The Ras-specific exchange factors mouse Sos1 (mSos1) and mSos2 are regulated differently: mSos2 contains ubiquitination signals absent in mSos1.

We have compared aspects of the mouse sos1 (msos1) and msos2 genes, which encode widely expressed, closely related Ras-specific exchange factors. Although an msos1 plasmid did not induce phenotypic changes in NIH 3T3 cells, addition of a 15-codon myristoylation signal to its 5' end enabled the resulting plasmid, myr-sos1, to induce approximately one-half as many foci of transformed cells as a v-H-ras control. By contrast, an isogenic myr-sos2 plasmid, which was made by fusing the first 102 codons from myr-sos1 at homologous sequences to an intact msos2 cDNA, did not induce focal transformation directly, although it could form foci in cooperation with c-H-ras. Pulse-chase experiments indicated that the half-life of Sos1 in NIH 3T3 cells was greater than 18 h, while that of Sos2 was less than 3 h. While in vitro-translated Sos1 was stable in a rabbit reticulocyte lysate, Sos2 was degraded in the lysate, as were each of two reciprocal chimeric Sos1-Sos2 proteins, albeit at a slower rate. In the lysate, Sos2 and the two chimeric proteins could be stabilized by ATPgammaS. Unlike Sos1, Sos2 was specifically immunoprecipitated by antiubiquitin antibodies. In a myristoylated version, the chimeric gene encoding Sos2 at its C terminus made a stable protein in NIH 3T3 cells and induced focal transformation almost as efficiently as myr-msos1, while the myristoylated protein encoded by the other chimera was unstable and defective in the transformation assay. We conclude that mSos2 is much less stable than mSos1 and is degraded by a ubiquitin-dependent process. A second mSos2 degradation signal, mapped to the C terminus in the reticulocyte lysate, does not seem to function under the growth conditions of the NIH 3T3 cells.

The magnetic moment of NiO nanoparticles determined by Mössbauer spectroscopy.

We have studied the magnetic properties of (57)Fe-doped NiO nanoparticles using Mössbauer spectroscopy and magnetization measurements. Two samples with different degrees of interparticle interaction were studied. In both samples the particles were characterized by high-resolution transmission electron microscopy and x-ray diffraction and found to be plate-shaped. Computer simulations showed that high-field Mössbauer data are very sensitive to the size of the uncompensated magnetic moment. From analyses of the Mössbauer spectra we have estimated that the size of the uncompensated magnetic moment is in accordance with a model based on random occupation of surface sites. The analyses of the magnetization data gave larger magnetic moments, but the difference can be explained by the different sensitivity of the two methods to a particle size distribution and by interactions between the particles, which may have a strong influence on the moments estimated from magnetization data.

Sensitivity of wild type and mutant ras alleles to Ras specific exchange factors: Identification of factor specific requirements.

We have investigated the productive interaction between the four mammalian Ras proteins (H-, N-, KA- and KB-Ras) and their activators, the mammalian exchange factors mSos1, GRF1 and GRP, by using a modified Saccharomyces cerevisiae whose growth is dependent on activation of a mammalian Ras protein by its activator. All four mammalian Ras proteins were activated with similar efficiencies by the individual exchange factors. The H-Ras mutant V103E, which is competent for membrane localization, nucleotide binding, intrinsic and stimulated GTPase activity as well as intrinsic exchange, was defective for activation by all factors tested, suggesting that the integrity of this residue is necessary for catalyzed exchange. However, when other H-Ras mutants were studied, some distinct sensitivities to the exchange factors were observed. GRP-mediated, but not mSos1-mediated, exchange was blocked in additional mutants, suggesting different structural requirements for GRP. Analysis of Ras-mediated gene activation in murine fibroblasts confirmed these results.

BALB/c mice infected with Brucella abortus express protracted polyclonal responses of both IgG2a and IgG3 isotypes.

A polyclonal IgG2a response dependent on the secretion of endogenous IFN-gamma has been demonstrated in BALB/c mice injected with killed whole cells of Brucella abortus [1]. Here we report intense and protracted polyclonal responses of IgG2a and also of IgG3 isotypes in BALB/c mice undergoing primary infections with B. abortus attenuated vaccine strain 19 or virulent strain 2308. Ratios of total serum Ig levels between infected mice and age matched controls were greater than 38 for IgG3 and greater than 12 for IgG2a between weeks 4 and 8 post-infection. Polyclonal increases of IgM and IgG1 that were proportionally much lower (ratios < 2 and < or = 3, respectively) also occurred in infected mice during this time. It is hypothesized that both IgG3 and IgG2a polyclonal responses required IFN-gamma, which was induced by B. abortus primarily in a T cell-independent fashion during the first weeks of infection, and from T cells thereafter.

