SIAH1介导了TRB3的泛素化和降解

目的 利用酵母回转实验和免疫共沉淀实验验证SIAH1和TRB3之间的相互作用并探讨其功能相关性..方法 将全长形式的TRB3基因和SIAH1基因分别克隆入酵母表达载体pDBLeu和pPC86中,共转化至MaV203酵母感受态细胞,验证其相互作用,然后分别构建至真核表达载体pCMV-Myc和pFLAG-CMV-2中,采用...     

Distinct roles of c-Abl and Atm in oxidative stress response are mediated by protein kinase C delta

c-Abl and Atm have been implicated in cell responses to DNA damage and oxidative stress. However, the molecular mechanisms by which they regulate oxidative stress response remain unclear. In this report, we show that deficiency of c-Abl and deficiency of ATM differentially altered cell responses to oxidative stress by induction of antioxidant protein peroxiredoxin I (Prx I) via Nrf2 and cell death, both of which required protein kinase C (PKC) delta activation and were mediated by reactive oxygen species. c-abl-/- osteoblasts displayed enhanced Prx I induction, elevated Nrf2 levels, and hypersusceptibility to arsenate, which were reinstated by reconstitution of c-Abl; Atm-/- osteoblasts showed the opposite. These phenotypes correlated with increased PKC delta expression in c-abl-/- osteoblasts and decreased PKC delta expression in Atm-/- cells, respectively. The enhanced responses of c-abl-/- osteoblasts could be mimicked by overexpression of PKC delta in normal cells and impeded by inhibition of PKC delta, and diminished responses of Atm-/- cells could be rescued by PKC delta overexpression, indicating that PKC delta mediated the effects of c-Abl and ATM in oxidative stress response. Hence, our results unveiled a previously unrecognized mechanism by which c-Abl and Atm participate in oxidative stress response.     

Comparison of pathways controlling toxicity in the eye and brain in Drosophila models of human neurodegenerative diseases

Most human neurodegenerative diseases have a number of common features, including adult onset, progressive degeneration of selected neuronal populations and formation of abnormal protein aggregates. Although these shared characteristics raise the possibility of conserved pathogenic mechanisms, the diverse clinical and pathological features of each disorder indicate significant differences. As a number of human neurodegenerative diseases have now been modeled in Drosophila, and genetic modifiers identified, we have been able to perform a genetic comparison of pathways controlling toxicity in these models. By directly comparing modifiers isolated in the models of polyglutamine diseases and in a Drosophila model of tauopathy, we find a final common pathway of cell death involving apoptosis. Among the polyglutamine diseases, protein folding and histone acetylation are common key mediators. In addition, two novel modifiers suggest shared pathways of toxicity among all the disorders. Cell-type specificity is a salient feature of all neurodegenerative diseases; however, most work to date in the Drosophila models have been performed in the retina. Therefore, we determined whether similar pathways of toxicity operate in neurons of the Drosophila brain. Many, but not all, retinal modifiers also modify toxicity in postmitotic neurons in the brain. Analysis of polyglutamine toxicity in the adult brain facilitated the identification of nicotinamide (vitamin B3), a vitamin with histone deacetylase inhibiting activity, as a potent suppressor of polyglutamine toxicity. These findings outline common pathways of neurotoxicity, demonstrate disease- and cell-type specific pathways and identify a common vitamin as a potential therapy in polyglutamine disorders.     

Construction and expression of hTNF-alpha fusion protein mediated by MMP1

This paper is aimed to present a research on fusion protein of human tumor necrosis factor-alpha (hTNF-alpha), matrix metalloproteinase 1 (MMP1), and foldon sequence using the methord of gene engineering. We transformed the recombinant plasmid, which contains the DNA sequences of hTNF-alpha, MMP1, and foldon sequence, into Rosetta2, and successfully induced the fusion protein to express under given conditions by isopropyl beta-D-1-Thiogalactopyranoside (IPTG). Then we purified the expression product through a glutathione S-transferase (GST) resin and collected the interested protein. This research may lay the groundwork for scientific research and clinical application.     

Adenoviral E1A protein nuclear import is preferentially mediated by importin alpha3 in vitro.

Nuclear import of some viral proteins depends on importin alpha proteins. However, no preferences of distinct alpha-importins for any viral protein import have been demonstrated. We used in vitro import assays and observed that all ubiquitously expressed human importin alpha isoforms mediate nuclear translocation of adenoviral E1A. Competition with nucleoplasmin suggests that importin alpha3 is the most efficient import mediator of E1A.     

