Validation of the BioPRYN enzyme-linked immunosorbent assay for detection of pregnancy-specific protein-B (PSPB) and diagnosis of pregnancy in American bison (Bison bison).

This study assessed the accuracy of the commercial BioPRYN® ELISA for the detection of pregnancy-specific protein-B (PSPB) using a single blood sample to determine pregnancy status in American bison (Bison bison). A total of 49 bison cows were used in the study, and sampled at two time-points during the gestation period, fall and spring, correlating with early- to mid-term gestation (average 62.9 days post-mating) and mid- to late-term gestation (average 229.2 days post-mating), respectively. Sensitivity of the test during early- to mid-term gestation sampling period (fall) was 87.1%, while specificity was 100%; sensitivity of the test during late-term gestation sampling period (spring) was 96.3%, while specificity remained at 100%. In total, the test showed a total sensitivity of 91.4%, specificity of 100% and total accuracy of 93.8%, similar to domestic cattle. Use of the single-sample BioPRYN® ELISA in American Bison for pregnancy diagnosis is economical and practical, minimizing animal handling time, frequency and subsequent stress while providing accurate results for pregnancy diagnosis at 62 days post-mating. This method should be considered over more traditional pregnancy diagnosis methods for use in managed bison herds.

State-selective binding peptides for heterotrimeric G-protein subunits: novel tools for investigating G-protein signaling dynamics.

Heterotrimeric G-proteins, comprising Galpha, Gbeta, and Ggamma subunits, are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This characteristic is achieved by the adoption of two principal states: an inactive state in which GDP-bound Galpha is complexed with the Gbetagamma dimer, and an active state in which GTP-bound Galpha is freed of its Gbetagamma binding partner. Structural studies have illustrated the basis for the distinct conformations of these states which are regulated by alterations in three precise 'switch regions' of the Galpha subunit. Discrete differences in conformation between GDP- and GTP-bound Galpha underlie its nucleotide-dependent protein-protein interactions (e.g., with Gbetagamma/receptor and effectors, respectively) that are critical for maintaining their proper nucleotide cycling and signaling properties. Recently, several screening approaches have been used to identify peptide sequences capable of interacting with Galpha (and free Gbetagamma) in nucleotide-dependent fashions. These peptides have demonstrated applications in direct modulation of the nucleotide cycle, assessing the structural basis for aspects of Galpha and Gbetagamma signaling, and serving as biosensor tools in assays for Galpha activation including high throughput drug screening. In this review, we highlight some of the methods used for such discoveries and discuss the insights that can be gleaned from application of these identified peptides.

Primary and secondary hemostatic functionalities of rehydrated, lyophilized platelets.

BACKGROUND: The rehydrated, lyophilized (RL) platelet (PLT) is being developed as a hemostatic infusion agent for the control of active bleeding. The key to the method for preparing RL PLTs is a mild aldehyde stabilization that allows for freezing and lyophilizing without cellular rupture. RL PLTs have been shown to be effective at rapidly controlling bleeding in animal models of cardiopulmonary bypass induced PLT dysfunction and washout thrombocytopenia, yet the rehydrated cells have proved to be safe with respect to induction of pathologic intravascular coagulation. STUDY DESIGN AND METHODS: In vitro and in vivo studies were performed to better understand the differential effect of the RL PLT manufacturing method on primary and secondary hemostatic processes. The functionality of the von Willebrand factor (VWF) receptor (glycoprotein Ib) complex, the PAR receptors, integrin-mediated aggregation (inside-out signaling), and surface membrane prothrombin to thrombin conversion systems were investigated. RESULTS: RL PLTs were found to retain native VWF-mediated adhesion and surface thrombin generation functions. In contrast, the coupling of thrombin receptors to integrin inside-out signaling was largely inhibited. CONCLUSION: These results suggest that RL PLTs may stop bleeding by forming primary hemostatic plugs and providing a localized source of thrombin for secondary hemostatic processes, yet do not build up occlusive pathologic clots possibly because integrin functions for forming PLT-PLT aggregates are partially inhibited.

