No description available.

While the poisonous effects of botulinum neurotoxin (BoNT) have been recognized since antiquity, the overall actions and mechanisms of effects of BoNT have been elucidated primarily over the past several decades. The general utility of BoNT is described in the paper, but the focus is mainly on the approaches towards negating the toxic effects of BoNT, and on the projection of an engineered BoNT molecule serving as a Trojan Horse to deliver a therapeutic load for treatment of a host of medical disorders. The BoNT molecule is configured with a binding domain, a zinc-dependent protease with specificity primarily for vesicular proteins, and a translocation domain for delivery of the metalloprotease into the cytoplasm. The anti-toxin approaches for BoNT include the use of vaccines, antibodies, block of BoNT binding or translocation, inhibition of metalloprotease activity, impeded translocation of the protease/catalytic domain, and inhibition of the downstream Src signaling pathway. Projections of BoNT as a therapeutic include its targeting to non-cholinergic nerves, also targeting to non-neuronal cells for treatment of hypersecretory disorders (e.g., cystic fibrosis), and treatment of hormonal disorders (e.g., acromegaly). Still in the exploratory phase, there is the expectation of major advances in BoNT neuroprotective strategies and burgeoning utility of engineered BoNTs as therapeutics.

Factors that determine the molecular basis of African horsesickness virus (AHSV) virulence are unclear. Several proteins and different events in the viral replication cycle together with different environmental factors are likely to contribute to the virulence phenotype. The aim of this investigation was to study the possible contribution of AHSV nonstructural protein NS3 to virus virulence. NS3 is a cytotoxic protein that localizes in areas of plasma membrane disruption and is assumed to be associated with the release of virus particles from infected cells. A conserved feature in all AHSV NS3 proteins is the presence of two hydrophobic domains. To investigate whether these hydrophobic domains interact with membranes, cell surface localization and membrane association studies were conducted on NS3 and the two hydrophobic domain mutant forms of NS3. Results indicated that mutations in either hydrophobic domain did not prevent membrane targeting but abolished membrane anchoring. This prevented cell surface localization and obviated the cytotoxic effect of NS3. AHSV NS3 is a highly variable protein. This level of variation may influence virulence properties. To compare the NS3 sequence variation level of South African AHSVs and bluetongue viruses (BTV/s) with those previously described, the NS3 protein sequences of field isolates, reference and vaccine strains were determined and analysed. The variation level of AHSV NS3 was found to be higher than previously reported. Comparison of the AHSV vaccine and field strain NS3 sequences revealed no obvious NS3 virulence marker. The level of AHSV NS3 sequence variation could distinguish between field isolates and vaccine strains and differentiate between sub-populations within a serotype. The inferred phylogeny of AHSV NS3 corresponded well with the described NS3 phylogenetic clusters with exception of AHSV-8 and one AHSV-6 encoded NS3. BTV NS3 inferred phylogeny indicated that the three BTV NS3 clusters that occur as geographically defined entities are all simultaneously present in S.A. This would suggest that BTV originated in southern Africa. To investigate the impact of minor NS3 variation on AHSV virulence, the membrane permeabilization properties of AHSV-2 vaccine and virulent NS3 proteins were compared. The AHSV-2 S10 genes were cloned and NS3 was expressed using the BAC-TO-BAC system. The membrane permeabilization effect of NS3 expression was monitored by calcium influx into insect cells. Expression of either the vaccine or virulent associated AHSV-2 NS3 protein equally increased membrane permeability. The NS3 sequence variation therefore had a limited influence on membrane permeabilization properties and the virulence status of the vaccine and virulent AHSV-2 strains in this study. The possible effect of larger NS3 sequence variation levels on AHSV virulence was investigated by NS3 associated properties such as virus release and membrane permeabilization. AHSV infections increased the permeability of cell membranes and the different strains had different virus release properties. Virus release was not exclusively related to increased membrane permeability of infected cells or to the yield of the virus in the infected cells. The level of NS3 variation may influence AHSV release mechanisms and membrane permeabilization properties thereby contributing to AHSV virulence properties. / Thesis (PhD (Genetics))--University of Pretoria, 2005. / Genetics / unrestricted

The Horse Heaven Hills uplift in south-central Washington consists of distinct northwest and northeast trends which merge in the lower Yakima Valley. The northwest trend is adjacent to and parallels the Rattlesnake-Wallula alignment (RAW; a part of the Olympic-Wallowa lineament). The northwest trend and northeast trend consist of aligned or en echelon anticlines and monoclines whose axes are generally oriented in the direction of the trend. At the intersection, folds in the northeast trend plunge onto and are terminated by folds of the northwest trend.

