Phylogenetic screening of the human genome: identification of differentially hybridizing repetitive sequence families

The phi-screen, a method of phylogenetic screening, can be employed to detect repetitive sequence families that differentially hybridize between closely related species. Such differences may involve sequence divergence or variations in copy number, including total presence versus absence of a family of repeated DNA. We present the results of a phi-screen comparing the human genome to that of the prosimian, Galago crassicaudatus. Three human repetitive families that are divergent or not present in galago have been detected. One of these families is described in detail; it is similar among the anthropoids but is present in a lower copy number and/or divergent form in prosimians. The family is clearly related to the transposon-like human element (THE) described by Paulson et al. (1985). THEs have long terminal repeats reminiscent of retroviruses but are unique in that they have no sequence similarity to known mammalian retroviruses. The sequence of a solo long terminal repeat, found unassociated with THE internal sequence, is presented. This family member, THE p2, is bordered by a 5-bp target-site repeat and is interrupted by the insertion of an Alu element. A solo THE element sequenced by Wiginton et al. (1986) contains an insertion of Alu at precisely the same position as does THE p2.      

DNA divergence in and around the alcohol dehydrogenase locus in five closely related species of Hawaiian Drosophila

The alcohol dehydrogenase (Adh) region from five planitibia subgroup species of Hawaiian picture-wing Drosophila has been cloned. A total of 15 kb of DNA in and around the Adh gene has been compared among the five species. Genetic distances were calculated to determine evolutionary relationships. These distances agree with previous distances determined by protein polymorphism and DNA hybridization techniques and can be interpreted in terms of specific island colonization and speciation (founder) events over the past 5 Myr. Examination of the restriction maps of the cloned Adh region from the five species shows many instances of small deletions, insertion of a transposable element in D. heteroneura, and the existence of a highly variable region on the 3' side of the Adh gene. Clustering relationships and rates of DNA change are calculated and compared with the relationship found for other species of Drosophila.      

Endoproteolytic cleavage of TUG protein regulates GLUT4 glucose transporter translocation

To promote glucose uptake into fat and muscle cells, insulin causes the translocation of GLUT4 glucose transporters from intracellular vesicles to the cell surface. Previous data support a model in which TUG traps GLUT4-containing vesicles and tethers them intracellularly in unstimulated cells and in which insulin mobilizes this pool of vesicles by releasing this tether. Here we show that TUG undergoes site-specific endoproteolytic cleavage, which separates a GLUT4-binding, N-terminal region of TUG from a C-terminal region previously suggested to bind an intracellular anchor. Cleavage is accelerated by insulin stimulation in 3T3-L1 adipocytes and is highly dependent upon adipocyte differentiation. The N-terminal TUG cleavage product has properties of a novel 18-kDa ubiquitin-like modifier, which we call TUGUL. The C-terminal product is observed at the expected size of 42 kDa and also as a 54-kDa form that is released from membranes into the cytosol. In transfected cells, intact TUG links GLUT4 to PIST and also binds Golgin-160 through its C-terminal region. PIST is an effector of TC10α, a GTPase previously shown to transmit an insulin signal required for GLUT4 translocation, and we show using RNAi that TC10α is required for TUG proteolytic processing. Finally, we demonstrate that a cleavage-resistant form of TUG does not support highly insulin-responsive GLUT4 translocation or glucose uptake in 3T3-L1 adipocytes. Together with previous results, these data support a model whereby insulin stimulates TUG cleavage to liberate GLUT4 storage vesicles from the Golgi matrix, which promotes GLUT4 translocation to the cell surface and enhances glucose uptake.     

The ubiquitin regulatory X (UBX) domain-containing protein TUG regulates the p97 ATPase and resides at the endoplasmic reticulum-golgi intermediate compartment

p97/VCP is a hexameric ATPase that is coupled to diverse cellular processes, such as membrane fusion and proteolysis. How p97 activity is regulated is not fully understood. Here we studied the potential role of TUG, a widely expressed protein containing a UBX domain, to control mammalian p97. In HEK293 cells, the vast majority of TUG was bound to p97. Surprisingly, the TUG UBX domain was neither necessary nor sufficient for this interaction. Rather, an extended sequence, comprising three regions of TUG, bound to the p97 N-terminal domain. The TUG C terminus resembled the Arabidopsis protein PUX1. Similar to the previously described action of PUX1 on AtCDC48, TUG caused the conversion of p97 hexamers into monomers. Hexamer disassembly was stoichiometric rather than catalytic and was not greatly affected by the p97 ATP-binding state or by TUG N-terminal regions in vitro. In HeLa cells, TUG localized to the endoplasmic reticulum-to-Golgi intermediate compartment and endoplasmic reticulum exit sites. Although siRNA-mediated TUG depletion had no marked effect on total ubiquitylated proteins or p97 localization, TUG overexpression caused an accumulation of ubiquitylated substrates and targeted both TUG and p97 to the nucleus. A physiologic role of TUG was revealed by siRNA-mediated depletion, which showed that TUG is required for efficient reassembly of the Golgi complex after brefeldin A removal. Together, these data support a model in which TUG controls p97 oligomeric status at a particular location in the early secretory pathway and in which this process regulates membrane trafficking in various cell types.     

