Cloning, sequence analysis and identification of a nonsense mutation-mediated mRNA decay of porcine GSTM2 gene

The glutathione S-transferase mu 2 gene （GSTM2） encodes a GST functioning in the elimination of electrophilic compounds and the regulation of cell growth. In this study, the sequence of porcine GSTM2 gene that contains the complete sequence encoding a protein of 218 amino acids was cloned. The deduced amino acid sequence shared 76%, 78% and 76% identity with that of human, mouse and rat, respectively, mRNA expression analysis showed that the porcine GSTM2 gene was expressed at a high level in liver and testis, at a medium level in longissimus dorsi muscle, adipose tissue, spleen and lung, at a low level in kidney, and at a very low level in heart and embryo. A nonsense mutation （CGA→TGA） resulted from C27T substitution in the fifth exon to produce a premature translation termination codon was identified, and it was discovered that nonsense-mediated mRNA decay might have an effect on the regulation of porcine GSTM2 gene expression. This polymorphism was analyzed in Large White, Landrace, Meishan and Qingping pig populations using the Taq I-polymerase chain reaction-restriction fragment length polymorphism method. The result showed that allele C had a higher frequency than allele T in each population.     

TNF-α和IL-13对人肺成纤维细胞胶原蛋白生成的影响

以胶原蛋白过量沉积为主要特征的纤维化是临床肺部疾患常见的病理现象。该研究利用RT-PCR技术检测不同剂量TNF-α和IL-13对人肺成纤维细胞IL-13Rα1、IL-13Rα2和Ⅰ型胶原蛋白转录水平的影响;ELISA检测细胞培养上清sIL-13Rα2分泌量;羟脯氨酸法定量分析各组肺成纤维细胞胶原蛋白生成情况。结果发现:在实验剂量条件下,TNF-α和IL-13对人肺成纤维细胞IL-13Rα1的表达无显著影响;两者均能不同程度地上调IL-13Rα2的表达;与对照组相比,TNF-α对胶原蛋白的表达有下调作用,IL-13则无显著影响。     

The branched actin nucleator Arp2/3 promotes nuclear migrations and cell polarity in the C. elegans zygote

Regulated movements of the nucleus are essential during zygote formation, cell migrations, and differentiation of neurons. The nucleus moves along microtubules (MTs) and is repositioned on F-actin at the cellular cortex. Two families of nuclear envelope proteins, SUN and KASH, link the nucleus to the actin and MT cytoskeletons during nuclear movements. However, the role of actin nucleators in nuclear migration and positioning is poorly understood. We show that the branched actin nucleator, Arp2/3, affects nuclear movements throughout embryonic and larval development in C. elegans, including nuclear migrations in epidermal cells and neuronal precursors. In one-cell embryos the migration of the male pronucleus to meet the female pronucleus after fertilization requires Arp2/3. Loss of Arp2/3 or its activators changes the dynamics of non-muscle myosin, NMY-2, and alters the cortical accumulation of posterior PAR proteins. Reduced establishment of the posterior microtubule cytoskeleton in Arp2/3 mutants correlates with reduced male pronuclear migration. The UNC-84/SUN nuclear envelope protein that links the nucleus to the MT and actin cytoskeleton is known to regulate later nuclear migrations. We show here it also positions the male pronucleus. These studies demonstrate a global role for Arp2/3 in nuclear migrations. In the C. elegans one-cell embryo Arp2/3 promotes the establishment of anterior/posterior polarity and promotes MT growth that propels the anterior migration of the male pronucleus. In contrast with previous studies emphasizing pulling forces on the male pronucleus, we propose that robust MT nucleation pushes the male pronucleus anteriorly to join the female pronucleus.     

