属植物过路黄和疏节过路黄的核型进行了首次报道。过路黄染色体数目为２ｎ＝２４,核型公式为２ｎ＝２４＝２ｍ＋４ｓｍ＋６ｓｔ＋１２ｔ,核型类型“３Ａ”;疏节过路黄染色体数目为２ｎ＝２２,核型公式为２ｎ＝２２＝４ｍ＋６ｓｍ＋１０ｔ,核型类型“３Ａ”。     

A multifrequency electron spin resonance study of T4 lysozyme dynamics.

Electron spin resonance (ESR) spectroscopy at 250 GHz and 9 GHz is utilized to study the dynamics and local structural ordering of a nitroxide-labeled enzyme, T4 lysozyme (EC 3.2.1.17), in aqueous solution from 10 degrees C to 35 degrees C. Two separate derivatives, labeled at sites 44 and 69, were analyzed. The 250-GHz ESR spectra are well described by a microscopic ordering with macroscopic disordering (MOMD) model, which includes the influence of the tether connecting the probe to the protein. In the faster "time scale" of the 250-GHz ESR experiment, the overall rotational diffusion rate of the enzyme is too slow to significantly affect the spectrum, whereas for the 9-GHz ESR spectra, the overall rotational diffusion must be accounted for in the analysis. This is accomplished by using a slowly relaxing local structure model (SRLS) for the dynamics, wherein the tether motion and the overall motion are both included. In this way a simultaneous fit is successfully obtained for both the 250-GHz and 9-GHz ESR spectra. Two distinct motional/ordering modes of the probe are found for both lysozyme derivatives, indicating that the tether exists in two distinct conformations on the ESR time scale. The probe diffuses more rapidly about an axis perpendicular to its tether, which may result from fluctuations of the peptide backbone at the point of attachment of the spin probe.     

Runners adjust leg stiffness for their first step on a new running surface.

Human runners adjust the stiffness of their stance leg to accommodate surface stiffness during steady state running. This adjustment allows runners to maintain similar center of mass movement (e.g., ground contact time and stride frequency) regardless of surface stiffness. When runners encounter abrupt transitions in the running surface, they must either make a rapid adjustment or allow the change in the surface stiffness to disrupt their running mechanics. Our goal was to determine how quickly runners adjust leg stiffness when they encounter an abrupt but expected change in surface stiffness that they have encountered previously. Six human subjects ran at 3 m s(-1) on a rubber track with two types of rubber surfaces: a compliant "soft" surface (ksurf = 21.3 kN m(-1) and a non-compliant "hard" surface (ksurf = 533 kN m(-1). We found that runners completely adjusted leg stiffness for their first step on the new surface after the transition. For example, runners decreased leg stiffness by 29% between the last step on the soft surface and the first step on the hard surface (from 10.7 kN m(-1) to 7.6 kN m(-1), respectively). As a result, the vertical displacement of the center of mass during stance ( approximately 7 cm) did not change at the transition despite a reduction in surface compression from 6 cm to less than 0.25 cm. By rapidly adjusting leg stiffness, each runner made a smooth transition between surfaces so that the path of the center of mass was unaffected by the change in surface stiffness.     

Exploring the conformational diversity of loops on conserved frameworks

Loops are structurally variable regions, but the secondary structural elements bracing loops are often conserved. Motifs with similar secondary structures exist in the same and different protein families. In this study, we made an all-PDB-based analysis and produced 495 motif families accessible from the Internet. Every motif family contains some variable loops spanning a common framework (a pair of secondary structures). The diversity of loops and the convergence of frameworks were examined. In addition, we also identified 119 loops with conformational changes in different PDB files. These materials can give some directions for functional loop design and flexible docking.     

Urothelial hinge as a highly specialized membrane: detergent-insolubility, urohingin association, and in vitro formation

Urothelial surface is covered by numerous plaques (consisting of asymmetric unit membranes or AUM) that are interconnected by ordinary looking hinge membranes. We describe an improved method for purifying bovine urothelial plaques using 2% sarkosyl and 25 mM NaOH to remove contaminating membrane and peripheral proteins selectively. Highly purified plaques interconnected by intact hinge areas were obtained, indicating that the hinges are as detergent-insoluble as the plaques. These plaque/hinge preparations contained uroplakins, an as yet uncharacterized 18-kDa plaque-associated protein, plus an 85-kDa glycoprotein that is known to be hinge-associated in situ. Examination of the isolated, in vitro-resealed bovine AUM vesicles by quick-freeze deep-etch showed that each AUM particle consists of a 16-nm, luminally exposed "head" anchored to the lipid bilayer via a 9-mm transmembranous "tail", and that an AUM plaque can break forming several smaller plaques separated by newly formed particle-free, hinge-like areas. These data lend support to our recently proposed three-dimensional model of mouse urothelial plaques. In addition, our findings suggest that urothelial plaques are dynamic structures that can rearrange giving rise to new plaques with intervening hinges; that the entire urothelial apical surface (both plaque and hinge areas) is highly specialized; and that these two membrane domains may be equally important in fulfilling some of the urothelial functions.     

鲫鱼视网膜亮度型水平细胞上不同视锥信号的相互作用:实验及模型

本文应用胞内记录和动态模型分析方法,研究了离体鲫鱼视网膜视锥驱动的高度型水平细胞（ＬＨＣ）上不同视锥信号的相互作用。实验表明,绿背景光的作用可以提高ＬＨＣ的红光反应,这种增强作用与绿敏锥的活动程度密切相关,模型分析表明,背景光 的作用谷氨酸介导的前馈性通路和ＧＡＢＡ介导的反馈性通路活动同时得以增强,水平细胞对光反应的增强效应不能为外泊性ＧＡＢＡ所消除。则其程度为前馈性通路和反馈性通路活动增加的相对     

Cultivation of HEK 293 cell line and production of a member of the superfamily of G-protein coupled receptors for drug discovery applications using a highly efficient novel bioreactor

A process of producing a receptor in HEK-293 cells used for the drug discovery program at Pfizer Inc. has been successfully developed with a novel BelloCell bioreactor to replace the conventional 2-D cell culturing devices including Cell Factories and roller bottles. A single BelloCell-500 has produced >1.4 × 10⁹ HEK-293 cells, which are equivalent to those produced by 12 roller bottles, with substantially easier operation, single inoculation, less inoculum, less medium consumption and better space utilization. The receptor expression levels are better than those obtained by the traditional process. 3.7 pmoles of radioligandY mg⁻¹ protein were attained in the bioreactor compared to 2.3 pmoles of radioligandY mg⁻¹ protein in roller bottles. This may be attributed to the three dimensional attachment during cell growth. A 92% cell recovery from the bioreactor has been attained using Acutase or Trypsin treatment followed by four washes. It has been proven to be a viable and efficient device to produce adherent cells and express target components of interest for drug discovery applications.