Lust, George (Fort Detrick, Frederick, Md.). Alterations of protein synthesis in arbovirus-infected L cells. J. Bacteriol. 91:1612-1617. 1966.-Cellular protein synthesis and ribonucleic acid (RNA) synthesis in mouse L cells were markedly depressed 1 hr after infection with Venezuelan equine encephalomyelitis virus. Host RNA and protein synthesis were inhibited more rapidly by the virus infection than by actinomycin D. In cells infected 4 hr, a cytoplasmic RNA polymerase was demonstrated which was absent in uninfected cells. At this time, deoxyribonucleic acid-directed RNA synthesis catalyzed by the nuclear RNA polymerase was inhibited in vitro in enzyme preparations from nuclei of virus-infected cells. For optimal activity, the cytoplasmic RNA polymerase required the four nucleoside triphosphates, Mg(++), and RNA. The enzyme was insensitive to actinomycin D and deoxyribonuclease, indicating that it catalyzed RNA-directed RNA synthesis. Attempts to purify the induced polymerase further were unsuccessful. Fresh preparations had to be used because the enzymatic activity was unstable.

Adenine Nucleotides↗

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Inorganic pyrophosphate and proteoglycan metabolism in cultured human articular chondrocytes and fibroblasts.

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Adult↗

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Origin of cartilage laminae in MRI.

To understand the origin of the laminated appearance of cartilage in MRI (the magic angle effect), microscopic MRI (mu MRI) experiments were performed at 14-microns pixel resolution on normal canine articular cartilage from the shoulder joints. Two-dimensional images of the spin-spin relaxation time (T2) of the cartilage-bone plug at two angles (0 degree and 55 degrees) were calculated quantitatively. A distinct T2 anisotropy was observed as a function of the cartilage tissue depth. The surface and the deep regions exhibit strong orientational dependence of T2, whereas the upper-middle region exhibits little orientational dependence of T2. These three mu MRI regions correspond approximately to the three histologic zones in cartilage tissue. The results from the bulk T2 measurements agreed with these mu MRI results. Our studies show that the laminated appearance of cartilage in MRI is caused by T2 anisotropy of the tissue. We further suggest that the molecular origin of the T2 anisotropy is the nuclear dipolar interaction. The structure of the cartilage tissue indicates that the collagen meshwork defines this T2 anisotropy. The results show that the T2 anisotropy provides an indirect but sensitive indicator for the orientation of macromolecular structures in cartilage. The clinical implications of this anisotropy are discussed.

Animals↗

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The effects of methylprednisolone on normal and monocyte-conditioned medium-treated articular cartilage from dogs and horses.

