A von Willebrand's factor genomic nucleotide variant and polymerase chain reaction diagnostic test associated with inheritable type-2 von Willebrand's disease in a line of german shorthaired pointer dogs.

Heritable, type-2 von Willebrand's disease (vWD) was studied in a line of German Shorthaired Pointers (GSPs) in which some members had a nucleotide variant in exon 28 of the von Willebrand factor (VWF) gene. A polymerase chain reaction (PCR) diagnostic test for the nucleotide variant was developed to establish the disorder's mode of inheritance and to eliminate it from the line. Thirty-six of the 49 GSPs in the line, 14 unrelated GSP controls, and 71 unrelated dogs of various breeds were tested for the presence of the variant nucleotide. All the dogs with a vWF antigen deficiency (<70% of normal) were either homozygous or heterozygous for the nucleotide variant. The variant was not located in any tested dog in the line or outside of the line with a vWF antigen value greater than 68%. Of the GSPs in the line tested, two were homozygous for the variant, 15 were heterozygous, and 19 were variant free. The collective evidence of this and other studies is consistent with the variant nucleotide being the cause of the type-2 vWD in this line of GSPs and German Wirehaired Pointers. The PCR diagnostic test for the variant nucleotide was successfully used to select and produce progeny that were variant free and vWD free. This test should be effective in the subsequent elimination of this same variant from other lines of dogs.

Enrichment of triadic and terminal cisternae vesicles from rabbit skeletal muscle.

An enriched triad and terminal cisternae preparation was achieved from skeletal muscle through alterations of the differential centrifugation and muscle homogenization protocols. Both yield and specific activity (pmoles of radioligand binding per mg protein) were optimized for (3)H-PN200-110 (transverse tubule marker) and (3)H-ryanodine (terminal cisternae marker) binding sites. By pelleting crude microsomes between 2,000 an 12,000 x g without any rehomogenizations, we improved both the yield and specific activity of transverse tubule and terminal cisternae markers in crude microsomes by approximately 4-fold to 1000-3000 pmoles binding sites (starting material: approximately 400 grams wet weight fast twitch skeletal muscle), with 10-15 pmoles/mg. Rehomogenization of the 1,000 x g pellet, which is typically discarded, allowed recovery of an additional 5000 pmoles PN200-110 binding sites and an additional 8000 pmoles ryanodine binding sites. Crude microsomes from the rehomogenized 1,000 x g pellets typically displayed specific activities of 20-25 pmoles binding/mg for both (3)H-PN200-110 and (3)H-ryanodine. Separation of crude microsomes on a sucrose gradient increased specific activity up to a maximum of 50 pmoles/mg in a specific fraction, a five- to ten-fold increase over standard triadic or terminal cisternae preparations. The mean specific activity for enriched triads was 30-40 pmoles/mg for both PN200-110 and ryanodine in pooled fractions, while pooled fractions of enriched terminal cisternae displayed low (3)H-PN200-110 binding (3-5 pmoles/mg) and high (3)H-ryanodine-specific activity (30-40 pmoles/mg).

Modulation of potassium channel gating by coexpression of Kv2.1 with regulatory Kv5.1 or Kv6.1 alpha-subunits.

We have determined the effects of coexpression of Kv2.1 with electrically silent Kv5.1 or Kv6.1 alpha-subunits in Xenopus oocytes on channel gating. Kv2.1/5.1 selectively accelerated the rate ofinactivation at intermediate potentials (-30 to 0 mV), without affecting the rate at strong depolarization (0 to +40 mV), and markedly accelerated the rate of cumulative inactivation evoked by high-frequency trains of short pulses. Kv5.1 coexpression alsoslowed deactivation of Kv2.1. In contrast, Kv6.1 was much less effective in speeding inactivation at intermediate potentials, had a slowing effect on inactivation at strong depolarizations, and had no effect on cumulative inactivation. Kv6.1, however, had profound effects on activation, including a negative shift of the steady-state activation curve and marked slowing of deactivation tail currents. Support for the notion that the Kv5.1's effects stem from coassembly of alpha-subunits into heteromeric channels was obtained from biochemical evidence of protein-protein interaction and single-channel measurements that showed heterogeneity in unitary conductance. Our results show that Kv5.1 and Kv6.1 function as regulatory alpha-subunits that when coassembled with Kv2.1 can modulate gating in a physiologically relevant manner.