Dissection of the HIV Vif Interaction with Human E3 Ubiquitin Ligase

The human immunodeficiency virus type 1 (HIV-1) protein Vif recruits the host E3 ubiquitin ligase, composed of cullin 5 (Cul5), Rbx2, Elongin B, and Elongin C (EloBC), to polyubiquitinate the antiviral protein APOBEC3G. Multiple regions in the C-terminal half of Vif interact with the E3 ligase. We have purified individual regions of Vif and investigated their thermodynamic contributions to the ligase assembly in vitro using isothermal titration calorimetry and fluorescence anisotropy. Our results quantify the high-affinity interactions between the Vif BC box and EloBC and between the Vif zinc finger and Cul5, as well as the modest interaction between the Vif cullin box and Cul5. Our purified Vif constructs also provide direct biochemical evidence that the Vif cullin box, containing the PPLP region, leads to the dimerization of Vif-EloBC complexes but not Cul5-Vif-EloBC complexes.HIV Vif antagonizes the human antiviral protein APOBEC3G by hijacking the human Elongin B/C (EloBC)-cullin-SOCS box (ECS)-type E3 ubiquitin ligase, resulting in the polyubiquitination of APOBEC3G and subsequently its proteasomal degradation. Canonical ECS-type ubiquitin ligases consist of a cullin scaffold protein to which adaptor and substrate receptor proteins bind at the N terminus. HIV Vif serves as a substrate receptor protein—its N terminus recruits APOBEC3G, while multiple C-terminal regions assemble with the E3 ligase (9, 13, 24). The E3 ligase interacting regions include a zinc finger (residues 100 to 140), a BC box (residues 141 to 154), and a cullin box (residues 155 to 176) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.(A) A sequence schematic of Vif showing the regions that interact with A3G, A3F, EloBC, and Cul5. (B) An illustration of the assembly of the Vif-E3 ubiquitin ligase. (C) A homology model of Vif-Cul5-EloBC, where the Vif BC box-EloBC is actual structural data (PDB ID 3DCG).Vif binds the cullin adaptor proteins EloB and EloC through the BC-box region (24). The BC box is a loop-helix motif with the consensus sequence (T/S)LxxxCxxx(V/L/I) (7), and it also exists in cellular proteins that interact with EloBC. While Vif does not fit this consensus perfectly, it still binds EloBC with high affinity, and this interaction is lost upon mutation or deletion of consensus BC-box residues (10, 24, 25). This interaction has been described previously for the cellular proteins VHL (15), SOCS2 (3), SOCS3 (1), SOCS4 (4), and recently HIV Vif (14).Both the Vif zinc finger and cullin box interact with the E3 ligase scaffold protein cullin 5 (Cul5) (11, 12, 20, 21). It has been established that the zinc finger is required for Vif to bind Cul5. Mutation of critical histidine or cysteine residues in this region or the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) abolishes the Vif-Cul5 interaction (8, 11, 25). The sequence of the Vif cullin box is not as conserved as those of cellular SOCS-box proteins, which have a defined structure and determine the specificities of their respective cullins (6). The role of the Vif cullin box is not clear, but it has been suggested to promote dimerization of Vif, involving the conserved PPLP region (22, 23), and has recently been implicated in APOBEC3G binding (5, 17). While its importance in Cul5 binding has been demonstrated in coimmunoprecipitation experiments (14), experimental data also exist showing that the Vif zinc finger alone still immunoprecipitates Cul5 (11, 21).To dissect the assembly of the Vif-E3 ubiquitin ligase, we quantified the binding interactions between various C-terminal Vif constructs, EloBC, and Cul5 by isothermal titration calorimetry (ITC) and fluorescence polarization (FP). We additionally probed the effects of the cullin box on Vif dimerization.     

Genetic evidence for functional dependency of p18Ink4c on Cdk4

The INK4 family of cyclin-dependent kinase (CDK) inhibitors negatively regulates cyclin D-dependent CDK4 and CDK6 and induces the growth-suppressive function of Rb family proteins. Mutations in the Cdk4 gene conferring INK4 resistance are associated with familial and sporadic melanoma in humans and result in a wide spectrum of tumors in mice, suggesting that INK4 is a major regulator of CDK4. Mice lacking the Cdk4 gene exhibit various defects in many organs associated with hypocellularity, whereas loss of the p18(Ink4c) gene results in widespread hyperplasia and organomegaly. To genetically test the notion that the function of INK4 is dependent on CDK4, we generated p18; Cdk4 double-mutant mice and examined the organs and tissues which developed abnormalities when either gene is deleted. We show here that, in all organs we have examined, including pituitary, testis, pancreas, kidney, and adrenal gland, hyperproliferative phenotypes associated with p18 loss were canceled. The double-mutant mice exhibited phenotypes very close to or indistinguishable from that of Cdk4 single-mutant mice. Mice lacking p27(Kip1) develop widespread hyperplasia and organomegaly similar to those developed by p18-deficient mice. The p27; Cdk4 double-mutant mice, however, displayed phenotypes intermediate between those of p27 and Cdk4 single-mutant mice. These results provide genetic evidence that in mice p18(Ink4c) and p27(Kip1) mediate the transduction of different cell growth and proliferation signals to CDK4 and that p18(Ink4c) is functionally dependent on CDK4.