Semaphorin 3E/collapsin-5 inhibits growing retinal axons

During development, the formation of neural networks is reflected by the oriented extension of neurites. Using retinal ganglion cells (RGCs) as a model, we identified the yet uncharacterized chick semaphorin Sema3E/collapsin-5 as a repulsive cue for outgrowing axons. Sema3E/collapsin-5 was highly regulated during retinal histogenesis, with peak expression during the period of intraretinal axon growth. Polymerase chain reaction analysis demonstrated Sema3E/collapsin-5 mRNA in retina layers, from which RGC axons are excluded. Neither isolated RGCs nor purified retinal Müller glia cells synthesized Sema3E/collapsin-5. Sema3E/collapsin-5 receptor sites were visualized by alkaline phosphatase fusion proteins in the axon-rich optic fiber layer. Time-lapse video recording of chick in vitro cultures revealed a growth cone collapsing activity of recombinant Sema3E/collapsin-5. This effect was specific for RGCs, since dorsal root ganglia (DRG) neurons of the peripheral nervous system were not affected. Comparison with Sema3A/collapsin-1 displayed a reciprocal specificity, because Sema3A/collapsin-1 hampered exclusively DRG but not RGC growth cones. The collapsing effect was mediated by low cGMP levels, but not cAMP, as revealed by a set of agonists. In summary, the data suggest a possible role of chick Sema3E/collapsin-5 in restricting growth of retinal ganglion cell axons to the optic fiber layer.     

E5 and E6/E7 of high-risk HPVs cooperate to enhance cancer progression through EMT initiation

It is estimated that 10–20% of human carcinogenesis is linked to virus infection including papillomaviruses (HPVs). Moreover, since metastatic cancer disease is a major cause of morbidity and mortality in cancer patients, the role of onco-viruses in cancer progression to a metastatic form is of particular interest. Recent studies reported that E5 and E6/E7 onco-proteins of high-risk HPVs could enhance cancer progression via the initiation of the epithelial–mesenchymal transition (EMT) event. Herein, we discuss the association between E5 as well as E6/E7 of high-risk HPV and cancer progression.     

Thermotropic phase behaviour of the pseudobinary mixtures of DPPC/C12E5 and DMPC/C12E5 determined by differential scanning calorimetry and ultrasonic velocimetry

The present paper reports on the phase behaviour of the pseudobinary aqueous mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/pentaethylene glycol monododecyl ether (C12E5) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine monohydrate (DMPC)/C12E5. Both systems exhibit a variety of mesophases, such as lamellar gel, liquid crystalline and micellar phases. The phase diagrams show peritectic and eutectic behaviours. The existence of a compound complex is established. From the phase diagrams, the temperature dependence of the solubilisation parameters is obtained. The phase diagrams, especially with respect to the solubilisation process were qualitatively explained assuming that the packing of the constituents plays a dominating role. Finally, differential scanning calorimetry and ultrasonic velocimetry are compared concerning their potentials to determine characteristics of phase transitions in pseudobinary phospholipid/surfactant mixtures.     

人乳头瘤病毒E5蛋白

E5是人乳头瘤病毒(human papillomavirus, HPV)表达的3个致瘤蛋白之一,它的致瘤能力较弱,在一些HPV相关的恶性肿痛经常缺乏E5表达.E5主要通过与表皮生长因子受体(epidermal growth factor receptor,EGFR)和V型H+-腺三磷酶(ATPase)等的相互作用,在肿瘤形成早期对细胞增殖、转化、免疫逃避等细胞行为发挥重要影响,促进宫颈上皮从HPV感染到恶性转变.     

The HPV16 E6/E7 oncogene sensitizes human ovarian surface epithelial cells to low-dose but not high-dose 5-FU and 5-FUdR

To evaluate the effect of HPV16 E6/E7 on drug sensitivity, primary human OSE cells were infected with HPV16 E6/E7 expressing retrovirus and then exposed to chemotherapeutic agents. Apoptosis induced by mitomycin C was dose-dependent in both primary OSE and E6E7/OSE cells. E6E7/OSE cells were more sensitive to mitomycin C than parental OSE cells. HPV16 E6/E7 also sensitized OSE cells to 5-FU and its derivative 5-FUdR, but only at low doses. This phenomenon was also observed in cervical cancer cells and was independent of thymidylate synthase, a target of thymine and thymidine analogues. We conclude that HPV16 E6/E7 specifically modulates the activity of 5-FU and 5-FUdR, and confers OSE cells hypersensitivity to low-dose but not high-dose 5-FU and 5-FUdR. Molecular analysis indicates that induction of p53 and p21, and suppression of pRB are associated with apoptosis induced by 5-FUdR and may partly explain the hypersensitivity of E6E7/OSE cells to low-dose 5-FUdR.     