A comparison of 5-minute povidone-iodine scrub and 1-minute povidone-iodine scrub followed by alcohol foam.

The purpose of this study was to determine if a 1-minute scrub with povidone-iodine followed by alcohol foam is as effective as a 5-minute scrub with povidone-iodine in reducing skin bacterial counts. A 1-minute scrub with povidone-iodine followed by alcohol foam and a 5-minute scrub with povidone-iodine was done. In the first study, cultures were obtained after 1 hour, and in the second study, cultures were obtained after 2 hours. Cultures were obtained by imprinting the first, second, and third fingers on nutrient agar plates. Bacterial counts were then obtained at 24 and 48 hours. The study involved two groups of 12 participants and a total of 37 patients over a period of 5 months. The results show that there was no significant difference between the number of colonies cultured for the 1-minute scrub compared with the 5-minute scrub for either the 1-hour or the 2-hour study. In fact, the total number of bacterial colonies was less after the 1-minute scrub with alcohol foam than after the standard 5-minute scrub in both the 1-hour group (10 vs. 18) and the 2-hour group (18 vs. 44).

Evaluation of chromogenic substrate assays for fibrinolytic analytes in dogs.

OBJECTIVE: To evaluate the ability of commercial, chromogenic kits designed to measure human fibrinolytic pathway components to measure the canine plasma fibrinolytic pathway enzymes, tissue plasminogen activator (tPA) and plasminogen (PLG), and their respective inhibitors, plasminogen activator inhibitor 1 (PAI) and alpha 2-antiplasmin (AP). ANIMALS: 20 healthy dogs of various ages and breeds. PROCEDURE: The commercial procedure was adapted to a microtitration plate. Standard curves were generated by use of a canine plasma pool. RESULTS: Modifications of the commercial kit consisted of change in incubation periods and the substitution of urokinase for the streptokinase. Plasminogen and AP procedures yielded intra- and interassay coefficients of variation (CV) ranging from 2 to 6.4%. The tPA activity gave an acceptable intra-assay CV of 4.2%, but an equivocal interassay CV of 18%. The PAI assay gave unacceptable intra-assay and interassay CV of 59 and 66%, respectively. CONCLUSIONS: Modifications of the commercial PLG and AP procedures were appropriate for use with fresh and frozen canine plasma. However, equivocal results were obtained for canine plasma tPA. Although the PAI assay was able to detect the inhibitor, it gave unacceptable quantifiable results. Human and canine plasma contained similar amounts of PLG and AP, but 25% more tPA was found in canine plasma than human plasma. CLINICAL RELEVANCE: With modifications, the commercial human PLG and AP chromogenic kits may serve to elucidate such canine fibrinolytic disorders as disseminated coagulopathy. The high cost of the chromogenic substrate limits its application.

Fibrinolytic activity in dogs after surgically induced trauma.

OBJECTIVE: To determine whether alterations in the fibrinolytic pathway analytes, plasminogen (PLG), tissue plasminogen activator, and alpha 2-antiplasmin are significant in dogs subjected to minor and major surgical trauma. ANIMALS: 18 dogs in 3 groups of 6 each. PROCEDURE: Plasma fibrinolytic pathway analytes were measured in dogs with trauma of ovariohysterectomy (minor trauma) or orthopedic surgery (major trauma) and halothane anesthesia (control group). A commercial procedure adapted to a microtitration plate was used to measure the analytes. Blood was obtained 24 hours before anesthesia, at extubation (0 hours), and again at 2, 24, and 48 hours after extubation. An analyte quality-control strategy was maintained. RESULTS: In the major trauma group, there was a significant, transient, postsurgical decrease in PLG activity at 0 and 24 hours and a return to presurgical values by 48 hours. The minor trauma group had a similar trend without significant changes, including an increase in PLG values at 48 hours that exceeded the reference range. Antiplasmin values changed significantly in the major trauma group only. Tissue plasminogen activator values remained within the reference range. CONCLUSIONS: Tissue plasminogen activator was not considered a clinical marker of interest for detection of alterations in fibrinolysis after trauma. In contrast, plasma PLG and alpha 2-antiplasmin values may be useful in the evaluation of hemostatic complications of surgery. CLINICAL RELEVANCE: Identification of altered fibrinolysis in dogs undergoing traumatic surgery may provide a baseline for preventive pre-and postsurgical hemostatic care.

