Replicon-helper systems from attenuated Venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo

A replicon vaccine vector system was developed from an attenuated strain of Venezuelan equine encephalitis virus (VEE). The replicon RNA consists of the cis-acting 5' and 3' ends of the VEE genome, the complete nonstructural protein gene region, and the subgenomic 26S promoter. The genes encoding the VEE structural proteins were replaced with the influenza virus hemagglutinin (HA) or the Lassa virus nucleocapsid (N) gene, and upon transfection into eukaryotic cells by electroporation, these replicon RNAs directed the efficient, high-level synthesis of the HA or N proteins. For packaging of replicon RNAs into VEE replicon particles (VRP), the VEE capsid and glycoproteins were supplied in trans by expression from helper RNA(s) coelectroporated with the replicon. A number of different helper constructs, expressing the VEE structural proteins from a single or two separate helper RNAs, were derived from attenuated VEE strains Regeneration of infectious virus was not detected when replicons were packaged using a bipartite helper system encoding the VEE capsid protein and glycoproteins on two separate RNAs. Subcutaneous immunization of BALB/c mice with VRP expressing the influenza HA or Lassa virus N gene (HA-VRP or N-VRP, respectively) induced antibody responses to the expressed protein. After two inoculations of HA-VRP, complete protection against intranasal challenge with influenza was observed. Furthermore, sequential immunization of mice with two inoculations of N-VRP prior to two inoculations of HA-VRP induced an immune response to both HA and N equivalent to immunization with either VRP construct alone. Protection against influenza challenge was unaffected by previous N-VRP immunization. Therefore, the VEE replicon system was characterized by high-level expression of heterologous genes in cultured cells, little or no regeneration of plaque-forming virus particles, the capability for sequential immunization to multiple pathogens in the same host, and induction of protective immunity against a mucosal pathogen.     

Cultural factors constraining the introduction of family planning among the Kassena-Nankana of northern Ghana

This study presents a focus group investigation of reasons why women in a rural, Sahelian community are reluctant to adopt family planning even when convenient services are made freely available. First, women opting to practice contraception must do so at considerable risk of social ostracism or familial conflict. Implementing individual preference is something that must be done without the support of others. Second, few women view personal decisions about contraceptives as theirs to make. Women and children are the property of the corporate family-kin and community militate against reproductive control. Third, although children are highly valued for a variety of economic, social, and cultural reasons, mortality risks remain extremely high. Low fertility imposes the unacceptable risk that a woman will have no surviving children at the end of her reproductive life. Taken together, these findings attest to the inadequacy of service strategies focused on the contribution of distribution, individual agency, or personal choice. Outreach should also build a sense of community legitimacy for the program, collective health action, and traditional leadership support for family planning behavior.     

Endoplasmic reticulum chaperones GRP78 and calreticulin prevent oxidative stress, Ca2+ disturbances, and cell death in renal epithelial cells

Activation of stress response genes can impart cellular tolerance to environmental stress. Iodoacetamide (IDAM) is an alkylating toxicant that up-regulates expression of hsp70 (Liu, H., Lightfoot, D. L., and Stevens, J. L. (1996) J. Biol. Chem. 271, 4805-4812) and grp78 in LLC-PK1 renal epithelial cells. Therefore, we used IDAM to determine the role of these genes in tolerance to toxic chemicals. Prior heat shock did not protect cells from IDAM but pretreatment with trans-4,5-dihydroxy-1,2-dithiane (DTTox), thapsigargin, or tunicamycin enhanced expression of the endoplasmic reticulum (ER) chaperones GRP78 and GRP94 and rendered cells tolerant to IDAM. Cells expressing a 524-base pair antisense grp78 fragment (pkASgrp78) had a diminished capacity to up-regulate grp78 and grp94 expression after ER stress. Protection against IDAM due to prior ER stress was also attenuated in pkASgrp78 cells suggesting that ER chaperones of the GRP family are critical for tolerance. Covalent binding of IDAM to cellular macromolecules and depletion of cellular thiols was similar in tolerant and na?ve cells. However, DTTox pretreatment blocked the increases in cellular Ca2+ and lipid peroxidation observed after IDAM treatment. Overexpressing the ER Ca2+-binding protein calreticulin prevented IDAM-induced cell death, the rise in cytosolic Ca2+, and oxidative stress. Although activation of the ER stress response did not prevent toxicity due to Ca2+ influx, EGTA-AM and ruthenium red both blocked cell death suggesting that redistribution of intracellular Ca2+ to the mitochondria may be important in toxicity. The data support a model in which induction of ER stress proteins prevents disturbances of intracellular Ca2+ homeostasis, thus uncoupling toxicant exposure from oxidative stress and cell death. Multiple ER stress proteins are likely to be involved in this tolerance response.     