Many progestin receptor-containing neurons in the guinea pig ventrolateral hypothalamus contain substance P: immunocytochemical evidence.

Behavioral and neuroanatomical evidence in rats suggests that substance P may mediate hormonally induced sexual receptivity. To determine if progestin receptor-containing neurons in the guinea pig hypothalamus also contained substance P, we used a double antibody, immunocytochemical technique. Ovariectomized, adult guinea pigs were estradiol-primed for one week. Two days prior to perfusion, colchicine was infused into the lateral ventricle. Substance P-immunoreactive neurons and progestin receptor-immunoreactive neurons were found throughout the hypothalamus and caudal preoptic area. However, substance P and progestin receptor immunoreactivity occurred in the same neurons almost exclusively in the ventrolateral area of the hypothalamus, an important site in the hormonal regulation of female sexual behavior in guinea pigs. Within this area, approximately 35% of the progestin receptor-immunoreactive neurons also contained substance P immunoreactivity. These neuroanatomical data are consistent with the hypothesis that substance P may mediate some of the neural actions of progesterone.

Characterization of monoclonal antibodies for the rapid detection of foodborne campylobacters.

The specificity of 97 monoclonal antibodies (MAbs) to the Campylobacter jejuni Lior serogroup 6 reference strain was assessed using an indirect enzyme linked immunosorbent assay (ELISA). Four MAbs, M316, M337, M357 and M637, reacted with whole cells of the C. jejuni, C. coli and C. lari reference strains of the 20 most common Lior serogroups and 25 recent C. jejuni and C. coli isolates, and did not react with most of the 42 other Campylobacter and non-Campylobacter spp. tested. Immunoblot analysis revealed that MAbs M337 and M357 reacted with a protein component with molecular mass of approximately 62 kiloDaltons (kDa) while M316 and M637 reacted with protein components of approximately 92 and 31 kDa, respectively. The detection limit of M357 in an indirect ELISA was 10(5) colony forming units. These four highly specific MAbs may be useful reagents of an immunoassay for the rapid detection of thermophilic campylobacters in foods and clinical samples.

Antibody dynamics in BRSV-infected Danish dairy herds as determined by isotype-specific immunoglobulins.

Using specific ELISAs, antibody levels of four different isotypes to bovine respiratory syncytial virus (BRSV) were determined in calves, following experimental BRSV infection. Most calves experienced an increase in the specific IgM and IgG1 titres about 6-10 days after infection with BRSV. The IgM titre was transient showing positive titres for only 5-10 days, while specific IgG1 was present for a longer time. IgA was detected concomitantly with IgM but at a lower level. Production of IgG2 anti-BRSV antibodies was detected from 3 weeks after infection. In two closed herds, repeated blood samplings were performed on young stock to analyse maternal immunity. The passively transferred antibodies were mainly of the IgG1 isotype and the half-life of IgG1 to BRSV was estimated to be 26.6 days. One of the herds had an outbreak of enzootic pneumonia, diagnosed to be caused by BRSV. Furthermore, another herd with acute BRSV was followed by weekly blood samples in six calves; in both herds IgM and IgG1 was detected shortly after the appearance of clinical signs. Serum samples from 50 Danish dairy herds (453 samples) were tested for immunoglobulins of the isotypes IgG1, IgG2 and IgM. The presence of antibodies to BRSV was widespread and more than 54% of the samples had BRSV antibodies of both the IgG1 and IgG2 isotypes indicating a high herd prevalence to BRSV. Test samples from two herds out of 50 were free from all isotypes to BRSV.

Bovine monoclonal antibody specific for Brucella abortus lipopolysaccharide.

The development of a bovine monoclonal antibody against Brucella abortus smooth lipopolysaccharide (BM-8) by interspecies fusion of bovine peripheral lymphocytes from an immunized cow and a murine plasmacytoma cell line is described. The twice cloned cell line secreted bovine IgG1 subclass antibody. Ascites fluid was prepared in pristane treated nu/nu mice by intraperitoneal injection. The pooled ascites fluid was purified by affinity chromatography and the functions of the antibody assessed in various serological tests. The BM-8 antibody did not agglutinate well at a neutral pH, however, under acid conditions it was efficient at agglutinating B. abortus cells. The antibody did not precipitate B. abortus LPS in double agar gel immunodiffusion but was very active in the direct complement fixation test and the indirect enzyme immunoassay, although it was unable to compete with a murine monoclonal antibody in a competitive enzyme immunoassay.