OBJECTIVE: To study in vitro (1) the dose-response relationships between proteoglycan metabolism in normal and corticosteroid-treated articular cartilage; (2) long-term proteoglycan metabolism after treatment of articular cartilage with corticosteroids; and (3) the effect of corticosteroids on proteoglycan metabolism in articular cartilage treated with monocyte-conditioned medium (MCM). STUDY DESIGN: Equine and canine articular cartilage explants were treated with corticosteroids and MCM. Proteoglycan synthesis and degradation were measured by radioactive labeling in short-term culture, and the long-term effect of corticosteroid treatment on proteoglycan metabolism was studied in normal explants. ANIMALS: Two young cross-breed horses and 3 young Labrador retrievers. METHODS: Equine articular cartilage explants were incubated in medium containing methylprednisolone sodium succinate (MPS) at 0, .001, .01, .1, 1, and 10 mg/mL (final concentration) for 1 day and then in fresh medium without MPS. Proteoglycan synthesis was measured by incorporation of sodium [35S]sulfate at 1, 3, 7, 10, and 13 days after initial treatment with MPS. Proteoglycan release was measured from separate explants prelabeled with sodium [35S]sulfate and treated similarly. Equine articular cartilage explants were treated with equine MCM simultaneously with, and 24 hours before MPS, at 0, 0.01, 0.1, 1, or 5 mg/mL for 72 hours. Proteoglycan synthesis and degradation in these explants was compared. Proteoglycan synthesis and degradation were measured similarly in canine articular cartilage explants treated simultaneously with canine MCM and MPS at 0, 0.001, 0.01, 0.1, 1 and 10 mg/mL for 72 hours. Equine articular cartilage explants treated with 0, 0.01, 0.1, 1, and 5 mg/mL of MPS for 72 hours were evaluated histologically. RESULTS: Proteoglycan synthesis in normal equine articular cartilage was severely depressed by 10 mg/mL MPS for 24 hours, and proteoglycan synthesis failed to recover after 13 days of culture in medium without MPS. Cartilage treated with 5 mg/mL MPS had pyknotic chondrocyte nuclei and empty lacunae. Concentrations of 1 and 0.1 mg/mL MPS depressed proteoglycan synthesis in normal equine cartilage explants. For these 2 concentrations, proteoglycan synthesis recovered 2 days after MPS removal and increased significantly (P < .05) 7 days after treatment with MPS compared with controls without MPS. Concentrations of 0.001 and 0.01 mg/mL MPS did not significantly affect proteoglycan synthesis in normal equine cartilage explants. Cumulative proteoglycan loss over 13 days in culture from normal equine explants treated for 24 hours with different concentrations of MPS was not significantly different between treatment groups at any time point. MCM significantly depressed proteoglycan synthesis in both canine and equine articular cartilage explants and significantly increased proteoglycan release. These effects were prevented in the canine explants by simultaneous treatment with MPS at 1 and 0.1 mg/mL, and proteoglycan release induced by MCM in equine articular cartilage was inhibited by 1 mg/mL MPS. CONCLUSIONS: Concentrations of 1.0 and 0.1 mg/mL MPS alleviated articular cartilage degradation in MCM-treated articular cartilage in vitro. These concentrations of MPS in contact with normal cartilage explants for 24 hours are unlikely to be detrimental in the long term to proteoglycan synthesis. The response of articular cartilage to MPS was affected by treatment with MCM so that results of experiments with normal articular cartilage explants may not reflect results obtained with abnormal cartilage. CLINICAL RELEVANCE: It may be possible to find an intraarticular concentration of corticosteroid that protects articular cartilage against cytokine-induced matrix degradation yet not have prolonged or permanent detrimental effects on chondrocyte matrix synthesis.

Analysis of Variance↗

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Random amplified polymorphic DNA (RAPD) sequences as markers for canine genetic studies.

Random amplified polymorphic DNA (RAPD) markers have been used in genetic studies of several plant and animal species. However, concerns exist about the reproducibility of RAPD-PCR reactions. Therefore, the use of specific 24mer primer pairs in standard PCR reactions has been suggested for reliable amplification of characterized polymorphic RAPD sequences. The purpose of this article is describe the application of the RAPD-PCR assay to genetic studies of dogs and to investigate the amplification of RAPD sequences with specific primer pairs. Of 240 decanucleotide primers tested by PCR, 34.6% resulted in amplification of at least one polymorphic fragment with samples of a Labrador retriever pedigree. We cloned and sequenced five of these RAPD fragments and synthesized specific 24mer primer pairs for each. Two primer pairs amplified a sequence exclusively from samples that were positive for the RAPD fragment, while three others amplified the respective sequence from all DNA samples. A new polymorphism was observed in the restriction digest products with Msel of one of the amplification products. None of the cloned sequences contains an open reading frame longer than 213 bases. Two sequences hybridized only to specific fragments of genomic DNA from samples that amplified the RAPD, the remaining three sequences hybridized to multiple sequences in all canine samples tested by Southern analysis. None of the five fragments hybridized to human or murine genomic DNA. Data suggested that RAPD sequences can be used as molecular markers in genetic studies of diseases in dogs. However, the use of specific primer pairs leads to loss of the RAPD polymorphism in three of five sequences tested.

Animals↗

PubMedDate not supplied

An outcrossed canine pedigree for linkage analysis of hip dysplasia.