No description available.

After ejaculation, mammalian spermatozoa must undergo a series of complex and poorly understood cellular events known as “capacitation” in order to be able to fertilize an oocyte. Among these, biochemical changes such as an increase in tyrosine phosphorylation of some sperm proteins have been correlated with the sperm capacity to fertilize an egg and found to be regulated by a cAMP dependent pathway. The influx of ions such as Ca2+ and HCO3- induce the activation of a soluble adenylyl cyclase (SACY) increasing the cAMP levels within the cell that leads to the activation of a protein kinase A (PKA), and a subsequent increase in protein tyrosine phosphorylation. This modification in sperm proteins seems to be essential for induction of a change in the motility pattern known as hyperactivation that enables the sperm to penetrate the zona pellucida of the oocyte and initiate fertilization. Since PKA is a serine/threonine kinase, it is not clear how it mediates protein tyrosine phosphorylation during sperm capacitation. Based on the finding that in somatic cells PKA activates c-Src, it has been proposed that the Src family of protein kinases (SFK) are the intermediate players involved in tyrosine phosphorylation induced by PKA activity. In order to better understand the molecular mechanisms involved in stallion sperm capacitation, the objectives of our study were: (1) To analyze PKA activity during stallion sperm capacitation (2) To evaluate the involvement of the Src family of protein kinases (SFK) on stallion sperm phosphorylation events associated with capacitation. Standard In Vitro Fertilization (IVF) has not been reproducibly successful in the horse. Recent data indicate that good fertilization rates may be achieved after treatment of sperm with procaine to induce hyperactivation. Our objectives were also to determine if drugs used in other species as well as procaine induce hyperactivation in stallion sperm and to evaluate biochemical changes such as protein tyrosine phophorylation.

No description available.

No description available.