Improved competitive enzyme immunoassay for the diagnosis of bovine brucellosis.

A modification of the competitive enzyme-linked immunosorbent assay (C-ELISA) for differentiating the antibody response of cattle vaccinated with Brucella abortus strain 19 and B. abortus infected cattle is described. This assay utilizes lipopolysaccharide as the antigen, immobilized on a polystyrene matrix, and a monoclonal antibody (M84) with specificity for an epitope of the O-polysaccharide. A goat anti-mouse IgG antibody-enzyme conjugate is used for detection. The specificity of the modified assay was 99.7% when 1446 sera from brucellosis free herds were tested and it correctly identified 636 sera from B. abortus infected cattle as positive, using a cut-off of 30% inhibition, for a sensitivity estimate of 100%. No reactions were noted among 261 sera from vaccinated cattle. However, in testing 1147 sera that gave positive reactions in the buffered plate antigen test, the indirect ELISA, the complement fixation test or a combination of these tests from the serum bank, 31 gave positive reactions. Twenty-seven of the 31 sera originated from recently vaccinated cattle. The overall specificity for sera from vaccinated cattle was 97.3%. Because of the sensitivity and specificity of this procedure and its ease of performance, it would be a reasonable alternative as a single assay for serological diagnosis of brucellosis.

Binding of bovine IgG2a and IgG2b allotypes to protein A, protein G, and Haemophilus somnus IgBPs.

Immunoglobulin binding proteins (IgBPs) are thought to be virulence factors which enable pathogens to evade the host's immune response. Since bovine IgG2 is important in protection against pyogenic infections, the binding characteristics of Staphylococcus aureus protein A (PrA), streptococcal protein G (PrG), or Haemophilus somnus high molecular weight IgBPs to the two bovine IgG2 allotypes were examined. For PrA or PrG binding of IgG2, guinea pig red blood cells coated with specific IgG2a or IgG2b antibodies were used in a competitive binding inhibition assay with unlabeled and horseradish peroxidase-labeled PrA or PrG. To determine which sizes of H. somnus. IgBPs bind to the two IgG2 allotypes, immunoblots with H. somnus culture supernatant were probed with anti-DNP IgG2a and IgG2b. This detects only Fc binding because anti-DNP does not cross-react with H. somnus antigens. Both IgG2 allotypes bound equally well to PrA and PrG. However, IgG2b but not IgG2a bound to H. somnus high molecular weight IgBPs. The lack of differential binding of bovine IgG2 allotypes to PrA and PrG means that these IgBPs can be considered to be unbiased reagents in assays for detection of bovine IgG2 or for immunoaffinity purification. The differential binding of H. somnus IgBPs to the IgG2 allotypes indicates that animals having one allotype may be more resistant to H. somnus infection than animals having the other allotype.

A review of enzyme immunoassay for detection of antibody to Brucella abortus in cattle.

Enzyme immunoassay has gained wide acceptance for serological diagnosis of bovine brucellosis because of its ability to detect antibody of all isotypes unlike the conventional tests. The indirect enzyme immunoassay, however, presents several parameters that require careful analysis. These parameters include the choice of antigen and antiglobulin-enzyme conjugate reagents for use in the assay, dealing with the large amount of data the semi-automatic or automatic assay can generate and the inter- and intralaboratory standardization and quality control. This review considers the various methods described in the literature and, briefly, how some of the problems have been overcome or how they might be dealt with.

Derivation of monoclonal antibodies against Brucella abortus antigens.

In vivo immunization, fusion, antibody detection, and cryopreservation procedures for monoclonal antibody production against antigens of Brucella abortus are described. Splenocytes from BALB/c mice immunized with irradiated B. abortus S2308 were fused with Sp2/O-Ag14 myeloma cells and 61 hybridomas secreting anti-Brucella antibodies were cloned. Hybridoma antibody synthesis was detected effectively and most efficiently by enzyme-linked immunosorbent assays. Antibodies from clones of hybridoma A23 reacted with S19 and S2308 whole bacterial cells, while hybridoma B49 reacted primarily with alkali--treated lipopolysaccharides of S19, S1119.3 and S2308. Cryopreservation of clones had no major effect on antibody synthesis. The application of monoclonal anti-Brucella antibodies in the differential diagnosis of bovine brucellosis is discussed.

Haemolysis in gel test for detecting bovine antibodies to Brucella abortus lipopolysaccharide.

