In this study, we described water-insoluble proteins extracted from the germinative regions (stratum internum and coronary band epithelium) and the cornified outer surface (stratum medium) of the equine hoof wall. Two major types of polypeptides were identified: the intermediate filaments (IF) and the IF-associated proteins. The IF, including keratins, composed a major portion of this fraction, had electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the range of 40 to 80 kDa, and reacted with acidic or basic keratinspecific monoclonal antibodies. Differences in the composition of keratins between germinative layers and the stratum medium were seen. Another less well-characterized group of polypeptides associated with the IF also were extracted with the water-insoluble polypeptide fraction. These associated proteins had an apparent molecular weight between 10 and 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and contained a higher percentage of sulfur-containing amino acids than did the IF. Water-insoluble protein fractions compared favorably with those found in other less-specialized keratinizing tissue with respect to size, immunoreactivity with monoclonal antibody, and amino acid composition.

Distal airway segments (ID, 3 to 4 mm; length, 5 mm) from 2 groups of horses were isolated and suspended in tissue baths filled with Krebs solution, aerated with 5% CO2 in oxygen and maintained at 37 C. Responses to exogenous acetylcholine, isoproterenol, or electrical field stimulation were compared. Control horses (n = 30) had no history of recurrent airway obstruction, whereas principal horses (n = 15) had recurrent airway obstruction and were studied during an acute episode of airway obstruction. Although the distal airways contracted in response to the cumulative half-logarithmic addition of acetylcholine (10(-10)M to 10(-3)M) in both groups, bronchi obtained from principals were less sensitive to acetylcholine than were bronchi obtained from controls. Tetrodotoxin-sensitive electrical field stimulation-induced contractions were observed in both groups of airways, but the tension achieved in principal bronchi was less than in controls. All electrical field stimulation-induced contractions were abolished by atropine, indicating that the only excitatory innervation of equine distal airways is through the parasympathetic system. To examine the effect of isoproterenol and determine inhibitory innervation, bronchi were precontracted with histamine. Electrical field stimulation did not cause relaxation of precontracted bronchi in either group, thus indicating that distal airways lack inhibitory innervation. Isoproterenol caused similar, dose-dependent relaxation in both groups.

Mucosa obtained from the cecum of healthy horses and incubated in vitro with 0.1 mM cycloleucine could accumulate this amino acid against an apparent concentration gradient after 60 and 120 minutes. Accumulation by the serosal (antiluminal) surface of the tissue was 3 times greater than accumulation by the mucosal (luminal) surface after 120 minutes (P < 0.001). Cycloleucine accumulation was significantly reduced by Na deprivation after 60 minutes (P < 0.05) and 120 minutes (P < 0.01) and by anoxic conditions after 120 minutes (P < 0.05). Transmucosal flux from mucosal to serosal surface of the tissue was significantly (P < 0.05) greater than the opposing flux, but both unidirectional fluxes were small and were largely attributed to passive processes. It was concluded that the most avid transport system for cycloleucine was on the serosal surface of the horse's cecal mucosa, and an active transport system was not evident on the mucosal surface. An active transport system for amino acids on the serosal surface could be explained by the need for crypt cells, the predominant epithelial cell type in the cecum, to obtain nutrients from blood, rather than from the intestinal lumen.

Sheets of mucosa from the jejunum of healthy horses were mounted in incubation chambers and bathed with Krebs-ringer bicarbonate solution. Changes in tissue function and histologic appearance were compared after the following conditions: (1) control conditions for 30 minutes with 95% O2/5% CO2 in the gas phase; (2) same conditions as control, except incubation with superoxide dismutase (300 U/ml) during the last 18 minutes; (3) anoxia for 15 minutes with 95% N2/5% CO2, followed by reoxygenation for 15 minutes; (4) same conditions as 3, except incubation with superoxide dismutase during reoxygenation; and (5) anoxia for 30 minutes. Anoxia reduced the accumulation of radiolabeled L-alanine and caused cell swelling, as indicated by an increase in tissue water and tissue Na contents. Reoxygenation improved the tissue's ability to accumulate L-alanine, but tissue swelling continued after this treatment. Tissue Na content and L-alanine accumulation were restored to control values by reoxygenation with superoxide dismutase in the bathing medium. The grade of structural damage, as indicated by separation of eptihelial cells from villi, was equally severe after all, but control, conditions. Superoxide dismutase had no effect on the tissue control conditions. Results of this study suggest that superoxide radicals are involved in the pathogenesis of reperfusion injury in equine jejunal mucosa and that this may be of clinical importance in cases of small intestinal strangulation obstruction.

