Association of gross virus-associated cell-surface antigen with liposomes.

Gross Cell-Surface Antigen (GCSAa) was obtained from W/Fu (C58NT)D lymphoma cells by Nonidet P40(NP40) or 3M KCl extraction and further purified by Sephadex G200 filtration. GCSAa was associated with lipids (dipalmitoylphosphatidycholine, cholesterol and dicetylphosphate, in molar ratios of 7:2:1) to form multilamellar liposomes. The amount of protein associated with liposomes was found to be proportional to the protein concentration of the sensitizing cellular extract and to the amount of phospholipids used and, under defined conditions, 22-55% of the protein of the cellular extract could be associated with liposomes. Analysis of disrupted sensitized liposomes showed that the GCSAa-specific activity of the liposome-associated proteins was quite similar to that of the proteins of the sensitizing cellular extract. Ultracentrifugation of disrupted liposomes showed that about 75% of the liposome-associated GCSAa activity was firmly associated with lipids and that little GCSAa was trapped within aqueous compartments between lipidic lamellae. 1.8--8.0% of the liposome-associated GCSAa was expressed at the liposome surface. No striking differences in degree of GCSAa association were found between liposomes sensitized by NP40 or by 3M KCl extracts. Storage experiments at +4 degrees C showed that GCSAa-sensitized liposomes were fairly stable

Measurement of Gross cell-surface antigen and p30 level in murine retrovirus-infected cell lines.

The level of Gross cell-surface antigen (GCSAa) expression at the surface of murine retrovirus-infected fibroblasts was determined by quantitative absorption of the anti-GCSAa activity of a serum produced in syngeneic W/Fu rats immunized against (C58NT)D lymphoma, and tested in a cytotoxicity assay against E male G2 lymphoma cells. While GCSAa was specifically expressed on Gross-type virus (G-MuLV)-induced lymphoma cells, and while G-MuLV and G-related MuLV induced a high level of GCSAa expression on murine fibroblasts, the Friend-Moloney-Rauscher (FMR) group viruses (FMR MuLV) and xenotropic isolates were also able to induce a high or intermediate level of GCSAa. Since GCSAa has been shown to be borne by glycosylated precursors of the viral nucleocapside (gp95gag and gp85gag), the amount of GCSAa expressed on these cells was compared to the level of cytoplasmic p30. In G- and G-related MuLV-infected cell lines, a significant relationship was found between the amount of GCSAa and the level of p30, whereas in FMR-MuLV or xenotropic virus-infected cells the amount of GCSAa varied independently of the p30 level. These results could explain the discrepancy in the specificity of expression of GCSAa in vivo and in vitro

Health reform requires policy capacity

Health reform requires policy capacity Pierre-Gerlier Forest 1 * , Jean-Louis Denis 2 , Lawrence D. Brown 3 , David Helms 4 Abstract Among the many reasons that may limit the adoption of promising reform ideas, policy capacity is the least recognized. The concept itself is not widely understood. Although policy capacity is concerned with the gathering of information and the formulation of options for public action in the initial phases of policy consultation and development, it also touches on all stages of the policy process, from the strategic identification of a problem to the actual development of the policy, its formal adoption, its implementation, and even further, its evaluation and continuation or modification. Expertise in the form of policy advice is already widely available in and to public administrations, to well-established professional organizations like medical societies and, of course, to large private-sector organizations with commercial or financial interests in the health sector. We need more health actors to join the fray and move from their traditional position of advocacy to a fuller commitment to the development of policy capacity, with all that it entails in terms of leadership and social responsibilit

Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells

Cell microparticles (MPs) released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5), and serotype 35 (HAdV35), respectively. We found that MPs derived from CHO cells (MP-donor cells) constitutively expressing CAR (MP-CAR) or CD46 (MP-CD46) were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR) were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins

Cytosolic 5'-triphosphate ended viral leader transcript of measles virus as activator of the RIG I-mediated interferon response.

International audienceBACKGROUND: Double stranded RNA (dsRNA) is widely accepted as an RNA motif recognized as a danger signal by the cellular sentries. However, the biology of non-segmented negative strand RNA viruses, or Mononegavirales, is hardly compatible with the production of such dsRNA. METHODOLOGY AND PRINCIPAL FINDINGS: During measles virus infection, the IFN-beta gene transcription was found to be paralleled by the virus transcription, but not by the virus replication. Since the expression of every individual viral mRNA failed to activate the IFN-beta gene, we postulated the involvement of the leader RNA, which is a small not capped and not polyadenylated RNA firstly transcribed by Mononegavirales. The measles virus leader RNA, synthesized both in vitro and in vivo, was efficient in inducing the IFN-beta expression, provided that it was delivered into the cytosol as a 5'-trisphosphate ended RNA. The use of a human cell line expressing a debilitated RIG-I molecule, together with overexpression studies of wild type RIG-I, showed that the IFN-beta induction by virus infection or by leader RNA required RIG-I to be functional. RIG-I binds to leader RNA independently from being 5-trisphosphate ended; while a point mutant, Q299A, predicted to establish contacts with the RNA, fails to bind to leader RNA. Since the 5'-triphosphate is required for optimal RIG-I activation but not for leader RNA binding, our data support that RIG-I is activated upon recognition of the 5'-triphosphate RNA end. CONCLUSIONS/SIGNIFICANCE: RIG-I is proposed to recognize Mononegavirales transcription, which occurs in the cytosol, while scanning cytosolic RNAs, and to trigger an IFN response when encountering a free 5'-triphosphate RNA resulting from a mislocated transcription activity, which is therefore considered as the hallmark of a foreign invader

Cytotoxic activity induced by crude extracts of Ganoderma lucidum (W. Curt.: Fr.) P. Karst. on mouse myeloma cancer cell-line

Ganoderma lucidum powder using hot water and methanol extraction methods indicated a twofold more active cytotoxic activity with IC50 of 44 ± 3.8 μg/ml in the latter method. The representative dose-response curves of the G. lucidum crude extracts on J558 cell-lines revealed that there were great similarities between the curves which reflected rapid killing activities. The percentage viability of the J558 cell exposed to these crude extracts was dose dependent only up to 150 μg/ml. After which, there was no significant reduction when the dose was increased to 200 or 400 μg/ml. The morphological alterations induced by the crude extract were examined under the phase contrast, fluorescent and electron microscopy. When J558 cells were treated with doses higher than 50 μg/ml of the crude extract, obvious morphological changes and apoptosis occurred after 72 h. At 400 μg/ml, most of the cells showed necrosis characterized as small fragments with uniformly stained red nuclei. The apoptotic and necrotic cells increased by 16.5 and 29.1%, respectively whereas the viable cells decreased by as much as 45.6. The mode of cell death via apoptosis was 3.6% higher than necrosis. However, these morphological changes were not observed in the case of 3T3 cells. Results obtained from scanning electron microscopy and transmission electron microscopy further confirmed the occurrence of various apoptotic and necrotic features