Canine hip dysplasia (CHD) is a prevalent, debilitating, polygenic disease characterized by hip subluxation and laxity which results in osteoarthritis. We are developing an informative pedigree for linkage analysis of CHD. The seven greyhound founders had excellent hip conformation with high dorsolateral subluxation scores (percentage of femoral head covered by the dorsal acetabulum in a weight-bearing position) of 66 +/- 4% (mean +/- SD averaged over both hips) and low hip distraction (laxity) indices of 0.14 +/- 0.08. Nine greyhounds bred on site had radiographic evidence of ossification in the capital femoral chondroepiphysis at 7.7 +/- 0.9 days of age. At 8 months of age they had a mean distraction index of 0.24 +/- 0.08 and dorsolateral subluxation score of 76 +/- 1%. Of the four dysplastic Labrador retriever founders, three had mean age at onset of capital femoral chondroeplphyseal ossification of 20 +/- 7 days of age n = 3) and a mean distraction index of 0.46 +/- 0.1 accompanied by hip osteoarthritis. Thirty-four F1s had mean onset of capital femoral ossification (10.7 +/- 4.0 days of age) and mean dorsolateral subluxation scores (61 +/- 12%) similar to the greyhound founders, but distraction indices (0.42 +/- 0.2) more similar to the Labrador retriever founders. One F1 had CHD radiographically but none of 20 F1s had osteoarthritis at necropsy at 10 months of age. These data suggested that maximum passive laxity (as measured by the distraction index) and normal osseous conformation (as indicated by a high dorsolateral subluxation score) were both dominant traits and were controlled by separate quantitative trait loci (QTL). Forty-three back-crosses between F1s with the highest hip laxity and greyhound founders had mean onset of capital femoral ossification at 9.9 +/- 2.6 days of age. Of 10 dogs in the backcross generation that have reached 8 months of age, 2 had palpable subluxation without marked CHD radiographically. The mean distraction index of these dogs was 0.36 +/- 0.16 and the dorsolateral subluxation score was 65 +/- 5%. Although dogs in the backcross generation that were three-quarter greyhound had a broad range of hip laxity, a protective effect of the greyhound QTLs for good osseous conformation has mitigated thus far against subluxation and CHD.

Animals↗

PubMedDate not supplied

Genetic structure of susceptibility traits for hip dysplasia and microsatellite informativeness of an outcrossed canine pedigree.

An outcrossed canine pedigree was developed for quantitative trait locus (QTL) mapping of hip dysplasia by breeding dysplastic Labrador retrievers to trait-free greyhounds. Measured susceptibility traits included age at onset of femoral capital chondroepiphyseal ossification (OSS), maximum hip distraction (laxity) index (DI), and the dorsolateral subluxation (DLS) score. The pedigrees consisted of 147 dogs representing four generations. For 59 dogs genotyped with 65 microsatellites, the median heterozygosity and polymorphic information content (PIC) values of the F(1) generation were 0.82 and 0.68, respectively. Seventy-seven percent of microsatellites had a PIC greater than 0.59 in the F(1)s. Ninety-six percent of alleles showed Mendelian inheritance. Based on marker informativeness, approximately 350 randomly selected markers would be required for genome-wide screening to obtain an average interval between informative markers of 10 cM. Heritability was estimated as 0.43, 0.5, and 0.61 for OSS, DI, and the DLS score, respectively. Biometric estimates of the mean (+/- variance) effective number of segregating QTLs was 1.2 (+/- 0.05), 0.8 (+/- 0.02), and 1.0 (+/- 0.03) for OSS, DI, and the DLS score, respectively. The distributions of simulated backcross trait data suggested that the loci controlling these traits acted additively and that the DI may be controlled by a major locus. When combined with previous power and quantitative genetic analyses, these estimates indicate that this pedigree is informative for QTL mapping of hip dysplasia traits.

Animals↗

PubMedDate not supplied

Effects of exercise and polysulfated glycosaminoglycan on the development of osteoarthritis in equine carpal joints with osteochondral defects.