Optimal reagent parameters for a haemolysis in gel test for detection of bovine anti-Brucella abortus antibody were established. Using an alkali-treated crude lipopolysaccharide antigen attached to J-negative bovine erythrocytes, 0.125 mg of antigen, 0.1 ml of erythrocytes and 0.4 ml of guinea pig complement were required to perform 15 tests with appropriate serum controls. The haemolysis in gel test and an IgM radioimmunoassay (RIA) procedure were compared for their ability to detect the onset of increased serum antibody levels and antibody levels in sera diluted to extinction. While the RIA was more sensitive in amount of antibody detected, the haemolysis in gel test was equally able to detect initiation of antibody synthesis.

Interaction of specifically purified isotypes of bovine antibody to Brucella abortus in the haemolysis in gel test and enzyme-linked immunosorbent assay.

The detection limits for purified bovine IgM, IgG1 and IgG2 antibody to Brucella abortus in the haemolysis in gel test and in an enzyme-linked immunosorbent assay (ELISA) were established. The limits for IgM, IgG1 and IgG2, respectively, were: 5500, 60 and 4850 ng of antibody in the lytic test and 2750, 3.5 and 16 ng in the ELISA test. Bovine IgG2 produced lysis only in the presence of fresh, whole bovine serum.

Bovine IgG2a antibodies to Haemophilus somnus and allotype expression.

Bovine IgG2a has been implicated in protection against pyogenic infections, including those caused by Haemophilus somnus. To further investigate the role of IgG2a in defense against H. somnus, IgG1 and IgG2a antibodies were purified from antiserum against an immunodominant 40 kDa outer membrane protein (p40) of H. somnus, which was previously shown to passively protect calves against H. somnus pneumonia. The passive protective capacity of anti-p40 IgG1 or IgG2a was evaluated in vivo in calves. Purified anti-p40 IgG1 or IgG2a was incubated with H. somnus for 15 min before intrabronchial inoculation of calves. Bacteria incubated with anti-p40 IgG1 or IgG2a were inoculated into one caudal lung lobe and bacteria incubated with IgG1 or IgG2a from the respective preimmunization serum were inoculated into the contralateral lobe. The volumes of pneumonia in the right and left lungs were determined 24 h later. The difference in volume of pneumonia with H. somnus preincubated in IgG1 pre- and postimmunization anti p40 was less (16 cm3, P = 0.298) than the difference in volume of pneumonia with H. somnus preincubated in IgG2a pre- and postimmunization anti p40 (30 cm3, P = 0.146). Although the differences in lesion size between pre- and postimmunization serum were not statistically significant, the trend suggests IgG2a may be more protective than IgG1. To examine this further, the peptide specificity of these IgG1 and IgG2a antibodies to p40 was examined. After limited proteolysis of p40, IgG2a antibodies reacted with 2 peptides not recognized by IgG1 antibodies. Other peptides were recognized by both isotypes. Since these studies suggested that IgG2a may be important in protection against infection, we then investigated some aspects of the role of the 2 IgG2a allotypes, A1 and A2. In retrospective studies of age differences in expression of IgG2a allotypes, no heterozygotes were detected in calves of 60 d old or less, and fewer heterozygotes were detected in calves 61-120 d old than in cattle older than 270 d (P < 0.01). In a subsequent prospective study of the time course of allotype expression, Holstein calves shown to be heterozygotes expressed the IgG2aA1 allotype early but the IgG2aA2 allotype was not usually detected until 3 to 4 mo of age. Thus, both the retrospective and the prospective studies showed age related differences in expression of the IgG2aA1 and A2 allotypes. This could have implication in protection.

An evaluation of selected screening tests for bovine paratuberculosis.

The objective of this study was to evaluate the performance of the lipoarabinomannan antigen enzyme-linked immunosorbent assay (LAM-ELISA), carbohydrate antigen complement fixation (CH-CFT), and protein D antigen agar gel immunodiffusion (D-AGID) tests for bovine paratuberculosis, relative to histopathology, and to culture and isolation of Mycobacterium paratuberculosis from tissues and feces. Samples for test evaluation were collected from four sources including blood and tissues from 400 cull cows at three abattoirs in Ontario, blood and feces from a paratuberculosis survey of cattle from 120 dairy farms in Ontario, a serum bank containing samples from cattle from Ontario and Québec, and a bank of sera from cattle from Pennsylvania and the northeastern United States. The data were analyzed using receiver operator characteristic curves, estimates of relative sensitivity and specificity, and kappa statistics of agreement between tests. The LAM-ELISA performed significantly better than both the CH-CFT and the D-AGID tests. The LAM-ELISA was better at predicting fecal shedding status than tissue infection. However, the LAM-ELISA also had limitations. When interpreted as positive or negative (+/-), at a critical optical density of 0.675, its sensitivity and specificity relative to bacteriology were 49% and 87% respectively. Although the serological tests examined in this study provided some information, they did not predict well the infection status of individual animals.

