Genome-wide association study of osteochondrosis in the tarsocrural joint of Dutch Warmblood horses identifies susceptibility loci on chromosomes 3 and 10.

Equine osteochondrosis is a developmental joint disease that is a significant source of morbidity affecting multiple breeds of horse. The genetic variants underlying osteochondrosis susceptibility have not been established. Here, we describe the results of a genome-wide association study of osteochondrosis using 90 cases and 111 controls from a population of Dutch Warmblood horses. We report putative associations between osteochondrosis and loci on chromosome 3 (BIEC2-808543; P = 5.03 × 10(-7) ) and chromosome 10 (BIEC2-121323; P = 2.62 × 10(-7) ).

Effects of GnRH immunization in sexually mature pony stallions.

Immunization against gonadotrophin releasing hormone (GnRH) was studied as an alternative for the commonly used surgical castration in stallions. Two GnRH vaccines comprising non-mineral oil adjuvants were evaluated for their potential to induce high antibody titers directed against GnRH and subsequent effects on reproductive characteristics. Twelve sexually mature male hemicastrated Shetland ponies were assigned to three groups. Group 1 and 2 were injected with 1mg peptide equivalent of G6k-GnRH-tandem-dimer conjugated to ovalbumin (OVA) in CoVaccine HT adjuvant (GnRH/CoVaccine) and in Carbopol (GnRH/Carbopol), respectively, and group 3 was injected with CoVaccine HT adjuvant without antigen (controls). After immunization no adverse effects were observed with respect to the injections sites or general health. Two weeks after the second vaccination antibody titers against GnRH increased rapidly in all animals of the GnRH/CoVaccine group, at the same time reducing serum testosterone levels maximally for the further duration of the experiment. In the GnRH/Carbopol group antibody responses and effects on testosterone levels were intermediate in two stallions and not apparent in the remaining stallions of this group. Semen evaluation showed that from 2 weeks after the second immunization onwards, sperm motility was affected in all stallions treated with GnRH/CoVaccine and one stallion treated with GnRH/Carbopol. Seven weeks after the second immunization, no semen could be collected from two stallions, one of each group, due to suppressed libido. Histological examination of the testes, 15 weeks after the initial immunization, demonstrated reduction in seminiferous tubuli diameters in all stallions of the GnRH/CoVaccine group and one stallion of the GnRH/Carbopol group. Furthermore, spermatogenesis was extremely disorganized in these stallions, as indicated by absence of the lumen in the seminiferous tubules, the absence of spermatozoa and spermatids in the tubular cross-sections and the impossibility to determine the stage of the tubular cross-sections. Testis size was also substantially reduced in three out of four stallions treated with GnRH/CoVaccine. The results demonstrate that two immunizations with G6k-GnRH-tandem-dimer-OVA conjugate in a suitable adjuvant such as CoVaccine HT caused a rapid and complete reduction of serum testosterone levels in sexually mature stallions, subsequently leading to reduced sperm motility and affected testis function, while no adverse reactions were observed after immunizations.

Effect of lateral heel wedges on sagittal and transverse plane kinematics of trotting Shetland ponies and the influence of feeding and training regimes.

