A casein kinase I activity is constitutively associated with Nck.

Nck is a 47-kDa cytosolic protein devoid of intrinsic catalytic activity and consisting of Src homology 2 and 3 (SH2 and SH3) domains organized as follows: SH3-SH3-SH3-SH2. Nck is believed to act as an adaptor protein mediating signal transduction initiated by receptor tyrosine kinases (RTKs). Through its SH2 domain, Nck recognizes a specific phosphotyrosine residue on RTKs or on protein substrates of RTKs like insulin receptor substrate-1, the major substrate of the insulin receptor, and through its SH3 domains it interacts with poorly characterized effector molecules. To identify novel proteins that might interact with Nck, we have used the amino-terminal segment of Nck encompassing its three SH3 domains in the yeast two-hybrid system. Among the polypeptides that associate with Nck, we have identified the gamma2 isoform of the serine/threonine casein kinase I (CKI-gamma2). In transformed rat hepatocytes overexpressing the insulin receptor (HTC-IR cells), serine/threonine protein kinase activity coimmunoprecipitates with Nck, an interaction mediated mainly by the third SH3 domain of Nck. This kinase activity is not apparently modulated by insulin, nor is it sensitive to staurosporine or heparin, and it does not use GTP as a phosphate donor. However the kinase activity coimmunoprecipitated with Nck is completely abolished by N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide, a specific inhibitor of casein kinase I. In an in vitro renaturation gel kinase assay, a protein kinase of 70-75 kDa was detected associated with the SH3 domains of Nck. Far Western analysis demonstrated that the SH3 domains of Nck bound directly to a cytosolic protein of 70-75 kDa. A rabbit polyclonal antibody raised against the C-terminal region of CKI-gamma2 protein kinase immunoprecipitated a single specific protein of 70-75 kDa from HTC-IR cell lysates and detected CKI-gamma2 among the proteins coimmunoprecipitated with Nck. These results support an in vivo interaction between Nck and CKI-gamma2 and suggest that CKI-gamma2 could be involved in signaling pathways downstream of RTKs.

Molecular cloning, physical mapping and cross-hybridization of the murine adenovirus type 1 and type 2 genomes.

Murine adenovirus (MAd) type 1 strain FL and type 2 strain K87 genomes were cloned into plasmid pAT153 as HindIII restriction fragments. The MAd-1 and MAd-2 DNA genomes, 30.10 kb and 34.71 kb in length respectively, were mapped using BglII, ClaI, EcoRI, HindIII and SphI restriction endonuclease cleavage sites. In view of the large differences found between the MAd-1 and MAd-2 genomes in terms of the number and location of restriction sites, cross-hybridization experiments were performed. Homologous DNA sequences were located on the MAd-1 and MAd-2 physical maps. Both viruses are also genetically related to human adenovirus type 2 (HAd-2). Nucleotide sequences shared by HAd-2 and the MAds code for structural proteins, which may explain the antigenic similarities between these viruses from different origins. Our results confirm the existence of two distinct adenovirus species in the mouse.

Low tumorigenicity of canine cells transformed by the human cytomegalovirus.

Dog embryo kidney cells transformed by the human cytomegalovirus (HCMV) were obtained after non-permissive infection or transfection with viral DNA digested by restriction endonuclease EcoR I. The transformed cells, growing rapidly and showing an unlimited division potential, could use medium with only 2% serum for growth, contained nuclear virus antigens, and formed small colonies (less than 0.2 mm) in agarose. From 40 mice inoculated with transformed canine cells, only one eventually developed a tumor. Results indicate that dog cells are immortalized but not tumorigenically transformed by the human cytomegalovirus.

Experimental polyvalent ISCOMs subunit vaccine induces antibodies that neutralize human and bovine respiratory syncytial virus.

The purpose of the present study was to evaluate experimentally, in guinea-pigs, the immunogenicity of respiratory syncytial (RS) virus subunit vaccines. Immunostimulating complexes (ISCOMs), made from the surface proteins of both human (Long) and bovine (A-51908) RS strains adsorbed to the adjuvant Quil A, were assayed for their capacity to induce neutralizing antibodies, in comparison to experimental live virus vaccines. Serums from animals vaccinated with either the human or bovine RS subunit vaccines were equally efficient in neutralizing human or bovine RS virus. ISCOMs prepared with bovine RS virus proteins were significantly (p less than 0.05%) more efficient than their human counterpart, in inducing neutralizing antibodies, suggesting their greater potential as a subunit vaccine.

Comparison of caprine, human and bovine strains of respiratory syncytial virus.

