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Biomedical subjects

A C Ward

Publications and source records attributed to A C Ward.

At least 55 records · Page 3Linked to original sources
PubMed1999

Pasteurellaceae isolated from tonsillar samples of commercially-reared American bison (Bison bison).

As commercial producers of American bison (Bison bison) become more numerous, concerns relative to bison health management increase. Since loss due to respiratory disease associated with Pasteurella and related Pasteurellaceae is a major concern for cattle producers, a study was conducted to determine what types of Pasteurellaceae are carried by bison to evaluate the potential of pneumonic pasteurellosis in bison herds where management practices are comparable to those used for cattle. Tonsillar biopsies, collected in May (n = 29) and August (n = 25) 1997 from 24- to 30-month-old bison bulls, at the time of slaughter were cultured for Pasteurellaceae. Pasteurella spp. were isolated from all the samples collected in May. These included isolates identified as P. haemolytica, trehalosi, testudinis, and multocida subsp. multocida a and multocida b. Actinobacillus spp. and Haemophilus somnus were also isolated from some samples. Pasteurella spp., haemolytica, trehalosi, and multocida subsp. multocida a, multocida b and septica, plus 2 nonspeciated indole-positive biotypes, U2 and U16, were isolated from the second group of tonsil samples. Most of these organisms, including P. haemolytica, P. multocida subsp., and H. somnus are associated with disease in domestic livestock and should be regarded as potential pathogens for bison, particularly in animals which become stressed by management practices commonly used with cattle such as herding, crowding, and shipping.

Animal Husbandry↗
PubMed1999

Characterization of putative Haemophilus somnus isolates from tonsils of American bison (Bison bison).

Three Haemophilus somnus isolates (2a, 3a, and 27b) and one H. somnus-like (13b) isolate from tonsils of commercially reared American bison were compared with 2 known H. somnus isolates from cattle, namely, 2336, shown to cause respiratory disease, and 129Pt, from the prepuce of an asymptomatic bull. All H. somnus isolates, but not the H. somnus-like isolate, required CO2 for growth. Biochemical utilization profiles were identical for bison and bovine H. somnus isolates with the exception of alpha-fucosidase production by isolate 3a. Isolate 27a varied from 2a, 2336 and 129Pt by hemolysis of bovine erythrocytes. Isolate 13b hemolyzed sheep but not bovine or bison erythrocytes and varied from other isolates in biochemical utilization tests. Outer membrane protein profiles of 2a, 3a and 27a were almost identical with those of bovine isolate 2336 and similar to that of 129Pt, but quite different from that of 13b. Western blots of bison isolates were similar to that of the virulent bovine 2336 isolate, including detection of high molecular mass antigens above 100 kDa and the 76 kDa antigens associated with bovine IgG2 Fc binding characteristic of virulent strains, as well as antigens of approximately 78, 60 and 40 kDa. Producers and veterinarians should be aware that H. somnus may be carried by bison and may have potential for causing diseases in bison similar to those described in cattle and sheep.

Animals↗
PubMed1999

Use of a polymerase chain reaction method to detect the leukotoxin gene lktA in biogroup and biovariant isolates of Pasteurella haemolytica and P trehalosi.

OBJECTIVE: To determine whether Pasteurella haemolytica and P trehalosi isolates possess the structural gene for Pasteurella leukotoxin lktA and whether beta-hemolytic activity of these isolates correlated with detection of the lktA gene. SAMPLE POPULATION: 147 P haemolytica isolates from 21 biovariant groups and 101 P trehalosi isolates from 7 biovariant groups. In addition, P multocida and organisms from 7 other genera were tested to establish specificity of the procedure. PROCEDURE: Isolates were observed for beta-hemolysis. A polymerase chain reaction (PCR) procedure was used to amplify the RTX domain of the Pasteurella lktA gene. RESULTS: The lktA gene was detected in 108 (44%) isolates, including 15 associated with respiratory tract disease. All but 2 (98%) of the isolates that had the lktA gene were beta-hemolytic when grown on sheep blood agar. The remaining 140 isolates were negative for the lktA gene and hemolytic activity. CONCLUSIONS AND CLINICAL RELEVANCE: Hemolytic activity of P haemolytica and P trehalosi isolates correlated with detection of the lktA gene for all but 2 isolates. However, 56% of isolates tested were negative for the lktA gene and beta-hemolytic activity. Leukotoxin production and secretion is a major virulence factor when other conditions are favorable for disease development. Therefore, identification of strains that possess the lktA gene may aid in the evaluation of the pathogenic potential of Pasteurella strains carried by wild and domestic animals.

