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J M Coll

Publications and source records attributed to J M Coll.

At least 37 records · Page 2Linked to original sources
PubMed1996

Phosphatidylserine binding to solid-phase rhabdoviral peptides: a new method to study phospholipid/viral protein interactions.

A new method is described for the study of phosphatidylserine binding to rhabdoviral peptides by using solid-phase assays. This new assay could probably be extended to study the interactions between host membrane phospholipid and viral proteins in other viruses. By using labeled and hydrated phosphatidylserine (PS), PS-binding to solid-phase 15-mer peptides (pepscan) could map putative phospholipid-binding regions of the glycoprotein G of viral haemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus. The major PS-binding region of 27 aa (aa82-109, p2) did not only bind PS, but also phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Extraction of the PS bound to solid-phase p2 by a variety of chemical compounds and competition experiments with several phospholipid-related compounds showed that PS-Binding to p2 was dependent on not only hydrophobic, but also ionic interactions, as suggested by prior work on phospholipid interactions in other rhabdoviruses. Saturation/competition experiments with labeled and cold PS, PE and PC also showed that the reaction probably takes place between high molecular weight aggregates of hydrated phospholipids and several molecules of solid-phase p2. This assay has been used previously to detect hydrophobic amino acid heptad-repeats in rhabdoviruses and when anti-p2 antibodies to VHSV were obtained they were capable of inhibiting VHSV-induced cell to cell fusion.

Amino Acid Sequence↗
PubMed1996

The human breast cell DNA synthesome: its purification from tumor tissue and cell culture.

In this report, we describe for the first time the isolation and purification of a multiprotein complex for DNA replication from MDA MB-468 human breast cancer cells. This complex, which we designate the DNA synthesome, fully supports the in vitro replication of simian virus 40 (SV40) origin-containing DNA in the presence of the viral large T-antigen. Since the SV40 virus utilizes the host's cellular proteins for its own DNA replication, our results indicate that the DNA synthesome may play a role not only in viral DNA synthesis but in human breast cell DNA replication as well. Our studies demonstrate that the following DNA replication proteins constitute the DNA synthesome: DNA polymerase alpha, DNA primase, DNA polymerase delta, proliferating cell nuclear antigen, replication protein A, replication factor C, DNA topoisomerases I, II, and DNA polymerase epsilon. In addition, we successfully isolated the DNA synthesome from human breast tumor tissue as well as from xenografts from nude mice injected with the human breast cancer cell line MCF-7. The DNA synthesome purified from the breast cancer tissues fully supports SV40 DNA replication in vitro. Furthermore, our results obtained from a novel forward mutagenesis assay suggest that the DNA synthesome isolated from a nonmalignant breast cell line mediates SV40 DNA replication by an error-resistant mechanism. In contrast, the DNA synthesome derived from malignant breast cells and tissue exhibited a lower fidelity for DNA synthesis in vitro. Overall, our data support the role of the DNA synthesome as mediating breast cell DNA replication in vitro and in vivo.

Animals↗
PubMed1995

Different peptides from hemorrhagic septicemia rhabdoviral proteins stimulate leucocyte proliferation with individual fish variation.

Trout leucocytes from most of the survivors of viral hemorrhagic septicemia virus (VHSV) infections were capable of in vitro proliferation (T-like response) when cultured in the presence of short synthetic peptides designed from the G and the N cDNA-derived protein sequences of VHSV, a virus with substantial economic impact in trout farms. In contrast, no significant proliferative responses were obtained for the above-mentioned peptides from leucocytes obtained from either noninfected or genetically VHSV-resistant trout. However, since the anamnestic recognition of particular peptides (epitopes) of the G and the N protein by trout leucocytes varies largely within the outbred trout population, larger VHSV protein fragments were also tested. The finding that recombinant G and N fragments carrying multiple epitopes are recognized by the majority of the individual trout surviving VHSV infections and with higher stimulation indexes suggests that the recombinant viral proteins could be used as vaccines given the outbred nature of the fish.

Amino Acid Sequence↗
PubMed1995

The glycoprotein G of rhabdoviruses.