A new continuous ovine kidney cell line allowing the growth of caprine, human and bovine respiratory syncytial virus was used to minimize host cell related variations for the direct comparison of the viral ultrastructures, serological relationships and structural protein profiles. Results show that all three strains are closely related although a closer relationship was found between bovine and caprine RS.

Further biological, serological and biochemical characterization of North American, European and Southeast Asian strains of bovine herpesvirus 1 compared with other alphaherpesvirinae members.

Hemagglutination activity, structural protein profiles and neutralization assays were used in a comparative study of bovine herpesvirus 1 strains from the U.S.A., Canada, Great Britain, Denmark and Malaysia with equine, feline and human herpesviruses in order to further characterize the bovine herpesvirus 1 hemagglutinin. Bovine herpesvirus 1 strains of different geographical origins all showed hemagglutinating activity for mouse erythrocytes; furthermore, feline herpesvirus 1 was also shown to hemagglutinate mouse erythrocytes. Analyses of partly purified viruses showed that a distinctive and specific polypeptides profile is associated with each species of herpesviruses used in our study; strains of bovine herpesvirus 1 from North America, Europe and Southeast Asia however, presented a remarkable similarity as to their electrophoretic protein patterns. A protein similar to the 97-kDa bovine viral hemagglutinin was not identified with the hemagglutinating feline herpesvirus. An important neutralization epitope on the bovine viral hemagglutinin was also not found on feline, equine and human herpesviruses but was identified on all bovine strains tested from North America, Europe and Southeast Asia stressing the importance of the bovine hemagglutinin for eventual prophylactic purposes.

Resistance to RadLV-induced leukemia: non-participation of splenic natural killer cells.

The phenotypic expression of genetically determined resistance to radiation leukemia virus (RadLV)-induced leukemia in mice has been shown to reside in the bone marrow. Because the bone marrow contains precursors of natural killer (NK) cells, known to play a role in retrovirally induced infections, and because these cells have been suggested as participating in resistance to radiation-induced leukemia, it was pertinent to establish whether their levels differed in strains of mice susceptible and resistant to leukemia. We therefore tested splenic NK cell levels in C57BL/Ka (susceptible) and B10.A(5R) (resistant) mice before viral inoculation, immediately after viral inoculation, and throughout the preleukemic period and showed that they were not different. This indicates that splenic NK cell levels have no bearing on the resistance to RadLV-induced leukemia and that other immune or non-immune mechanisms must be sought.

Genotypic differences between the mouse adenovirus strains FL and K87.

Restriction endonuclease analysis was used to compare the genome of mouse adenovirus (MAd) strains FL and K87. Large differences were found between the Kpn I, PaeR7, Pvu I, Sal I and Sma I restriction profiles of the prototype strains. MAd-FL and MAd-K87 thus represent two distinct species of mouse adenovirus.

Genome typing of mouse adenoviruses.

Restriction endonuclease cleavage site analysis was used to differentiate between mouse adenovirus (MAV) types 1 and 2 strains. Viral DNA of suitable purity and quantity for multiple enzymatic digestions was obtained from cloned CMT-93 mouse tumor cells infected with each type of MAV. Clear differences between the MAV-1 (FL) and MAV-2 (K87) genomes were observed after cleavage with restriction enzymes such as BglII, EcoRI, and PaeR7. Fast electrophoresis of DNA fragments in miniature agarose slab gels allowed rapid and unequivocal identification of the MAV strains. This relatively simple and accurate method should be quite useful to determine the different modes of transmission of mouse adenoviruses and their presence in various animal populations.

Serological relationship between mouse adenovirus strains FL and K87.

Three-week-old outbred mice were inoculated intraperitoneally or orally and intranasally with the FL or K87 strains of mouse adenovirus and bled at intervals after infection. Serum was tested by both the complement fixation and indirect immunofluorescence tests for reactivity with either virus antigen. A unilateral relationship was detected between FL and K87 strains. Serum from mice given the FL strain of virus reacted in both tests with FL and K87 antigens. Serum from mice given the K87 strain reacted only with the homologous antigen. Serum antibody titers were generally higher in the immunofluorescence test than in the complement fixation test. These observations stress the need to use both FL and K87 antigens for specific serologic diagnosis of adenovirus infection in mouse colonies.

Protection from mouse hepatitis virus type 3-induced acute disease by an anti-nucleoprotein monoclonal antibody. Brief report.

Fusion of MHV-3-immune splenocytes from MHV-3-resistant A/J murine strain, with NS myeloma cells produced several hybridomas. Among eight hybridoma clones, the 1E7A4H1 clone secreted kappa IgG2a apparently directed against the nucleoprotein of the MHV-3 virion. The monoclonal antibody was able to neutralize the in vitro cytopathic effect of MHV-3 on cultured L2 cells, and was detected by indirect immunofluorescence on MHV-3-infected cultured YAC cells. In addition, it conferred a significant protection against MHV-3-induced acute disease, if injected intraperitoneally to C57BL/6 mice before inoculation with MHV-3.