Animals↗
PubMed1998

Direct binding of Shc, Grb2, SHP-2 and p40 to the murine granulocyte colony-stimulating factor receptor.

Granulocyte colony-stimulating factor (G-CSF) mediates the proliferation, differentiation and activation of cells in the granulocytic lineage. However, knowledge about the specific signaling pathways utilized by the G-CSF receptor (G-CSF-R) upon ligand binding remains limited. In this report, we show rapid phosphorylation of Shc upon stimulation of NFS-60 cells with G-CSF, and inducible association of Shc and Grb2 with the G-CSF-R in these cells. Using a tyrosine-phosphorylated GST-G-CSF-R fusion we demonstrate that Shc, Grb2 and SHP-2 directly bind the receptor via their respective SH2 domains, suggesting multiple routes of MAPK activation from the G-CSF-R are possible. In addition, we have identified an unknown p40 molecule which is associated with the G-CSF-R transiently following G-CSF stimulation, and a constitutively-associated p37 molecule.

Adaptor Proteins, Signal Transducing↗
PubMed1998

The Src-like tyrosine kinase Hck is activated by granulocyte colony-stimulating factor (G-CSF) and docks to the activated G-CSF receptor.

Activation of the granulocyte colony-stimulating factor receptor (G-CSF-R) leads to tyrosine-phosphorylation of multiple cytoplasmic components. To date, the kinases Jak1, Jak2, Tyk2, Lyn, and Syk have been implicated in this process. However, it is unknown if other kinases might be involved in the diverse responses from the G-CSF-R, which include mitogenesis, survival, differentiation, and functional activation of responsive cells. The hematopoietic cell kinase (Hck) is a member of the Src-family of kinases known to be expressed in cells of the granulocytic lineage. It also interacts with the gp130 subunit of the LIF/IL-6 receptors, which is closely related to the G-CSF-R, and so represents a good candidate for mediating at least some of the downstream signaling from the G-CSF-R. Therefore, we investigated the activation of Hck by the G-CSF-R in intact cells as well as in vitro. These studies revealed recruitment of Hck to activated G-CSF-R, mediated by direct binding via its SH2 domain to multiple phosphotyrosines of the receptor. In addition, we show that Hck becomes activated upon G-CSF treatment and is, in turn, able to phosphorylate the G-CSF-R, indicating a clear functional and physical involvement in G-CSF signaling.

Animals↗
PubMed1998

Perturbed granulopoiesis in mice with a targeted mutation in the granulocyte colony-stimulating factor receptor gene associated with severe chronic neutropenia.

Mutations in the granulocyte colony-stimulating factor (G-CSF) receptor gene are found in a number of patients with severe chronic neutropenia predisposed to acute myeloid leukemia. These mutations result in the absence of the C-terminal domain of the G-CSF-R, a region which has been implicated in differentiation signaling. We generated mice with an equivalent mutation (gcsfr-triangle Delta715) by homologous and Cre-mediated recombination in embryonic stem cells. Both wt/triangle Delta715 and triangle Delta715/triangle Delta715 mice have significantly reduced numbers of blood neutrophils compared with their wt/wt littermates. However, under continuous G-CSF administration mutant mice develop peripheral neutrophil counts that significantly exceed those of wild-type littermates. These findings indicate that depending on G-CSF levels in mice, the triangle Delta715 mutation can contribute both to neutropenia and to neutrophilia.

Animals↗
PubMed1998

cAMP enhances CSF-1-induced ERK activity and c-fos mRNA expression via a MEK-dependent and Ras-independent mechanism in macrophages.

