Cyclooxygenase-2 (COX-2) expression in canine intracranial meningiomas.

Meningiomas are the most common canine intracranial tumour. Neurologic disability and death from treatment failure remain problematic despite current surgical and radiotherapeutic treatments for canine intracranial meningiomas. Cyclooxygenase-2 (COX-2) over-expression has been demonstrated in multiple canine malignancies, and COX-2 inhibitory treatment strategies have been shown to have both preventative and therapeutic effects in spontaneous and experimental models of cancer. The purpose of this study was to evaluate COX-2 expression in canine intracranial meningiomas. Immunohistochemical and Western blot (WB) analyses showed COX-2 expression in multiple tissues of the normal canine brain, and 87% (21/24) of intracranial meningiomas studied were immunoreactive to COX-2. No significant associations between COX-2 immunoreactivity and tumour grade were identified. Further studies are required to elucidate the physiologic roles of constitutive COX-2 expression in the central nervous system as well as its participation in meningioma tumourigenesis.

B-cell lymphoma in the peripheral nerves of a cat.

A B-cell, Burkitt-type lymphoma, diffusely affecting the peripheral nerves and intramuscular nerve branches was diagnosed in a 4-year-old domestic shorthair cat with a chronic progressive history of flaccid tetraparesis and generalized muscle atrophy. There was no evidence of cranial nerve, central nervous system, radicular, bone marrow, splenic, or lymph node involvement. The cat tested negative for feline retroviruses and a wide variety of herpes viruses, including Epstein-Barr virus. The clinical manifestation of this case was similar to the chronic polyneuropathic variant of human diffuse neurolymphomatosis; a condition most commonly caused by an axonopathy resulting from infiltration of peripheral nerves with non-Hodgkin's lymphoma.

Gene expression analysis of the acute phase response using a canine microarray.

The safety of pharmaceuticals is typically assessed in the dog and rat prior to investigation in humans. As a result, a greater understanding of adverse effects in these preclinical testing species would improve safety assessment. Despite this need, there is a lack of tools to examine mechanisms and identify biomarkers in the dog. To address this issue, we developed an Affymetrix-based oligonucleotide microarray capable of monitoring the expression of thousands of canine genes in parallel. The custom canine array contains 22,774 probe sets, consisting of 13,729 canine and 9045 human-derived probe sets. To improve cross-species hybridization with human-derived probes, the detection region was moved from the variable 3' UTR to the more homologous coding region. Testing of this strategy was accomplished by comparing hybridization of naive dog liver RNA to the canine array (coding region design) and human U133A array (standard 3' design). Although raw signal intensity was greater with canine-specific probe sets, human-derived probes detected the expression of additional liver transcripts. To assess the ability of this tool to detect differential gene expression, the acute phase response was examined in beagle dogs given lipopolysaccharide (LPS). Hepatic gene expression 4 and 24 h post-LPS administration was compared to gene expression profiles of vehicle-treated dogs (n=3/group). Array data was consistent with an acute inflammatory response, with transcripts for multiple cytokines and acute phase proteins markedly induced 4 h after LPS challenge. Robust changes in the expression of transcripts involved with glucose homeostasis, biotransformation, and extracellular matrix remodeling were observed 24 h post-dose. In addition, the canine array identified several potential biomarkers of hepatic inflammation. Strong correlations were found between gene expression data and alterations in clinical chemistry parameters such as serum amyloid A (SAA), albumin, and alkaline phosphatase (ALP). In summary, this new genomic tool successfully detected basal canine gene expression and identified novel aspects of the acute phase response in dog that shed new light on mechanisms underlying inflammatory processes.

Antidotes--inappropriate timely availability.