Analysis of DNA interband regions 3A5/A6, 3C5-6/c7 and 60E8-9/E10 of Drosophila melanogaster polytene chromosomes]

Using electron microscopic (EM) data on the formation of a novel band from the P-element material after its insertion in the interband and the procedure of P-target rescue, DNA interband regions 3A5/A6, and 60E8-9/E10 of Drosophila melanogaster polytene chromosomes were cloned and sequenced. EM analysis of the 3C region have shown that the formation of the full-size 3C5-6/C7 interband requires a 880-bp DNA sequences removed by deletion Df(1)faswb. A comparison of DNA sequences of six bands, two of which were obtained in the present work and four were described earlier, demonstrated the uniqueness of each of them in the Drosophila genome and heterogeneity of their molecular organization. Interband 60E8-9/E10 contains gene rpl19 transcribed throughout the development, in particular in salivary glands. In the other interbands examined 5' and 3' nontranslated gene regions are located. These results suggest that Drosophila interbands may contain both housekeeping genes and regulatory sequences of currently inactive genes from adjacent bands.     

Identification of phosphorylation sites on the E3 ubiquitin ligase UBR5/EDD

UBR5 (ubiquitin protein ligase E3 component n-recognin 5)/EDD (E3 ligase identified by differential display) is an E3 ubiquitin ligase that is a potential biomarker for poor prognosis for recurrent, platinum-resistant ovarian cancer. UBR5 has a role in the DNA damage response and many such proteins are regulated by phosphorylation. UBR5 is a 309 kDa nuclear phosphoprotein that we previously identified as a substrate of the MAP kinase ERK2. With its 477 potential phosphorylation sites, little is known about UBR5 phosphorylation and how it may regulate protein function. Currently, thirty-four sites of phosphorylation on UBR5 have been reported in the literature, mostly identified by large scale proteomics studies of tissues or of cells after various treatments; however, no studies have specifically targeted the identification of UBR5 phosphorylation sites. In this study, we used Liquid Chromatography-Mass Spectrometry (LC-MS/MS) to obtain a total sequence coverage of 64.3% from combining tryptic and GluC digests on UBR5 isolated from transfected COS-1 cells. We identified 24 sites of phosphorylation, 18 of which are novel sites. This data enhances our knowledge of UBR5 phosphorylation and provides a framework for the study of how phosphorylation affects UBR5 function.     

19F NMR relaxation studies on 5-fluorotryptophan- and tetradeutero-5-fluorotryptophan-labeled E. coli glucose/galactose receptor

Summary ¹⁹F NMR relaxation studies have been carried out on a fluorotryptophan-labeled E. coli periplasmic glucose/galactose receptor (GGR). The protein was derived from E. coli grown on a medium containing a 50:50 mixture of 5-fluorotryptophan and [2,4,6,7-²H₄]-5-fluorotryptophan. As a result of the large -isotope shift, the two labels give rise to separate resonances, allowing relaxation contributions of the substituted indole protons to be selectively monitored. Spin-lattice relaxation rates were determined at field strengths of 11.75 T and 8.5 T, and the results were analyzed using a model-free formalism. In order to evaluate the contributions of chemical shift anisotropy to the observed relaxation parameters, solid-state NMR studies were performed on [2,4,6,7-²H₄]-5-fluorotryptophan. Analysis of the observed ¹⁹F powder pattern lineshape resulted in anisotropy and asymmetry parameters of =–93.5 ppm and =0.24. Theoretical analyses of the relaxation parameters are consistent with internal motion of the fluorotryptophan residues characterized by order parameters S² of 1, and by correlation times for internal motion 10^-11 s. Simultaneous least squares fitting of the spin-lattice relaxation and line-width data with ⁱ set at 10 ps yielded a molecular correlation time of 20 ns for the glucose-complexed GGR, and a mean order parameter S²=0.89 for fluorotryptophan residues 183, 127, 133, and 195. By contrast, the calculated order parameter for FTrp²⁸⁴, located on the surface of the protein, was 0.77. Significant differences among the spin-lattice relaxation rates of the five fluorotryptophan residues of glucose-complexed GGR were also observed, with the order of relaxation rates given by: R _inf1F ^sup183 >R _inf1F ^sup127 R _inf1F ^sup133 R _inf1F ^sup195 >R _inf1F ^sup284 . Although such differences may reflect motional variations among these residues, the effects are largely predicted by differences in the distribution of nearby hydrogen nuclei, derived from crystal structure data. In the absence of glucose, spin-lattice relaxation rates for fluorotryptophan residues 183, 127, 133, and 195 were found to decrease by a mean of 13%, while the value for residue 284 exhibits an increase of similar magnitude relative to the liganded molecule. These changes are interpreted in terms of a slower overall correlation time for molecular motion, as well as a change in the internal mobility of FTrp²⁸⁴, located in the hinge region of the receptor.Abbreviations FTrp D,L-5-fluorotryptophan - GGR glucose/galactose receptor protein - R_1F spin-lattice relaxation rate of fluorine - R_1F(H) spinlattice relaxation rate of the fluorine nuclei in normal (nondeuterated) fluorotryptophan residues - R_1F(D) spin-lattice relaxation rate of the fluorine in [2,4,6,7-²H₄]-5-fluorotryptophan To whom correspondence should be addressed.     

