Biomedical subjects

K D Clinkenbeard

Publications and source records attributed to K D Clinkenbeard.

At least 37 records · Page 2Linked to original sources

PubMed1990

Effects of Pasteurella haemolytica leukotoxin on neutrophils from white-tailed deer and several exotic ruminant species.

Pasteurella haemolytica leukotoxin is a pore-forming cytolysin which acts as a virulence factor in pasteurellosis of domestic ruminants. Leukocytes from cattle, sheep and goats are susceptible to leukotoxin-induced lysis; however, leukocytes from non-ruminant species so far tested are resistant to leukotoxin-induced lysis. Neutrophils obtained from three white-tailed deer, four Saiga antelope, an Addra gazelle, a Grant's gazelle and a Sable antelope were tested for susceptibility to the lytic effects of P. haemolytica leukotoxin using lactate dehydrogenase release. Results were compared to those obtained using neutrophils from a steer and cultured bovine lymphoma cells. Neutrophils obtained from all these ruminants, except the Addra gazelle, were susceptible to P. haemolytica leukotoxin. Individual variation among the Saiga and the deer did not appear to be due to the percentages of neutrophils or the percentage of contaminating erythrocytes in the cell preparations.

Animals↗

PubMed1990

Molecular aspects of virulence of Pasteurella haemolytica.

Pasteurella haemolytica is represented by two biotypes (A and T), 15 serotypes, and numerous untypable strains. Specific biotypes and serotypes are associated with fibrinous pleuropneumonia (pneumonic pasteurellosis) in cattle, sheep, and goats, septicemic pasteurellosis in lambs, and mastitis in ewes. Four virulence factors have been associated with P. haemolytica: fimbriae, a polysaccharide capsule, endotoxin [lipopolysaccharide (LPS)], and leukotoxin (LKT). The interactions of these virulence factors with components of the pulmonary alveolus are discussed as a model for the pathogenesis of pasteurellosis. Fimbriae on P. haemolytica may enhance colonization of the upper respiratory tract. The capsule of P. haemolytica varies in composition among serotypes. It inhibits complement-mediated serum killing as well as phagocytosis and intracellular killing of P. haemolytica. The capsule enhances neutrophil directed migration and adhesion of P. haemolytica to alveolar epithelium. Pasteurella haemolytica LPS can alter bovine leukocyte functions, by dose-dependent inhibition or augmentation; it is directly toxic to bovine endothelium; it modifies cardiopulmonary hemodynamics; and it elevates circulatory prostanoids, serotonin, cAMP, and cGMP. Leukotoxin is produced by all known serotypes and many untypable strains. Leukotoxin is a poreforming cytolysin that affects ruminant leukocytes and platelets by altering function at low levels but causing lysis at high levels.

Animals↗

PubMed1990

Pasteurella haemolytica lipopolysaccharide-induced arachidonic acid release from and neutrophil adherence to bovine pulmonary artery endothelial cells.

Bovine pulmonary artery endothelial cells (BPAEC) were labeled with 3H-arachidonic acid. Exposure of the labeled BPAEC to Pasteurella haemolytica lipopolysaccharide (LPS) resulted in a time- and dose-dependent release of radioactivity. The release was inhibited by 5 mM indomethacin, but inhibition was not caused by less than or equal to 500 microM indomethacin or hydrocortisone, which suggests that the release was caused primarily by a mechanism other than cyclooxygenase or phospholipase A2 metabolism of arachidonic acid. Pasteurella haemolytica LPS also caused increased adherence of bovine neutrophils to BPAEC through independent effects on both cell types. The increased adherence was inhibited by treatment of either cell type with cycloheximide or actinomycin D prior to LPS exposure, indicating that de novo protein synthesis was required in both cell types to promote the LPS-induced adherence. Lipopolysaccharide may be an important factor in neutrophil-mediated effects in pneumonic pasteurellosis by causing increased neutrophil adherence and, thus, the vascular sequestration of neutrophils. Together, these experiments provide additional evidence for the involvement of LPS in pneumonic pasteurellosis. Moreover, they provide evidence of LPS-induced endothelial activation, which could have broad ramifications in the inflammatory and immune responses of pneumonic pasteurellosis.