Minimal determinants for binding activated G alpha from the structure of a G alpha(i1)-peptide dimer.

G-proteins cycle between an inactive GDP-bound state and an active GTP-bound state, serving as molecular switches that coordinate cellular signaling. We recently used phage display to identify a series of peptides that bind G alpha subunits in a nucleotide-dependent manner [Johnston, C. A., Willard, F. S., Jezyk, M. R., Fredericks, Z., Bodor, E. T., Jones, M. B., Blaesius, R., Watts, V. J., Harden, T. K., Sondek, J., Ramer, J. K., and Siderovski, D. P. (2005) Structure 13, 1069-1080]. Here we describe the structural features and functions of KB-1753, a peptide that binds selectively to GDP x AlF4(-)- and GTPgammaS-bound states of G alpha(i) subunits. KB-1753 blocks interaction of G alpha(transducin) with its effector, cGMP phosphodiesterase, and inhibits transducin-mediated activation of cGMP degradation. Additionally, KB-1753 interferes with RGS protein binding and resultant GAP activity. A fluorescent KB-1753 variant was found to act as a sensor for activated G alpha in vitro. The crystal structure of KB-1753 bound to G alpha(i1) x GDP x AlF4(-) reveals binding to a conserved hydrophobic groove between switch II and alpha3 helices and, along with supporting biochemical data and previous structural analyses, supports the notion that this is the site of effector interactions for G alpha(i) subunits.

A bifunctional Galphai/Galphas modulatory peptide that attenuates adenylyl cyclase activity.

Signaling via G-protein coupled receptors is initiated by receptor-catalyzed nucleotide exchange on Galpha subunits normally bound to GDP and Gbetagamma. Activated Galpha . GTP then regulates effectors such as adenylyl cyclase. Except for Gbetagamma, no known regulators bind the adenylyl cyclase-stimulatory subunit Galphas in its GDP-bound state. We recently described a peptide, KB-752, that binds and enhances the nucleotide exchange rate of the adenylyl cyclase-inhibitory subunit Galpha(i). Herein, we report that KB-752 binds Galpha(s) . GDP yet slows its rate of nucleotide exchange. KB-752 inhibits GTPgammaS-stimulated adenylyl cyclase activity in cell membranes, reflecting its opposing effects on nucleotide exchange by Galpha(i) and Galpha(s).

Structure of Galpha(i1) bound to a GDP-selective peptide provides insight into guanine nucleotide exchange.

Heterotrimeric G proteins are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This characteristic is achieved by the adoption of two principal states: an inactive, GDP bound state and an active, GTP bound state. Under basal conditions, G proteins exist in the inactive, GDP bound state; thus, nucleotide exchange is crucial to the onset of signaling. Despite our understanding of G protein signaling pathways, the mechanism of nucleotide exchange remains elusive. We employed phage display technology to identify nucleotide state-dependent Galpha binding peptides. Herein, we report a GDP-selective Galpha binding peptide, KB-752, that enhances spontaneous nucleotide exchange of Galpha(i) subunits. Structural determination of the Galpha(i1)/peptide complex reveals unique changes in the Galpha switch regions predicted to enhance nucleotide exchange by creating a GDP dissociation route. Our results cast light onto a potential mechanism by which Galpha subunits adopt a conformation suitable for nucleotide exchange.

Outbreak of salmonellosis in a zoologic collection of lorikeets and lories (Trichoglossus, Lorius, and Eos spp.).

