Determining prevalence of bluetongue and epizootic hemorrhagic disease viruses in mule deer in Arizona (USA) using whole blood dried on paper strips compared to serum analyses

We investigated the feasibility of using whole blood dried on paper strips as a means to collect antibody prevalence data for the epizootic hemorrhagic disease viruses (EHDV) and bluetongue viruses (BTV) from hunter-harvested male mule deer (Odocoileus hemionus) in October 2002 from Arizona, USA. We compared antibody prevalence estimates in mule deer from paired paper strip and serum samples. Prevalence data obtained from elution of dried blood on paper strips proved to be consistent with results from serum in 94% of the samples tested. The paper strip method allows easy collection of blood from dead animals, with a smaller amount of blood being needed for analyses. Also, samples do not need to be refrigerated before analyses. We also used serum samples to determine hemorrhagic disease (HD) serotype exposure status of mule deer harvested from 4 distinct areas in Arizona. Antibodies to BTV and EHDV were identified in 3 of the 4 areas, with positive results to EHDV-1, EHDV-2, BTV-10, and BTV-11 being most common. Many animals did not have antibodies against the BTV serotypes. Exposure varied geographically and potentially with elevation. Hemorrhagic disease viruses commonly infect Arizona mule deer, except on the Kaibab Plateau in northern Arizona.     

Comparison of Lactate, Base Excess, Bicarbonate, and pH as Predictors of Mortality after Severe Trauma in Rhesus Macaques (Macaca mulatta)