Shoulder joint instability after primary arthroplasty

Inhibitors of enzymatic amplification in serum may cause false-negative results for direct detection of hepatitis C virus (HCV) by polymerase chain reaction (PCR). This study was undertaken to demonstrate the importance of the internal control in a PCR assay for detection of HCV-RNA to monitor false-negatives due to inhibitors. HCV-RNA was extracted using RNA extraction kit (SepaGene RV-R, Sanko Junyaku) and a prototype instrument for automated specific capture of HCV-RNA with probes and magnetic bead/fluid separation (Roche Molecular Systems). The extracted HCV-RNA and internal control were detected by an automated PCR machine (Cobas Amplicor, Roche Diagnostic Systems). Addition of hemoglobin (up to 4.5 g/l) to the sera followed by RNA extraction with SepaGene RV-R had no inhibitory effect on the detection of either HCV-RNA or the internal control. In contrast, addition of heparin to the sera showed an inhibitory effect with a dose-dependent manner on the detection of both HCV-RNA and the internal control, with a greater effect at lower copy number of HCV. When HCV-RNA was extracted by the automated system, the inhibitory effect of heparin was successfully eliminated. In the assays of 65 serum samples positive for anti-HCV antibodies, positivity for the internal control indicated efficient amplification and validated 14 negative and two equivocal results for detection of HCV-RNA. Detection of the internal control was negatively correlated with viral copy number in sera suggesting competitive inhibition of high viral copy number on amplification of the internal control. Extraction, co-amplification and detection of the internal control appears useful for estimating effects of inhibitors on amplification in each assay for the detection of HCV-RNA, and for evaluating efficacy of RNA extraction methods.     

Interaction of the baculovirus anti-apoptotic protein p35 with caspases. Specificity, kinetics, and characterization of the caspase/p35 complex

The anti-apoptotic protein p35 from baculovirus is thought to prevent the suicidal response of infected insect cells by inhibiting caspases. Ectopic expression of p35 in a number of transgenic animals or cell lines is also anti-apoptotic, giving rise to the hypothesis that the protein is a general inhibitor of caspases. We have verified this hypothesis by demonstrating that purified recombinant p35 inhibits human caspase-1, -3, -6, -7, -8, and -10 with kass values from 1.2 x 10(3) to 7 x 10(5) (M-1 s-1), and with upper limits of Ki values from 0.1 to 9 nM. Inhibition of 12 unrelated serine or cysteine proteases was insignificant, implying that p35 is a potent caspase-specific inhibitor. Mutation of the putative inhibitory loop to favor caspase-1 resulted in a substantial decline in caspase-3 inhibition, but minimal changes in caspase-1 inhibition. The interaction p35 with caspase-3, as a model of the inhibitory mechanism, revealed classic slow-binding inhibition, with both active-sites of the caspase-3 dimer acting equally and independently. Inhibition resulted from complex formation between the enzyme and inhibitor, which could be visualized under nondenaturing conditions, but was dissociated by SDS to give p35 cleaved at Asp87, the P1 residue of the inhibitor. Complex formation requires the substrate-binding cleft to be unoccupied. Taken together, these data revealed that p35 is an active-site-directed inhibitor highly adapted to inhibiting caspases.     