Rhabdoviruses show an RNA-containing helically-wound nucleocapsid either enclosed by or enclosing a membrane M protein, surrounded by a lipid bilayer through which dynamic protein trimers made up of non-covalently associated monomers of glycoprotein G (G) project outside. Mature monomeric rhabdoviral G has more than 500 amino acids, 2-6 potential glycosylation sites, 12-16 highly conserved cysteine residues, 2-3 stretches of a-d hydrophobic heptad-repeats, a removed amino terminal hydrophobic signal peptide, a close to the carboxy terminal hydrophobic transmembrane sequence and a carboxy terminal short hydrophylic cytoplasmic domain. Association-dissociation between monomers-trimers and displacement of the trimers along the plane of the lipid membrane, are induced by changes in the external conditions (pH, temperature, detergents, etc.). Throughout conformational changes the G trimers are responsible for the virus attachment to cell receptors, for low-pH membrane fusion and for reacting with host neutralizing monoclonal antibodies (MAbs). Antigenic differences could exist between monomers and trimers, which may have implications for future vaccine developments. The family Rhabdoviridae is made up of the Lyssavirus (rabies), the Vesiculovirus (vesicular stomatitis virus, VSV) and many rhabdoviruses infecting fish, plants, and arthropod insects. All these reasons make the G of rhabdoviruses an ideal subject to study comparative virology and to investigate new vaccine technologies.

Amino Acid Sequence↗
PubMed1995

Heptad-repeat sequences in the glycoprotein of rhabdoviruses.

Two or three regions containing three or more successive newly defined heptads of a-d hydrophobic amino acid repeats have been located in the cDNA-derived amino acid sequences of glycoprotein G of all rhabdoviruses examined (rabies, vesicular stomatitis, fish, and plant rhabdoviruses) by computer search. These new heptad-repeats differ from those previously reported in other viruses because of the presence of all the hydrophobic amino acids in positions a or d, and because they are not predicted to form coiled coils by current methods and thus they have not been detected previously in any rhabdoviruses. The two or three heptad-repeat regions were the only parts of the glycoprotein with at least three successive heptad-repeats in all the rhabdoviral sequences studied and had low sequence variability among the members of each of the rhabdoviral genius but show no sequence similarity among the different genus. All these newly detected heptad repeats were in the vicinity of some of the higher hydrophobic regions in each of the rhabdovirus genera studied and were found mostly, but not always, outside the extra amino acid sequences that occur in the longer insect or plant rhabdovirus glycoprotein G. The correspondence of position and structure of these heptad-repeats among all the rhabdoviruses suggests its participation in common function(s), most probably related to viral fusion with cellular membranes.

Amino Acid Sequence↗
PubMed1995

Synthetic peptides reveal a phospholipid binding domain in the glycoprotein of VHSV, a salmonid rhabdovirus.

Using phosphatidylserine (PS) binding to solid-phase synthetic 15-aa peptides, which covered the full length glycoprotein G of a salmonid rhabdovirus, viral haemorrhagic septicaemia virus (VHSV), evidence is presented showing the mapping of its major phospholipid-binding region. Three overlapping peptides were the dominant but not exclusive, reactive peptides that defined the phospholipid-binding main region. A 28-aa synthetic peptide (p2, aa 82-109), defined by the sequences of the 3 above-mentioned peptides, contained a putative alpha-helix domain with 3 consecutive hydrophobic amino acid a-d heptad-repeats (amphipathic alpha-helix), and 2 arginines at its carboxy terminal part. This peptide showed a higher apparent specific activity of PS-binding than the 15-aa peptides. Only native, denatured or recombinant fragment G4 viral glycoprotein G showed PS-binding. This did not occur for any of the other VHSV proteins tested. The highest specific activity of PS-binding, however, was found for purified VHSV. PS-binding to purified VHSV was abolished by any VHSV treatment that removed the glycoprotein G from the virions and was partially inhibited by anti-p2 mouse antibodies. It was higher at pH 5.6 than at pH 7.6. The identification of the fish rhabdovirus main phospholipid binding domain allowed some preliminary comparative sequence studies that showed that p2-like sequences exist in all rhabdoviruses.

Amino Acid Sequence↗
PubMed1994

Recombinant protein fragments from haemorrhagic septicaemia rhabdovirus stimulate trout leukocyte anamnestic responses in vitro.

This work shows that viral protein fragments are capable of stimulating fish anamnestic immunological responses in leukocytes from the rainbow trout (Oncorhynchus mykiss, W.). Recombinant protein fragments of glycoprotein and nucleoprotein from the rhabdovirus causing viral haemorrhagic septicaemia of trout (VHSV), were cloned and expressed in Escherichia coli, Yersinia ruckeri (a trout pathogen) and Saccharomyces cerevisiae. The recombinant protein fragments stimulated anamnestic responses in leukocyte cultures derived from the anterior kidney of survivors of VHSV infection but not from uninfected trout. Two types of stimulatory anamnestic responses were detected, (i) a stimulation of lymphoproliferation as measured by thymidine incorporation assays and (ii) an increase in number, spreading and size of cells as determined by fibrin-clot and/or flow cytometry techniques. The evidence presented suggests that both adherent and non-adherent trout cell populations are needed for the immunological response to VHSV in this primitive vertebrate. The possible use of in vitro lymphoproliferation assays as a preliminary screening method for candidate fish vaccines prior to their testing in vivo is discussed.