Animals↗

PubMed1989

Effects of Pasteurella haemolytica leukotoxin on isolated bovine neutrophils.

P. haemolytica leukotoxin caused rapid leakage of intracellular K+ (greater than 90% in 30 sec) from and cell swelling (approximately 100% increase in 15 min) of isolated bovine neutrophils. Incubation media made hypertonic by the addition of raffinose, dextran or inulin (carbohydrates with mol. wts of greater than or equal to 505) prevented leukotoxin-induced cell swelling, but not K+ leakage. Assuming that leukotoxin acts as a transmembrane molecular sieve, then the leukotoxin-induced functional transmembrane pore size in bovine neutrophil plasma membranes is slightly smaller than the molecular size of raffinose, i.e. 1.2 nm. Morphologically, leukotoxin caused bovine neutrophils to swell, lose their membrane ruffling, develop a finely porous surface, and form large plasma membrane defects. Exposure of neutrophils to leukotoxin caused slower (5-50 min) leakage of 80% of the cellular L-lactate: NAD oxidoreductase (E.C. 1.1.27, lactate dehydrogenase, LDH). Leukotoxin-induced K+ leakage and cell swelling developed in Ca2+-free medium, whereas leakage of lactate dehydrogenase develop only in medium containing Ca2+ and was inhibited by the addition of ethylene glycol-bis(B-aminoethyl ether)N,N,N1,N1-tetraacetic acid (EGTA). This sequence of leukotoxin-induced changes in neutrophils is compatible with the mechanism of action of pore-forming cytolysins.

Animals↗

PubMed1989

Serum IgG and IgM antibody response in cattle to antigens of Pasteurella haemolytica.

The serum IgG and IgM antibody responses of 48 cattle vaccinated with live Pasteurella haemolytica (LIVE), formalin-killed P. haemolytica in Freund's incomplete adjuvant (FIA), or formalin-killed P. haemolytica in aluminum hydroxide adjuvant (ALH) to a variety of P. haemolytica antigens were evaluated. Enzyme-linked immunosorbent assays (ELISAs) were used to determine the sequential and day 21 IgG and IgM antibody responses to whole P. haemolytica (WB), a capsular carbohydrate-protein subunit (CPS) extracted from the organism, P. haemolytica capsular carbohydrate (CC), and P. haemolytica leukotoxin (LT). LIVE and FIA vaccinates developed generally higher IgG and IgM responses to all antigens compared to ALH vaccinates. LIVE vaccinates developed IgG responses to LT which were significantly higher (P less than 0.05) than all other vaccinates. In contrast, FIA vaccinates developed significantly higher IgG responses to CPS than all other vaccinates. On the basis of the ELISA results, similar or cross reacting antigenic sites were present in preparations containing surface antigens (WB, CPS and CC), but not LT. Disease resistance, as determined by experimental lesions induced in the 48 calves by transthoracic challenge with P. haemolytica, was significantly greater in the LIVE and FIA vaccinates compared with ALH vaccinates. No significant difference in resistance was detected between LIVE and FIA vaccinates. Lesions in ALH vaccinates were not significantly different than those in phosphate-buffered saline (PBS) controls. Increased IgG responses to all antigens were significantly associated with resistance to experimental disease; however, IgG responses to CPS were most highly correlated with resistance. The only IgM response which was significantly correlated with resistance was the response to CPS. These studies indicate that serum IgG antibody responses to various surface antigens of P. haemolytica, as well as LT, can enhance resistance to experimental pneumonic pasteurellosis. Serum IgM responses, however, do not appear to play a major role in resistance to experimental disease.