Comparison of Ca2+ loading and retention in isolated skeletal muscle triads and terminal cisternae.

Ca2+ loading and retention were examined in isolated skeletal muscle triads and terminal cisternae to determine 1) whether excessive loading altered the response of triads to depolarization-induced Ca2+ release and 2) whether these vesicles were similar in their ability to load and retain Ca2+. A mixture of triads and terminal cisternae was loaded with variable amounts of Ca2+ and then subjected to maximal depolarization. Ca2+ release was monitored by changes in extravesicular fura 2 fluorescence using 340/380-nm excitation and 510-nm emission wavelengths. The amount of Ca2+ released from triads due to maximal depolarization increases with increasing Ca2+ loads until a maximal response is obtained, indicating triad saturation. At pH 7, triadic vesicles preferentially loaded and retained Ca2+ at low Ca2+ loads, but, with increasing loads, nontriadic vesicles began to retain Ca2+. At pH 6.5, which should close all open uncoupled ryanodine receptors, triadic and isolated terminal cisternae vesicles loaded and retained Ca2+ in a similar manner. A population of triads, which have some uncoupled ryanodine receptors, did not retain their loaded Ca2+ at pH 7.0 but did retain Ca2+ at pH 6.5; this resulted in a doubling of the amount of Ca2+ released on maximal depolarization after loading at pH 6.5.

The voltage dependence of depolarization-induced calcium release in isolated skeletal muscle triads.

We demonstrate for the first time in this study that triadic vesicles derived from skeletal muscle display a voltage dependence of depolarization-induced calcium release similar to that found in intact muscle. We confirm previous studies by Dunn (1989) which demonstrated that changes in extravesicular potassium induced membrane potential changes in isolated transverse tubules with the voltage sensitive dye DiSC(3)-5. Depolarization-induced calcium release was studied in isolated triadic vesicles through similar changes in extravesicular [K] while clamping extravesicular Ca++ to submicromolar concentrations. The amplitude of fast phase of calcium release, identified as depolarization-induced calcium release, varied with the percentage of transverse tubules in the preparation (determined through 3H-PN200-110 specific activity) and different levels of depolarization. Threshold activation of calcium release was obtained with a 40.5 mV potential change; maximal calcium release was obtained with a 75 to 81 mV potential change. Boltzmann fits to the normalized depolarization induced calcium release plotted against the membrane potential change yielded a voltage dependence (k = 4.5 mV per e-fold change) very similar to that found in intact muscle (k = 3-4 mV per e-fold change; Baylor, Chandler & Marshall 1978, 1983; Miledi et al., 1981). Substitution of methanesulfonate for propionate as the impermeant ion or addition of valinomycin in the depolarizing solutions had little effect on the voltage dependence of calcium release.

Inheritance of diabetes mellitus in Keeshond dogs.

The genetic aspects of inherited, insulin-dependent diabetes mellitus of Keeshond dogs were studied retrospectively and in a prospective mating program. The symbol dm was used to designate the gene that causes hypoplasia of the islets of Langerhans. The retrospective study disclosed 4 diabetic dogs; prospective outcross, backcross, and inbred matings disclosed 49 diabetic dogs. Outcrossing demonstrated that the diabetic phenotype was displayed readily against a genetic background of a breed other than the Keeshond. In dogs with the dm/dm genotype, onset of diabetes was most frequent before the dog was 6 months old, but did occur in some older dogs. The dm genotype was best described as autosomal recessive.

Electromyographic evaluation of adult Labrador retrievers with type-II muscle fiber deficiency.

Labrador Retrievers with type-II muscle fiber deficiency were examined electrodiagnostically. Electromyographic changes consisted of positive sharp waves, fibrillation potentials, bizarre high-frequency discharges, and, rarely, myotonic-like discharges. Fasciculation potentials were recorded infrequently. Fibrillation potentials and bizarre high-frequency discharges were the most commonly observed electromyographic changes. Bizarre high-frequency discharges were prominent in muscles of the head and neck, proximal muscles of the thoracic limbs, and the thoracolumbar paraspinal musculature. Marked abnormalities were not observed in the motor nerve conduction velocity. Decremental responses of the evoked compound muscle action potential to repetitive nerve stimulation were not observed.