REASONS FOR PERFORMING STUDY: Lateral heel wedges are used to treat horses and ponies with patella fixation or bone spavin. However, these therapies are purely empirically based and lack scientific evidence. OBJECTIVES: Lateral heel wedges would change joint motion in the sagittal, but mainly in the transversal planes, in healthy horses. This effect would be increased by restricted feeding and decreased by extra training. METHODS: A group of 24 Shetland ponies age 3 years was used, as foals had been assigned to restricted and ad libitum (ad lib) feeding, and low and high level training groups of 6 animals each. An experienced judge evaluated passive patella luxation in the square standing pony, using a score of 0 (normal) to 4 (stationary patella luxation). The motion of the markers, glued to the skin covering skeletal landmarks on the left fore- and hindlimbs, was recorded 3 dimensionally at a frequency of 300 Hz using a modified CODA-3 apparatus while trotting on a treadmill at a speed of 3.0 m/sec, before and directly after 5 degrees lateral heel wedges had been applied to the hindlimbs. After data analysis, the kinematic variables in the sagittal and transversal plane, under these 3 conditions (wedge, feeding, training), were compared statistically using a multivariate repeated measures analysis, general linear model (P < 0.05). RESULTS: In the sagittal plane, an acute change in hind hoof conformation resulted in a less animated trot with a less protracted forelimb and less hindlimb flexion. This is similar, although less pronounced, to the decrease in limb flexion reported previously as a result of restricted feeding. More specifically, lateral heel wedges resulted in significant changes in the transversal plane angles of all joints in the hindlimb. The stifle joint became maximally 1.8 degrees more adducted just before the end of the stance phase, while the tarsal joint was 2.9 degrees and fetlock joint 4.7 degrees more abducted (P < 0.05). In the restricted feeding group, stifle joint adduction was 85 degrees and tarsal joint abduction 5.6 degrees larger than in the ad libitum feeding group (P < 0.05). The patella luxation score was also significantly higher in this group (1.8) compared to ponies fed ad libitum (0.9). CONCLUSIONS: The acute effects of lateral heel wedges on the equine locomotor system in the transversal plane movement relieve tension from the medial patellar ligament and decrease pressure on the medial side of the tarsal joint. However, the fetlock joint experiences considerably more out of plane stress. Poor body condition resulted in a 2x worse patella luxation score, while the effect on stifle and tarsal joint movement in the transversal plane was almost 5x and 2x larger, respectively, than a lateral wedge. POTENTIAL RELEVANCE: The clinical importance of general body condition for maintaining lateral stability in the equine hindlimbs is established, but future research may prove that wedges are beneficial to treat patella fixation and bone spavin in the long term.

Influence of liver copper status of mare and newborn foal on the development of osteochondrotic lesions.

REASON FOR PERFORMING STUDY: To elucidate the highly contentious role of copper in the pathogenesis of osteochondrosis. HYPOTHESIS: There would be no relationship between liver copper concentration of mares and foals and incidence of radiographically detectable osteochondrotic lesions in foals and yearlings was tested. METHODS: Liver copper concentration was assessed in biopsies taken within 4 days after birth from both mares and foals and from the same foals at age 5 months. Biopsies were taken in the standing, sedated animal under ultrasonographic guidance. Radiographs were taken of both hocks (lateromedial, dorsoplantar and dorsomedial-plantarolateral oblique views) and stifles (lateromedial and caudolateral-craniomedial oblique views) at ages 5 and 11 months and scored for the presence and severity of osteochondrotic lesions. RESULTS: Copper concentrations in newborn foals were high with a large variation (351 +/- 201 mg/kg DM). They declined until reaching values comparable to those in mature animals at 5 months (20 +/- 8 mg/kg DM; mares: 19 +/- 20 mg/kg DM). Radiographic osteochondrotic lesions decreased in number and severity from 5 to 11 months. This pattern was more predominant in the stifle than in the hock, as has been described previously. CONCLUSIONS: There was no relationship between foal or mare liver copper concentration and osteochondrosis status at either 5 or 11 months. However, osteochondrotic lesions in foals with low-level copper status at birth decreased significantly less in number and severity than those in foals with high-level copper status at birth. POTENTIAL RELEVANCE: It is concluded that copper is not likely to be an important factor in the aetiopathogenesis of osteochondrosis, but this study indicates that there may be a significant effect of high copper status on the natural process of repair of early lesions.

Jumping characteristics of naïve foals: lead changes and description of temporal and linear parameters.

