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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
PubMed1998

Phospholipid interactions of a peptide from the fusion-related domain of the glycoprotein of VHSV, a fish rhabdovirus.

Previous studies mapped a p2 domain (aa 82-109) which binds phosphatidylserine (PS) (Estepa and Coll, 1996a) and contains three contiguous hydrophobic amino acid heptad repeats followed by a positively charged stretch (Coll, 1995b) in the glycoprotein G of the viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus. Anti-p2 antibodies inhibited low-pH VHSV-induced fusion (Estepa and Coll, 1997) and low-pH PS binding to VHSV (Estepa and Coll, 1996a). We report here further studies on the interaction of the synthetic peptide p2 with phospholipid vesicles. The synthetic p2 peptide was able to mediate aggregation, lipid mixing, and leakage of contents only with negatively charged phospholipid vesicles and in a concentration-dependent manner. As shown by its effect on lipid phase transitions deduced from data with fluorescence polarization and differential scanning calorimetry, the p2 peptide becomes inserted into the hydrophobic negatively charged phospholipid vesicle bilayers. In addition, data based on circular dichroism showed that the p2 peptide folds as a structure with a high content of beta-sheets stabilized by interaction with anionic phospholipids. These studies are potentially relevant to viral fusion in VHSV.

Amino Acid Sequence↗
PubMed1998

Purification of the glycoprotein G from viral haemorrhagic septicaemia virus, a fish rhabdovirus, by lectin affinity chromatography.

A new method for the isolation of glycoprotein G from viral haemorrhagic septicaemia virus (VHSV), a fish rhabdovirus, was developed by using affinity chromatography with immobilized Concanavalin A (ConA). The glycoprotein G was isolated from detergent solubilized concentrated virions and from large-volume virion-free supernatants from VHSV infected cells (soluble form). The purity achieved was higher than 85%. The estimated recovery of the initial glycoprotein G present in the virions was between 20 and 50%. These glycoprotein G preparations showed the presence of about 30% of trimers by ultracentrifugation, reacted with antibodies to the phosphatidylserine binding domain (p2) in a pH-dependent manner by ELISA and bound phosphatidylserine in a pH-dependent manner by solid-phase binding assays. These data suggest that ConA purified glycoprotein G conserved most of its native properties and conformation.

Animals↗
PubMed1997

An in vitro method to obtain T-lymphocyte-like cells from the trout.

We describe a methodology to obtain from the trout T-lymphocyte-like cell cultures showing in vitro antigen-dependent cell (ADC) proliferation. ADC cultures were developed from each of 3 outbred rainbow trout that survived two consecutive viral haemorrhagic septicaemia virus (VHSV) infections (an important salmonid fish disease in Europe). As stimulating antigen, we used a yeast recombinant form of the glycoprotein G (G4) of VHSV. No similar ADC cultures could be obtained from non-infected control trout kidney cells incubated in the presence of G4, confirming previously published observations. For long-term culture, and to overcome the absence of inbred trout populations, autologous haematopoietic adherent (Ad) cells were obtained and maintained from each individual trout kidney and periodically used as viral antigen-presenting cells (called G4-pulsed Ad cells) to autologous ADC cultures. The methodology to obtain trout ADC lines constitutes a new tool that would facilitate further studies on the comparative and developmental immunology of lower vertebrates. It could also be applied to research on fish vaccination and viral antigen-presentation mechanisms.

Animals↗
PubMed1997

Synthetic peptides from the heptad repeats of the glycoproteins of rabies, vesicular stomatitis and fish rhabdoviruses bind phosphatidylserine.

This work follows up on observations previously published concerning phosphatidylserine (PS) binding properties of synthetic peptides (p2) from the hydrophobic heptad repeats of the glycoprotein of viral haemorrhagic septicemia (VHS) rhabdovirus and the presence of similar repeats in the sequences of the glycoproteins of four separate rhabdoviruses. Similar p2-like peptides are now synthesized according to the corresponding cDNA sequences of infectious haematopoietic necrosis (IHN), rabies and vesicular stomatitis (VSV) viruses and shown to bind phosphatidylserine (PS) by solid-phase as well as from liquid-phase assays. The PS-binding peptides located in the amino-terminal part of the glycoproteins contained 3-5 contiguous heptad repeats (abcdefg) of hydrophobic amino acids (aa) in positions a and d followed by a short aa stretch containing positively charged aa and not belonging to the heptad repeats. The rhabdoviral PS-binding regions had low sequence variability among the members of each of the rhabdoviral genus but show no sequence similarity among the different genera.