Evaluation of an enzyme-labeled antiglobulin test for anti-Brucella immunoglobulin G among 3 cattle populations.

Antibody to smooth Brucella abortus lipopolysaccharide antigen on the surface of polystyrene tubes was detected with peroxidase-labeled antibody against bovine immunoglobulin G. The enzyme-labeled antiglobulin test (ELAT) activity of samples was expressed in arbitrary units/0.01 ml by reference to a standard curve based on tests of dilutions of a positive serum pool. Reactions greater than 3.0 U/0.01 ml were classified positive because specificity at this level was 99.8% (417/418 samples correctly classified negative) with agglutination test-negative sera from 33 Brucella-free herds. Results of the ELAT were compared with results of agglutination tests and the complement-fixation test (CFT), using 430 sera from cattle in 7 infected herds. Activity of greater than 5.0 ELAT U/0.01 ml was detected in all 54 sera classified as positive (titer greater than 1:10) by the CFT, including 5 sera classified as negative by the tube agglutination test. Sera from 8 nonvaccinated cows in the infected herds reacted only by the ELAT, whereas reactions were obtained with 25 and 5 sera by only agglutination tests and the CFT, respectively. The ELAT and CFT results were in agreement for 25 of 26 sera from agglutination test-reactor cattle in herds of unknown status. Comparisons of milk ring and whey agglutination tests with the whey ELAT on 146 quarter samples from cows in an infected herd revealed no ELAT activity greater than or equal to 1.0 U/0.01 ml in the 73 samples considered negative by the 2 other tests. Samples (n = 47) that contained greater than or equal to 1.0 ELAT U/0.01 ml included all (n = 40) samples with milk ring or whey agglutination titers greater than or equal to 1:16 and greater than or equal to 32, respectively, and 7 samples that gave weaker reactions to the latter tests.

Serodiagnosis of ovine paratuberculosis, using lipoarabinomannan in an enzyme-linked immunosorbent assay.

The use of lipoarabinomannan (LAM; obtained from Mycobacterium paratuberculosis) in and ELISA (LAM-ELISA) to test 75 sheep sera from a paratuberculosis-infected flock resulted in an approximate threefold increase in sensitivity (from 23.5% to 70.6%), compared with the use of Annau's polysaccharide in a complement fixation test (P-CFT). Even after manipulation of the LAM-ELISA cut-off value to produce a specificity of 100% to match that of the P-CFT, the sensitivity still was approximately twofold greater than that of the P-CFT. Anti-bovine monoclonal antiglobulin-enzyme conjugates matched commercially available anti-ovine polyclonal antiglobulin-enzyme conjugates with respect to sensitivity and specificity. False-positive results were found to be less frequent after combining 2 serodiagnostic tests, LAM-ELISA and D antigenagar gel immunodiffusion, resulting in an increase in specificity from 88.1% to 95.2%. The repeatability of true seropositive and seronegative results was found to be 89.5% and 91.1%, respectively, for sera obtained less than or equal to 1 month prior to slaughter and 91.7% and 95.5%, respectively, for reanalysis of sera obtained at the time of slaughter.

Hemoparasitism, humoral immunodeficiency, and an IgG1 fragment in a cow.

An adult cow with nonregenerative anemia was found to be infected with Eperythrozoon wenyoni and Trypanosoma theileri. Further laboratory testing revealed hypoalbuminemia, hypogammaglobulinemia, decreased numbers of circulating B lymphocytes, and presence of a serum immunoglobulin G1 fragment. Reduced B-lymphocyte regions in lymphoid tissue, evidence of chronic nephritis, and infection with Fasciola hepatica and Sarcocystis spp were found at necropsy. The cause of the acquired humoral immunodeficiency and the serum immunoglobulin G1 fragment was undetermined.

Enzyme immunoassay for detection of Mycoplasma bovis antigens in bull semen and preputial washings.

A capture enzyme immunoassay was developed for the detection of Mycoplasma bovis antigens in bull semen or preputial washings. IgG prepared from rabbits immunised with M bovis was passively adsorped to 96-well polystyrene plates. This antibody captured M bovis antigens which were then detected by using an IgG preparation from an immunised cow and murine monoclonal antibody to the bovine L-chain conjugated with horseradish peroxidase. The sensitivity of the assay was approximately 200 colour changing units (ccu)/ml and the specificity was excellent in that other species of mycoplasma, ureaplasma or acholeplasma did not react. A blind study of bull semen experimentally contaminated with M bovis detected all specimens with more than 200 ccu/ml.