Exploration of glycoprotein structures: Sequences and consequences

The vast expansion in our knowledge of the detailed molecular structure of many glycoproteins has been achieved by purpose-designed methodologies. In this review, principal experimental methods for gaining fundamental evidence are assessed with respect to well-known iV-glycoproteins (e.g. ribonucleases), 0,N-glycoproteins (e.g. glycophorin A) and O-glycoproteins (e.g. submaxiilary gland mucins and some sulfated forms). Relevant properties of N-carbohydrate-peptide links (N-acetylglucosaminyl-asparagine) and O-linkages (N-acetylgalac-tosaminyl-Thr (or -Ser)) are reviewed and the classification and structure of more elaborate glycoproteins is outlined. Key reactions, both chemical and enzymic, effect the cleavage of N- and O-oligosaccharide side-chains from core protein, and subsequent calibrated chromatographic analysis can permit the identification of certain oligosaccharide groupings characteristic of glycoforms (families of glycoproteins different only in the oligosaccharide chain lengths and bond structures). Basic sequencing of individual oligosaccharides is generally achieved by chemical methods (e.g. methylation analysis, degradation with periodate) and by the action of well-characterised specific exo- and endoglycosidases. Degradation of a glycoprotein chemically (e.g. by cyanogen bromide) or enzymically by endopeptidases to glycopeptides provides means for locating oligosaccharide position(s) on the core protein. The presence of O-sulfate ester groups on oligosaccharide side-chains presents further challenging problems for sequencing investigations. Some correlations between oligosaccharide sequences and biological function are considered. While, in the main, the best-known glycoprotein structures are for those of mammalian origin (e.g. Igs), evidence points to other areas of interest in plants (especially yeasts), invertebrates and micro-organisms. Allusion is made to current comparative and fundamental physico-chemical techniques, especially gel permeation chromatography, membrane-transfer, FAB-MS and NMR (NOE, COSY, etc.).     

Intermuscular variation in tenderness: association with the ubiquitous and muscle-specific calpains

The biochemistry of intermuscular variation in tenderness is not fully understood. To investigate the role of the calpains in this process we performed two experiments using bovine and ovine species. In the bovine experiment, two distinct muscles, longissimus thoracis et lumborum (LT) and psoas major (PM), were used. In the ovine experiment, four muscles, LT, PM, semimembranosus (SM), and semitendinosus (ST), were used. Muscles were sampled at death for the determination of the steady-state mRNA level of calpains and calpastatin and the activities of calpain 1, 2, and calpastatin. Muscles were also sampled to determine the temporal changes in pH, tenderness, and the activity of the ubiquitous calpain system during postmortem aging. The results of the relative rate of tenderization in both species was found to be related to muscle type; LT had the highest value in both species. Within species, the mRNA steady-state levels of calpain 1 and calpastatin were similar in various bovine and ovine muscles. Bovine calpain 2 mRNA level was significantly lower in the LT than in the PM. Ovine calpain 2 mRNA level was lower, but not significantly different, in the LT compared to the other muscles. The mRNA level of bovine calpain 3 was significantly higher in the LT muscle than in the PM. In the ovine, the mRNA level of calpain 3 was highest in the LT, followed by SM, PM, and ST. Results on the activity of the ubiquitous calpain system in various muscles at death were dependent on muscle type and species. Temporal changes in the activity of calpains and calpastatin during the first 24 h of postmortem aging were similar in the muscles studied: calpain 1 and calpastatin declined significantly and calpain 2 remained relatively unchanged. The temporal changes in muscle pH in both experiments indicated that the extent and rate of pH decline during aging was related to muscle type. Correlation analysis between the relative rate of tenderization and mRNA expression of calpains revealed a strong relationship with calpain 3 in both species.     

Osteomyelitis secondary to trauma involving the proximal end of the radius in horses: five cases (1987-2001)