Amino Acid Sequence↗
PubMed1997

Mapping specific protein-protein interactions within the core component of the breast cell DNA synthesome.

We have previously described the isolation and characterization of an intact multiprotein complex for DNA replication, designated the DNA synthesome, from human breast cancer cells and biopsied human breast tumor tissue. The purified DNA synthesome was observed to fully support DNA replication in vitro. We had also proposed a model for the breast cell DNA synthesome, in which DNA polymerases alpha, delta, and epsilon, DNA primase, and replication factor C (RF-C) represent members of the core component, or tightly associated, proteins of the complex. This model was based on the observed fractionation, chromatographic, and sedimentation profiles for these proteins. We report here that poly(ADP-ribose)polymerase (PARP) and DNA ligase 1 are also members of the breast cell DNA synthesome core component. More importantly, in this report we present the results of coimmunoprecipitation studies that were designed to map the protein-protein interactions between several members of the core component of the DNA synthesome. Consistent with our proposed model for the breast cell DNA synthesome, our data indicate that DNA polymerases alpha and delta, DNA primase, RF-C, as well as proliferating cell nuclear antigen (PCNA), tightly associate with each other in the complex, whereas DNA polymerase epsilon, PARP, and several other components were found to interact with the synthesome via a direct contact with only PCNA or DNA polymerase alpha. The association of PARP with the synthesome core suggests that this protein may serve a regulatory function in the complex. Also, the coimmunoprecipitation studies suggest that the three DNA polymerases alpha, delta, and epsilon all participate in the replication of breast cell DNA. To our knowledge this is the first report ever to describe the close physical association of polypeptides constituting the intact human breast cell DNA replication apparatus.

Breast↗
PubMed1996

Fast neutralization/immunoperoxidase assay for viral haemorrhagic septicaemia with anti-nucleoprotein monoclonal antibody.

An enzyme-immunohistochemical procedure was employed to facilitate neutralization/diagnostic tests for viral haemorrhagic septicaemia virus (VHSV), a significant pathogen in trout farms throughout Europe. The method described can be used for trout or mice antibodies; increases speed (1 day), simplicity, and minimizes the use of reagents compared to other neutralization assays. Furthermore, the test requires a minimum handling of the cell cultures under sterile conditions, decreasing frequent contamination due to the non-sterile conditions of the fish pathological samples. Foci of 5-20 infected epithelioma papillosum carp (EPC) cells are detected and counted with an inverted microscope in under 16 h after infection of EPC monolayers using a high titre anti-N VHSV monoclonal antibody (MAb) 2C9. MAb 2C9 recognizes different viral haemorrhagic septicaemia virus serotypes and VHSV isolates from different host species (trout, salmon and barbel) and Spanish geographical locations. The high titre and specificity of MAb 2C9 favour its conjugation to peroxidase and also make it possible to use in direct immunoperoxidase staining of the VHSV infected EPC monolayers. This neutralization/immunoperoxidase assay should improve diagnostics that use currently agarose or methylcellulose plaque reduction neutralization assays.

Animals↗
PubMed1996

Pepscan mapping and fusion-related properties of the major phosphatidylserine-binding domain of the glycoprotein of viral hemorrhagic septicemia virus, a salmonid rhabdovirus.