This study assessed the effects of postoperative exercise and intra-articular polysulfated glycosaminoglycan (PSGAG) on the repair of osteochondral defects in the carpal joints of ponies. Eighteen ponies with normal carpi had osteochondral defects (mean dimensions 2.4 cm x 0.9 cm) created arthroscopically on the dorsal aspect of the distal articular surface of the radial carpal bone. The ponies were randomized (while balancing for age [range, 2 to 15 years; median, 5.0 years]) to two groups--nine ponies were exercised and nine were stall confined. Beginning at surgery, six ponies in each group received five weekly intra-articular injections of PSGAG (250 mg) in one joint and lactated Ringer's solution in the contralateral joint; the remaining three ponies in each group received lactated Ringer's solution in both joints. The incremental exercise schedule on a circular, rotating walker was begun six days after surgery and occurred twice daily, reaching a maximum of 0.7 miles of walking and 2.7 miles of trotting by the third postoperative month. The effects of treatment on the joint tissues were determined by weekly lameness examinations and measurement of the range of carpal joint motion, carpal radiographs at six and 17 weeks after surgery, synovial fluid analysis, and cytologic evaluation of alcohol-fixed synovial fluid specimens at weeks 1 through 4 and week 17, and histology of the synovial membrane. Ultrasound images of the carpi were acquired before operation and at weeks 1, 2, 4, 8, 10, 13, and 17. Ponies were euthanatized 17 weeks after surgery. Exercise, without medication, caused more lameness throughout the study compared with no exercise. Exercised, nonmedicated ponies had the greatest limitation to carpal flexion (more painful joints), and nonexercised, nonmedicated (control) ponies had the least limitation to flexion. Radiographic scores indicated that the exercised, nonmedicated ponies had significantly (p < .05) more signs of osteoarthritis than exercised, medicated and control ponies. Ultrasonographic measurements indicated that exercise, without medication, caused the greatest increase in combined measurement of the joint capsule thickness and synovial fluid accumulation at all postoperative times. Synovial lining cell numbers in the synovial fluid from exercised ponies were significantly (p < .05) higher than in nonexercised ponies at week 1, and this trend continued at weeks 4 and 17 (p < .1). There were significantly (p < .05) more morphologic abnormalities in the synovial lining cells from exercised than from nonexercised ponies at week 17. Medication with PSGAG enabled exercised carpal joints to be flexed significantly further from weeks 2 through 6 compared with nonmedicated joints.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMedDate not supplied

Dorsolateral subluxation of hip joints in dogs measured in a weight-bearing position with radiography and computed tomography.

OBJECTIVE: develop a radiographic procedure to measure dorsolateral subluxation (DLS) of the femoral head in canine coxofemoral (hip) joints in a weight-bearing position. STUDY DESIGN: DLS measured on a radiographic projection was compared with DLS measured on computed tomography (CT) images of hip joints in a weight-bearing position. ANIMALS: A total of 24 dogs of varying ages were examined including Labrador retrievers, greyhounds, and Labrador-greyhound crossbreeds. METHODS: Anesthetized dogs were placed in sternal recumbency in a kneeling position in a foam rubber mold. The stifles were flexed and adducted with the femora perpendicular to, and in contact with, the table. To test for DLS, dogs were imaged in this weight-bearing position (DLS test) with routine radiography and CT. For each hip, the DLS score was determined by measuring the percentage of the femoral head medial to the lateralmost point of the cranial acetabular rim on the dorsoventral radiographic projection and the lateralmost point of the central, dorsal acetabular rim on the CT image. Higher DLS scores indicated better coverage of the femoral head by the acetabulum. DLS scores were compared with the distraction index (DI) by grouping joints according to their probability of developing osteoarthritis (OA) as predicted by the DI. RESULTS: The DLS score in the new position ranged from 29% to 71% for radiography and 15% to 59% for CT. Joints classified as OA unsusceptible had a mean score of 64% +/- 1.5% for radiography and 55% +/- 0.8% for CT (n = 10); hip joints having a high probability of developing OA had a score of 39% +/- 2.6% for radiography and 26% +/- 1.9% for CT (n = 8). When the DLS test was repeated on the same dogs at a different time, the intraclass correlation coefficient for the DLS score on the radiographs was 0.85 (left hip) and 0.89 (right hip). There was a strong correlation (r = .89 for both hips) between the DLS score measured on the weight-bearing radiograph and the CT image. A strong correlation also was observed between the DLS score and the DI (r = -.87). The DLS scores for OA unsusceptible joints and joints with a high probability of developing OA were significantly different (P < .05). CONCLUSIONS AND CLINICAL RELEVANCE: The DLS test can be performed with CT or routine radiography to measure variable amounts of DLS in weight-bearing hip joints oriented similarly to those of a standing dog. After additional long-term follow-up studies evaluating the development of OA and breed effects are performed, the DLS method may prove useful in studies of normal and abnormal hip joint development related to canine hip dysplasia.