Social group housing of rhesus macaques at biomedical facilities is advocated to improve the psychologic wellbeing of these intelligent and social animals. An unintended outcome of social housing in this species is increased intraspecific aggression resulting in cases of severe multiple trauma and posttraumatic shock. The metabolic correlates of oxygen debt are likely important quantifiers of the severity of posttraumatic shock and may serve as useful guides in the treatment of these cases. The purpose of this retrospective study was to evaluate venous blood lactate, base excess, bicarbonate, and pH as predictors of mortality. These 4 variables were assessed in 84 monkeys with severe traumatic injury and shock. Data were available from blood samples collected prior to resuscitation therapy and the day after resuscitation therapy. The pre- and postresuscitation therapy levels of the variables then were tested for association with 6-d survival. When measured prior to resuscitation therapy, all variables were strongly correlated with each other and had a statistically significant association with survival. No single variable had both strong specificity and high sensitivity when measured prior to resuscitation therapy. Survival analysis showed that as the number of categorical indicators of acidosis increased, 6-d survival decreased. Analysis of the 4 variables after resuscitation therapy indicated that lactate was the only variable significantly associated with survival in our study.Many research facilities maintain populations of rhesus macaques in group housing that allows them to engage in social behaviors that are normal for the species, such as grooming, play, and huddling with conspecifics.²¹ An unintended consequence of social housing is intraspecific aggression. The trauma, specifically crush injuries from multiple bite wounds to the limbs and face, that may result from this aggression can be severe and often leads to significant morbidity and mortality if ignored or poorly treated.¹⁷ Major multiple trauma injuries result in sudden physical and metabolic alterations that can progress to organ dysfunction, organ failures, and even death. In addition to multiple severe skin wounds, the hemorrhage and obligatory edema that occur within the injured soft tissues have significant effect on circulating blood volume, resulting in intravascular volume depletion and hypovolemic shock. The associated decreased organ perfusion and impaired oxygen delivery result in regional hypoxia and anaerobic metabolism. The end-products of anaerobic metabolism are 2 ATP molecules and pyruvate, which is converted to lactic acid. Prolonged and severe tissue hypoperfusion results in the generation of large quantities of hydrogen ions (H⁺) from lactic acid, resulting in metabolic acidosis.^2,9Serum markers of metabolic acidosis may be measured as part of the critical care diagnostic plan to assess the severity of injury, determine treatment efficacy, and provide prognostic information. The most common of these markers include lactate, base excess (or base deficit), bicarbonate, and pH. Technologic advances in point-of-care testing have made rapid laboratory assessment of these markers accurate, practical, and affordable for veterinary medical facilities. However, no studies to date have validated markers of metabolic acidosis as predictors of mortality in rhesus macaques. Nor has the significance of these values been examined when used in combination or when they provide conflicting data.Lactate, an end product of anaerobic metabolism, can be measured directly by many point-of-care analyzers. As calculated by most point-of-care analyzers, base excess is determined by using measured carbon dioxide, pH, and serum bicarbonate values and represents the concentration of titratable base minus the concentration of titratable acid needed to normalize the pH of a liter of blood to physiologic levels. A decreased base excess is thought to represent the presence of unmeasured anions. In acute trauma cases, the primary unmeasured anion is assumed to be lactate.⁴ Therefore, base excess usually is viewed as a surrogate marker for lactic acidosis.^9,11 Bicarbonate is a buffer for serum hydrogen ions released during anaerobic metabolism. As metabolic acidosis worsens, bicarbonate levels decrease. In humans, bicarbonate and base excess are strongly correlated.^8,10 Compared with lactate, base excess, and bicarbonate, serum pH is less clinically relevant for assessing metabolic acidosis in human patients.^6,9,19 This difference most likely is due to the extensive compensatory physiologic mechanisms in place to maintain normal pH. pH is a direct measure of acidemia, whereas lactate, base excess, and bicarbonate are common means of characterizing acidosis.⁹Multiple studies have been published in the human medical literature that validate the ability of lactate, base excess, bicarbonate, and pH for predicting morbidity and mortality among trauma and surgical patients.^12,14,20,23 However, no single value has proven superior to the others in these regards,⁹ and the validity of these measurements has not been demonstrated in nonhuman primates. Several human studies have noted the predictive value of the initial lactate level.^1,3,16 Other studies have demonstrated outcome is better predicted by other variables²² or have shown no correlation between lactate level and mortality.¹³During hospitalization, the validity of these markers may be confounded by the rapid intravenous infusion of large volumes of resuscitation fluids and electrolytes administered to increase circulatory volume in shock patients. The confounding affects of shock therapy are important because these markers of metabolic acidosis frequently are used by clinicians to guide resuscitation. For example, the administration of large quantities of exogenous lactate (massive infusion of lactated Ringers solution) has been shown to increase lactate to levels significantly greater than those expected to result from the shock process alone.^7,18 However, this elevation of lactate is transient and not associated with acidosis, because the end products of lactate metabolism are bicarbonate and glucose.^7,24 In addition, the administration of sodium bicarbonate during resuscitation likely would confound the utility of bicarbonate measurements. The administration of sodium bicarbonate would similarly confound the utility of base excess as an endpoint for resuscitation, because bicarbonate is used in the calculation of base excess.For the past 7 y, serial measurements of these systemic markers of metabolic acidosis have been used to guide the need for and response to resuscitation of nonhuman primate cases at our institution, the Oregon National Primate Research Center. Although the human medical literature and our experience in practice indicate that these measurements are valuable for case management, no previous scientific studies have validated them as indicators of morbidity or mortality in nonhuman primates. The purpose of this retrospective study was to evaluate venous blood lactate, base excess, bicarbonate, and pH values, obtained before and after treatment of shock, as predictors of 6-d mortality after trauma in rhesus macaques.     

Discovery of a novel and potent series of thieno[3,2-b]pyridine-based inhibitors of c-Met and VEGFR2 tyrosine kinases

A series of thieno[3,2-b]pyridine-based inhibitors of c-Met and VEGFR2 tyrosine kinases is described. The compounds demonstrated potency with IC₅₀ values in the low nanomolar range in vitro while the lead compound also showed in vivo activity against various human tumor xenograft models in mice. Further exploration of this class of compounds is underway.     

The Rous sarcoma virus Env glycoprotein contains a highly conserved motif homologous to tyrosine-based endocytosis signals and displays an unusual internalization phenotype