The binding of labeled phosphatidylserine (PS) to a collection of synthetic 15-mer peptides covering full-length glycoprotein G (G) of viral hemorrhagic septicemia virus (VHSV), a salmonid rhabdovirus, showed three dominant overlapping reactive peptides. This major PS-binding region was contained in a 28-mer peptide (p2; aa 82-109) with consecutive hydrophobic amino acid a-d heptad repeats (putative amphipathic alpha-helix) and 2 carboxy-terminal arginines. This 28-mer peptide showed a 10-fold higher apparent specific activity for PS binding than the 15-mer peptides. Binding to PS was also detected with virion-purified protein G but was not detected with other viral proteins. The highest apparent specific activity for PS binding was found with purified VHSV particles by both solid-phase and liquid assays. In contrast to the pH-independent PS binding to peptide p2, binding to virions was optimal at pH 5.6. PS binding to purified VHSV was greatly reduced by protease or detergent treatments that removed protein G, by treatment at pH 7.6, or by anti-p2 mouse antibodies at pH 5.6. The PS-binding region seems to be related to viral-host cell fusion since anti-p2 mouse antibodies inhibited VHSV-infected cell to cell fusion (fusion from within) and the pH profile of the VHSV-infected cell to cell fusion was similar to the pH profile of PS binding to VHSV. Comparative analysis showed that sequences similar to the major PS-binding domain of VHSV were also present in other fish rhabdoviruses and in rabies and vesicular stomatitis viruses.

Amino Acid Sequence↗
PubMed1996

The human cell multiprotein DNA replication complex (MRC): the effect of camptothecin on its ability to support in vitro DNA synthesis.

PURPOSE: We have previously reported on the isolation and characterization of a multiprotein DNA replication complex (MRC) from HeLa cells that fully supports in vitro DNA replication. Based upon its ability to replicate DNA in a cell-free environment (devoid of other cellular processes) the MRC may serve as a unique model system for investigating the mechanisms of action of anticancer drugs that directly affect DNA synthesis. The experiments described in this report were performed to establish whether the MRC could serve as a model system to examine in detail the mechanism of action of camptothecin, a DNA topoisomerase I inhibitor. METHODS: We examined the effects of increasing concentrations of camptothecin on HeLa cell survival, intact HeLa cell DNA synthesis and MRC-mediated in vitro DNA replication. We also performed topoisomerase I assays in the presence of increasing concentrations of camptothecin to study the direct effects of the agent on MRC-associated topoisomerase I activity. Furthermore, we employed an SDS precipitation assay to measure the formation of MRC-associated topoisomerase I-cleavable complexes in the presence of increasing concentrations of camptothecin. RESULTS: We found a close correlation between the IC50 values for intact HeLa cell DNA synthesis (0.15 microM) and MRC-mediated in vitro DNA synthesis (0.05 microM). Similarly, we found that 0.05 microM camptothecin inhibited MRC-associated topoisomerase I activity by approximately 50%. In addition, we found that the formation of MRC-associated topoisomerase I-cleavable complexes increased linearly with increasing concentrations of camptothecin. CONCLUSIONS: The data presented in this report support the use of the MRC as a model system to study the mechanism of action of camptothecin. We anticipate that future studies with the MRC will help elucidate the cellular consequences of camptothecin-cleavable complex formation.

Antineoplastic Agents, Phytogenic↗
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

Applications of monoclonal antibodies in aquaculture.

Monoclonal antibodies (MAbs) currently are being applied to the study of fish immunology and fish infectious diseases. MAbs to fish immunoglobulins (Igs) have helped isolate fish Igs, identify heavy and light chain variants in fish Ig, study the ontogeny of B lymphocytes, and improve techniques for the measurement of fish Ig and specific antibodies (Abs). MAbs have been obtained against several leucocyte surface antigens and are being used as markers for different subsets of fish leukocytes: neutrophils, non-specific cytotoxic cells and cells responsible for the mixed leucocyte reaction. The sensitivity and specificity of many immunoassays for identifying fish pathogens have been improved by the use of MAbs. Further improvement of these tests is currently being attempted by the use of MAbs together with the polymerase chain reaction (PCR). Epizootiological studies of fish diseases are beginning to emerge from the use of these reagents and techniques. In the near future these new methods should detect low levels of pathogens in adult carriers and perhaps in eggs, thus helping to prevent the dissemination of fish diseases. MAbs to fish pathogens are also being used for passive immunization studies as well as for conformational probes in the development of genetically engineered vaccines.

Journal Article↗
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↗