The selection of foals as future showjumpers remains a subjective process based on qualitative parameters; and hence, frequently suffers from disparity in the criteria used by experts in the field. A detailed biomechanical description of foals while jumping would be most helpful in providing a better basis for the accurate assessment of their future athletic ability. The Qualisys Pro Reflex system was used to capture 3-dimensional kinematics of 41 Dutch Warmblood foals age 6 months free jumping a vertical fence, preceded by a cross pole fence. The left lead was the most preferred lead for both the fore- and hindlimbs, from the landing following the cross poles to the first move-off stride after clearing the vertical fence. The foals displayed a high incidence of rotary gallop during both the jump stride (divided into take-off, jump suspension and landing) and the first move-off stride, while change of lead was frequently observed during jump suspension. At the take-off side of the fence, the trailing forelimb in the last approach stride was placed furthest from the fence, whereas the trailing hindlimb at take-off was placed closest (P<0.05). At the landing side, the trailing forelimb was the closest and the leading hindlimb of the move-off stride 1 was the furthest (P<0.05). The trailing forelimb in the approach stride 1 had a significantly longer stance phase duration than the leading forelimb. At landing, the leading forelimb stance phase lasted longer than that of the trailing forelimb (P<0.05). The hindlimbs did not differ in their stance phase duration at take-off. The height reached by the hooves above the fence top was significantly greater in the hind limbs (P<0.05). In addition, the hindlimbs (97.1 +/- 2.6%) shortened more than the forelimbs (92.6 +/- 5.7%) (P<0.05). It is concluded that the overall jumping technique of foals is similar to that reported in literature for mature horses. If the patterns are consistent throughout the rearing period, the quantitative analysis of the kinematics of free jumping foals may provide a valid quantitative basis for early selection.

Heart rate and heart rate variability during a novel object test and a handling test in young horses.

Forty-one Dutch Warmblood immature horses were used in a study to quantify temperamental traits on the basis of heart rate (HR) and heart rate variability (HRV) measures. Half of the horses received additional training from the age of 5 months onwards; the other half did not. Horses were tested at 9, 10, 21 and 22 months of age in a novel object and a handling test. During the tests, mean HR and two heart variability indices, e.g. standard deviation of beat-to-beat intervals (SDRR) and root mean square of successive beat-to-beat differences (rMSSD), were calculated and expressed as response values to baseline measures. In both tests, horses showed at all ages a significant increase in mean HR and decrease in HRV measures, which suggests a marked shift of the balance of the autonomic nervous system towards a sympathetic dominance. In the novel object test, this shift was more pronounced in horses that had not been trained. Furthermore, statistical analysis showed that the increase in mean HR could not be entirely explained by the physical activity. The additional increase in HR, the nonmotor HR, was more pronounced in the untrained horses compared to the trained. Hence, it is suggested that this nonmotor HR might be due to the level of emotionality. HR variables showed consistency between years, as well as within the second year. These tests bring about a HR response in horses, part of which may indicate a higher level of emotionality; and horses show individual consistency of these HR variables over ages. Therefore, it is concluded that mean HR and HRV measures used with these tests quantify certain aspects of a horse's temperament.

TAF(II)170 interacts with the concave surface of TATA-binding protein to inhibit its DNA binding activity.

The human RNA polymerase II transcription factor B-TFIID consists of TATA-binding protein (TBP) and the TBP-associated factor (TAF) TAF(II)170 and can rapidly redistribute over promoter DNA. Here we report the identification of human TBP-binding regions in human TAF(II)170. We have defined the TBP interaction domain of TAF(II)170 within three amino-terminal regions: residues 2 to 137, 290 to 381, and 380 to 460. Each region contains a pair of Huntington-elongation-A subunit-Tor repeats and exhibits species-specific interactions with TBP family members. Remarkably, the altered-specificity TBP mutant (TBP(AS)) containing a triple mutation in the concave surface is defective for binding the TAF(II)170 amino-terminal region of residues 1 to 504. Furthermore, within this region the TAF(II)170 residues 290 to 381 can inhibit the interaction between Drosophila TAF(II)230 (residues 2 to 81) and TBP through competition for the concave surface of TBP. Biochemical analyses of TBP binding to the TATA box indicated that TAF(II)170 region 290-381 inhibits TBP-DNA complex formation. Importantly, the TBP(AS) mutant is less sensitive to TAF(II)170 inhibition. Collectively, our results support a mechanism in which TAF(II)170 induces high-mobility DNA binding by TBP through reversible interactions with its concave DNA binding surface.

The gene for human TATA-binding-protein-associated factor (TAFII) 170: structure, promoter and chromosomal localization.