Animals↗

PubMed1989

Transmembrane pore size and role of cell swelling in cytotoxicity caused by Pasteurella haemolytica leukotoxin.

Pasteurella haemolytica A1 leukotoxin causes rapid (5 to 15 min) leakage of intracellular K+ and cell swelling and slower (15 to 60 min), Ca2+-dependent formation of large plasma membrane defects (congruent to 100 nm) and leakage of lactate dehydrogenase from bovine lymphoma cells (BL3 cells) (K. D. Clinkenbeard, D. A. Mosier, A. L. Timko, and A. W. Confer, Am. J. Vet. Res., in press). Incubation of BL3 cells in medium made hypertonic by inclusion of 75 mM sucrose blocked leukotoxin-induced cell swelling, formation of large plasma membrane defects, and leakage of lactate dehydrogenase but did not block leukotoxin-induced leakage of intracellular K+. Carbohydrates with molecular weights less than that of sucrose, e.g., mannitol, did not block leukotoxin-induced cell swelling of BL3 cells. Increasing the concentration of mannitol to twice that of sucrose still resulted in no protective effect. Assuming that leukotoxin acts as a transmembrane molecular sieve, then the functional transmembrane pore size formed by leukotoxin in BL3 cells is slightly less than the size of sucrose, i.e., 0.9 nm. Exposure of BL3 cells to leukotoxin for 15 or 45 min followed by the addition of hypertonic sucrose to the incubation medium reversed leukotoxin-induced cell swelling and prevented further leakage of lactate dehydrogenase. Leukotoxin-induced leakage of lactate dehydrogenase required both cell swelling and Ca2+-dependent processes. The Ca2+-dependent steps can occur before or concurrent with cell swelling.

Animals↗

PubMed1989

Pasteurella haemolytica antigens associated with resistance to pneumonic pasteurellosis.

Antigens associated with whole Pasteurella haemolytica biotype A serotype 1, a capsular carbohydrate-protein extract of the organism, and P. haemolytica leukotoxin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antigens of the electrophoresed preparations were detected by Western blotting (immunoblotting) with sera from cattle which were either nonvaccinated or vaccinated with live or killed P. haemolytica vaccines and had variable degrees of resistance to experimental pneumonic pasteurellosis. Distinct, easily recognizable antigens of these preparations were identified, and the antibody responses to these antigens were quantified by densitometry. To determine their importance to disease resistance, we then compared antibody responses with experimental lesion scores. Antibody reactivity to surface antigens which were significantly correlated with resistance and present in two or more of the preparations were detected at 86, 66, 51, 49, 34, 31, and 16 kilodaltons (kDa). Of these, antibody responses to antigens at 86, 49, and 31 kDa appeared most important based on their concentration and significance levels. Antibody reactivity to leukotoxin antigens which were significantly correlated with resistance and common with important surface antigens were detected at 86, 66, and 49 kDa. Antibody responses to unique leukotoxin antigens which were significantly correlated with resistance were present at 92 and 58 kDa.

Animals↗

PubMed1989

Effects of Pasteurella haemolytica leukotoxin on cultured bovine lymphoma cells.

Leukotoxin activity from culture supernatants of Pasteurella haemolytica serotype 1 in logarithmic growth phase caused rapid (less than 5 min) release of intracellular K+, uptake of extracellular Ca2+, and swelling of cultured bovine lymphoma cells (BL3 cells). Release of 51CrO4(2-) and lactate dehydrogenase (LDH) from BL3 cells began after 15 minutes of incubation with leukotoxin at 37 C and was completed between 60 and 120 minutes of incubation. In addition, leukotoxin exposure of BL3 cells resulted in cell aggregation and adherence to glass surfaces. Scanning electron microscopy indicated that after 10 minutes of leukotoxin exposure, BL3 cells increased in size, and large membrane defects developed between 20 and 60 minutes of exposure. The rate of release of LDH from leukotoxin-exposed BL3 cells was proportional to the amount of leukotoxin added. At high cell concentrations, the activity of LDH released at completion was directly proportional to the amount of leukotoxin added. Leukotoxin-induced release of LDH required a divalent cation, whereas K+ release and cell swelling did not. The addition of Ca2+, Mn2+, and Ba2+ resulted in increased leukotoxin-induced release of LDH. Divalent cation concentrations of 0.5 to 2.5 mM resulted in 50% of maximal stimulation. Ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid blocked increased release of LDH caused by Ca2+ addition, but had no effect on K+ release or cell swelling. Leukotoxin action on BL3 cells (K+ release, cell swelling, Ca2+ uptake, and release of LDH) was prevented by incubation at 4 C.