Animals↗
PubMed1993

Characterization of a soluble hemagglutinin induced in African swine fever virus-infected cells.

Hemadsorption (Had) of erythrocytes to the surface of African swine fever virus (ASFV)-infected cells is a well-known phenomenon but hemagglutination of pig erythrocytes in the supernatant of ASFV-infected cells has not been reported before. We report here the discovery of a pig erythrocyte-agglutinating activity released to the in vitro cell culture medium by cells infected with some isolates of ASFV. This finding allowed the identification and characterization of a soluble hemagglutinin (HA) molecule that could be separated from the ASFV particles either by ultracentrifugation or by gel-permeation chromatography. The HA was inactivated by agents known to affect protein conformation such as heat, beta-mercaptoethanol, urea, and guanidine isothiocyanate. Glycosylation seemed to be of importance since treatment of HA with glycosidase F inhibited the hemagglutinating activity and HA could be partially purified by affinity chromatography on immobilized concanavalin A. When native it had an estimated molecular weight of 300 kDa by gel-permeation chromatography yielding 51-kDa protein monomers under denaturing conditions as identified by immunoblotting. Preliminary attempts to correlate the induced anti-HA serum antibodies with viremia or infection-inhibition serum antibodies after infection of pigs with attenuated ASFV or immunization with purified HA are also reported.

African Swine Fever↗
PubMed1993

The in vitro infection of the hematopoietic stroma of trout kidney by hemorrhagic septicemia rhabdovirus.

Viral hemorrhagic septicaemia virus (VHSV) infected the hematopoietic stromal cells (7,8) derived from pronephritic tissue of the rainbow trout, Oncorhynchuss mykiss, W., at their ninth passage in vitro. Viral infection resulted in the development of lytic cytopathic effects on confluent in vitro tridimensional network stromal cell cultures. Replication of VHSV in the stromal cell cultures was demonstrated by the increase in infectivity by epithelioma papulosum cyprini (EPC) cell culture assays and by the increase of the nucleoprotein antigen of VHSV by ELISA. By using anti-VHSV monoclonal antibodies (MAbs), flow cytometry studies demonstrated that only the infected stromal cells contained cytoplasmic viral antigens. The lytic infection of trout hematopoietic stromal cells in vitro could be relevant to the hemorrhagic pathology seen in the kidney of fish infected with VHSV.

Animals↗
PubMed1993

Enhancement of fish mortality by rhabdovirus infection after immunization with a viral nucleoprotein peptide.

A similar sequence to a mouse immunodominant CTL peptide (SYVLQGN, single-letter amino acid code, conserved amino acids underlined) identified in the nucleoproteins of several strains of vesicular stomatitis virus (VSV) (37) was found in the nucleoproteins of viral hemorrhagic septicemia virus (VHSV) of salmonid fish (GYVYQGL in VHSV 07.71 and GYVYQGS in VHSV Makah) and not in the nucleoproteins of other rhabdoviruses. The in vivo immunization of fingerling salmonid fish (rainbow trout Onchorynchus mykiss, W) with this VHSV peptide and their subsequent challenge with VHSV resulted in the enhancement rather than in the reduction of fingerling trout mortality. Possible implications for the development of subunit vaccines against VHSV are discussed.

Amino Acid Sequence↗
PubMed1992

Neutralizing-enhancing monoclonal antibody recognizes the denatured glycoprotein of viral haemorrhagic septicaemia virus.

Two monoclonal antibodies (MAbs), 1H10 and 1F10, have been selected against the denatured glycoprotein of the viral haemorrhagic septicaemia virus (VHSV) of salmonids by immunoblotting. Three reference VHSV serotypes (F1, F2, 23.75) and five VHSV isolates from either different host species (trout, salmon, barbel) or geographical locations in Spain reacted with both MAbs by ELISA. In vitro neutralization of all VHSV serotypes but not infectious haematopoietic necrosis virus (IHNV) was obtained only with MAb 1H10. However, when the MAb 1H10 (but not the non-neutralizing Mab 1F10) concentration was below 100 micrograms/ml rather than neutralizing VHSV infectivity, plaque counts increased 2-3-fold. MAb 1H10 is unique in that it has both, an in vitro enhancing infectivity effect (not described yet for VHSV) and it recognizes denatured G protein in contrast to other previously described neutralizing MAbs against VHSV.

Animals↗
PubMed1992

In vitro studies and in vivo immunisation with the first viral haemorrhagic septicaemia viruses isolated in Spain compared to international reference serotypes.

