Recognition of the E1kE1f cholinesterase genotype in a family segregating three rare genes, E1k, E1f and E1a

The first identification of the cholinesterase variant E1kE1f is reported from a family study. The evidence is based on the determination of enzymic activity, dibucaine, fluoride and RO2 numbers. Three individuals appear to have this genotype, and family evidence is not at variance with our conclusions. All three individuals will be sensitive to suxamethonium.     

In Vitro and In Vivo Biology of Recombinant Adenovirus Vectors with E1, E1/E2A,or E1/E4 Deleted

Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.Replication-deficient human adenoviruses (Ad) have been widely investigated as ex vivo and in vivo gene delivery systems for human gene therapy. The ability of these vectors to mediate the efficient expression of candidate therapeutic or vaccine genes in a variety of cell types, including postmitotic cells, is considered an advantage over other gene transfer vectors (3, 28, 49). However, the successful application of currently available E1-defective Ad vectors in human gene therapy has been hampered by the fact that transgene expression is only transient in vivo (2, 15, 16, 33, 36, 46). This short-lived in vivo expression of the transgene has been explained, at least in part, by the induction in vivo of cytotoxic immune responses to cells infected with the Ad vector. Studies with rodent systems have suggested that cytotoxic T lymphocytes (CTLs) directed against virus antigens synthesized de novo in the transduced tissues play a major role in eliminating cells containing the E1-deleted viral genome (56–58, 61). Consistent with the concept of cellular antiviral immunity, expression of transgenes is significantly extended in experimental rodent systems that are deficient in various components of the cellular immune system or that have been rendered immunocompromised by administration of pharmacological agents (2, 33, 37, 48, 60, 64).Based on the assumption that further reduction of viral antigen expression may lower the immune response and thus extend persistence of transgene expression, previous studies have investigated the consequences of deleting both E1 and an additional viral regulatory region, such as E2A or E4. The E2A region encodes a DNA binding protein (DBP) with specific affinity for single-stranded Ad DNA. The DNA binding function is essential for the initiation and elongation of viral DNA synthesis during the early phase of Ad infection. During the late phase of infection, DBP plays a central role in the activation of the major late promoter (MLP) (for a recent review, see reference 44). The E4 region, located at the right end of the viral genome, encodes several regulatory proteins with pleiotropic functions which are involved in the accumulation, splicing, and transport of early and late viral mRNAs, in DNA replication, and in virus particle assembly (reviewed in reference 44). The simultaneous deletion of E1 and E2A or of E1 and E4 should therefore further reduce the replication of the virus genome and the expression of early and late viral genes. Such multidefective vectors have been generated and tested in vitro and in vivo (9, 12, 17, 19–21, 23, 24, 26, 34, 40, 52, 53, 59, 62, 63). Recombinant vectors with E1 deleted and carrying an E2A temperature-sensitive mutation (E2Ats) have been shown in vitro to express much smaller amounts of virus proteins, leading to extended transgene expression in cotton rats and mice (19, 20, 24, 59). To eliminate the risks of reversion of the E2Ats point mutation to a wild-type phenotype, improved vectors with both E1 and E2A deleted were subsequently generated in complementation cell lines coexpressing E1 and E2A genes (26, 40, 63). In vitro analysis of human cells infected by these viruses demonstrated that the double deletion completely abolished viral DNA replication and late protein synthesis (26). Similarly, E1/E4-deleted vectors have been generated in various in vitro complementation systems and tested in vitro and in vivo (9, 17, 23, 45, 52, 53, 62). These studies showed that deletion of both E1 and E4 did indeed reduce significantly the expression of early and late virus proteins (17, 23), leading to a decreased anti-Ad host immune response (23), reduced hepatotoxicity (17, 23, 52), and improved in vivo persistence of the transduced liver cells (17, 23, 52).Interpretation of these results is difficult, however, since all tested E1- and E1/E4-deleted vectors encoded the bacterial β-galactosidase (βgal) marker, whose strong immunogenicity is known to influence the in vivo persistence of Ad-transduced cells (32, 37). Moreover, the results described above are not consistent with the conclusions from other studies showing, in various immunocompetent mouse models, that cellular immunity to Ad antigens has no detectable impact on the persistence of the transduced cells (37, 40, 50, 51). Furthermore, in contrast to results of earlier studies (19, 20, 59), Fang et al. (21) demonstrated that injection of E1-deleted/E2Ats vectors into immunocompetent mice and hemophilia B dogs did not lead to an improvement of the persistence of transgene expression compared to that with isogenic E1-deleted vectors. Similarly, Morral et al. (40) did not observe any difference in persistence of transgene expression in mice injected with either vectors deleted in E1 only or vectors deleted in both E1 and E2A. Finally, the demonstration that some E4-encoded products can modulate transgene expression (1, 17, 36a) makes the evaluation of E1- and E1/E4-deleted vectors even more complex when persistence of transgene expression is used for direct comparison of the in vivo persistence of cells transduced by the two types of vectors.The precise influence of the host immune response to viral antigens on the in vivo persistence of the transduced cells, and hence the impact of further deletions in the virus genome, therefore still remains unclear. To investigate these questions, we generated a set of isogenic vectors with single deletions (AdE1°) and double deletions (AdE1°E2A° and AdE1°E4°) and their corresponding complementation cell lines and compared the biologies and immunogenicities of these vectors in vitro and in vivo. To eliminate any possible influence of transgene-encoded products on the interpretation of the in vivo results, we used E1-, E1/E2A-, and E1/E4-deleted vectors with no transgenes.     

