Localization of extracellular matrix receptors in ICGN mice, a strain of mice with hereditary nephrotic syndrome.

Fibrotic degeneration was examined in the kidneys of ICR-derived glomerulonephritis (ICGN) mice, a novel inbred mouse line with a hereditary nephrotic syndrome of unknown etiology considered to be a good model of human idiopathic nephrotic syndrome. In the present study, we histochemically revealed changes in accumulation of extracellular matrix (ECM) components and in localization of integrins, cellular receptors for ECM, in the kidneys of ICGN mice with the progression of renal failure. Excessive accumulation of basement membrane (laminin and collagen IV) and interstitial (type III collagen) ECM components were demonstrated in the glomeruli and tubulointerstitum of ICGN mice. Marked deposition of type I collagen and tenascin was seen only in the glomeruli of ICGN mice but not in those of ICR mice as normal controls. Increased expression of integrin alpha1-, alpha2-, alpha5- and beta1-subunits in glomeruli with fibrotic degeneration and abnormal distribution of alpha6-subunit were noted in the kidneys of ICGN mice. Excessive laminin, a ligand of alpha6beta1-integrin, was demonstrated on the tubular basement membrane, but alpha6-subunit diffusely disappeared on the basal side of the tubular epithelial cells. We presumed that abnormal integrin expression in renal tubules causes epithelial cell detachment, and consequently tubular nephropathy, and results in disorder of ECM metabolism causing excessive accumulation of ECM components in the kidneys of ICGN mice.     

Participation of H1-receptors in histamine-induced contraction and relaxation of horse coronary artery in vitro.

The mechanisms of histamine-induced contraction and relaxation were investigated in rings isolated from a middle part of the left descending coronary arteries of horses. Intact and endothelium-denuded preparations were compared. Rings of horse coronary arteries contracted in response to histamine in a concentration dependent manner, but some of them relaxed with lower concentrations and contracted with higher concentrations. Removal of the endothelium abolished the relaxation and potentiated the contraction. The pD2 values were 4.70 +/- 0.08 in the rings with intact endothelium and 4.95 +/- 0.08 in endothelium-denuded rings. Histamine-induced contractions in intact and denuded preparations were not affected by an H2-antagonist, cimetidine, but were inhibited by an H1-antagonist, diphenhydramine in non-competitive manner in the rings with endothelium and in competitive manner in denuded rings. After precontraction with PGF2 alpha or norepinephrine, histamine relaxed preparations with intact endothelium (pD2 value, 7.80 +/- 0.11), although histamine-induced relaxations were not observed in denuded preparations. The relaxation was competitively inhibited by diphenhydramine. Relaxing response was significantly attenuated by methylene blue, quinacrine, L-nitro-arginine, gossypol and AA861 but not by indomethacin. These results suggest that the histamine-induced contraction and relaxation in horse coronary arteries are mediated mainly by H1-receptors in the smooth muscle and endothelium, respectively, and H1-receptor activation of endothelial cells may liberate vasodilator substances.     

Purification and properties of pyrethroid carboxyesterase in rat liver microsome

Pyrethroid carboxyesterase which hydrolyzes the esters of chrysanthemumic acid was purified from rat liver microsome by cholic acid solubilization, ammonium sulfate fractionation, heat treatment, and DEAE-Sephadex A-50 column chromatography. The 45-fold purified enzyme (38% yield) is likely to consist of single protein, as evidenced by polyacrylamide gel disc electrophoresis and Sephadex G-100 column chromatography, and had a molecular weight of approximately 74,000 and a K_m of 0.21 mM. It is susceptible to inhibition by organophosphates and carbamate insecticides and insensitive to pCMB, mercuric ion, and cupric ion. It is capable of hydrolyzing trans isomers of synthetic pyrethroids much more rapidly (five to ten times) than the cis counterparts. The purified pyrethroid carboxyesterase is apparently identical in nature with malathion carboxyesterase and with p-nitrophenyl acetate carboxyesterase.     

Characterization of a wild-type strain of Francisella tularensis isolated from a cat.

Francisella tularensis type A is the primary cause of tularemia in animals and humans in North America. The majority of research on F. tularensis has been done with the attenuated live vaccine strain (LVS), which is a type B, but very few wild-type F. tularensis strains have been characterized. A gram-negative coccobacillus that was isolated in pure culture from the lungs of a cat that died after being lost for 5 days was received for identification at the Virginia-Maryland Regional College of Veterinary Medicine Teaching hospital. The isolate (strain TI0902) was not identified (or was misidentified) by commercial identification systems; however, it was identified as F. tularensis subspecies tularensis (type A) by sequencing a portion of the 16S ribosomal RNA gene. Furthermore, repetitive extragenic palindromic sequences-polymerase chain reaction amplified a 4-kb DNA fragment from TI0902 that was characteristic of F. tularensis type A but not type B. The electrophoretic profile of the lipopolysaccharide of strain TI0902 was identical to that of the LVS by Western blotting with antiserum to LVS. The protein-enriched outer membrane of strain TI0902 contained 6-8 proteins, which were similar in molecular size to those from the LVS. Electron microscopy of negatively stained and alcian blue-stained LVS and TI0902 cells showed that both strains were coccobacillary in shape and may be encapsulated. However, after mouse challenge, the TI0902 strain was clearly more virulent than the LVS strain. Results of this study indicate that the genotype and phenotype of wild-type F. tularensis type A strain TI0902 is similar, but not identical, to that of the LVS strain. Further studies will help determine whether pathogenesis and host-pathogen interactions are also similar between the 2 strains.     