During August 2001, a syndrome characterized by acute lethargy and dyspnea was observed in a population of 45 lorikeets and lories in an open-air zoologic exhibit. The first death occurred on August 10, and within the next 12 days, nine more birds died (22% mortality rate). Hepatomegaly, reddening and congestion of the lungs, and injection of the serosal surface of the intestines were the common gross findings. Histologic changes, including fibrinonecrotic hepatitis and splenitis, bacterial emboli (liver, spleen, lung, kidney, proventriculus), pulmonary congestion and hemorrhage, and enteritis, were indicative of an acute, overwhelming bacterial septicemia. Salmonella typhimurium, with the same antibiogram, was isolated from four birds. Several birds had attacked and killed a snake on July 24, and Salmonella serogroup B (untypeable) was isolated from intestine and kidney samples of a garter snake caught in the open-air exhibit on August 28. Salmonella was also isolated from environmental samples of the exhibit but not from food preparation areas. After antimicrobial therapy, Salmonella spp. was not isolated from the surviving birds. The source of Salmonella in this outbreak remains unknown, but infection either directly or indirectly from snakes in the exhibit is possible. Contact between captive psittacine populations and reptiles should be avoided to prevent the risk of salmonellosis.

An Epstein-Barr-related herpesvirus from marmoset lymphomas.

Epstein-Barr virus (EBV) is implicated in the development of human B cell lymphomas and carcinomas. Although related oncogenic herpesviruses were believed to be endemic only in Old World primate species, we now find these viruses to be endemic in New World primates. We have isolated a transforming, EBV-related virus from spontaneous B cell lymphomas of common marmosets (Callithrix jacchus). Sequencing of two-thirds of the genome reveals considerable divergence from the genomes of EBV and Old World primate EBV-related viruses, including differences in genes important for virus-induced cell growth transformation and pathogenesis. DNA related to the C. jacchus herpesvirus is frequently detected in squirrel monkey peripheral blood lymphocytes, indicating that persistent infection with EBV-related viruses is prevalent in both New World primate families. Understanding how these more divergent EBV-related viruses achieve similar biologic outcomes in their natural host is likely to provide important insights into EBV infection, B cell growth transformation, and oncogenesis.

Fatal lymphoproliferative disease associated with a novel gammaherpesvirus in a captive population of common marmosets.

BACKGROUND AND PURPOSE: Callitrichids (marmosets and tamarins) are extremely susceptible to experimental tumor induction by herpesviruses native to other primate species. A colony of common marmosets developed a syndrome of weight loss, inappetence, diarrhea, and in several animals, palpable abdominal masses. METHODS: Marmosets in the colony were subjected to histologic examination and serologic testing for Epstein-Barr virus (EBV). The DNA from tumors that developed in the marmosets was subjected to consensus primer polymerase chain reaction (PCR) analysis designed to amplify conserved regions of herpesvirus genomes. RESULTS: The mesenteric lymph nodes and intestinal mucosa were consistently infiltrated by principally B lymphocytes, which often obliterated the normal architecture. Of 84 clinically normal marmosets, 52 were seropositive for EBV. The tumor DNA contained previously unreported herpesvirus sequences closely related to but distinct from those of EBV, Herpesvirus papio, and these lymphocryptovirus, a novel gammaherpesvirus. Results of PCR analysis of circulating lymphocytes from EBV-positive, clinically normal marmosets were negative for EBV antibodies and were positive for marmoset lymphocryptovirus; PCR analysis of circulating lymphocytes from EBV-negative marmosets yielded negative results for EBV and this novel marmoset lymphocryptovirus. CONCLUSION: This novel gammaherpesvirus possibly associated with tumor development may have important management implications for captive callitrichids.

Ferrets. Clinical pathology and sample collection.

Diagnostic sampling and interpretation in the ferret are similar to other small mammals in many ways. There are several aspects of clinical pathology and diagnostic procedures, however, that are unique to the ferret. This article is designed to assist the clinician in obtaining and interpreting diagnostic samples for the ferret. Methods for blood and urine collection, bone marrow and organ aspirations, and organ biopsy techniques are discussed.

Urgent care of the pet rabbit.