Low molecular weight polysulfated glycosaminoglycan (PSGAG) stimulated net collagen and glycosaminoglycan synthesis by normal and arthritic equine fetlock cartilage tissues in organ culture. Arthritic tissues were more sensitive to PSGAG stimulation. The rates of cartilage-specific type-II collagen and chondroitin sulfate-rich glycosaminoglycan synthesis by confluent chondrocyte cell cultures obtained from normal and arthritic equine cartilage tissues were increased by 25 and 50 mg of PSGAG/ml. Cells from arthritic cartilage were also more sensitive to the presence of PSGAG. In addition, concentrations of PSGAG (25 and 50 mg/ml) approximate to those in synovial fluid after intra-articular injection of 250 mg of PSGAG inhibited the rate of collagen and glycosaminoglycan degradation in cell culture. These findings suggest that PSGAG may have a role in the healing of mild cartilage degeneration by encouraging the production of replacement hyaline matrix materials, while delaying their subsequent degradation. In contrast, growth of cell cultures was inhibited by PSGAG, suggesting that these compounds may fail to stimulate chondrocyte replication, a prerequisite for tissue regeneration. Nonetheless, these observations provide direct evidence of a truly chondroprotective role for low molecular weight PSGAG in the treatment of equine degenerative joint disease.

The influence of cortisol on estrogen synthesis by the bovine placenta and the importance of the delta 4 and delta 5 pathway for estrogen production were investigated. For experiment 1, portions of fetal villi (200 mg) were incubated for 48 hours with 0, 10, 100, and 1,000 ng of cortisol/ml with [3H]androstenedione (3H-A) or [3H]pregnenolone (3H-P5). Villi were also incubated for 4, 28, and 52 hours with or without cortisol (500 ng/ml) and with 3H-A or 3H-P5 (experiment 2). The conversion of various [3H]steroid metabolites such as A, P5, 17 alpha-OH-pregnenolone (17 alpha-OH-P5), progesterone (P4), 17 alpha-OH-P4, cholesterol (chol), and chol plus lipoprotein (500 micrograms/ml) into estrogen was measured during a 4-hour incubation (experiment 3). In experiment 1, cortisol increased conversion of 3H-A and 3H-P5 into estrogen by 3 to 41% and 7 to 34%, respectively, in a dose-dependent manner (P < 0.05). In experiment 2, times of incubation did not influence conversion of 3H-A into estrogen, which, however, was increased significantly (P < 0.05) over all times of incubation by administration of 500 ng of cortisol/ml. Conversion of 3H-P5 into estrogen increased over time of incubation and was stimulated by cortisol (P < 0.05). However, there was no interaction between cortisol treatment and time of incubation. In experiment 3, conversion of 3H-A, 3H-P5, and 3H-17 alpha-OH-P5 into estrogen was greater than the conversion of the other precursors tested. Mean conversion of 3H-A, 3H-P5, 3H-17 alpha-OH-P5, 3H-P, 3H-17 alpha-OH-P4, 3H-chol, and 3H-chol plus lipoprotein was 23%, 10.6%, 11.0%, 1.8%, 1.8%, 0.3% and 0.7%, respectively. Our results suggest that, in cows, the delta 5 pathway is the preferred pathway for placental estrogen synthesis and that cortisol directly stimulates estrogen production, probably by activating enzymes involved in this pathway.

Ninety-one equine aortic and cranial mesenteric arterial segments were evaluated ultrasonographically in a water bath. On the basis of pathologic evidence of verminous arteritis, arterial segments were classified into 4 categories, and the ultrasonographic characteristics of each group were evaluated. Normal arteries (class 1) were ultrasonographically characterized by a smooth luminal surface layer and uniform wall thickness and echogenicity. Arteries with only histopathologic evidence of verminous arteritis (class 2) were ultrasonographically characterized by a smooth luminal surface layer, uniform thickness, uniform echogenicity, and the presence of a hyperechoic luminal layer. Arteries with both gross and histopathologic evidence of verminous arterities (class 3) were characterized ultrasonographically by an irregular luminal surface layer, varying wall thickness, varying wall echogenicity, and the presence of a hyperechoic luminal layer. The ultrasonographic characteristics of arteries with luminal thrombosis (class 4) were an irregular luminal surface, varying wall thickness, and nonuniform echogenicity.