Animals↗

PubMedDate not supplied

Exclusion of melanocortin-1 receptor (mc1r) and agouti as candidates for dominant black in dogs.

The domestic dog exhibits a variety of coat colors that encompass a wide range of variation among different breeds. Very little is known about the molecular biology of dog pigmentation; current understanding is based mostly on traditional breeding experiments, which in some cases have suggested genetic interactions that are different from those reported in other mammals. We have examined the molecular genetics of dominant black, a uniform coat color characteristic of black Labrador retrievers or Newfoundlands that has been proposed to be caused by either variation in the melanocortin-1 receptor gene (Mc1r) or by variation in the Agouti gene (A). We identified several coding polymorphisms within Mc1r and several simple sequence repeat polymorphisms closely linked to A, and examined their inheritance in a Labrador retriever x greyhound cross that segregates dominant black. No single Mc1r allele was found consistently in animals carrying dominant black, and neither Mc1r nor A cosegregated with dominant black. These results refine our understanding of mammalian coat color inheritance and suggest that dominant black coat color in dogs is caused by a gene not previously implicated in pigment type switching.

Agouti Signaling Protein↗

PubMedDate not supplied

Cytology of polychrome-stained equine synovial fluid smears. Comparison with clinical findings, histologic specimens, Wright-Giemsa-stained smears and outcome.

Polychrome-stained equine synovial fluid specimens from 34 normal joints and 129 joints with clinical abnormalities were examined cytologically. The smears from joints with abnormalities were categorized as within normal limits (4.7%), slight abnormality (27.9%), proliferative synovitis (21.7%), neutrophilic pattern (20.2%), elongated cell pattern (10.1%), other moderate to marked abnormality (11.6%) and unsatisfactory (3.9%). Cytologic abnormalities that were not restricted to a single category included spindle cells, crystals, stellate cells and cartilage fragments. Multinucleate cells and mononucleate cells with dense cytoplasm and a delicate periphery were seen in smears from cases with clinical diagnoses of osteochondrosis or fracture; interpretation of these cells as osteoclasts and their mononucleate precursors was supported by positive staining with tartrate-resistant acid phosphatase. Smears within the same cytologic category were not found to correspond with a single clinical diagnosis. The identification of several cytologic patterns in cases with the same clinical diagnosis suggests that multiple stages of disease were sampled. Except in cases with the cytologic neutrophilic pattern, there was not a consistent relationship between the histologic features in synovial biopsy specimens and the cytologic findings; the morphologic variation within synovial membrane sections and between sections from different locations was sometimes marked. When compared with air-dried, Wright-Giemsa-stained smears, the polychrome-stained smears were more sensitive in the detection of cytologic abnormalities and were less often falsely negative or unsatisfactory. Following surgery, cases with clinical diagnoses of osteochondrosis (29 cases) and fracture (25 cases) were analyzed according to clinical outcome and cytologic category. While 80% of the horses with proliferative synovitis in cytologic specimens were sound, only 67% of those with the elongated cell pattern, 50% of those with slight abnormality and 33% of those with other moderate to marked abnormality were sound. A statistically significant relationship (P less than .02) was found in cases with a diagnosis of osteochondrosis: animals with a proliferative synovitis pattern were almost three times as likely to be sound as compared to those with slight abnormality. These findings indicate that polychrome-stained equine synovial fluid smears (1) provide information that is different from that found in corresponding histologic sections and (2) are superior to air-dried, Wright-Giemsa-stained smears for cytologic examination. The polychrome-stained equine synovial fluid smears were found to provide information supportive of clinical, radiographic and prognostic data.

Animals↗

PubMedDate not supplied

Effect of infection on protein and nucleic acid synthesis in mammalian organs and tissues.

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Animals↗