Emergency and critical care principles are similar for all mammals; however, the physiology and natural behavior of rabbits create an animal that is easily stressed and requires specialized handling techniques. This article reviews diagnostic and therapeutic techniques, nutritional support, and pain management for urgent care of pet rabbits. Common differential diagnoses for emergencies are briefly reviewed by the clinical presentation. A table of drug dosages used for urgent care is provided.

Regaining space by using elastic orthodontic separators.

When an extensive carious lesion is present, interproximal space may be lost due to drifting of adjacent teeth. This may encroach upon the space needed to restore the tooth to proper occlusal and interproximal contours. Although the space may be regained with conventional orthodontics, this may not be an option in some cases due to financial or technical considerations. The outlined clinical technique uses elastic orthodontic separators in conjunction with an incrementally modified acrylic crown to regain lost interproximal space. The results of this technique were achieved rapidly and allowed for the placement of a physiologically contoured restoration. This procedure is technically simple, provides quick results, and is less expensive for the patient than conventional orthodontics.

Effects of mydriatic agents in cockatoos, African gray parrots, and Blue-fronted Amazon parrots.

OBJECTIVE: To compare, in psittacines, the mydriatic effects of several topically applied curariform, sympathomimetic, and parasympatholytic drugs with and without the addition of surface-acting penetrating agents. DESIGN: Prospective, randomized controlled trial. ANIMALS: 10 adult cockatoos (Cacatua sulphurea subspecies), 2 adult African gray parrots (Psittacus erithacus), and 3 adult Blue-fronted Amazon parrots (Amazona aestiva). PROCEDURE: Three curariform drugs (d-tubocurarine, pancuronium, and vecuronium bromide) and 2 autonomic drugs (atropine and phenylephrine hydrochloride) were evaluated. Drugs were tested with and without the addition of a surface-acting penetrating agent, either saponin or benzalkonium chloride. The agent that resulted in the most significant change in pupillary diameter with the fewest systemic side effects in the cockatoos then was evaluated for its effects in the African gray parrots and the Blue-fronted Amazon parrots. During each drug trial, 1 eye was randomly selected to receive the control drug (0.9% NaCl), and the opposite eye was selected to receive the test drug. Each pupil was videotaped 5 (cockatoos only), 15, 30, 45, 60, and 75 minutes after treatment. Pupil diameters were measured by use of a computerized image analysis system. Data for pupil size were analyzed by means of repeated measures ANOVA. RESULTS: Vecuronium without the addition of a surface-acting penetrating agent produced the most consistent and greatest pupillary dilatation in all 3 species with the fewest systemic side effects. CLINICAL IMPLICATIONS: Vecuronium is potentially a clinically useful, topical mydriatic agent for use in avian species. Documented differences in the prevalence of systemic side effects between species suggests that caution should be applied when applying this drug bilaterally.

Outcome after open-heart surgery in infants and children.

We have studied the neurodevelopmental outcome of 104 consecutive unselected children who underwent open-heart surgery from 1987 through 1989. Survivors had formal neurologic and psychometric examinations after 2 years of age. Mean IQ was 90, and 78% had scores above 70. Cerebral palsy occurred in 22%. Deep hypothermia for longer than 45 minutes was associated with IQ less than 85 (P < .001) and later cerebral palsy (P = .02). Those less than 1 month old at operation had a median IQ of 96, and 25% had cerebral palsy. Median IQ for survivors of hypoplastic left heart syndrome was 66, only one had an IQ above 70, and 57% had cerebral palsy. Median IQ for transposition of great arteries was 109, only one was less than 85, and all had normal neurologic examinations. Those between 1 and 6 months of age at operation had a median IQ of 93, with 64% above 85, and 5% had cerebral palsy. Those older than 6 months had a median IQ of 99, with 70% above 85, and 13% had cerebral palsy. For infants less than 1 month old at operation, a strong association existed between outcome, type of lesion, and duration of deep hypothermia (P < .01), although not in all cases. In those older than 1 month at operation, no association existed between outcome and any study variable. Although the majority of children have an uneventful outcome after open-heart surgery, a significant morbidity exists. This is related to several factors, including type of lesion and duration of hypothermia, particularly in neonates; preoperative congenital and acquired lesions; and possible perioperative cerebrovascular events.