Analysis of DNA Interband Regions 3A5/A6, 3C5-6/C7, and 60E8-9/E10 in Polytene Chromosomes of Drosophila melanogaster

Using electron microscopic (EM) data on the formation of a novel band from theP-element material after its insertion in the interband and the procedure of P-target rescue, DNA interband regions 3A5/A6, 3C5-6/C7, and 60E8-9/E10 of Drosophila melanogasterpolytene chromosomes were cloned and sequenced. EM analysis of the 3C region have shown that the formation of the full-size 3C5-6/C7 interband requires a 880-bp DNA sequence removed by deletion Df(1)fa ^swb. A comparison of DNA sequences of six bands, two of which were obtained in the present work and four were described earlier, demonstrated the uniqueness of each of them in the Drosophilagenome and heterogeneity of their molecular organization. Interband 60E8-9/E10 contains gene rpl19transcribed throughout the development, in particular in salivary glands. In the other interbands examined 5" and 3" nontranslated gene regions are located. These results suggest that Drosophilainterbands may contain both housekeeping genes and regulatory sequences of currently inactive genes from adjacent bands.     

NT5E Mutations That Cause Human Disease Are Associated with Intracellular Mistrafficking of NT5E Protein

Ecto-5′-nucleotidase/CD73/NT5E, the product of the NT5E gene, is the dominant enzyme in the generation of adenosine from degradation of AMP in the extracellular environment. Nonsense (c.662C→A, p.S221X designated F1, c.1609dupA, p.V537fsX7 designated F3) and missense (c.1073G→A, p.C358Y designated F2) NT5E gene mutations in three distinct families have been shown recently to cause premature arterial calcification disease in human patients. However, the underlying mechanisms by which loss-of-function NT5E mutations cause human disease are unknown. We hypothesized that human NT5E gene mutations cause mistrafficking of the defective proteins within cells, ultimately blocking NT5E catalytic function. To test this hypothesis, plasmids encoding cDNAs of wild type and mutant human NT5E tagged with the fluorescent probe DsRed were generated and used for transfection and heterologous expression in immortalized monkey COS-7 kidney cells that lack native NT5E protein. Enzyme histochemistry and Malachite green assays were performed to assess the biochemical activities of wild type and mutant fusion NT5E proteins. Subcellular trafficking of fusion NT5E proteins was monitored by confocal microscopy and western blot analysis of fractionated cell constituents. All 3 F1, F2, and F3 mutations result in a protein with significantly reduced trafficking to the plasma membrane and reduced ER retention as compared to wild type protein. Confocal immunofluorescence demonstrates vesicles containing DsRed-tagged NT5E proteins (F1, F2 and F3) in the cell synthetic apparatus. All 3 mutations resulted in absent NT5E enzymatic activity at the cell surface. In conclusion, three familial NT5E mutations (F1, F2, F3) result in novel trafficking defects associated with human disease. These novel genetic causes of human disease suggest that the syndrome of premature arterial calcification due to NT5E mutations may also involve a novel “trafficking-opathy”.