OBJECTIVE: To determine clinical, radiographic, and scintigraphic abnormalities in and treatment and outcome of horses with trauma-induced osteomyelitis of the proximal aspect of the radius. DESIGN: Retrospective study. ANIMALS: 5 horses. PROCEDURE: Data collected from the medical records included signalment; history; horse use; degree of lameness; radiographic, ultrasonographic, and scintigraphic findings; treatment; and outcome. RESULTS: Duration of lameness prior to referral ranged from 14 to 60 days. Mean severity of lameness was grade 3 of 5, and all horses had a single limb affected. All horses had signs of pain during elbow joint manipulation and digital palpation over the lateral aspect of the proximal end of the radius. Radiographic lesions consisted of periosteal proliferation, osteolysis, and subchondral bone lysis. Scintigraphy in 3 horses revealed intense pharmaceutical uptake diffusely involving the proximal end of the radius. Two horses had sepsis of the elbow joint. All horses were treated with antimicrobials long-term; 1 horse was also treated by local perfusion of the radial medullary cavity through an indwelling cannulated screw. At follow-up, all horses had returned to their previous function. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that osteomyelitis of the proximal end of the radius can result from a traumatic injury to the antebrachium. Because lesions may be an extension of septic arthritis, a thorough examination of the wound area and elbow joint is recommended. Prolonged systemic antimicrobial treatment can result in a successful outcome.     

Assessment of the accuracy of computed tomography for measurement of normal equine pituitary glands

OBJECTIVE: To describe the anatomic features of the pituitary gland region in horses via computed tomography (CT) and determine the accuracy of CT for estimating normal equine pituitary gland dimensions. ANIMALS: 25 adult horses with no clinical signs of pituitary disease. PROCEDURE: Transverse CT images and gross transverse tissue sections were compared in 2 horses. Contrast-enhanced CT of the pituitary gland region was performed postmortem in 23 horses with 4 slice thickness and interval settings (10-mm contiguous or overlapping slices and 4-mm contiguous or overlapping slices). Gross and CT estimates of pituitary gland dimensions were compared via ANOVA. Accuracy of CT estimates was calculated with gross pituitary gland measurements as the known value. RESULTS: Pituitary glands were located between the temporomandibular joints and had contrast enhancement. Mean gross dimensions were length, 2.11 cm; width, 2.16 cm; height, 0.98 cm; and volume, 2.66 cm3. Gross measurements and CT estimates of pituitary gland length from 10-mm contiguous and overlapping slices did not differ. Gross measurements and CT estimates of pituitary gland width from 4-mm contiguous and overlapping slices did not differ. Estimates of height and volume from all CT techniques differed from gross measurements. Accuracies for CT estimates were length, 88 to 99%; width, 81 to 92%; height, 58 to 71%; and volume, 43 to 55%. CONCLUSIONS AND CLINICAL RELEVANCE: Accuracy of estimates of pituitary gland dimension in horses varied with CT scanning technique; via CT estimates of length and width of glands were more accurate than estimates of height or volume.     

Effect of anticonvulsant dosages of potassium bromide on thyroid function and morphology in dogs

A placebo-controlled experiment was performed to evaluate the effect of potassium bromide on the canine thyroid gland. Basal total thyroxine, free thyroxine, and basal thyrotropin serum concentrations were evaluated over a 6-month period in potassium bromide-treated and control dogs. A thyrotropin-releasing hormone stimulation test was also performed in all dogs at the beginning and conclusion of the study. Thyroid histopathology was compared between treated and control dogs at the end of the study. No difference was detected in any parameter between the two groups at the end of the study. A decline in thyroid hormone concentrations over the course of the study did occur in both groups of dogs. Potassium bromide does not appear to have a significant effect on canine thyroid function or morphology.     

In vitro biomechanical study of rotational stabilizers of the canine elbow joint

OBJECTIVE: To develop a model for measuring rotary stability of the canine elbow joint and to evaluate the relative contribution of the anconeal process (AN), lateral collateral ligament (LCL), and medial collateral ligament (MCL). SAMPLE POPULATION: 18 forelimbs from 12 canine cadavers. PROCEDURE: Forelimbs were allocated to 3 experimental groups (6 forelimbs/group). Each intact forelimb was placed in extension at an angle of 135 degrees and cycled 50 times from -16 degrees (pronation) to +28 degrees (supination) in a continuous manner at 2.0 Hz. Cycling was repeated following sectioning of the structure of interest (group 1, AN; group 2, LCL; and group 3, MCL). Torque at -12 degrees (pronation) and +18 degrees (supination) was measured for each intact and experimentally sectioned limb. A Student t test was performed to compare torque values obtained from intact verses experimentally sectioned limbs and for comparison with established criteria for differentiation of primary (> or = 33%), secondary (10 to 33%), and tertiary rotational stabilizers (< 10%). RESULTS: In pronation, the AN was the only primary stabilizer (65%). For supination, the LCL was a primary stabilizer (48%), AN was a secondary stabilizer (24%), and MCL was a tertiary stabilizer (7%). CONCLUSIONS AND CLINICAL RELEVANCE: With the elbow joint in extension at an angle of 135 degrees, the AN is a primary rotational stabilizer in pronation, and the LCL is a primary stabilizer in supination. Disruption of the AN or LCL may affect rotary range of motion or compromise stability of the elbow joint in dogs.     

