Eimeria tenella, E. necatrix, E. acervulina, E. maxima, and E. brunetti: potent anticoccidial activity of an uridine analog, 1-(beta-D-ribofuranosyl)-2(1H)-pyrazinone 4-oxide

The anticoccidial activity of an uridine analog, 1-(beta-D-ribofuranosyl)-2(1H)-pyrazinone 4-oxide (emimycin riboside), against five species of chicken Eimeria was tested individually in battery experiments. With 16 ppm of the compound in feed, marked anticoccidial activity was obtained against Eimeria tenella, E. necatrix, E. acervulina, E. maxima, and E. brunetti. The last named species was more drug-sensitive than the others--dietary levels of at least 8 ppm of the drug exhibited good protection and eliminated practically all clinical signs. The battery tests with delayed and restricted medications showed that emimycin riboside affected the development of parasites in first and second generation schizogony of the life cycle of E. tenella.     

Synthesis of methyl dl-(1,3/2,4,5)- and dl-(1,3,4/2,5)-2,3,4,5-tetrahydroxycyclohexane-1-carboxylates

Two new diastereoisomers of the pseudo-hexuronate methyl 2,3,4,5-tetrahydroxycyclohexane-1-carboxylate, having (1,3/2,4,5)- (8) and (1,3,4/2,5)-configurations (16), have been synthesised from the readily available bromo-lactone (1) of the endo-adduct of furan and acrylic acid. Treatment of 1 with 20% hydrogen bromide in acetic acid at 80° resulted in regioselective cleavage of the 1,4-anhydro ring to give dl-(1,3,5/2,4)-2,3-diacetoxy-4,5-dibromocyclohexane-1-carboxylic acid (2). Debromination of 2 with zinc dust gave dl-(1,3/2)-2,3-diacetoxycyclohex-4-ene-1-carboxylic acid (5). The methyl ester (6) of 5 was oxidised with osmium tetraoxide and hydrogen peroxide, followed by acetylation, to give the tetra-acetate of 8. Epoxidation of 6 gave two isomeric epoxides, each of which gave 16 on hydrolysis followed by O-deacetylation.     

Negative regulation of the bovine papillomavirus E5, E6, and E7 oncogenes by the viral E1 and E2 genes.

Papillomaviruses induce benign squamous epithelial lesions that infrequently are associated with uncontrolled growth or malignant conversion. The virus-encoded oncogenes are clearly under negative regulation since papillomaviruses can latently infect cells and since different levels of viral oncogene expression are seen within the layers of differentiating infected epitheliomas. We used bovine papillomavirus type 1 (BPV-1) to investigate the mechanisms involved in the negative regulation of transformation. We found that the following two distinct and interacting mechanisms negatively regulate BPV-1 transformation effected by virally encoded trans-acting factors: (i) E2 repressors suppress transformation by the E6 and E7 oncogenes, and (ii) E1 and the E2 transactivator suppress transformation by the E6, E7, and E5 oncogenes. These systems interact in that the E2 repressors function to relieve the transformation suppression effected by the E1 and E2 transactivator genes. A BPV-1 mutant that lacked E2 repressors and E1 had greatly augmented transformation capacity. Analysis of this mutant revealed that the enhanced transformation was due to expression of the E6 and E7 genes in the absence of E5, revealing a previously unappreciated potency and synergy for the BPV-1 E6 and E7 oncogenes.     

Platelet aggregation. II. Adenyl cyclase, prostaglandin E1, and calcium

In exploration of the proposal that prostaglandin E1 (PGE1) inhibits platelet aggregation via stimulation of adenyl cyclase, the temporal relationship of adenosine cyclic 3',5' monophosphate (cyclic AMP) synthesis and inhibition of ADP-induced aggregation in response to PGE1 was studied. The requirement for calcium in aggregation led to the investigation of the effects of calcium ions on platelet adenyl cyclase activity. PGE1 stimulated the synthesis of cyclic AMP from adenosine-5'-triphosphate-8-14-C by platelet membrane fractions and also increased cyclic AMP synthesis in intact platelets previously incubated for 2 hours with adenosine-14-C. The accumulation of cyclic AMP increased signficiantly at low concentrations of PGE1 and reached a maximum at about 1 mug. Regardless of the inducing agent, calcium ions are an absolute requirement for the aggregation of platelets.     