Inhibition of MAPK by elevated intracellular cAMP has often been correlated with suppression of growth factor-induced proliferation. However, in murine bone marrow-derived macrophages (BMM) we show that the cAMP analogue, 8-bromo cAMP (8BrcAMP) (1mM), despite being a dramatic G1 phase proliferation inhibitor, increased ERK activity both in the absence and presence of CSF-1; these increases were blocked by PD98059 (100 microM) suggesting MEK dependence. In contrast, CSF-1-stimulated p21Ras activity was blocked by 8BrcAMP thus correlating with the inhibition of proliferation. This is the first report to indicate that elevated intracellular cAMP can activate ERK activity while inhibiting proliferation and the data support the concept in CSF-1-treated macrophages of Ras-independent activation of ERK activity. It was also found that the acute but not the sustained elevation of c-fos mRNA expression due to 8BrcAMP was also MEK dependent indicating that there are separate pathways controlling c-fos mRNA expression in BMM.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

Biovariants of isolates of Pasteurella from domestic and wild ruminants.

A total of 608 bacterial isolates previously identified as Pasteurella haemolytica biotypes A and 3, P. trehalosi, and P. multocida, were separated into 73 distinct biovariants using 21 phenotypic characteristics. The largest group (54%) of wildlife isolates was identified as biogroup 2 and biogroup 2 variants. Biogroup 2 and biogroup 2 variants accounted for only 17% of isolates from domestic ruminants and were all from sheep. In contrast, 43% of isolates from domestic ruminants were identified as biogroup 1 and biogroup 1 variants, whereas only 6% of isolates from wildlife were identified in these groups. The majority of biogroup 1 isolates from wild ruminants were from 1 group of bighorn sheep in Arizona that were geographically separated from other wildlife sampled. Similarly, 1 biogroup 2 variant, 2E, was cultured only from free-ranging Dall sheep in Alaska. Twelve percent of domestic isolates and 6% of wildlife isolates were indole positive. The remaining isolates from wildlife (33%) and domestic animals (30%) were distributed among 53 distinct biovariants. None of these individual biovariants represented >4% of the total isolates. Phenotypic characterization was valuable for distinguishing between isolates from different hosts and from different geographic areas and can be used to assist in epidemiologic studies.

Animals↗
PubMed1997

cAMP suppresses p21ras and Raf-1 responses but not the Erk-1 response to granulocyte-colony-stimulating factor: possible Raf-1-independent activation of Erk-1.

The cAMP analogue 8-bromo-cAMP (8BrcAMP) inhibits granulocyte-colony-stimulating factor (G-CSF)-stimulated DNA synthesis in myeloid NFS-60 cells. We examined the effect of 8BrcAMP addition on the G-CSF-stimulated extracellular signal-related protein kinase 1 (Erk-1), p21ras and Raf-1 activation. The Erk-1 activity was not down-regulated by the increase in intracellular cAMP levels, whereas p21ras and Raf-1 activities were, suggesting that Erk-1 activity might not be dependent on upstream p21ras and/or Raf-1 activity in this system. To explore this possibility further, we sought to determine whether there were downstream substrates of Raf-1 that were distinguishable from those of Erk-1 by using two-dimensional SDS/PAGE analysis of the protein phosphorylation patterns of NFS-60 cell cytosolic extracts treated with exogenous Raf-1 or Erk-1 in the presence of [gamma-32P]ATP. The two phosphorylation patterns were found to have many differences. To gain further insights into the possible relevance of these phosphorylation patterns and as an approach to exploring in more detail the inhibitory effect of 8BrcAMP, two-dimensional SDS/PAGE analysis was performed on the cytosolic extracts of 32P-labelled NFS-60 cells treated with G-CSF, in the absence or presence of 8BrcAMP. It was found that the phosphorylated proteins whose appearance was specific to the action of exogenous Raf-1 were sensitive to the action of 8BrcAMP in vivo, whereas those whose appearance was specific to the action of exogenous Erk-1 alone, or common to the actions of Raf-1 and Erk-1, were 8BrcAMP-insensitive. The results are consistent with a Raf-1-independent pathway for Erk-1 activation in G-CSF treated myeloid cells, and a number of potential downstream substrates of these kinases have been identified for further characterization.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

Bioprocess supervision: neural networks and knowledge based systems.