Absence of linkage of apparently single gene mediated ADHD with the human syntenic region of the mouse mutant Coloboma.

Attention deficit disorder (ADHD) is a complex biobehavioral phenotype which affects up to 8% of the general population and often impairs social, academic, and job performance. Its origins are heterogeneous, but a significant genetic component is suggested by family and twin studies. The murine strain, coloboma, displays a spontaneously hyperactive phenotype that is responsive to dextroamphetamine and has been proposed as a genetic model for ADHD. Coloboma is a semi-dominant mutation that is caused by a hemizygous deletion of the SNAP-25 and other genes on mouse chromosome 2q. To test the possibility that the human homolog of the mouse coloboma gene(s) could be responsible for ADHD, we have carried out linkage studies with polymorphic markers in the region syntenic to coloboma (20p11-p12). Five families in which the pattern of inheritance of ADHD appears to be autosomal dominant were studied. Segregation analysis of the traits studied suggested that the best fitting model was a sex-influenced, single gene, Mendelian pattern. Several genetic models were evaluated based on estimates of penetrance, phenocopy rate, and allele frequency derived from our patient population and those of other investigators. No significant linkage was detected between the disease locus and markers spanning this chromosome 20 interval.

Physical examination and radiographic analysis to detect dental and mandibular bone resorption in cats: a study of 81 cases from necropsy.

The mandibles of 81 randomly selected cats were examined clinically and radiographically for dental calculus, periodontitis, odontoclastic resorptive lesions (FORL) and osteoporosis. Extent of calculus increased with increasing age. Prevalence and severity of periodontitis and FORL increased with increasing age. Osteoclastic resorption and/or osteoporosis increased with age, advancing from rostral to caudal in cortical bone. No significant correlation of periodontitis with FORL could be demonstrated.

Previously apparently undescribed syndrome: shallow orbits, ptosis, coloboma, trigonocephaly, gyral malformations, and mental and growth retardation.

We describe 2 children with severe ptosis, trigonocephaly, broad nasal bridge, and major brain malformation. A total of 8 children have been reported who share most of these findings. Two of the individuals have had identical pericentric inversions involving chromosome 2p12-q14. These cases appear to represent a unique malformation syndrome.

Familial leuconychia, knuckle pads, hearing loss, and palmoplantar hyperkeratosis: an additional family with Bart-Pumphrey syndrome.

A family with five members who have variable findings of leuconychia, knuckle pads, hearing loss, and palmoplantar hyperkeratosis is described. The findings in these subjects are compared with those noted in previously reported patients with Bart-Pumphrey syndrome. The range of disorders which include knuckle pads as part of the phenotype is reviewed.

Congenital contractures, ectodermal dysplasia, cleft lip/palate, and developmental impairment: a distinct syndrome.

Brothers were affected with severe congenital contractures, multiple cutaneous manifestations of ectodermal dysplasia, cleft lip/palate, and psychomotor and growth impairment. High resolution prometaphase chromosomes were normal. Molecular studies of DNA markers, closely flanking the X-linked hypohidrotic ectodermal dysplasia locus, did not show evidence of a submicroscopic deletion from the Xq12-q13 region. The parents and a normal sister exhibited none of these findings. This constellation of anomalies appears to represent a unique AR or XLR syndrome.

Identical twins with Weissenbacher-Zweymüller syndrome and neural tube defect.

Neurologic abnormalities have been described only once previously in a child with Weissenbacher-Zweymüller syndrome (WZS), a rare skeletal dysplasia, evident neonatally. We report on identical twin male infants with skeletal findings typical of WZS, including small size at birth, proximal limb shortness, mid face hypoplasia, and myopia. In addition, twin B had a parieto occipital encephalocele while twin A had a meningocele at the same location. Twin B has had significant delays in development and hearing loss.