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.     

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.     

Rubella virus contains one capsid protein and three envelope glycoproteins, E1, E2a, and E2b.

We have analyzed the structure of rubella virus proteins labeled metabolically with [35S]methionine, [3H]mannose, and [3H]glucosamine or externally with [3H]borohydride after galactose oxidase treatment. Four structural proteins, with MrS of about 58,000 (E1), 47,000 (E2a), 42,000 (E2b), and 33,000 (C), were resolved on sodium dodecyl sulfate-polyacrylamide gels. Tryptic peptide maps obtained from [35S]methionine-labeled proteins indicated that E1 and C were unrelated to each other and to E2a and E2b, whereas the latter two gave similar, if not identical, maps. E1, E2a, and E2b were associated with the envelope and were located externally on the virus particle, whereas the C protein was associated with the RNA in the nucleocapsid. Solubilization of the virus with Triton X-100, followed by removal of the nucleocapsid and the detergent, resulted in the formation of soluble envelope protein complexes (rosettes) containing E1, E2a, and E2b. Although external labeling with [3H]borohydride and metabolic labeling with [3H]glucosamine suggested that all three proteins were glycosylated, only E1 and E2b were efficiently labeled with [3H]mannose. It is thus possible that the difference in migration between E2a and E2b is due to differences in glycosylation. Analysis by immunoprecipitation and sodium dodecyl sulfate-gel electrophoresis of intracellular [35S]methionine-labeled structural proteins synthesized in the presence and absence of tunicamycin supported the conclusion that E1 and E2 are glycoproteins. Unglycosylated E1 and E2 had an Mr of about 53,000 and 30,000, respectively.     

The adenylate cyclase of rheumatoid synovial fluid macrophages is more sensitive for dl-5E, 19,14-di dehydro-carbo-prostacyclin, a stable prostacyclin analogue, than for prostaglandin E2

dl-5E, 19,14-di dehydro-carbo-prostacyclin (DDH-carbo PGI2), a stable prostacyclin (PGI2) derivative, but not prostaglandin (PG) E2, stimulated the adenylate cyclase of synovial fluid macrophages, isolated from rheumatoid patients with an active synovitis, in a dose dependent manner (10-1000 ng/ml). DDH-carbo PGI2 also stimulated synovial macrophage cAMP synthesis when injected into the knee joint. Exogenous arachidonic acid (AA) had little effect on cyclic-AMP (cAMP) formation or PGI2 release (assayed as 6ketoPGF1 alpha). It stimulated, however, the release of PGE2 and, to a lesser extent, thromboxane (Tx) A2 (measured as TxB2).     

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.     

Effect of prostaglandins E1, E2 and F2alpha on neutrophil aggregation.

Recently we have found that chemotactic factors stimulate neutrophils in suspension to aggregate. Because of an obvious analogy to platelet aggregation, we examined the influence of three prostaglandins on this process. Prostaglandins E1, E2 and F2alpha alone did not cause aggregation of the neutrophils but were able to partially inhibit the aggregation response induced by the synthetic chemotactic tripeptide, formyl-methionyl-leucyl-phenylalanine. The minimal inhibitory concentrations for prostaglandins E1, E2 and F2alpha were 10(-7), 10(-6) and 10(-5)M, respectively. These results are similar to those found for the prostaglandin-induced inhibition of platelet aggregation. It may be, therefore, that neutrophil aggregation, like platelet aggregation, is modulated by intracellular prostaglandins and other products of arachidonic acid metabolism.     

Mode of Action of Colicins of Types E1, E2, E3, and K

The effect of colicins on deoxyribonucleic acid and protein synthesis, and also their effect on the ability of T4 phage to replicate in Escherichia coli K-12, were studied. Colicins of type K inhibited deoxyribonucleic acid synthesis, protein synthesis, and phage growth. Among colicins of type E, there was an absolute correlation between mode of action and subdivision into types E(1), E(2), and E(3).     

Simple sequence repeat (SSR) markers linked to E1, E3, E4, and E7 maturity genes in soybean.