Strips of trachealis muscle were dissected from the mid-cervical portion of the trachea from horses that were free of respiratory tract disease. The epithelium and mucosa were removed from one group of tissues and were left intact in a second group of tissues. Each tissue was suspended in a bath filled with Krebs-bicarbonate solution that was aerated with 5% CO2 in oxygen and maintained at 37 C. Isometric tension was continuously recorded. The contractile response to square-wave electrical stimulations increased as frequency (3, 5, 10, 15, 20, 25, and 30 Hz), voltage (10, 15, 18, and 25 V), and pulse duration (0.2, 0.5, 1.0, 1.5, and 2.0 ms) increased in tissues with the epithelium and mucosa intact. A stimulus of 18 V, 20 Hz, and 0.5 ms induced maximal contraction. Atropine (10(-6) M) abolished the response to 18 V and 0.5 ms at all frequencies. The increase in active isometric tension was concentration dependent when acetylcholine (10(-9) to 10(-4) M) was added to the baths in 0.5-logarithmic increments. Tissues that were contracted in response to acetylcholine (10(-5) M) had a concentration-dependent decrease in active isometric tension when isoproterenol was added to the baths in 0.5-logarithmic increments (10(-9) to 10(-4) M). The contraction and relaxation curves were qualitatively similar, but quantitatively different in tissues with and without the epithelium and mucosa. Removing the epithelium and mucosa increased the contractile response to acetylcholine at bath concentrations of 3.1 X 10(-7) M and 10(-6) M. The presence of epithelium and mucosa enhanced the magnitude of isoproterenol-induced relaxations. We concluded that electrical stimulation released acetylcholine from isolated equine trachealis muscle, that isoproterenol induced relaxation of the trachealis muscle, and that the magnitude of the responses to exogenous agonists depended on the presence of epithelium and mucosa.

Strips of trachealis muscle were dissected from the mid-cervical portion of the trachea of horses that were free of respiratory tract disease, and the overlying epithelium and mucosa were removed. Muscle strips were suspended in tissue baths that were filled with Krebs-bicarbonate solution, aerated with 5% CO2 in oxygen and maintained at 37 C. Isometric tension was continuously recorded. The increase in active isometric tension was concentration dependent when acetylcholine (10(-9) to 10(-4) M) or histamine (10(-9) to 10(-4) M) was added to the tissue baths in 0.5-logarithmic increments. When the tissues were contracted with acetylcholine (3.1 X 10(-6) M) or histamine (10(-4) M), the decrease in active isometric tension was concentration dependent when isoproterenol (10(-9) to 10(-4) M) or salbutamol (10(-9) to 10(-4) M) was added to the tissue baths in 0.5-logarithmic increments. There was no difference between the response to isoproterenol and salbutamol when tissues from the same horses were compared whether the tissues were contracted in response to acetylcholine (3.1 X 10(-6) M) or histamine (10(-4) M). Relaxation was antagonized by 10(-6) M) propranolol. The degree of relaxation obtained in these muscle strips was considerably less than that reported from other species' tracheal muscle strips that had the epithelium and mucosa intact. We concluded that equine tracheal smooth muscle contains beta-adrenoceptors that can be stimulated by either a mixed beta-1, beta-2 agonist or a selective beta-2 agonist.

Anaplasma marginale was propagated in a tick cell line derived from Dermacentor variabilis embryos. The rickettsial organism was identified and monitored in culture by transmission electron microscopy and the indirect immunofluorescence technique, using specific monoclonal antibodies. Inoculation of the embryonic tick cell line with midguts of infected adult ticks (culture 1), nymphal ticks (culture 2) and adult ticks that were infected as nymphs and dissected as adults (culture 3) resulted in 3 continuous cultures of A marginale. Culture 1 had been maintained through 22 passages over a 11-month period; cultures 2 and 3 had been maintained for 18 passages over a 9-month period. Growth of A marginale in the cell line began in the area of the nuclear membrane at approximately 4 days after inoculation or transfer. Thereafter, the organisms were observed in inclusions scattered throughout the cytoplasm of the host cells. Maximal growth of the organism occurred at 7 to 14 days, after which numbers of inclusions rapidly decreased to minimal or undetectable levels. The organism began new cycles of growth with each 1:5 to 1:10 split and transfer of the host cells. Electron microscopy of recently infected cells revealed a morphology of the organism that closely resembled that observed in marginal bodies of infected erythrocytes. After several passages, A marginale organisms had a varied morphology and resembled the organism described in midgut cells of naturally infected ticks. Substitution of adult bovine serum for fetal bovine serum and adjustment of the pH of the medium from 6.9 to 7.4 resulted in several-fold increases in amount of growth and reduced the period required to reach maximal growth to a predictable time of 5 to 7 days. The importance and potential of this method of continuous laboratory propagation of A marginale are discussed.