Quantitative studies of erythropoiesis in the clinically normal, phlebotomized, and feline leukemia virus-infected cat.

Erythropoiesis was evaluated in 5 cats at base line with normal PCV and then in the same cats with anemia induced by phlebotomy and in 5 other cats with nonregenerative anemia from community-acquired feline leukemia virus (FeLV) infection. The hematologic evaluation included complete blood cell and reticulocyte counts, marrow morphologic features, determination of serum erythropoietin concentrations by radioimmunoassay, ferrokinetic studies, and in vitro marrow culture of early erythroid progenitors (erythroid burst-forming units; BFU-E) and late erythroid progenitors (erythroid colony-forming units; CFU-E). Phlebotomized cats developed marrow erythroid hyperplasia and an increased reticulocyte count. Ferrokinetic studies revealed an increase in plasma iron turnover from 1.4 to 3.8 mg of Fe/dl of blood/day and RBC use from 50.4% to 78.5%. The mean CFU-E number and CFU-E/BFU-E ratio increased after phlebotomy, but the increase was not significant (P greater than 0.05). Serum erythropoietin values did increase significantly. In FeLV-infected cats, a nonregenerative anemia was demonstrated by marrow erythroid hypoplasia and a low total reticulocyte count. An increased percentage of rubriblasts and prorubricytes was observed in 4 of the 5 cats. Although serum erythropoietin values were high (321 +/- 123 mU/ml vs normal 14 +/- 1 mU/ml), ferrokinetic data revealed decreased erythropoiesis. Marrow culture studies in the FeLV-infected cats also revealed low numbers of BFU-E and CFU-E, but normal numbers of granulocyte-macrophage progenitors remained. Seemingly, the FeLV infection impaired the ability of feline marrow to respond physiologically to anemia.

Serum and tissue enzyme profiles of goats.

Serum enzyme activities of sorbitol dehydrogenase, glutamate dehydrogenase, gamma glutamyltransferase, alkaline phosphatase, aspartic aminotransferase, and creatine kinase, were measured in five clinically normal mixed-breed goats. Tissue activities of these enzymes were also measured in two goats. These basal serum values were then used to determine the response to treatment with carbon tetrachloride (CCl4). The basal value for serum and hepatic tissue sorbitol dehydrogenase were appreciably greater for goats than previously reported for sheep and cattle. The change in the above serum enzymes after CCl4 treatment resembled that in sheep, but the amount of sorbitol dehydrogenase increase was less than that in sheep. This study established basal tissue and serum enzyme activity values and demonstrated the efficacy of the use of changes in serum S.D.H. and G.D.H. activity as indicators of acute hepatopathy in goats.

Urinary indices for differentiation of prerenal azotemia and renal azotemia in horses.

The urine urea nitrogen/plasma urea nitrogen ratio (Uun/Pun), urine creatinine/plasma creatinine ratio (Ucr/Pcr), urine osmolality/plasma osmolality ratio (Uosm/Posm), and fractional excretion of filtered sodium (FENa) were evaluated in 16 horses with acute azotemia to ascertain the significance of each index in the differentiation of prerenal azotemia from renal azotemia. Renal azotemia was diagnosed when renal biopsy or postmortem histologic examination demonstrated evidence of organic renal disease or when azotemia was found in the presence of isosthenuria. The diagnosis of prerenal azotemia was based on the absence of renal histologic lesions or stabilization of blood urea nitrogen and serum creatinine soon after therapy. In 10 horses with renal azotemia, Uun/Pun was 2.1-14.3, Ucr/Pcr was 2.6-37.0, Uosm/Posm was 0.8-1.7, and FENa was 0.08-10.0. In 6 horses with prerenal azotemia, Uun/Pun was 15.2-43.7, Ucr/Pcr was 51.2-241.5, Uosm/Posm was 1.7-3.4, and FENa was 0.02-0.50. The values for each of these indices differed significantly between the 2 groups of horses (P less than 0.05). It was concluded that these indices were of value in the early classification of renal failure in the horse and that this information could be utilized in planning of therapy of acute azotemia in the horse.