The TATA-binding protein (TBP) plays a central role in eukaryotic transcription and forms protein complexes with TBP-associated factors (TAFs). The genes encoding TAF(II) proteins frequently map to chromosomal regions altered in human neoplasias. TAF(II)170 of B-TFIID is a member of the SF2 superfamily of putative helicases. Members of this superfamily have also been implicated in several human genetic disorders. In this study we have isolated human genomic clones encoding TAF(II)170 and we show that the gene contains 37 introns. Ribonuclease-protection experiments revealed that TAF(II)170 has multiple transcription start sites, consistent with the observation that the promoter lacks a canonical TATA box and initiator element. Deletion analysis of the promoter region showed that a fragment of 264 bp is sufficient to direct transcription. In addition, we determined the chromosomal localization by two independent methods which mapped the gene to human chromosome 10q22-q23 between the markers D10S185 and WI-1183. The region surrounding these markers has been implicated in several human disorders.

Cloning of the cDNA for the TATA-binding protein-associated factorII170 subunit of transcription factor B-TFIID reveals homology to global transcription regulators in yeast and Drosophila.

The human transcription factor B-TFIID is comprised of TATA-binding protein (TBP) in complex with one TBP-associated factor (TAF) of 170 kDa. We report the isolation of the cDNA for TAFII170. By cofractionation and coprecipitation experiments, we show that the protein encoded by the cDNA encodes the TAF subunit of B-TFIID. Recombinant TAFII170 has (d)ATPase activity. Inspection of its primary structure reveals a striking homology with genes of other organisms, yeast MOT1, and Drosophila moira, which belongs to the Trithorax group. Both homologs were isolated in genetic screens as global regulators of pol II transcription. This supports our classification of B-TFIID as a pol II transcription factor and suggests that specific TBP-TAF complexes perform distinct functions during development.

A new class of tobacco chitinases homologous to bacterial exo-chitinases displays antifungal activity.

A novel chitinase gene of tobacco was isolated and characterized by DNA sequence analysis of a genomic clone and a cDNA clone. Comparative sequence analysis of both clones showed an identity of 94%. The proteins encoded by these sequences do not correspond to any of the previously characterized plant chitinases of classes I-IV and are designated as class V chitinases. Comparison of the chitinase class V peptide sequence with sequences in the Swiss Protein databank revealed significant sequence similarity with bacterial exo-chitinases from Bacillus circulans, Serratia marcescens and Streptomyces plicatus. It was demonstrated that class V chitinase gene expression is induced after treatment of tobacco with different forms of stress, like TMV-infection, ethylene treatment, wounding or ultraviolet irradiation. Two related chitinase class V proteins of 41 and 43 kDa were purified from Samsun NN tobacco leaves inoculated with tobacco mosaic virus. The proteins were purified by Chelating Superose chromatography and gel filtration. In vitro assays demonstrated that class V chitinases have endo-chitinase activity and exhibit antifungal activity toward Trichoderma viride and Alternaria radicina. In addition, it was shown that class V chitinase acts synergistically with tobacco class I beta-1,3-glucanase against Fusarium solani germlings.

The effect of diabetes regulation on platelet release, fibrinolysis and coagulation tests, before and after stimulation with DDAVP.

In this longitudinal study we measured beta-TG, PF4, fibrinolytic activity (extrinsic and euglobulin fraction), fibrinogen, FVIII RAg and FVIII Rcof before and after i.v. DDAVP (FPA was only measured before DDAVP) in 20 patients with diabetes mellitus. These parameters were measured on three occasions: phase I: during disregulation, phase II: after three weeks of strict control, phase III: after nine weeks of good control. Twenty-two healthy volunteers served as normal controls. No significant differences related to metabolic control were found for beta-TG, PF4, FPA and fibrinogen. There was no change after i.v. DDAVP administration. Fibrinolytic activity showed a significant increase after i.v. DDAVP. Baseline values and post-DDAVP increase were not significantly different from our normal controls. FVIII RAg and FVIII Rcof were both significantly elevated in diabetes mellitus. Both increased significantly after DDAVP. The FVIII RAg release (delta FVIII RAg) was significantly less in the diabetics. Fibrinolytic activity, FVIII RAg and FVIII Rcof are independent of the degree of metabolic control in patients with diabetes.