Animals↗

PubMed1989

Direct effects of Pasteurella haemolytica lipopolysaccharide on bovine pulmonary endothelial cells in vitro.

Bovine pulmonary artery cells in cell culture were exposed to lipopolysaccharide (LPS) purified from Pasteurella haemolytica serotype Al. This resulted in severe membrane damage, which caused a time- and dose-dependent release of lactate dehydrogenase that was first detected 4 hours after exposure and reached a maximal mean release of 67% after 24 hours of exposure to 1 micrograms of LPS/ml. Mean release of 51chromium followed by a similar pattern and reached a maximum of 61% following 24 hours of exposure to 10 micrograms of LPS/ml. Morphologically, endothelial cells responded to LPS by marked cell membrane retraction, the formation of numerous cytoplasmic blebs, and ruffling of the cell membrane. Subsequently, the cells became round and detached. Cell detachment reached a mean of 95% following 8 hours of exposure to 1 micrograms of LPS/ml. These studies demonstrated that P haemolytica LPS is capable of causing direct damage to bovine pulmonary arterial endothelial cells, which may be important in the pathogenesis of bovine pneumonic pasteurellosis.

Animals↗

PubMed1988

Disseminated histoplasmosis in dogs: 12 cases (1981-1986).

Diarrhea, intestinal blood loss, anemia, and lethargy were predominant clinical findings in 12 dogs with disseminated histoplasmosis. Young dogs were affected most commonly, with 6 dogs being 1 to 3 years old. A diagnosis of disseminated histoplasmosis was established on the basis of histologic or cytologic detection of Histoplasma organisms in intestinal or rectal mucosa in 7 dogs, in circulating leukocytes in 5 dogs, in bone marrow in 3 dogs, and in multiple tissues at necropsy in 1 dog (4 dogs had Histoplasma organisms detected in greater than 1 site). Anemia was detected in 10 dogs (PCV less than 20% in 3 dogs), and the anemia was inadequately regenerative or nonregenerative in 7. Hypoalbuminemia was detected in 9 dogs, and serum albumin concentrations were low (less than 1.0 g/dl) in 4 of the 9 dogs. Of 5 dogs treated with ketoconazole, 2 were in remission for greater than or equal to 1 year. Corticosteroid therapy may have exacerbated the disease in 4 dogs. Histoplasma infection of multiple organs was detected in 5 necropsied dogs.

Animals↗

PubMed1988

Ehrlichiosis and polyarthritis in three dogs.

Three dogs admitted for evaluation of lameness were determined to be infected with a neutrophilic strain of Ehrlichia. Ehrlichia morulae were detected in low numbers in both synovial fluid and blood neutrophils. The dogs had rapid clinical improvement after appropriate antibiotic therapy. A diagnosis of canine ehrlichiosis easily could have been missed if morulae had not been identified. Ehrlichiosis should be considered in acutely lame dogs residing in areas enzootic for ehrlichiosis. A careful search for Ehrlichia morulae within synovial fluid and blood neutrophils should be performed. An E canis titer determination also may be helpful.

Animals↗

PubMed1988

Identification of Histoplasma organisms in circulating eosinophils of a dog.