Canine models of Duchenne muscular dystrophy and their use in therapeutic strategies

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder in which the loss of dystrophin causes progressive degeneration of skeletal and cardiac muscle. Potential therapies that carry substantial risk, such as gene- and cell-based approaches, must first be tested in animal models, notably the mdx mouse and several dystrophin-deficient breeds of dogs, including golden retriever muscular dystrophy (GRMD). Affected dogs have a more severe phenotype, in keeping with that of DMD, so may better predict disease pathogenesis and treatment efficacy. Various phenotypic tests have been developed to characterize disease progression in the GRMD model. These biomarkers range from measures of strength and joint contractures to magnetic resonance imaging. Some of these tests are routinely used in clinical veterinary practice, while others require specialized equipment and expertise. By comparing serial measurements from treated and untreated groups, one can document improvement or delayed progression of disease. Potential treatments for DMD may be broadly categorized as molecular, cellular, or pharmacologic. The GRMD model has increasingly been used to assess efficacy of a range of these therapies. A number of these studies have provided largely general proof-of-concept for the treatment under study. Others have demonstrated efficacy using the biomarkers discussed. Importantly, just as symptoms in DMD vary among patients, GRMD dogs display remarkable phenotypic variation. Though confounding statistical analysis in preclinical trials, this variation offers insight regarding the role that modifier genes play in disease pathogenesis. By correlating functional and mRNA profiling results, gene targets for therapy development can be identified.     

Solution structure and backbone dynamics of an N-terminal ubiquitin-like domain in the GLUT4-regulating protein, TUG

The GLUT4-regulating protein, TUG, functions to retain GLUT4-containing membrane vesicles intracellularly and, in response to insulin stimulation, releases these vesicles to the cellular exocytic machinery for translocation to the plasma membrane. As part of our on going effort to describe the molecular basis for TUG function, we have determined the tertiary structure and characterized the backbone dynamics for an N-terminal ubiquitin-like domain (TUG-UBL1) using NMR spectroscopy. A well-ordered conformation is observed for residues 10-83 of full-length TUG, and confirms a beta-grasp or ubiquitin-like topology. Although not required for in vitro association with GLUT4, the functional role of the TUG-UBL1 domain has not yet been described. We undertook a limited literature review of similar N-terminal UBL domains and noted that a majority participate in protein-protein interactions, generally functioning as adaptor modules to physically associate the over all activity of the protein with a specific cellular process, such as the ubiquitin-proteasome pathway. In consistent fashion, TUG-UBL1 is not expected to participate in a covalent protein modification reaction as it lacks the characteristic C-terminal diglycine ("GG") motif required for conjugation to an acceptor lysine, and also lacks the three most common acceptor lysine residues involved in polyubiquitination. Additionally, analysis of the TUG-UBL1 molecular surface reveals a lack of conservation of the "Ile-44 hydrophobic face" typically involved in ubiquitin recognition. Instead, we speculate on the possible significance of a concentrated area of negative electrostatic potential with increased backbone mobility, both of which are features suggestive of a potential protein-protein interaction site.     

Germ band retraction as a landmark in glucose metabolism during <Emphasis Type="Italic">Aedes aegypti</Emphasis> embryogenesis

Background  

The mosquito A. aegypti is vector of dengue and other viruses. New methods of vector control are needed and can be achieved by a better understanding of the life cycle of this insect. Embryogenesis is a part of A. aegypty life cycle that is poorly understood. In insects in general and in mosquitoes in particular energetic metabolism is well studied during oogenesis, when the oocyte exhibits fast growth, accumulating carbohydrates, lipids and proteins that will meet the regulatory and metabolic needs of the developing embryo. On the other hand, events related with energetic metabolism during A. aegypti embryogenesis are unknown.     

大鼠心肌缺血再灌注损伤模型的实验研究

目的 制备一种新型的心肌急性缺血再灌注损伤模型,以探讨一种更符合临床实际需求的实验方法.方法 将20只雌性SD(Sprague-Dawley)大鼠随机分成2组(对照组、实验组),采用结扎主动脉根部引起心肌缺血5min再灌注30 min建立心肌急性缺血再灌注模型;通过应用透射电镜观察心肌细胞超微结构的改变,同时检测心肌组织匀浆丙二醛(Maleic Dialdehyde,MDA)含量、超氧化物歧化酶(Superoxide Dismutase,SOD)活力.结果 透射电镜下超微结构显示实验组较对照组明显加重了心肌组织结构和线粒体的损害;实验组心肌组织MDA明显高于对照组(P<0.01),而SOD明显低于对照组(P<0.01).结论 本实验成功建立了方法简便、易于操作、取材范围广泛的心肌缺血再灌注损伤模型,为心肌缺血再灌注损伤研究提供了一种更为可行的模型.