Cell-free translation of adenovirus 2 E1a- and E1b-specific mRNAs and evidence that E1a-related polypeptides are produced from E1a-E1b overlapping mRNA

We have characterized the polypeptides translated in vitro by mRNAs of early region 1 (E1) of human adenovirus (Ad) type 2. Poly (A+) polyribosomal RNA was isolated from early Ad2-infected cells, the viral specific mRNAs were selected by hybridization to Ad2 E1a and E1b DNA, and the mRNAs were translated in vitro using [35S]methionine as a labeled precursor with a rabbit reticulocyte lysate. E1a-selected mRNA was translated to the 45-58-kDa cluster of polypeptides. We show here that E1b-selected mRNA can also be translated to the 45-58-kDa cluster of polypeptides in addition to the major 19-kDa polypeptide. The E1b 58-kDa polypeptide was produced only at a low level unless E1b mRNA is fractionated before translation to enrich for the 58-kDa mRNA. Translation of E1b region-selected mRNAs that have been fractionated by size shows that the 22 S mRNA fraction is translated to at least the 53-58-kDa E1a-related polypeptides as well as to E1b 58- and 19-kDa polypeptides. Our experiments suggest that the 22 S mRNA fraction includes E1a-E1b overlapping mRNA which was translated to E1a-related polypeptides as well as E1b 22 S mRNA. When compared by two-dimensional gel electrophoresis and by tryptic peptide mapping, the cluster of polypeptides translated from E1a-selected mRNA and the cluster translated from E1b-selected mRNA were distinguishable. A possible explanation for this is discussed, based upon splicing sites of the E1a-E1b overlapping mRNA which would result in an amino acid sequence with a COOH-terminal end slightly different from that of E1a polypeptides.     

Structures of the SUMO E1 provide mechanistic insights into SUMO activation and E2 recruitment to E1

E1 enzymes facilitate conjugation of ubiquitin and ubiquitin-like proteins through adenylation, thioester transfer within E1, and thioester transfer from E1 to E2 conjugating proteins. Structures of human heterodimeric Sae1/Sae2-Mg.ATP and Sae1/Sae2-SUMO-1-Mg.ATP complexes were determined at 2.2 and 2.75 A resolution, respectively. Despite the presence of Mg.ATP, the Sae1/Sae2-SUMO-1-Mg.ATP structure reveals a substrate complex insomuch as the SUMO C-terminus remains unmodified within the adenylation site and 35 A from the catalytic cysteine, suggesting that additional changes within the adenylation site may be required to facilitate chemistry prior to adenylation and thioester transfer. A mechanism for E2 recruitment to E1 is suggested by biochemical and genetic data, each of which supports a direct role for the E1 C-terminal ubiquitin-like domain for E2 recruitment during conjugation.     

Phosphatidylethanolamide derivatives of prostaglandins E1 and E2.

Prostaglandins E1 (PGE1) and E2 (PGE2) have been coupled with the amine group of phosphatidylethanolamine (PE) by means of dicyclohexylcarbodiimide. These complexes basically mimic the relaxant and contractile effects of the corresponding free prostaglandins (PGs) on various smooth muscle preparations, but exhibit a delayed onset of action and a lower affinity for the PG receptors. The complexes are comparable with the free, parent PGs, in their intrinsic activities. The same holds true for the effects on blood pressure and on the motility of the uterus in situ. The PGE2-PE complex is hydrolysed to release obviously free PGE2 by cell-free homogenates prepared from various tissues, but not by blood plasma. The PGE2-PE complex is immunologically indistinguishable from the free PGE2.     

Isolation of 19,20-dehydroprostaglandins E1 and E2 in human seminal fluid and further studies on 18,19-dehydroprostaglandins E1 and E2

Human seminal fluid was recently found to contain 18,19-dehydroprostaglandins E1 and E2 (E. H. Oliw, H. Sprecher, and M. Hamberg, (1986) J. Biol. Chem. 261, 2675-2683). In the present study, the cis and trans isomers of 18,19-dehydroprostaglandins E1 and E2 were prepared by incubation of microsomes of ram vesicular glands and glutathione with the precursor fatty acids, 8(Z),11(Z),14(Z),18(E/Z)-eicosatetraenoic acids, and 5(Z),8(Z),11(Z),14(Z),18(E/Z)-eicosapentaenoic acids, and used as references to characterize the 18,19-dehydroprostaglandins of human seminal fluid. Based on separation by reversed-phase high-performance liquid chromatography, capillary gas chromatography-mass spectrometry, and ozonolysis of the (-)-menthoxycarbonyl derivatives and on comparison with the authentic compounds, human seminal fluid was found to contain both the cis and trans isomers of 18,19-dehydroprostaglandins E1 and E2. Furthermore, human seminal fluid contained two related compounds, viz. 19,20-dehydroprostaglandins E1 and E2. The structures of these compounds were established by conversion into the corresponding prostaglandin B compounds, by mass spectrometric analysis and by chemical degradation by oxidative ozonolysis, which afforded, inter alia, 2(S)-hydroxy-adipic acid.     