Disseminated histoplasmosis was diagnosed in a 10-year-old dog that had chronic diarrhea, weight loss, fever, and anemia. The diagnosis was based on detection of Histoplasma organisms in circulating neutrophils, monocytes, and eosinophils. The dog had severe histoplasmal fungemia, which may have been caused by treatment with prednisolone.

Animals↗

PubMed1987

Renal concentrating ability in dehydrated hyponatremic dogs.

Eleven hyponatremic dogs were unable to concentrate their urine during periods of severe dehydration and azotemia. When normonatremia was reestablished in eight of the dogs, their renal concentrating ability returned. Six dogs, including the 3 dogs in which normonatremia was not reestablished, died or were euthanatized; renal lesions were not found during postmortem examination. Two dogs had hypoadrenocorticism, which has been documented as a cause of hyponatremia and impaired renal concentrating ability. Two dogs had gastrointestinal disease, which has been documented as a cause of hyponatremia, but not of impairment of renal concentrating ability. All dogs without hypoadrenocorticism had clinical and clinicopathologic indications of blood loss, which has not been documented as a cause of hyponatremia or impairment of renal concentrating ability. Hyponatremia (less than 120 mEq/L) was induced by chronic blood removal in a dog maintained on a low-sodium diet. During the period of hyponatremia, the dog became azotemic, hypotensive, and severely dehydrated; renal concentrating ability was impaired. We conclude that hyponatremia may be caused by hemorrhage, but irrespective of the cause, hyponatremia impairs renal concentrating ability.

Adrenal Cortex Diseases↗

PubMed1987

Disseminated histoplasmosis in cats: 12 cases (1981-1986).

Anemia, weight loss, lethargy, fever, anorexia, and interstitial lung disease were the predominant clinical findings in 12 cats with disseminated histoplasmosis. Some cats were examined because of dysfunction or lesions of bone, eyes, or skin. In most cases, the clinical signs were observed by the owner for 4 weeks or less before seeking veterinary care. Young cats were most commonly affected, with 7 of the 12 cats less than or equal to 1 year old. Identification of Histoplasma organisms in bone marrow aspirates was used to confirm the diagnosis of histoplasmosis in 11 of the 12 cats. Histoplasma infection of multiple organs was found at necropsy. In this study, disseminated histoplasmosis had a higher prevalence in cats than in dogs at the same veterinary medical teaching hospital. Feline disseminated histoplasmosis was not associated with FeLV infection. Treatment was attempted in 7 of the 12 cats.

Animals↗

PubMed1987

Hematologic values in horses and interpretation of hematologic data.

Normal reference ranges and pertinent background information on equine hematology are presented and briefly discussed. Diagnostic interpretation of hematologic data is discussed and three diagnostic algorithms and two diagnostic tables are provided to facilitate the use of the presented information for diagnosis. Two cases are presented and the information presented in the article is used to interpret the case data.

Animals↗

PubMed1987

Collection and evaluation of equine peritoneal and pleural effusions.

This article discusses collection, slide preparation, culture technique, fluid analysis and evaluation, and cytologic evaluation of peritoneal and pleural effusions. The morphologic characteristics of various effusions are described, and the physical characteristics (volume, color, turbidity) of effusions are discussed. An algorithm for classifying effusions as transudates, modified transudates, or exudates is included, and each category is discussed.

Animals↗

PubMed1975

3-Hydroxy-3-methylglutaryl coenzyme A synthase. Evidence for an acetyl-S-enzyme intermediate and identification of a cysteinyl sulfhydryl as the site of acetylation.