Supervision of highly non-linear and time variant bioprocesses is of considerable importance to bioindustries as a means of achieving improved productivity and reduced process variability. Artificial intelligence methodologies, including artificial neural networks and knowledge based systems, can contribute significantly to the achievement of these objectives.

Biosensing Techniques↗
PubMed1997

Changes in the hemagglutinin molecule of influenza type A (H3N2) virus associated with increased virulence for mice.

The H3N2 influenza virus A/Philippines/82 (Phil82) and its bovine serum-resistant mutant, Phil82/BS, were used to investigate factors that influence virulence of influenza virus for mice. Phil82/BS, which lacks the high-mannose oligosaccharide at residue 165 of the hemagglutinin (HA) molecule, was found to replicate to a much higher titer in mouse lung than the parent Phil82, and had acquired lethality for mice. Further adaptation of Phil82/BS by sequential lung passage in mice yielded a strain of greater virulence, Phil82/BS/ML 10, in which a change at residue 246 of HA resulted in loss of a second potential glycosylation site. Phil82 is highly sensitive to neutralization in vitro by murine serum- and lung-associated mannose-binding lectins (collectins). Characterization of the two mutant viruses indicated that resistance to murine collectins can account for the enhanced virulence of Phil82/BS but not for the further increase in virulence of Phil82/BS/ML10. Evidence is presented that residue 246 is not in fact glycosylated in Phil82/BS HA, nor presumably in the parent Phil82 virus. The HA molecule of Phil82/BS/ML10 displayed functional differences from Phil82/BS, including a change in the optimum pH of fusion and a minor change in receptor-binding specificity, which may allow improved efficiency of replication in the mouse lung.

Animals↗
PubMed1997

Evaluation of Streptomyces species-groups by pyrolysis mass spectrometry.

Curie-point pyrolysis mass spectrometry was used to evaluate the taxonomic integrity of three subclusters, provisionally labelled S. albidoflavus, S. anulatus and S. halstedii, which formed a species-group in an extensive numerical phenetic survey of the genus Streptomyces. Excellent agreement was found between the results of the triplicate analyses of each strain while the duplicated strains clustered adjacent to one another. Sequential principal component-canonical variates analysis of the experimental data collected on the 32 representative organisms showed that the S. albidoflavus strains formed a distinct group. This result taken together with earlier chemical, molecular and numerical taxonomic data indicates that the S. albidoflavus subcluster corresponds to a distinct species. In contrast, the subclusters equated with S. anulatus and S. halstedii were found to be heterogeneous and hence in need of further study. However, it is evident from the present investigation that Curie-point pyrolysis mass spectrometry provides a rapid and reproducible way of evaluating the taxonomic integrity of Streptomyces species-groups.

Mass Spectrometry↗
PubMed1997

Long-term identification of streptomycetes using pyrolysis mass spectrometry and artificial neural networks.

Sixteen reference strains and thirteen fresh isolates of three putatively novel Streptomyces species were examined six times over twenty months using pyrolysis mass spectrometry to examine the long-term reproducibility of the procedure. The reference strains and new isolates were correctly identified using information in each of the datasets and operational fingerprinting, but direct statistical comparison of the datasets for strain identification was unsuccessful between datasets. Artificial neural networks were also used to identify the strains held in the datasets. Neural networks trained with pyrolysis mass spectra from a single dataset were found to successfully identify the reference strains and fresh isolates in that dataset but were unable to identify many of the strains in the other datasets. However, a neural network trained on representative pyrolysis mass spectra from each of the first three datasets were found to identify the reference strains and fresh isolates in those three datasets and in the three subsequent datasets. Therefore, artificial neural network analysis of pyrolysis mass spectrometric data can provide a rapid, cost-effective, accurate and long-term reproducible way of identifying and typing microorganisms.