The first five viral haemorrhagic septicaemia virus (VHSV) isolates found in Spain were examined for in vitro growth characteristics, neutralisation by trout antiserum and immunisation challenge of trout fingerlings by water immersion. The viruses had come from different host species in various geographical locations in different years. Three reference VHSV serotypes (F1, F2 and 23.75) were also included in the study. There appeared to be little relationship between the in vivo delay of mortality or the protection of the immunised trout after challenge and the in vitro characteristics studied. In contrast to the in vitro results, the in vivo delay of mortality suggested a closer relationship of the Spanish VHSV isolates to the F2 serotype than to the F1 or the 23.75 serotypes. If the final protection figures are analysed, however, there could be three or four groups of viruses.

Animals↗
PubMed1992

Mitogen-induced proliferation of trout kidney leucocytes by one-step culture in fibrin clots.

This work describes a trout kidney leucocyte mitogen-stimulation assay using fibrinogen/thrombin. Cloning was obtained by only one step instead of by two steps, as required by the agar method described to date. Cultures were stimulated not only with phytohemagglutinin (PHA) and Escherichia coli lipopolysaccharides (LPS) but also with Concanavalin A (Con A) and six LPS from aquatic pathogenic bacteria. The number of colony-forming cells detected, and their morphological type depended on the mitogen, the time of incubation and the trout. PHA was the best inducer of trout kidney leucocyte colony formation followed by Con A, giving rise to four different homogeneous types of colonies formed by large-nucleated cells, cells with eccentric nuclei, multinucleated cells and lymphocytes.

Animals↗
PubMed1992

Susceptibility of trout kidney macrophages to viral hemorrhagic septicemia virus.

Viral hemorrhagic septicemia virus (VHSV) lysed the macrophages from rainbow trout kidney cultures either isolated by plastic adherence or stimulated with purified glycoprotein G from VHSV. The trout macrophages supported the replication of VHSV as tested by cell culture and by sandwich ELISA of the supernatants from infected cultures. VHSV-infected macrophages showed a decrease in both acridine-orange fluorescence and average size. Immunofluorescence studies with flow cytometry showed positive membrane staining with monoclonal antibodies (MAbs) anti-N and anti-G VHSV. These findings open the possibility of using trout macrophages as presenting cells to study the possible existence of helper or cytotoxic epitopes relevant to the protection of trout against VHSV.

Animals↗
PubMed1992

Detection of hemorrhagic septicemia virus of salmonid fishes by use of an enzyme-linked immunosorbent assay containing high sodium chloride concentration and two noncompetitive monoclonal antibodies against early viral nucleoproteins.

Inclusion of high-ionic strength buffers helped us to develop a sandwich ELISA to detect hemorrhagic septicemia virus (HSV) in cell culture and infected trout tissue extracts. For maximal sensitivity of 0.1 to 0.2 ng/well/100 microliters or about 10 to 50 TCID50/well/100 microliters, trout extracts were diluted 1:1 and assayed for the earliest synthesized nucleoprotein N. Simultaneous binding of the N protein from HSV in the sample to the wells coated with monoclonal antibody (2D5 against the N protein) and to the peroxidase-labeled monoclonal antibody (2C9 against the N protein) proceeded during a 2-hour incubation at 20 to 22 C (room temperature). The response was linear between 6 to 60 ng/well of purified virus. Monoclonal antibodies used were noncompetitive with each other and reacted with F1, F2, 23.75, and 5 Spanish isolates of HSV, but not with infectious hematopoietic necrosis or infectious pancreatic necrosis viruses. Tissue specimens with low content of HSV virus may now be assayed directly without use of cell culture, rapidly, and with high precision, during the acute phase of the disease in salmonid fishes.

Animals↗
PubMed1991

The free nucleocapsids of the viral haemorrhagic septicaemia virus contain two antigenically related nucleoproteins.

A protein of 34 kDa (Nx) was induced in vitro by the infection of fish cell cultures with the rhabdovirus agent of viral haemorrhagic septicaemia (VHS) of the trout. This protein only appeared as a major component in concentrated or intracellular labeled VHS virus but not in purified VHS or in the related infectious haematopoietic necrosis virus. That Nx protein is antigenically related to the nucleoprotein of purified virus was shown by its reaction with four anti-nucleoprotein monoclonal antibodies (at least 3 of them reacting non competitively against different epitopes) and by immunoprecipitation with polyvalent international reference sera. The Nx protein was shown to be specifically associated with free non-infective particles isolated by ultracentrifugation which were confirmed to be nucleocapsids by electron microscopy.

Animals↗