Canine TIMP-2: purification, characterization and molecular detection

Matrix metalloproteinases (MMPs), which degrade tissues in health and disease are under the control of the tissue inhibitors of MMPs, the TIMPs. TIMP-2 is particularly important for control of MMP-2 and both have been implicated in many pathological processes from arthritis to tumour invasion. This study characterized and detected TIMP-2 from canine cells; including synovial fibroblasts and three tumour-derived canine cell lines, K1, K6 and DH82. Gelatin zymography demonstrated that pro-MMP-2 is produced by synovial fibroblasts and the three cells lines. Reverse zymograms showed that all the cell sources tested secrete both TIMP-1 and TIMP-2. The 22 kDa band was purified and n-terminal amino acid sequencing showed it to be highly homologous to equine and human TIMP-2. Analysis of purified canine MMP-2 and MMP-9 showed that TIMP-2 is associated, and co-purifies with MMP-2. Polymerase chain reaction, using consensus primers, was used to detect TIMP-2 mRNA from the cell sources and proved positive in all cases. This work highlights the importance of TIMP-2 as the main inhibitor for MMP-2 and, therefore, opens the possibilities of targeting TIMP-2 for therapeutic intervention against connective amino acid tissue degradation in a range of diseases.     

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.     

Lidocaine in the horse: its pharmacological effects and their relationship to analytical findings

Lidocaine is a local anaesthetic agent that is widely used in equine medicine. It is also an Association of Racing Commissioners International (ARCI) Class 2 foreign substance that may cause regulators to impose substantial penalties if residues are identified in post race urine samples. Therefore, an analytical/pharmacological database was developed for this drug. Using our abaxial sesamoid local anaesthetic model, the highest no-effect dose (HNED) for the local anaesthetic effect of lidocaine was determined to be 4 mg. Using enzyme-linked immunosorbent assay (ELISA) screening, administration of the HNED of lidocaine to eight horses yielded peak serum and urine concentrations of apparent lidocaine of 0.84 ng/mL at 30 min and 72.8 ng/mL at 60 min after injection, respectively. These concentrations of apparent lidocaine are readily detectable by routine ELISA screening tests (LIDOCAINE ELISA, Neogen, Lexington, KY). ELISA screening does not specifically identify lidocaine or its metabolites, which include 3-hydroxylidocaine, dimethylaniline, 4-hydroxydimethylaniline, monoethylglycinexylidine, 3-hydroxymonoethylglycinexylidine, and glycinexylidine. As 3-hydroxylidocaine is the major metabolite recovered from equine urine, it was synthesized, purified and characterized, and a quantitative mass spectrometric method was developed for 3-hydroxylidocaine as recovered from horse urine. Following subcutaneous (s.c.) injection of the HNED of lidocaine, the concentration of 3-hydroxylidocaine recovered from urine reached a peak of about 315 ng/mL at 1 h after administration. The mean pH of the 1 h post dosing urine samples was 7.7, and there was no apparent effect of pH on the amount of 3-hydroxylidocaine recovered. Within the context of these experiments, the data suggests that recovery of less than 315 ng/mL of 3-hydroxylidocaine from a post race urine sample is unlikely to be associated with a recent local anaesthetic effect of lidocaine. Therefore these data may be of assistance to industry professionals in evaluating the significance of small concentrations of lidocaine or its metabolites in postrace urine samples. It should be noted that the quantitative data are based on analytical methods developed specifically for this study, and that methods used by other laboratories may yield different recoveries of urine 3-hydroxylidocaine.     

The frequent detection of a treponeme in bovine digital dermatitis by immunocytochemistry and polymerase chain reaction

A study was carried out to determine whether spirochaetes are frequently associated with digital dermatitis in United Kingdom (UK) dairy cattle. Histopathological examination of lesions using a silver stain showed a large number of unidentified spirochaete-like organisms present in digital dermatitis hoof skin tissue in all examined biopsies. Immunocytochemical staining demonstrated that spirochaetes in skin lesions were identified by polyclonal antisera to Borrelia burgdorferi, Treponema denticola and Treponema vincentii (again all biopsies were positively stained), whereas monoclonal antibodies to B. burgdorferi and any Treponema pallidum did not stain any organisms in all biopsies. A PCR of 16S rRNA, previously shown to be specific for a new treponeme, was employed and produced positive results from 82.4% of digital dermatitis tissues. It is concluded that this spirochaete (or related spirochaetes), which is similar to human oral treponemes, is frequently associated with, and may be responsible for, pathological changes in digital dermatitis.     

Clinical management of the cancer patient. Taking a biopsy

Good biopsy protocol depends on excellent interaction with diagnostic pathologists. This article reviews the essentials of diagnostic appraisal, specimen preparation, biopsy interpretation, proper reporting, and implementation of biopsy results in case management. The emphasis is on biopsy of malignancy.     

Short-rotation woody crops and phytoremediation: Opportunities for agroforestry?

Agroforestry Systems - Worldwide, fuelwood demands, soil and groundwater contamination, and agriculture's impact on nature are growing concerns. Fast growing trees in short rotation woody crop...     

Predicting spring phenology and frost damage risk of Betula spp. under climatic warming: a comparison of two models

Timing of bud burst and frost damage risk for leaves of Betula spp. in response to climatic warming in Finland was examined with two models. In the first model, ontogenetic development in spring was triggered by an accumulation of chilling temperatures. The second model assumed an additional signal from the light climate. The two models gave radically different estimates of frost damage risk in response to climate warming. The chilling-triggered model forecast a significant and increasing risk with increased warming, whereas the light-climate-triggered model predicted little or no risk. The chilling-triggered model is widely applied in phenological research; however, there is increasing experimental evidence that light conditions play a role in the timing of spring phenology. Although it is not clear if the light response mechanisms are appropriately represented in our model, the results imply that reliance on a light signal for spring development would afford a degree of protection against possible frost damage under climate warming that would not be present if chilling were the sole determinant. Further experimental tests are required to ascertain the light-related mechanisms controlling phenological timing, so that credible model extrapolations can be undertaken.     

Sapstain fungi on <Emphasis Type="Italic">Pinus radiata</Emphasis> logs – from New Zealand Forest to export in Japan

Supply of clear, unstained logs from an export country is important economically as well as in reducing the biosecurity risk to an importing country such as Japan. Although conditions found within the holds of ships containing logs are thought to be ideal for rapid colonization of sapstain fungi, no research has been conducted. Research-focussed log export trials were designed to determine the extent of sapstain colonisation at specific points in the processing of logs from harvesting to arrival at the export destination. Two trials were established, in the austral summer and winter, in which mature Pinus radiata logs were harvested in New Zealand and shipped to export ports in Japan. Data loggers placed above and below deck of the ships recorded microclimatic conditions. Nine species of sapstain fungi were isolated from logs during the summer trial; the most common species isolated were Ophiostoma floccosum, Ophiostoma querci, and Ophiostoma setosum. In contrast, ten sapstain species were detected during the winter trial, with Sphaeropsis sapinea, O. querci, O. floccosum, and O. setosum most commonly isolated. This research was the first successful attempt at measuring visual sapstain development and isolating sapstain fungi from the time of harvesting to arrival at an export destination.