Humans↗
PubMed1997

Application of neural networks to the analysis of pyrolysis mass spectra.

Pyrolysis mass spectrometry is a data rich analysis technique now becoming widely applied in microbiology. Data analysis is a key step in the exploitation of the technique and the application of neural network analysis to pyrolysis mass spectrometric data offers new opportunities for classification, identification and inter-strain comparison of microorganisms in biotechnology and clinical microbiology. The use of a supervised neural network for the identification of members of a streptomycete species-group is described.

Mass Spectrometry↗
PubMed1997

A highly discriminatory method for the direct comparison of two closely related bacterial populations by pyrolysis mass spectrometry.

The ability of pyrolysis mass spectrometry (Py-MS) to discriminate between paired groups of closely related bacteria has been examined. The technique was challenged with increasing levels of biological difference; from that between different subcultures of the same isolate of Staphylococcus aureus, through that between separate isolates of the same strain and eventually to that between different staphylococcal species and Streptococcus pyogenes. By using an analytical method in which the spectral data from any two groups can be directly compared at a time, it was shown that Py-MS was capable of measuring statistically significant differences between staphylococci and Streptococcus pyogenes, between different Staphylococcus species and between two isolates of the same strain of S. aureus from different sources. Two subcultures of the same isolate were found to be indistinguishable. The differing magnitude of the Py-MS-derived differences corresponded to the "biological differences", being much larger between staphylococci and streptococci than between staphylococcal species and only just statistically significant between isolates of the same strain from different sources. The technique described allows the assessment of the magnitude and significance of Py-MS-derived differences between any two bacterial populations.

Mass Spectrometry↗
PubMed1997

Virulence of influenza A virus for mouse lung.

The experimental infection of mouse lung with influenza A virus has proven to be an invaluable model for studying the mechanisms of viral adaptation and virulence. These investigations have identified critical roles for the haemagglutinin (HA) and matrix (M) genes of the virus in determining virulence for mouse lung. For the HA gene, the loss of glycosylation sites from the encoded polypeptide or changes which may affect the pH of HA-mediated endosome fusion have been observed following adaptation. These alterations also have the potential to impact on receptor specificity, beta inhibitor sensitivity and activation cleavage which may act in concert to account for the increased virulence of adapted strains. For the M gene, two specific changes in the M1 protein have been identified in strains adapted to, or virulent for, mouse lung. These changes are likely to affect pH-dependent association/dissociation of M1 with the viral ribonucleoprotein, and control virulence as well as growth. The role of other genes in mouse lung virulence remains unknown.

Adaptation, Biological↗
PubMed1997

Identification of phosphoproteins specific to granulocyte colony-stimulating factor-mediated signaling using 2D-SDS-PAGE.

Like other cytokines, granulocyte colony-stimulating factor (G-CSF) activates a complex array of signal transduction pathways involving multiple kinases and phosphatases. We sought to identify phosphoproteins specific to G-CSF signaling. Using 2D-SDS-PAGE of 32P-labeled cytosolic extracts, we compared phosphoprotein patterns of NFS-60 cells treated with G-CSF or interleukin-3 (IL-3). We also compared the patterns found after stimulation of M-NFS-60 cells with macrophage-CSF (M-CSF). A large number of phosphoproteins were found that were specific for the G-CSF response. Their distribution contrasted with that of Erk-1-related spots, identified by Western blotting, which were common to G-CSF, M-CSF (CSF-1), and IL-3 responses. The activation of Erk-1 by these cytokines was confirmed by in vitro kinase assays. The 2D-SDS-PAGE approach was also used to demonstrate that a series of unrelated G1 phase inhibitors of the mitogenic action of G-CSF elicited both common and diverse protein phosphorylation changes in G-CSF-treated NFS-60 cells that were not dependent on the inhibition of Erk-1 activity, as demonstrated by both in vitro kinase assays and 2D-SDS-PAGE. Therefore, 2D-SDS-PAGE has potential to dissect both the signal transduction pathways lying downstream of the G-CSF receptor (and of the receptors for other CSFs) and also the site of action of proliferation inhibitors.

Animals↗