Involvement of the MP1 scaffold protein in ERK signaling regulation during Drosophila wing development

Mitogen-activated protein kinase (MAPK) cascades are evolutionary conserved transduction pathways involved in many cellular processes. Kinase modules are associated with scaffold proteins that regulate signaling by providing critical spatial and temporal specificities. Some of these scaffold proteins have been shown to be conserved, both in sequence and function. In mouse, the scaffold MP1 (MEK Partner 1) forms a signaling complex with MEK1 and ERK1. In this work, we focus on Drosophila MP1 (dMP1). We show that dMP1 is expressed ubiquitously during embryonic and larval development. By in vitro and in vivo experiments, we show that dMP1 is located in the cytoplasm and the nuclei, and that it interacts with MEK and ERK. Genetic studies with transgenic Drosophila lines allowing either dMP1 over-expression or dMP1 down-regulation by RNA interference highlight dMP1 function in the control of cell differentiation during development of the Drosophila wing.     

Hypertonicity-induced transmitter release at Drosophila neuromuscular junctions is partly mediated by integrins and cAMP/protein kinase A

The frequency of quantal transmitter release increases upon application of hypertonic solutions. This effect bypasses the Ca²⁺ triggering step, but requires the presence of key molecules involved in vesicle fusion, and hence could be a useful tool for dissecting the molecular process of vesicle fusion. We have examined the hypertonicity response at neuromuscular junctions of Drosophila embryos in Ca²⁺-free saline. Relative to wild-type, the response induced by puff application of hypertonic solution was enhanced in a mutant, dunce , in which the cAMP level is elevated, or in wild-type embryos treated with forskolin, an activator of adenylyl cyclase, while protein kinase A (PKA) inhibitors decreased it. The response was also smaller in a mutant, DC0 , which lacks the major subunit of PKA. Thus the cAMP/PKA cascade is involved in the hypertonicity response. Peptides containing the sequence Arg-Gly-Asp (RGD), which inhibit binding of integrins to natural ligands, reduced the response, whereas a peptide containing the non-binding sequence Arg-Gly-Glu (RGE) did not. A reduced response persisted in a mutant, myospheroid , which expresses no integrins, and the response in DC0 was unaffected by RGD peptides. These data indicate that there are at lease two components in the hypertonicity response: one that is integrin mediated and involves the cAMP/PKA cascade, and another that is not integrin mediated and does not involve the cAMP/PKA cascade.     

Synergistic regulation of vertebrate muscle development by Dach2, Eya2, and Six1, homologs of genes required for Drosophila eye formation

We have identified a novel vertebrate homolog of the Drosophila gene dachshund, Dachshund2 (Dach2). Dach2 is expressed in the developing somite prior to any myogenic genes with an expression profile similar to Pax3, a gene previously shown to induce muscle differentiation. Pax3 and Dach2 participate in a positive regulatory feedback loop, analogous to a feedback loop that exists in Drosophila between the Pax gene eyeless (a Pax6 homolog) and the Drosophila dachshund gene. Although Dach2 alone is unable to induce myogenesis, Dach2 can synergize with Eya2 (a vertebrate homolog of the Drosophila gene eyes absent) to regulate myogenic differentiation. Moreover, Eya2 can also synergize with Six1 (a vertebrate homolog of the Drosophila gene sine oculis) to regulate myogenesis. This synergistic regulation of muscle development by Dach2 with Eya2 and Eya2 with Six1 parallels the synergistic regulation of Drosophila eye formation by dachshund with eyes absent and eyes absent with sine oculis. This synergistic regulation is explained by direct physical interactions between Dach2 and Eya2, and Eya2 and Six1 proteins, analogous to interactions observed between the Drosophila proteins. This study reveals a new layer of regulation in the process of myogenic specification in the somites. Moreover, we show that the Pax, Dach, Eya, and Six genetic network has been conserved across species. However, this genetic network has been used in a novel developmental context, myogenesis rather than eye development, and has been expanded to include gene family members that are not directly homologous, for example Pax3 instead of Pax6.     

Defense mechanisms in clients and non-clients as mediated by gender and sex-role

This study examined defense mechanism utilization patterns of psychotherapy clients vs. individuals without mental health history, by administering the Defense Mechanism Inventory to 104 subjects. Concurrently, the mediating influence of gender and sex-role on defense mechanism utilization was assessed. Results indicated that gender and sex-role were significant in mediating the use of Turning against Self defenses and Turning against Other defenses, respectively. More importantly, however, psychotherapy clients differed from non-clients in their increased use of Projection and their decreased use of Principalization. Use of Projection was mediated further by a gender by sex-role interaction, which complicates the interpretation of the presence of Projection in an individual for the clinician.     