The cytoplasmic domains of retroviral transmembrane (TM) glycoproteins contain conserved sequence motifs that resemble tyrosine-based (YXXO-type) endocytosis signals. We have previously described a mutant Rous sarcoma virus (RSV) Env protein, Env-mu26, with an L165R mutation in the membrane-spanning domain (MSD) of TM, that exhibited dramatically decreased steady-state surface expression (G. L. Davis and E. Hunter, J. Cell Biol. 105:1191-1203, 1987; P. B. Johnston, J. Y. Dong, and E. Hunter, Virology 206:353-361, 1995). We now demonstrate that the tyrosine of the Y(190)RKM motif in the RSV TM cytoplasmic domain is crucial for the mu26 phenotype and is part of an efficient internalization signal in the context of a mutant MSD. In contrast, despite the presence of the Y(190)RKM motif, wild-type RSV Env is constitutively internalized at a slow rate (1.1%/min) more characteristic of bulk uptake during membrane turnover than of active clustering into endocytic vesicles. The mu26 mutation and two MSD mutations that abrogate palmitoylation of TM resulted in enhanced Env endocytosis indicative of active concentration into coated pits. Surprisingly, an Env-Y190A mutant was apparently excluded from coated pits since its uptake rate of 0.3%/min was significantly below that expected for the bulk rate. We suggest that in RSV Env an inherently functional endocytosis motif is silenced by a counteracting determinant in the MSD that acts to prevent clustering of Env into endocytic vesicles. Mutations in either the cytoplasmic tail or the MSD that inactivate one of the two counteracting signals would thus render the remaining determinant dominant.     

Association of Moraxella ovis with keratoconjunctivitis in mule deer and moose in Wyoming

Six cases of infectious keratoconjunctivitis (IKC) in mule deer (Odocoileus hemionus) and moose (Alces alces) in Wyoming (USA) were investigated during fall and winter of 1995 and 1996. Excessive lacrimation, mucopurulent conjunctivitis, keratitis, and corneal opacity were observed in mule deer. Moose had severe mucopurulent conjunctivitis, keratitis, and corneal ulceration. Hemolytic, non-piliated Moraxella ovis was isolated from two mule deer and two moose. We attempted to reproduce IKC in three mule deer fawns using an isolate of M. ovis from a clinically affected mule deer. These fawns did not develop clinical signs of infection and the bacterium was not reisolated from inoculated deer. Inoculated deer may not have developed clinical signs because deer were not exposed to ultraviolet light or mechanical insult before inoculation. In addition, the isolate used for inoculation may have lost virulence factors through passage, or M. ovis may not have been the primary pathogen responsible for clinical disease in the natural cases of IKC we investigated. The etiology of IKC in free-ranging wild ruminants remains poorly understood.     

Strongyloides robustus and the northern sympatric populations of northern (Glaucomys sabrinus) and southern (G. volans) flying squirrels

Within North America, northern (Glaucomys sabrinus) and southern (Glaucomys volans) flying squirrels occupy distinct ranges with limited overlap. Sympatry in northern latitudes coincides with northern hardwood vegetation from Minnesota to New England. Strongyloides robustus is an intestinal parasite that infects both species but appears to be deleterious only to northern flying squirrels. As a result, S. robustus could be a critical determinant of flying squirrel population characteristics in at least some areas of sympatry. However, cold weather could potentially limit the distribution of S. robustus in northern climates. Therefore, we assessed fecal samples from both flying squirrel species to determine the presence of the nematode in Wisconsin. Strongyloides robustus was found in 12 flying squirrel scat samples and infected 52% of southern flying squirrels and 11% of northern flying squirrels. Prevalence of S. robustus infection for northern flying squirrels was substantially lower than previously reported from more southern regions. This is the northernmost documentation of S. robustus in flying squirrels and the first documentation of S. robustus parasitizing flying squirrels in Wisconsin.     

An assay for adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase

Adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the conversion of glucose 1-phosphate and adenosine 5'-triphosphate to ADP-glucose and pyrophosphate. We present a radioactive assay of this enzyme with a higher signal/noise ratio. After stopping the reaction that uses [14C]glucose 1-phosphate as a substrate, the ADP-[14C]glucose formed as a product is converted to [14C]glycogen by the addition of glycogen synthase and nonradioactive glycogen as primer. The final product is precipitated and washed, and the radioactivity is measured in a scintillation counter. The [14C]glucose 1-phosphate that did not react is easily eliminated during the washes. We have found that this assay produces much lower blanks than previously described radioactive methods based on binding of ADP-[14C]glucose to O-(diethylaminoethyl)-cellulose paper. In addition, we tested the kinetic parameters for the effectors of the Escherichia coli ADP-Glc PPase and both assays yielded identical results. The presented method is more suitable for Km or S(0.5) determinations of ADP-Glc PPases having high apparent affinity for glucose 1-phosphate. It is possible to use a higher specific radioactivity to increase the sensitivity at lower concentrations of [14C]glucose 1-phosphate without compromising the blanks obtained at higher concentrations.     