Non-steroidal anti-inflammatory drugs (NSAIDs) are a mainstay of the management of pain and inflammation associated with musculoskeletal disorders and systemic inflammation in horses. The most utilized NSAIDs in equine practice are non-selective cyclooxygenase (COX) inhibitors, such as flunixin meglumine and phenylbutazone, which act through global inhibition of prostaglandin synthesis and release. While non-selective COX inhibitors are effective as anti-inflammatory agents, they are mired with complications with prolonged or high-dose use, particularly in critically ill patients. Therefore, non-selective COX-inhibitors have been displaced by selective COX-2 inhibitors for many practitioners due to the perceived reduced risk of gastrointestinal complications. It should be noted, however, that the use of COX-2 selective inhibitors in horses is not without risk. Due to the potential for significant adverse events in horses with critical illness treated with traditional NSAIDs, there is clinical need for safe, and effective anti-inflammatories and anti-pyretics for administration in these patients. The studies presented in this dissertation explore the pharmacokinetics, efficacy, and safety of acetaminophen in adult horses for use in musculoskeletal pain and pyrexia. In the first study, the pharmacokinetics and efficacy of oral acetaminophen at two different doses (20 mg/kg and 30 mg/kg) were examined in an experimentally induced lameness model and the analgesic efficacy of acetaminophen was compared to placebo and the non-selective COX inhibitor phenylbutazone. Acetaminophen when administered at 30 mg/kg produced a more rapid onset of greater improvement in subjective lameness scores and heart rate compared to other treatments in this model, and therefore would be more suitable as a monotherapy than acetaminophen dosed at 20 mg/kg. Acetaminophen dosed at 30 mg/kg resulted in a more rapid improvement in lameness scores than phenylbutazone at 2.2 mg/kg and was equivalent to phenylbutazone in lameness score reduction. However, results of this study necessitated further evaluation of the pharmacokinetics and safety of repeated oral dosing of acetaminophen at 30 mg/kg orally every 12 hours to determine clinical utility. In the second study, the pharmacokinetics, efficacy, and safety of oral acetaminophen (30 mg/kg) were examined in adult horses with naturally occurring chronic lameness. In that study, following 21 days of twice daily oral dosing at 30 mg/kg, acetaminophen was found to be safe with no evidence of gastric ulceration or hepatopathy in horses. Acetaminophen at 30 mg/kg twice daily for 21 days provided transient improvement in subjective and objective lameness evaluation when compared to baseline evaluation; however, the study concluded that acetaminophen may not be suitable as a monotherapy for management of moderate to severe orthopedic pain in horses In the third study, the pharmacokinetics and efficacy of oral acetaminophen (30 mg/kg) was examined in adult horses with experimentally induced endotoxemia when compared to placebo and the nonselective COX inhibitor flunixin meglumine. That study found that acetaminophen was superior to placebo and not statistically different from flunixin meglumine in reducing rectal temperature in adult horses with experimentally induced endotoxemia and may be an option for antipyresis in clinical cases, particularly when administration of traditional NSAIDs is contraindicated. Furthermore, acetaminophen administered at 30 mg/kg orally to adult horses with experimentally induced endotoxemia is an effective antipyretic but is unlikely to provide any alteration in systemic inflammatory response. / Doctor of Philosophy / Non-steroidal anti-inflammatory drugs (NSAIDs) are a mainstay of the management of pain and fever in horses. The most used NSAIDs in horses are the non-selective cyclooxygenase (COX) inhibitors flunixin meglumine and phenylbutazone, which are powerful anti-inflammatory agents. However, there are several complications, such as gastric ulcers and kidney damage, that are associated with these drugs – particularly in sick or dehydrated horses. Therefore, to reduce the risk of these complications, COX-2 selective inhibitors, such as firocoxib, have been developed for use in horses. However, there are still safety concerns with COX-2 inhibitors, and a safe, effective option for oral treatment of pain and fevers in horses is still needed. The studies presented in this dissertation explores the use, safety, and pharmacology of acetaminophen in adult horses for treating orthopedic pain and fever. In the first study, the pharmacokinetics and efficacy of oral acetaminophen at two different doses (20 mg/kg and 30 mg/kg) were examined in horses with pressure applied to the bottom of their hoof. The efficacy of acetaminophen at improving lameness in horses was compared to placebo and the non-selective COX inhibitor phenylbutazone. Acetaminophen when administered at 30 mg/kg produced a more rapid onset of greater improvement in lameness and lower heart rate compared to other treatments in this model, and therefore would be more suitable than acetaminophen at a lower dose of 20 mg/kg. Acetaminophen dosed at 30 mg/kg also resulted in a more rapid improvement in lameness scores than phenylbutazone at 2.2 mg/kg and was equivalent to phenylbutazone in lameness score reduction. However, results of this study necessitated further evaluation of the pharmacokinetics and safety of repeated oral dosing of acetaminophen at 30 mg/kg orally every 12 hours to determine if it was clinically useful in horses with orthopedic pain. In the second study, the pharmacokinetics, efficacy, and safety of oral acetaminophen (30 mg/kg) were examined in adult horses with naturally occurring chronic lamenesses (such as arthritis and navicular syndrome). In that study, following 21 days of twice daily oral dosing at 30 mg/kg, acetaminophen was found to be safe with no evidence of gastric ulcers or liver dysfunction in horses. Acetaminophen at 30 mg/kg twice daily for 21 days provided short-lived improvement in lameness when compared to each horse's baseline evaluation; however, the study concluded that acetaminophen may not be suitable alone for management of moderate to severe orthopedic pain in horses In the third study, the pharmacokinetics and efficacy of oral acetaminophen (30 mg/kg) was examined in adult horses with experimentally induced fever when compared to placebo and the nonselective COX inhibitor flunixin meglumine. That study found that acetaminophen was better than placebo and similar to flunixin meglumine in reducing fever in adult horses with experimentally induced fever and may be a suitable option for fever management in clinical cases.

The adipose-derived stromal vascular fraction (SVF) is a promising new therapy for equine flexor tendonitis. This heterogeneous population of cells may improve tendon healing via the production of growth and chemotactic factors capable of recruiting endogenous stem cells and increasing extracellular matrix production by tendon fibroblasts (TFBL). The purpose of this study was to evaluate the ability of adipose-derived cells (ADC) culture expanded from the SVF to act as trophic mediators in vitro. We hypothesized that ADCs would produce growth and chemotactic factors important in tendon healing and capable of inducing cell migration and matrix protein gene expression. Superficial digital flexor tendons and adipose tissue were harvested from eight adult horses and processed to obtain SVF cells, ADCs and TFBLs. Adipose-derived cells and TFBLs were grown in monolayer culture for growth factor quantification, to produce conditioned media for microchemotaxis, and in co-culture for quantification of matrix protein gene expression by TFBLs. Growth factor gene expression by SVF cells was significantly greater than in ADCs or TFBLs. Co-culture of TFBLs and ADCs resulted in modest up-regulation of matrix protein expression (collagen types I and III, decorin, and cartilage oligomeric matrix protein) by TFBLs. Media conditioned by ADCs induced ADC migration in a dose dependent manner. These findings support the role of both SVF and ADCs as trophic mediators in tendon regeneration. The differences detected in gene expression between SVF cells and ADCs indicate that additional studies are needed to evaluate the changes that occur during culture of these cells. / Master of Science