Homogeneous liver 3-hydroxy-3-methylglutaryl coenzyme A synthase, which catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA, also carries out: (a) a rapid transacetylation from acetyl-CoA to 31-dephospho-CoA and (b) a slow hydrolysis of acetyl-CoA to acetate and CoA. Transacetylation and hydrolysis occur at 50 and 1 percent, respectively, the rate of the synthasecatalyzed condensation reaction. It appears that an acetyl-enzyme intermediate is involved in the transacetylase and hydrolase reactions of 3-hydroxy-3-methylglutaryl-CoA synthase, as well as in the over-all condensation process. Covalent binding to the enzyme of a [14C]acetyl group contributed by [1(-14)C]acetyl-CoA is indicated by migration of the [14C]acetyl group with the dissociated synthase upon electrophoresis in dodecyl sulfate-urea and by precipitation of [14C]acetyl-enzyme with trichloroacetic acid. At 0 degrees and a saturating level of acetyl-CoA, the synthase is rapidly (less than 20 s) acetylated yielding 0.6 acetyl group/enzyme dimer. Performic acid oxidation completely deacetylates the enzyme, suggesting the site of acetylation to be a cysteinyl sulfhydryl group. Proteolytic digestion of [14C]acetyl-S-enzyme under conditions favorable for intramolecular S to N acetyl group transfer quantitatively liberates a labeled derivative with a [14C]acetyl group stable to performic acid oxidation. The labeled oxidation product is identified as N-[14C]acetylcysteic acid, thus demonstrating a cysteinyl sulfhydryl group as the original site of acetylation. The ability of the acetylated enzyme, upon addition of acetoacetyl-CoA, to form 3-hydroxy-3-methylglutaryl-CoA indicates that the acetylated cysteine residue is at the catalytic site.

Acetylation↗

PubMed1975

Intracellular localization of the 3-hydroxy-3-methylglutaryl coenzme A cycle enzymes in liver. Separate cytoplasmic and mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A generating systems for cholesterogenesis and ketogenesis.

Acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl coenzyme synthase which comprise the 3-hydroxy-3-methylglutaryl-CoA-generating system(s) for hepatic cholesterogenesis and ketogenesis exhibit dual mitochondrial and cytoplasmic localization. Twenty to forty per cent of the thiolase and synthase of avian and rat liver are localized in the cytoplasmic compartment, the remainder residing in the mitochondria. In contrast, 3-hydroxy-3 methylglutaryl-CoA lyase, an enzyme unique to the "3-hydroxy-3-methylglutaryl-CoA cycle" of ketogenesis, appears to be localized in the mitochondrion. The small proportion, 4 to 8 percent, of this enzyme found in the cytoplasmic fraction appears to arise via leakage from the mitochondria during cell fractionation in that its properties, pI and stability, are identical to those of the mitochondrial lyase. These results are consistent with the view that ketogenesis which involves all three enzymes, acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA lyase, occurs exclusively in the mitochondrion, whereas cholesterogenesis, a pathway which involves only the 3-hydroxy-3-methylglutaryl-CoA synthesizing enzymes, is restricted to the cytoplasm. Further fractionation of isolated mitochondria from chicken and rat liver showed that all three of the 3-hydroxy-3-methylglutaryl-CoA cycle enzymes are soluble and are localized within the matrix compartment of the mitochondrion. Likewise, cytoplasmic acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase are soluble cytosolic enzymes, no thiolase or synthase activity being detectable in the microsomal fraction. Chicken liver mitochondrial 3-hydroxy-3methylglutaryl-CoA synthase activity consists of a single enzymic species with a pI of 7.2, whereas the cytoplasmic activity is composed of at least two species with pI values of 4.8 and 6.7. Thus it is evident that the mitochondrial and cytoplasmic species are molecularly distinct as has been shown to be the case for the mitochondrial and cytoplasmic acetoacetyl-CoA thiolases from avian liver (Clinkenbeard, K. D., Sugiyama, T., Moss, J., Reed, W. D., and Lane, M. D. (1973) J. Biol. Chem. 248, 2275). Substantial mitochondrial 3-hydroxy-3-methylglutaryl-CoA lyase activity is present in all tissues surveyed, while only liver and kidney possess significant mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity. Therefore, it is proposed that tissues other than liver and kidney are unable to generate acetoacetate because they lack the mitochondrial synthase.

Adenosine Monophosphate↗