Association of racing performance with specific abnormal radiographic findings in Thoroughbred yearlings sold in Texas

The objective of this prospective study was to determine the association between presence of abnormal radiographic findings attributed to orthopedic disorders of development (ARF) with subsequent performance among yearlings in Texas. The study population was 348 yearlings offered for sale at the Texas Summer Yearling Sale during 2002 and 2003 for which presale radiographs of limbs were available for review. Radiographs of yearlings were reviewed and presence of ARF were recorded. Additionally, the following data were recorded for each horse: identifiers [name of dam, name of sire, hip number, and name (when available)], year of sale, state of birth, and sales price. Subsequent performance data were obtained for each horse at the conclusion of their 2^nd and 3^rd years of life. Results indicated that no ARF was significantly associated with performance outcomes. Lesions of the proximal dorsal aspect of the sagittal ridge of the 3^rd metacarpus/metatarsus resulted in significantly lower sales prices than either horses without this ARF or horses without any ARF. In conclusion, multiple radiographic abnormalities of Thoroughbred yearlings were not associated with reduced performance during the first 2 years of the racing careers of horses studied. Presence of some abnormalities, however, may be associated with reduced sales price.     

A comparison of Riley encased methodology and traditional techniques for planting red mangroves (Rhizophora mangle)

The effectiveness of encasement and traditional techniques for planting red mangroves (Rhizophora mangle) in moderate to high wave energy environments was assessed. The three encasement types were the halflength PVC pipes, fulllength PVC pipes, and bamboo pipes. Plantings were conducted in August, 1997 at two locations in the Indian River Lagoon, Florida: Sebastian and Rocky Point. Furthermore, plantings were conducted in November, 1997 using fulllength encasements and conventional planting. Results indicate that seedlings planted within fulllength PVC encasements had the highest survivorship and growth because of their protection from waves and currents. Failure of seedlings within bamboo encasements seemed to be caused by insufficient light exposure. When comparing the two locations, a significantly greater growth was observed at the Sebastian location than at the Rocky Point location for the planting conducted in November, but not for those planted in August. No significant difference was observed in seedling survival when comparing those planted in August and November. However, there was a significantly greater growth in mangroves planted in August. With the exception of width of surf zone, there was no significant difference in the selected environmental parameters between the two locations.     

INTER‐ AND INTRAFRACTION MOTION FOR STEREOTACTIC RADIOSURGERY IN DOGS AND CATS USING A MODIFIED BRAINLAB FRAMELESS STEREOTACTIC MASK SYSTEM

Precise and accurate patient positioning is necessary when doing stereotactic radiosurgery (SRS) to ensure adequate dosing to the tumor and sparing of normal tissues. This prospective cross‐sectional study aimed to assess feasibility of a commercially available modified frameless SRS positioning system for use in veterinary radiotherapy patients with brain tumors. Fifty‐one dogs and 12 cats were enrolled. Baseline and verification CT images were acquired. The verification CT images from 32 dogs and five cats had sufficient images for fusion to baseline CT images. A rigid box‐based fusion was performed to determine interfraction motion. Forty‐eight dogs and 11 cats were assessed for intrafraction motion by cine CT. Seventy percent of dogs and 60% of cats had interfraction 3D vector translational shifts >1 mm, with mean values of 1.9 mm in dogs, and 1.8 mm in cats. In dogs muscle wasting was weakly correlated with translational shifts. The maximum angular interfraction motion observed was 6.3° (roll), 3.5° (pitch), and 3.3° (yaw). There was no correlation between angular interfraction motion and weight, brachycephaly, or muscle wasting. Fifty‐seven percent of dogs and 50% of cats had respiration‐related intrafraction motion. Of these, 4.5% of dogs and 10% of cats had intrafraction motion >1 mm. This study demonstrates the modified Brainlab system is feasible for SRS in dogs and cats. The smaller cranial size and difference in anatomy increases setup uncertainty in some animals beyond limits usually accepted in SRS. Image‐guided positioning is recommended to achieve clinically acceptable setup accuracy (<1 mm) for SRS.