Distinct compartmentalization of dentin matrix protein 1 fragments in mineralized tissues and cells

Dentin matrix protein 1 (DMP1) has been shown to be critical for the formation of dentin and bone. However, the precise pathway by which DMP1 participates in dentinogenesis and osteogenesis remains to be clarified. DMP1 is present in the extracellular matrix of dentin and bone as processed NH(2)- and COOH-terminal fragments. The NH(2)-terminal fragment occurs as a proteoglycan, whereas the COOH-terminal fragment is highly phosphorylated. The differences in biochemical properties suggest that these fragments may have different tissue and cell distribution in association with distinct functions. In this study, we analyzed the distribution of the NH(2)- and COOH-terminal fragments of DMP1 in tooth, bone, osteocytes as well as MC3T3-E1 and HEK-293 cells. Immunohistochemical analyses were performed using antibodies specific to the NH(2)- or COOH-terminal region of DMP1. Clear differences in the distribution of these fragments were observed. In the teeth and bone, the NH(2)-terminal fragment was primarily located in the nonmineralized predentin and cartilage of the growth plate, while the COOH-terminal fragment accumulated in the mineralized zones. In osteocytes, the NH(2)-terminal fragment appeared more abundant along cell membrane and processes of osteocytes, while the COOH-terminal fragment was often found in the nuclei. This pattern of distribution in cellular compartments was further confirmed by analyses on MC3T3-E1 and HEK-293 cells transfected with a construct containing DMP1 cDNA. In these cell lines, the COOH-terminal fragment accumulated in cell nuclei, while the NH(2)-terminal fragment was in the cytosol. The different distribution of DMP1 fragments indicates that these DMP1 variants must perform distinct functions.     

活性维生素D3对大鼠肺纤维化中蛋白激酶D1介导的抗氧化通路的影响

目的:探讨肺纤维化发生发展中蛋白激酶D1(PKD1)介导的线粒体抗氧化通路的作用以及活性维生素D3在纤维化过程中对该通路的影响。方法:雄性SD大鼠随机分成对照组、模型组和治疗组。应用实时荧光定量PCR和免疫组织化学分别从mRNA和蛋白质水平检测大鼠肺组织中PKD1、核转录因子(NF-κB)和锰超氧化物歧化酶(MnSOD)的表达。结果:在第14天,治疗组和模型组中PKD1、NF-κB和MnSOD的表达量都显著低于对照组,而治疗组中3种因子的表达量又明显高于模型组;在第21天,3种因子在模型组和治疗组中的表达量明显高于对照组。在第28天,3种因子在模型组和治疗组中的表达量与对照组相比两两之间均没有差异。结论:PKD1-MnSOD线粒体抗氧化通路在博莱霉素引起的大鼠肺纤维化早期发挥重要作用,活性维生素D3能够上调这一抗氧化通路,具有一定的抗氧化作用,对大鼠肺纤维化的发生发展具有一定的抑制作用。     

A macaque model for studying mechanisms controlling oocyte development and maturation in human and non-human primates.

A model to study mechanisms controlling nuclear and cytoplasmic maturation of primate oocytes is being developed in our laboratory. The high incidence of pregnancy failure in women following in-vitro fertilization (IVF) may be partly attributed to inadequate cytoplasmic maturation of oocytes. Advancement of knowledge of mechanisms controlling primate oocyte maturation would have important implications for treatment of human infertility, and would potentially increase numbers of viable non-human primate embryos for biomedical research. Use of a non-human primate model to study oocyte and embryo biology avoids legal, ethical and experimental limitations encountered in a clinical situation. Using this model, the meiotic and developmental capacity of oocytes from three sources have been compared: (i) in-vivo matured oocytes from monkeys stimulated with follicle-stimulating hormone (FSH) and human chorionic gonadotrophin, (ii) in-vitro matured oocytes from monkeys primed with FSH, and (iii) in-vitro matured oocytes from non-stimulated monkeys. This work demonstrates that oocyte developmental competence is likely acquired both during follicle development, before meiotic resumption, and during meiotic progression, concurrent with nuclear maturation. Potential causes of developmental failure of in-vitro matured oocytes, implications for human infertility, and future strategies to study the regulation of primate oocyte maturation are discussed.