Prediction of "aggregation-prone" and "aggregation-susceptible" regions in proteins associated with neurodegenerative diseases

Increasing evidence indicates that many peptides and proteins can be converted in vitro into highly organised amyloid structures, provided that the appropriate experimental conditions can be found. In this work, we define intrinsic propensities for the aggregation of individual amino acids and develop a method for identifying the regions of the sequence of an unfolded peptide or protein that are most important for promoting amyloid formation. This method is applied to the study of three polypeptides associated with neurodegenerative diseases, Abeta42, alpha-synuclein and tau. In order to validate the approach, we compare the regions of proteins that are predicted to be most important in driving aggregation, either intrinsically or as the result of mutations, with those determined experimentally. The knowledge of the location and the type of the "sensitive regions" for aggregation is important both for rationalising the effects of sequence changes on the aggregation of polypeptide chains and for the development of targeted strategies to combat diseases associated with amyloid formation.     

Mycobacterial lipoarabinomannan: an extraordinary lipoheteroglycan with profound physiological effects

Detailed structural and functional studies over the last decade have led to current recognition of the mycobacterial lipoarabinomannan (LAM) as a phosphatidylinositol anchored lipoglycan with diverse biological activities. Fatty acylation has been demonstrated to be essential for LAM to maintain its functional integrity although the focus has largely been on the arabinan motifs and the terminal capping function. It has recently been shown that the mannose caps may be involved not only in attenuating host immune response, but also in mediating the binding of mycobacteria to and subsequent entry into macrophages. This may further be linked to an intracellular trafficking pathway through which LAM is thought to be presented by CD1 to subsets of T-cells. The implication of LAM as major histocompatibility complex (MHC)-independent T-cell epitope and the ensuing immune response is an area of intensive studies. Another recent focus of research is the biosynthesis of arabinan which has been shown to be inhibitable by the anti- tuberculosis drug, ethambutol. The phenomenon of truncated LAM as synthesized by ethambutol resistant strains provides an invaluable handle for dissecting the array of arabinosyltransferases involved, as well as generating much needed structural variants for further structural and functional studies. It is hoped that with more systematic investigations based on clinical isolates and human cell lines, the true significance of LAM in the immunopathogenesis of tuberculosis and leprosy can eventually be explained.      

Analysis of the cleavage site of the human immunodeficiency virus type 1 glycoprotein: requirement of precursor cleavage for glycoprotein incorporation.

Endoproteolytic cleavage of the glycoprotein precursor to the mature SU and TM proteins is an essential step in the maturation of retroviral glycoproteins. Cleavage of the precursor polyprotein occurs at a conserved, basic tetrapeptide sequence and is carried out by a cellular protease. The glycoprotein of the human immunodeficiency virus type 1 contains two potential cleavage sequences immediately preceding the N terminus of the TM protein. To determine the functional significance of these two potential cleavage sites, a series of mutations has been constructed in each site individually, as well as in combinations that altered both sites simultaneously. A majority of the mutations in either potential cleavage site continued to allow efficient cleavage when present alone but abrogated cleavage of the precursor when combined. Despite being transported efficiently to the cell surface, these cleavage-defective glycoproteins were unable to initiate cell-cell fusion and viruses containing them were not infectious. Viruses that contained glycoproteins with a single mutation, and that retained the ability to be processed, were capable of mediating a productive infection, although infectivity was impaired in several of these mutants. Protein analyses indicated that uncleaved glycoprotein precursors were inefficiently incorporated into virions, suggesting that cleavage of the glycoprotein may be a prerequisite to incorporation into virions. The substitution of a glutamic acid residue for a highly conserved lysine residue in the primary cleavage site (residue 510) had no effect on glycoprotein cleavage or function, even though it removed the only dibasic amino acid pair in this site. Peptide sequencing of the N terminus of gp41 produced from this mutant glycoprotein demonstrated that cleavage continued to take place at this site. These results, demonstrating that normal cleavage of the human immunodeficiency virus type 1 glycoprotein can occur when no dibasic sequence is present at the cleavage site, raise questions about the specificity of the cellular protease that mediates this cleavage and suggest that cleavage of the glycoprotein is required for efficient incorporation of the glycoprotein into virions.