Evaluation of ponatinib in vitro effect in three canine mast cell tumor cell lines expressing FGFR-1, PDGFR-α, and VEGFR-2.

Ponatinib is a broad-spectrum tyrosine kinase inhibitor that targets numerous receptor tyrosine kinases (RTKs), including but not limited to fibroblast growth factor receptor (FGFR)-1, platelet derived growth factor receptor (PDGFR)-α, and vascular endothelial growth factor receptor (VEGFR)-2. This study evaluated the expression of FGFR-1, PDGFR-α, and VEGFR-2 in three canine mast cell tumor (MCT) cell lines (CM-MC1, VI-MC1, CoMS) and the effects of ponatinib on these MCT cell lines. Quantitative RT-PCR confirmed the expression of FGFR-1, PDGFR-α, and VEGFR-2 in the three MCT cell lines. Ponatinib exhibited dose- and time-dependent cytotoxicity in MCT cell lines via MTT assay. The IC50 for 24, 48, and 72 h across the three cell lines ranged from 38.47 nM to 103.3 nM, which is clinically comparable to dose ranges established for humans. Significantly increased apoptosis in each cell line was seen between 12 and 18 h after treatment with IC50 of ponatinib via Annexin-V and Caspase-3/7 assays. These data suggest that ponatinib could be a possible therapeutic agent for canine MCTs. Further studies are needed to investigate the prognostic value of FGFR-1, PDGFR-α, and VEGFR-2 in canine MCTs.

Characterization of toasted cereal flours from the Canary Islands (gofios).

In this work, different types of gofio produced in the Canary Islands were characterized on the basis of physico-chemical parameters as well as spectral features. An overall of 7 physico-chemical parameters (moisture, ash, pH, Cu, Fe, Mn and Zn) were determined in 64 samples from different cereals and legumes, and IR spectra of all samples were registered. The chemometric processing of both, metal ions contents determined by atomic absorption spectrophotometry, and the MIR spectra, with various techniques including cluster analysis, principal component analysis, and discriminant analysis enabled the accurate characterization and classification of the gofio samples according to the grain used in their obtaining process.

Invited Review: pathophysiology of cardiac muscle contraction and relaxation as a result of alterations in thin filament regulation.

Cardiac muscle contraction depends on the tightly regulated interactions of thin and thick filament proteins of the contractile apparatus. Mutations of thin filament proteins (actin, tropomyosin, and troponin), causing familial hypertrophic cardiomyopathy (FHC), occur predominantly in evolutionarily conserved regions and induce various functional defects that impair the normal contractile mechanism. Dysfunctional properties observed with the FHC mutants include altered Ca(2+) sensitivity, changes in ATPase activity, changes in the force and velocity of contraction, and destabilization of the contractile complex. One apparent tendency observed in these thin filament mutations is an increase in the Ca(2+) sensitivity of force development. This trend in Ca(2+) sensitivity is probably induced by altering the cross-bridge kinetics and the Ca(2+) affinity of troponin C. These in vitro defects lead to a wide variety of in vivo cardiac abnormalities and phenotypes, some more severe than others and some resulting in sudden cardiac death.

Rapid activation of neutral sphingomyelinase by hypoxia-reoxygenation of cardiac myocytes.

Elevated levels of oxygen free radicals have been implicated in the pathways of reperfusion injury to myocardial tissue. The targets for free radicals may include specific as well as random intracellular components, and part of the cellular response is the induction of extracellularly activated and stress-activated kinases. The intermediate signals that initiate these stress responses are not known. Here we show that one of the earliest responses of cardiac myocytes to hypoxia and reoxygenation is the activation of neutral sphingomyelinase and accumulation of ceramide. Ceramide increased abruptly after reoxygenation, peaking at 10 minutes with 225+/-40% of the control level. Neutral sphingomyelinase activity was induced with similar kinetics, and both activities remained elevated for several hours. c-Jun N-terminal kinase (JNK) was also activated within the same time frame. Treatment of cardiac myocytes with extracellular ceramides also activated JNK. Pretreating cells with antioxidants quenched sphingomyelinase activation, ceramide accumulation, and JNK activation. Ceramide did not accumulate in reoxygenated nonmuscle fibroblasts, and JNK was not activated by reoxygenation in these cells. The results identify neutral sphingomyelinase activation as one of the earliest responses of cardiac myocytes to the redox stress imposed by hypoxia-reoxygenation. The results are consistent with a pathway of ceramide-mediated activation of JNK.

Induction of a herpesvirus saimiri small RNA AU binding factor (AUBF70) activity and lymphokine mRNAs by T cell mitogens.

Herpesvirus saimiri (H. saimiri) can transform T lymphocytes and cause lymphoid tumors in rabbits and New World monkeys. H. saimiri-immortalized T cells express IL-2 and IL-4. The putative oncogenes of a group C strain of H. saimiri have been mapped to a region of the unique L-DNA which includes genes encoding four U-like small nuclear RNAs (HSUR1-HSUR4). Jurkat T cells express a 70 kD RNA binding factor (AUBF70) which binds HSUR2. Here we examined AUBF70 expression in resting and mitogen-stimulated human peripheral blood T cells and its sequence specificity and subcellular distribution. Band-shift assays demonstrated that resting human T cells express low amounts of AUBF70 which is induced by mitogen treatment. IL-2 and IL-4 mRNAs were co-induced with AUBF70 suggesting that AUBF70 is a positive regulator of lymphokine gene expression. Normal resting, mitogen-stimulated, and leukemic Jurkat T cells all express AUBF70 with virtually identical V8 proteolytic enzyme digestion patterns. Northern blots demonstrated that HSUR1 and HSUR2 are localized both in the nucleus and cytoplasm. HSUR2 accumulate in the cytoplasm in the presence of actinomycin D, which is consistent with re-transport of HSURs to the nucleus by (an) unstable factor(s). We hypothesize that HSUR1 and 2 transport AUBF70 from the cytoplasm to the nucleus; in the nucleus, AUBF70 binds and stabilizes lymphokine transcripts. Increased stability of lymphokine mRNAs could contribute to oncogenic transformation induced by H. saimiri.

Antisense DNA inhibition of tumor growth induced by c-Ha-ras oncogene in nude mice.

Antisense DNA has shown an ability to target specific oncogene transcripts and inhibit their expression in cells, but the degree to which sustained treatment can suppress total levels of an oncogenic product and alter tumorigenesis in vivo remains to be determined. In this study, NIH-3T3 cells transformed by the activated c-Ha-ras oncogene from T24 human bladder cancer cells were treated for 3 consecutive days in vitro with an antisense DNA pentadecamer complementary to a target in the 5'-flanking region of the c-Ha-ras RNA transcript. Following antisense DNA treatment, a portion of the cells was lysed for measurement of RAS p21 while the remaining cells were evaluated for tumorigeneity by injection s.c. into athymic nude mice at a dose of 5 x 10(5) cells/mouse. The 3 days of treatment with the anti-c-Ha-ras DNA reduced RAS p21 cellular levels by more than 90% while a nonspecific control DNA reduced p21 levels by approximately 20%. Tumor growth of cells treated with anti-c-Ha-ras DNA was significantly reduced for up to 14 days following the end of treatment and implantation into the mice whereas the nonspecific control DNA had no significant effect. These effects on tumor growth were evident in two different strains of nude mice and in both males and females. It is suggested that the pronounced decrease in RAS p21 levels produced by anti-c-Ha-ras DNA resulted in a reversal of the transformed phenotype, and it is this reversal which accounts for the prolonged inhibition of tumorigenesis following antisense DNA treatment.