Bibliometric analysis of the original articles published in the Revista Espa?ola de Anesthesiología y Renimación in 10 years (1987-1996)

OBJECTIVE: To describe the original research articles published in Revista Espa?ola De Anestesiología y Reanimación (REAR) from 1987 through 1996, as well as to characterize the citations included in those articles. MATERIAL AND METHODS: The 299 articles published as original research in REAR over the past 10 years (1987 through 1996) were analyzed. The bibliographic aspects examined were coauthorship (authors/paper index), citations per article, isolation in function of language of publication of references, degree of obsolescence of articles based on year of references cited ("half-life"), self-citation and degree of dispersion of citations. RESULTS: The authors/paper index was 5.16 +/- 1.62. No statistically significant difference was found in number of authors over the 10-year study period. Mean number of references cited per article was 24.05 +/- 12.02. We found statistically significant differences for 1993 and the period 1987 to 1988, and 1994 and the year 1987 (p < 0.001). The "half-life" of articles was 6 when analyzing on a year-by-year basis; this index ranged from 5.5 to 7, with no significant annual differences. REAR articles accounted for 4.02% of all citations. English was the most frequent language of cited publications, with 6,240 references (86.8%), followed by Spanish with 621 (8.64%), French with 223 (3.1%) and German with 74 (1.03%). Of the 7,191 references analyzed, 6,447 (89.65%) were of scientific journals. Books are the second most commonly cited type of document, with 623 (8.66%) citations. Analyzing journals cited 25 or more times, we found that 74.19% of the articles (4,783/6,447) had been published in 5.3% of the journals (36/678). Seven journals of anesthesia, which represented 1.03% of all journals (7/678) appeared in 52.81% of references of this type (3,405/6,447). CONCLUSIONS: The number of authors of original research articles published in REAR in the last 10 years was high. Spanish authors in anesthesiology cite mainly literature in English; use up-to-date sources of information, mainly journals; and take a large proportion of information from a small number of journals, which are those of greatest international impact in our specialty.     

CO2 and gadopentetate dimeglumine as alternative contrast agents for malfunctioning dialysis grafts and fistulas

BACKGROUND: Hemodialysis grafts and native fistulas are frequently evaluated angiographically utilizing iodinated contrast material to determine the cause of malfunction. Occasionally, patients are not able to receive iodinated contrast material due to a history of previous severe allergic reaction or concern that iodinated contrast material could worsen renal function requiring premature initiation of permanent dialysis. We set out to test the feasibility of gadopentetate dimeglumine as an alternative contrast agent in conjunction with carbon dioxide (CO2) angiography in the evaluation and treatment of hemodialysis grafts and native fistulas in patients who have a contraindication to iodinated contrast material. METHODS: Six patients with a malfunctioning hemodialysis graft and native fistula were evaluated. Four patients were successfully evaluated using carbon dioxide and gadopentetate dimeglumine. Two additional patients underwent balloon angioplasty using gadopentetate dimeglumine alone as the alternative contrast agent. RESULTS: All six patients successfully were evaluated and treated using gadopentetate dimeglumine either alone or as a supplement to CO2 angiography. Five of these patients had lesions successfully treated using gadopentetate dimeglumine alone or in combination with CO2 as the angiographic contrast agents. One patient underwent a successful diagnostic angiogram using gadopentetate dimeglumine and CO2 as alternative contrast agents and was subsequently treated with surgical revision. The gadopentetate dimeglumine angiograms identified the arterial anastomosis and more clearly identified stenotic lesions and venous outflow anatomy compared to carbon dioxide angiograms. CONCLUSION: Gadopentetate dimeglumine is useful as an alternative contrast agent in conjunction with CO2 in patients with malfunctioning hemodialysis grafts and fistulas, who have a contraindication to the administration of iodinated contrast material.     