Soybean near isogenic lines (NILs), contrasting for maturity and photoperiod sensitivity loci, were genotyped with approximately 430 mapped simple sequence repeats (SSRs), also known as microsatellite markers. By analysis of allele distributions across the NILs, it was possible to confirm the map location of the Dt1 indeterminate growth locus, to refine the SSR mapping of the T tawny pubescence locus, to map E1 and E3 maturity loci with molecular markers, and to map the E4 and E7 maturity loci for the first time. Molecular markers flanking these loci are now available for marker-assisted breeding for these traits. Analysis of map locations identified a putative homologous relationship among four chromosomal regions; one in the middle of linkage group (LG) C2 carrying E1 and E7, one on LG I carrying E4, one at the top of LG C2, at which there is a reproductive period quantitative trait locus (QTL), and the fourth on LG B1. Other evidence suggests that homology also exists between the E1 + E7 region on LG C2 and a region on LG L linked to a pod maturity QTL. Homology relationships predict possible locations in the soybean genome of additional maturity loci, as well as which maturity loci may share a common evolutionary origin and similar mechanism(s) of action.     

Electrophoretic and immunologic characterization of proteins of merozoites of Eimeria acervulina, E. maxima, E. necatrix, and E. tenella.

Merozoites of Eimeria acervulina, Eimeria maxima, Eimeria necatrix, and Eimeria tenella were compared by gel electrophoresis, western-blotting with chicken antiserum, indirect fluorescent antibody reactions, and antiserum neutralization. Merozoites from the 4 species had dissimilar patterns of proteins and antigens in soluble and membrane fractions. Coomassie blue staining of SDS-PAGE gels revealed 16-22 protein bands depending on the species of merozoite but only 3 bands per species in the membrane fractions. Homologous and heterologous antisera recognized 5-12 soluble fraction bands and 3-7 membrane fraction bands on immunoperoxidase-stained western blots, depending on the species. When antisera from infected chickens were used in an indirect fluorescent antibody reaction, the merozoites of E. tenella and E. necatrix had a strong reaction with homologous and heterologous antisera. Merozoites of E. acervulina and E. maxima reacted with homologous antisera but had a weak or no reaction with heterologous antisera. Chicken antiserum against E. tenella had no effect on the viability of E. tenella merozoites when they were inoculated into chicken embryos.     

The structure of human apolipoprotein E2, E3 and E4 in solution 1. Tertiary and quaternary structure

Three recombinant apoE isoforms fused with an amino-terminal extension of 43 amino acids were produced in a heterologous expression system in E. coli. Their state of association in aqueous phase was analyzed by size-exclusion liquid chromatography, sedimentation velocity and sedimentation equilibrium experiments. By liquid chromatography, all three isoforms consisted of three major species with Stokes radii of 4.0, 5.0 and 6.6 nm. Sedimentation velocity confirmed the presence of monomers, dimers and tetramers as major species of each isoform. The association schemes established by sedimentation equilibrium experiments corresponded to monomer-dimer-tetramer-octamer for apoE2, monomer-dimer-tetramer for apoE3 and monomer-dimer-tetramer-octamer for apoE4. Each of the three isoforms exhibits a distinct self-association pattern. The apolipoprotein multi-domain structure was mapped by limited proteolysis with trypsin, chymotrypsin, elastase, subtilisin and Staphylococcus aureus V8 protease. All five enzymes produced stable intermediates during the degradation of the three apoE isoforms, as described for plasma apoE3. The recombinant apoE isoforms, thus, consist of N- and C-terminal domains. The presence of the fusion peptide did not appear to alter the apolipoprotein tertiary organization. However, a 30 kDa amino-terminal fragment appeared during the degradation of the recombinant apoE isoforms resulting from cleavage in the 273-278 region. This region, not accessible in plasma apoE3, results from a different conformation of the C-terminal domain in the recombinant isoforms. A specific pattern for the apoE4 C-terminal domain was observed during the proteolysis. The region 230-260 in apoE4, in contrast to that of apoE3 and apoE2, was not accessible to proteases, probably due to the existence of a longer helix in this region of apoE4 stabilized by an interdomain interaction.