Bone marrow phenotype determines genetic resistance to RadLV-induced leukemia in radiation chimeric mice.

The development of RadLV-induced T-cell leukemia is a multistep process which evolves along the bone marrow-thymus axis. This process has been shown to be under the control of resistance and susceptibility genes. The relative importance of bone marrow and thymic phenotypes in this genetic control have not been established. We have constructed radiation chimeras with bone marrow from susceptible C57BL/Ka and thymus from resistant B10.A(5R) mice (and vice versa). The rate of leukemia development in the various groups indicates that the phenotype of the bone marrow and not that of the thymus determines the expression of resistance or susceptibility.

Bovine herpesvirus 1: strain comparison of polypeptides and identification of a neutralization epitope on the 90-kilodalton hemagglutinin.

The intracellular and structural polypeptides of the Los Angeles and Cooper 1 reference strains of bovine herpesvirus 1, together with 12 other Canadian field isolates, were analyzed by polyacrylamide gel electrophoresis. Although a few minor differences were noted among some isolates in regard to intracellular viral protein content, analysis of partly purified virus showed strikingly similar polypeptide profiles among 19 proteins with molecular masses of 14 to 145 kilodaltons (kDa). Moreover, a neutralizing monoclonal antibody produced against the Cooper 1 strain also neutralized all of the other 13 strains tested in this study and immunoprecipitated the major 90-kDa glycoprotein. A second monoclonal antibody with a high hemagglutination inhibition titer prevented hemagglutination of other strains tested and also reacted against the 90-kDa glycoprotein by immunoprecipitation, indicating that this glycoprotein is responsible for the hemagglutinating activity of the viral particle and carries an important neutralization epitope.

An enteric coronavirus of the rabbit: detection by immunoelectron microscopy and identification of structural polypeptides.

The immunoelectron microscopy (IEM) technique has been used for the detection of a rabbit enteric coronavirus (RECV). Immune serum was prepared in guinea pigs; the viral antigen used for the immunization procedure was obtained from the caecum of a sick rabbit, concentrated by centrifugation and purified on Percoll gradient. In order to identify the viral particles used in the immunization procedure, the protein pattern of the particles was determined by electrophoresis and compared with the pattern of other known coronaviruses. Analysis of structural polypeptides of the purified viral particles revealed a pattern similar to that reported for other coronaviruses. These polypeptides cross reacted with two other coronavirus specific immune sera (IBV and TGE). IEM assay of fecal samples collected from healthy and sick rabbits showed the presence of immune aggregates in specimens from both sick and healthy rabbits. Those aggregates contained viral particles sharing morphological characteristics with other coronaviruses. Furthermore, IEM assay was shown to be more sensitive than a direct EM procedure to detect coronavirus particles in rabbit feces. This assay also allowed the detection of a larger number of chronic carriers.

Serologic study on the prevalence of murine viruses in five Canadian mouse colonies.

A serologic study was made of five Canadian mouse colonies to determine the prevalence of antibodies to 11 murine viruses. A total of 139 sera from the five colonies were evaluated by complement fixation or hemagglutination inhibition methods. Viral antibodies were present in all five colonies. Antibodies to pneumonia virus of mice, minute virus of mice, Theiler's encephalomyelitis virus, and reovirus type 3 were found in all five colonies. K-virus antibodies were present in four colonies. Polyoma virus antibodies were found in two colonies and Sendai virus antibodies in two other colonies. Mouse hepatitis virus antibodies were present at a low prevalence in three of the five colonies. No antibodies to adenovirus, lymphocytic choriomeningitis virus, and ectromelia were detected in any of the colonies.

[Formation of spheriods by cultured human diploid cells]. / Formation de sphéroïdes par des cellules diploïdes humaines en culture.

When human lung diploid fibroblasts are grown in micro-wells in presence of the organic buffer HEPES in an atmosphere of 5% of CO2, the cells migrate and form spheroids. Histologic examination reveals the presence of fibroblastic and epithelioid cells in these spheroids. Signs of degeneration are seen in both cell types under the electron microscope. Following dissociation of the spheroids and their subculture in absence of HEPES, the cells reverse to their original fibroblast-like morphology and grow in monolayer.

Effect of anti-mouse macrophage serum on murine cytomegalovirus infection.

Rabbit anti-mouse macrophage serum (AMS) was used to study the role played by macrophages against murine cytomegalovirus infection. Treatment of mice with AMS enhanced morbidity and mortality following virus infection. These results are discussed in relation to the role of macrophages against virus infections.