Inhibition of human angiogenin by DNA aptamers: nuclear colocalization of an angiogenin-inhibitor complex

Specific ligands (aptamers) for angiogenin were selected from a 72-mer oligodeoxynucleotide library consisting of 28 randomized positions flanked by two constant regions of 22 residues each. From a starting pool of approximately 10(14) molecules, 19 angiogenin-binding ligands were obtained. Among them, two oligonucleotides showed significant inhibition of the ribonucleolytic activity of angiogenin with apparent Kis of 0.65 and 0.60 micro M, respectively. One of them was shortened on the basis of its secondary structure to provide a 45-mer oligonucleotide that retained much of the inhibitory properties of the parent molecule. It inhibits both the angiogenic and cell proliferative activities of angiogenin but does not interfere with its nuclear translocation in human endothelial cells. Importantly, the inhibitor is cotranslocated to the nucleus with angiogenin in a approximately 1:1 stoichiometric ratio. These results demonstrate that the inhibition of angiogenin-induced cell proliferation and angiogenesis by the oligonucleotide is due to suppression of the ribonucleolytic activity of angiogenin, an event that occurs most likely within the cell nucleus.     

Adipose tissue omega-3 and omega-6 fatty acid content and breast cancer in the EURAMIC study. European Community Multicenter Study on Antioxidants, Myocardial Infarction, and Breast Cancer

Three experiments examined the effect of either withdrawal from diazepam, or repeated treatment with the convulsant, pentylenetetrazol (PTZ), on behaviour and seizure threshold. The behaviours measured were on the elevated plus maze and in the four-plate test; seizure threshold was measured as dose of PTZ infused via the tail vein to the first clonic twitch. In experiment 1, we examined the effect of either single or repeated withdrawal from diazepam using a procedure in which the drug was administered SC in a slow release depot. Three cycles of withdrawal from diazepam were compared to a single withdrawal experience. A single withdrawal from diazepam following chronic treatment gave rise, 72 h following the last dosing, to behavioural changes, suggestive of anxiety, in both tests, but did not result in a reduced convulsant threshold. In contrast, repeated withdrawal resulted in a reduction in sensitivity in several measures of anxiety, but sensitised the mice to the convulsive effects of the PTZ. The unexpected failure to find an increased sensitivity to a convulsive agent following a single withdrawal from SC diazepam was examined in experiment 2. The seizure threshold following a single withdrawal of mice which had received diazepam chronically IP in aqueous vehicle was significantly reduced relative to vehicle-treated controls, whereas that of animals receiving the same dose SC in oil, was not. It is argued that the difference may arise from the animals treated repeatedly with IP diazepam unintentionally experiencing repeated withdrawal, since the half-life of the drug by this route is short. In experiment 3, repeated sub-convulsant PTZ treatment reduced the convulsant threshold (the dose of PTZ required to give rise to the first clonic twitch), but had no significant effect on the behavioural measures of anxiety compared to a single dose of PTZ or vehicle controls. The results suggest that repeated withdrawal from chronic treatments with diazepam sensitises mice to convulsant stimuli in a manner resembling the effects of repeated administration of sub-convulsant doses of PTZ, but that neither repeated PTZ nor repeated diazepam withdrawal results in increased sensitivity to anxiogenic stimuli; rather, repeated withdrawal from diazepam may reduce the susceptibility of mice to behavioural measures of anxiety.     

A review of the effects of manipulation of the cysteine residues of rat aryl sulfotransferase IV

Aryl sulfotransferase IV from rat liver has the broad substrate range that is characteristic of the enzymes of detoxication. With the standard assay substrates, 4-nitrophenol and 3'-phosphoadenosine 5'-phosphosulfate (PAPS), sulfation is optimum at pH 5.4 whereas the reaction is minimal in the physiological pH range. These properties preclude a physiological function for this cytosolic enzyme. Partial oxidation of the enzyme, however, results not only in an increase in the rate of sulfation but also in a shift of the pH optimum to the physiological pH range. The mechanism for this dependence on the redox environment involves oxidation at Cys66, the cysteine residue that is conserved throughout the phenol sulfotransferase family. As documented by mass spectroscopic methods, oxidation by GSSG leads to the formation of an internal disulfide between Cys66 and Cys232; for mutants at Cys232, the oxidation product is a mixed disulfide of Cys66 and glutathione. Both of these disulfide species activate the enzyme and allow it to function at a pH optimum in the physiological range. The activated enzyme differs from the reduced form by a more circumscribed substrate spectrum. All five mutants, in which each of the cysteines of the sulfotransferase subunit have been changed to serine, are catalytically active. Only Cys66 is required for the redox response.