The aim of this study was to determine the availability of antidotes to poisons in Wales and the South West of England. A stocklist of antidotes that are available to accident and emergency departments was requested and was compared with recommendations from the International Programme on Chemical Safety (IPCS). Chief pharmacists were invited to complete a short questionnaire regarding knowledge of existing guidelines. Thirty-four of 43 centres replied (response rate 77%). No department held all 36 antidotes (mean 13, range 7-33). All departments held antidotes that were frequently used. Ninety-one percent of departments held one cyanide antidote. Eighty-eight percent held one heavy metal chelating agent. The remaining antidotes were variably stocked. New agents such as 4-methylpyrazole, hydroxocobalamin and the heavy metal chelating agents DMSA and DMPS were infrequently held. Twenty of 34 chief pharmacists were unfamiliar with existing UK guidelines. A trend exists whereby larger departments stocked more antidotes. Some antidotes to poisons are not available in a timely fashion in Wales and the South West of England. There is a lack of awareness of existing guidelines. New recommendations relevant to clinical need and local practice should ideally be developed.

Basolateral proteinase-activated receptor (PAR-2) induces chloride secretion in M-1 mouse renal cortical collecting duct cells.

1. Using RT-PCR, Northern blot analysis, and immunocytochemistry, we confirmed renal expression of proteinase-activated receptor (PAR-2) and demonstrated its presence in native renal epithelial and in cultured M-1 mouse cortical collecting duct (CCD) cells. 2. We investigated the effects of a PAR-2 activating peptide (AP), corresponding to the tethered ligand that is exposed upon trypsin cleavage, and of trypsin on M-1 cells using patch-clamp, intracellular calcium (fura-2) and transepithelial short-circuit current (ISC) measurements. 3. In single M-1 cells, addition of AP elicited a concentration-dependent transient increase in the whole-cell conductance. Removal of extracellular Na+ had no effect while removal of Cl- prevented the stimulation of outward currents. The intracellular calcium concentration increased significantly upon application of AP while a Ca2+-free pipette solution completely abolished the electrical response to AP. 4. In confluent monolayers of M-1 cells, apical application of AP had no effect on ISC whereas subsequent basolateral application elicited a transient increase in ISC. This increase was not due to a stimulation of electrogenic Na+ absorption since the response was preserved in the presence of amiloride. 5. The ISC response to AP was reduced in the presence of the Cl- channel blocker diphenylamine-2-carboxylic acid on the apical side and abolished in the absence of extracellular Cl-. 6. Trypsin elicited similar responses to those to AP while application of a peptide (RP) with the reverse amino acid sequence of AP had no effect on whole-cell currents or ISC. 7. In conclusion, our data suggest that AP or trypsin stimulates Cl- secretion by Ca2+-activated Cl- channels in M-1 CCD cells by activating basolateral PAR-2.

Regulation of ATP-sensitive K+ channel by membrane-bound protein phosphatases in rat principal tubule cell.

The role of membrane-bound protein serine/threonine phosphatases (PP) in modulating the renal ATP-sensitive K+ (KATP) channel was examined using the patch-clamp technique in principal cells of rat cortical collecting duct. In the absence of ATP, channel activity rapidly (11.2 s) declines (channel "rundown") upon excision of the membrane patches into control bath solutions (1 mM Mg2+, Ca2+ free). Both orthovanadate (5 mM), a broad-spectrum inhibitor of phosphatases except for Ca(2+)-dependent PP (PP-2B), and okadaic acid (OA, 1 microM), a potent inhibitor of PP types 1 and 2A (PP-1 and PP-2A), significantly slowed channel rundown. Removal of Mg2+ from the bath also slowed the rundown process. Incubation of cells with OA in the absence of Mg2+ or with orthovanadate in ATP-free solution maintained channel activity at levels of approximately 70% of control values for 3 min after membrane excision. In contrast, Ca2+ (0.1 mM) and calmodulin (1 microM) in the presence of 1 mM Mg2+, a condition in which PP-2B is stimulated, had no significant effect on the channel activity that persisted in the presence of OA and orthovanadate. Application of exogenous PP-2A (1 U/ml) to the cytosolic side of membrane in inside-out patches significantly inhibited channel activity to 35.0% of control, but the inhibitory-effects of PP-1 (1 U/ml) and PP-2B (20 micrograms/ml) were minor. These results suggest that rundown of the renal KATP channel after membrane excision results mainly from dephosphorylation of the channel or an associated protein by membrane-bound phosphatases.(ABSTRACT TRUNCATED AT 250 WORDS)