Effect of dasatinib in a xenograft mouse model of canine histiocytic sarcoma and in vitro expression status of its potential target EPHA2

Canine histiocytic sarcoma (HS) is an aggressive and highly metastatic tumor. Previously, the kinase inhibitor dasatinib was shown to have potent growth inhibitory activity against HS cells in vitro, possibly via targeting the EPHA2 receptor. Here, the in vivo effect of dasatinib in HS cells was investigated using a xenograft mouse model. Moreover, the expression status of EPHA2 was examined in six HS cell lines, ranging from insensitive to highly sensitive to dasatinib. In the HS xenograft mouse model, dasatinib significantly suppressed tumor growth, as illustrated by a decrease in mitotic and Ki67 indices and an increase in apoptotic index in tumor tissues. On Western blot analysis, EPHA2 was only weakly detected in all HS cell lines, regardless of sensitivity to dasatinib. Dasatinib likely results in the inhibition of xenograft tumor growth via a mechanism other than targeting EPHA2. The findings of this study suggest that dasatinib is a targeted therapy drug worthy of further exploration for the treatment of canine HS.     

Pharmacokinetic indices for cefovecin after single‐dose administration to adult sea otters (Enhydra lutris)

Seven sea otters received a single subcutaneous dose of cefovecin at 8 mg/kg body weight. Plasma samples were collected at predetermined time points and assayed for total cefovecin concentrations using ultra‐performance liquid chromatography and tandem mass spectrometry. The mean (±SD) noncompartmental pharmacokinetic indices were as follows: C_Max (obs) 70.6 ± 14.6 μg/mL, T_{Max (obs)} 2.9 ± 1.5 h, elimination rate constant (k_el) 0.017 ± 0.002/h, elimination half‐life (t_1/2kel) 41.6 ± 4.7 h, area under the plasma concentration‐vs.‐time curve to last sample (AUC_last) 3438.7 ± 437.7 h·μg/mL and AUC extrapolated to infinity (AUC_0→∞) 3447.8 ± 439.0 h·μg/mL. The minimum inhibitory concentrations (MIC) for select isolates were determined and used to suggest possible dosing intervals of 10 days, 5 days, and 2.5 days for gram‐positive, gram‐negative, and Vibrio parahaemolyticus bacterial species, respectively. This study found a single subcutaneous dose of cefovecin sodium in sea otters to be clinically safe and a viable option for long‐acting antimicrobial therapy.     

Polymorphism in promoter region of growth hormone receptor is associated with potential production capacity of insulin‐like growth factor‐1 in pre‐pubertal Holstein heifers

Insulin‐like growth factor‐1 (IGF‐1) is one of the important factors for growth, milk production and reproductive functions and mainly released from the liver in response to growth hormone (GH) via GH receptor (GHR) in cattle. Recently, some single nucleotide polymorphisms (SNPs) were identified in the bovine GHR gene. Some GHR‐SNPs were shown to be related to plasma IGF‐1 concentration in cattle. Hence, the capacity to IGF‐1 production in the liver might be affected by GHR‐SNP and associated with performance in the future. This study examined whether GHR‐SNP is associated with IGF‐1 production in the liver of pre‐pubertal heifers. In 71 Holstein calves, blood samples for genomic DNA extraction were obtained immediately after birth. To genotype the GHR‐SNPs in the promoter region, polymerase chain reaction (PCR) products were digested with restriction enzyme NsiI (cutting sites: AA, AG and GG). All heifers at 4 months of age were intramuscularly injected with 0.4 mg oestradiol benzoate. Blood samples were obtained from the jugular vein just before (0 h) and 24 h after injection. The number of AA, AG and GG at the NsiI site was 0, 17 and 54 respectively. In AG and GG, plasma GH concentrations were higher pre‐injection than 24 h post‐injection (p < 0.01). Moreover, plasma GH concentrations in AG post‐injection were higher than in GG (p < 0.05). In contrast, the GG genotype exhibited higher plasma IGF‐1 concentrations in pre‐injection than post‐injection (p < 0.01), although oestradiol did not change IGF‐1 concentration in the AG genotype. We conclude that the GG polymorphism in the promoter region of GHR is associated with a higher potential capacity of IGF‐1 production in the liver of cattle.