Cloning of Thermomonospora fusca genes coding for beta 1-4 endoglucanases E1, E2 and E5

Thermomonospora fusca chromosomal DNA was partially digested with EcoRI and fragments in the size range from 4 to 15 kb were isolated, ligated into lambda gtWES.lambda B arms, packaged, and the recombinant phages plated on Escherichia coli. The plaques were screened for carboxymethyl cellulase (CMCase) activity by a gel overlay procedure, and 25 plaques were positive among the 15,000 plaques that were screened. Positive phages were amplified and used to prepare infected E. coli extracts which were assayed for CMCase activity before and after treatment with antisera prepared against five purified T. fusca beta 1-4 endoglucanases (E1-E5). One phage produced an enzyme that was inhibited by E1 antiserum, nine of the phages produced enzymes that were inhibited by E2 antiserum, 14 produced enzymes that were inhibited by E5 antiserum and the enzyme produced by the other phages was not inhibited by any of the five antisera. The DNA insert present in the phage coding for E1 was cut into a number of different fragments which were subcloned into E. coli first using lambda gtWES.lambda B and then plasmid pBR322. The smallest active subclone, pTE12, contained a 3.1-kb insert. The insert present in one of the phages coding for E2 was also subcloned and the smallest active subclone pTE23 contained a 2-kb insert. E. coli HB101 containing plasmid pTE12 or pTE23 produced enzymes that were identical to E1 and E2, respectively, in all the properties tested.     

Prostaglandin E antagonist activity of 11-deoxy-16,16-trimethyleneprostaglandin E1

11-Deoxy-16,16-trimethyleneprostaglandin E1 is a potent inhibitor of prostaglandin E-induced contractions of the gerbil colon. The antagonism is directed specifically against the prostaglandin E receptor and is not manifested when contractions are induced by either prostaglandin F2 alpha or acetylcholine.     

E1 on the move

E1 enzymes activate ubiquitin or related proteins and pass them to E2 enzymes. A recent structure and associated biochemical studies (Huang et al., 2005) show how an E1 binds its cognate E2 and indicates that large, conformational changes will be an integral component of the E1 reaction cycle.     

Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins.

Infection with adenovirus mutants carrying either point mutations or deletions in the coding region for the 19-kDa E1B gene product (19K protein) causes degradation of host cell and viral DNAs (deg phenotype) and enhanced cytopathic effect (cyt phenotype). Therefore, one function of the E1B 19K protein is to protect nuclear DNA integrity and preserve cytoplasmic architecture during productive adenovirus infection. When placed in the background of a virus incapable of expressing a functional E1A gene product, however, E1B 19K gene mutations do not result in the appearance of the cyt and deg phenotypes. This demonstrated that expression of the E1A proteins was responsible for inducing the appearance of the cyt and deg phenotypes. By constructing a panel of viruses possessing E1A mutations spanning each of the three E1A conserved regions in conjunction with E1B 19K gene mutations, we mapped the induction of the cyt and deg phenotypes to the amino-terminal region of E1A. Viruses that fail to express conserved region 3 (amino acids 140 to 185) and/or 2, (amino acids 121 to 185) or nonconserved sequences between conserved regions 2 and 1 of E1A (amino acids 86 to 120) were still capable of inducing cyt and deg. This indicated that activities associated with these regions, such as transactivation and binding to the product of the retinoblastoma susceptibility gene, were dispensable for induction of E1A-dependent cytotoxic effects. In contrast, deletion of sequences in the amino terminus of E1A (amino acids 22 to 107) resulted in extragenic suppression of the cyt and deg phenotypes. Therefore, a function affected by deletion of amino acids 22 to 86 of E1A is responsible for exerting cytotoxic effects in virally infected cells. Furthermore, transient high-level expression of the E1A region using a cytomegalovirus promoter plasmid expression vector was sufficient to induce the cyt and deg phenotypes, demonstrating that E1A expression alone is sufficient to exert these cytotoxic effects and that other viral gene products are not involved. Finally, placing E1A expression under the control of a strong promoter did not alter the requirement for E1B in the transformation of primary cells. One possibility is that the E1B 19K protein is required to overcome the cytotoxic effects of E1A protein expression and thereby enable primary cells to become transformed.