We report the elucidation of a mechanism of apoptosis induction in breast cancer (MCF-7) cells by an L-amino acid oxidase (LAAO), Rusvinoxidase, purified from the venom of Daboia russelii russelii. Peptide mass fingerprinting analysis of Rusvinoxidase, an acidic monomeric glycoprotein with a mass of ~57 kDa, confirmed its identity as snake venom LAAO. The enzymatic activity of Rusvinoxidase was completely abolished after two cycles of freezing and thawing; however, its cytotoxicity toward MCF-7 cells remained unaffected. Dose- and time-dependent induction of apoptosis by Rusvinoxidase on MCF-7 cells was evident from changes in cell morphology, cell membrane integrity, shrinkage of cells and apoptotic body formation accompanied by DNA fragmentation. Rusvinoxidase induced apoptosis in MCF-7 cells by both the extrinsic (death-receptor) and intrinsic (mitochondrial) signaling pathways. The former pathway of apoptosis operated through activation of caspase-8 that subsequently activated caspase-7 but not caspase-3. Rusvinoxidase-induced intrinsic pathway of apoptosis was accompanied by a time-dependent depolarization of the mitochondrial membrane through the generation of reactive oxygen species, followed by a decrease in cellular glutathione content and catalase activity, and down-regulation of expression of anti-apoptotic proteins Bcl-XL and heat-shock proteins (HSP-90 and HSP-70). Rusvinoxidase treatment resulted in increase of the pro-apoptotic protein Bax, subsequently leading to the release of cytochrome c from mitochondria to the cytosol and activating caspase-9, which in turn stimulated effector caspase-7. Rusvinoxidase at a dose of 4 mg/kg was non-toxic in mice, indicating that it may be useful as a model for the development of peptide-based anticancer drugs.

Objectives of these studies were to determine the effects of flunixin meglumine (FM) administration on early embryonic mortality and circulating PG and cortisol concentrations in transported and non-transported cows. Cows (n = 483) from 3 locations were used to evaluate the effects of transportation and FM approximately 14 d after AI on the establishment of pregnancy and serum concentrations of progesterone, PGF metabolite (PGFM), and cortisol. Treatments were transport (n = 129), transport + FM (n = 128), no transport (n = 130), and no transport + FM (n = 96). Multiparous cows (n = 224) were used at 2 locations, and nulliparous cows (n = 259) were used at 1 location. The no transport + FM treatment was used at only 2 locations. Flunixin meglumine (approximately 1.1 mg/kg of BW; i.m.) was administered before the cows were separated into transportation groups. Transportation included 4 to 6 h of transportation, without calves, via semitractor trailer. Nontransported cows remained penned, with their calves in adjacent pens, during the same period as the transported cows. Blood samples were collected from all cows before and after treatment and, at 2 locations, approximately 3 h after the onset of treatment. Location affected AI pregnancy rate (P < 0.01). Treatment effects, although not significant (P = 0.16), were of a magnitude to be considered practically important. Cows that received transportation + FM tended (P = 0.07) to have greater AI pregnancy rates (74%) than those that did not receive FM (66%), irrespective of transportation. Cortisol concentration was greater (P < 0.05) for transported cows than for nontransported cows. Cows receiving FM had greater (P < 0.05) AI pregnancy rates than non-FM cows (71 vs. 61%, respectively). Cows receiving transportation had lower (P < 0.01) mean PGFM concentrations than nontransported cows (45.4 vs. 54.6 pg/mL, respectively), and cows receiving FM had lower (P < 0.01) mean PGFM concentrations than non-FM cows (39.4 vs. 60.6, respectively). We conclude that transportation of cows approximately 14 d after AI increased serum cortisol concentrations but did not affect AI pregnancy rates. However, treatment of cows with FM increased AI pregnancy rates, irrespective of whether they were transported.

Crossbred, multiparous beef cows (n = 178 in Year 1; n = 148 in Year 2) were used to evaluate the effects of Cu, Zn, and Mn supplementation and source on reproduction, mineral status, and performance in grazing cattle in eastern Colorado over a 2-yr period. Cows were stratified by expected calving date, age, BW, BCS, and liver mineral status and assigned to the following treatments: 1) control (no supplemental Cu, Zn, or Mn); 2) organic (ORG; 50% organic and 50% inorganic Cu, Zn, and Mn); and 3) inorganic (ING; 100% inorganic CuSO4, ZnSO4, and MnSO4). Free-choice mineral feeders were used to provide current NRC-recommended concentrations of Cu, Zn, and Mn from 82 d (Year 1) and 81 d (Year 2) before the average calving date of the herd through 110 d (Year 1) and 135 d (Year 2) after calving. At the end of Year 1, supplemented cows had greater liver Cu (P < 0.01), Zn (P < 0.05), and Mn (P < 0.01) concentrations compared with controls, whereas liver Cu concentration was greater (P < 0.01) in ORG vs. ING cows. At the end of Year 2, supplemented cows had greater (P < 0.01) liver Cu concentrations relative to controls, whereas control cows had greater (P < 0.02) liver Mn concentration than did supplemented cows. In Year 1, pregnancy rate to AI in control cows did not differ (P = 0.47) from supplemented cows, but there was a trend (P < 0.08) for pregnancy rate to be higher for ORG than ING cows. In Year 2, supplemented cows had a higher (P < 0.02) pregnancy rate to AI than controls. In both years, when cows were inseminated after an observed estrus, supplemented cows had a higher (P < 0.04) pregnancy rate than did controls. Also, for both years, overall 60-d pregnancy rate tended (P = 0.10) to be higher for supplemented cows than for controls. In Year 1, kilograms of calf weaned per cow exposed was greater (P < 0.02) in controls than in supplemented cows, and kilograms of calf weaned per cow exposed was greater (P < 0.01) in ING than ORG treatments. However, in Year 2, kilograms of calf weaned per cow exposed was greater (P < 0.02) in controls than in supplemented cows, and tended (P = 0.09) to be greater in ORG than ING treatments. Results indicate that supplementation and source of trace minerals affected mineral status and kilograms of calf weaned per cow exposed in grazing beef cows. Supplementation also improved pregnancy rate to AI compared with cows not supplemented with Cu, Zn, or Mn for more than 1 yr. Furthermore, mineral source may influence pregnancy rate to AI.

Seven nonlactating mature Angus cows (4 to 10 yr old) were used to examine the effects of fish meal supplementation on plasma and endometrial fatty acid composition. Cows were fed a corn silage-based diet supplemented with either fish meal, a rich source of the n-3 fatty acids, eicosapentaenoate and docosahexaenoate (n = 3; 5.1% of dietary DM), or corn gluten meal (n = 4; 8.5% of dietary DM) for approximately 64 d. Cows were given 25 mg of PGF2alpha (i.m.) on d 11 and 25 of supplementation to synchronize estrous cycles. On d 18 postestrus of the second estrous cycle, cows were slaughtered, and caruncular endometrium was dissected from uteri immediately after slaughter. Jugular blood samples were collected immediately before supplementation was initiated (d 0) and at 7-d intervals for 35 d of the study. Plasma eicosapentaenoic and docosahexaenoic acids did not differ between treatment groups on d 0 (P > 0.10); however, these fatty acids were greater in cows supplemented with fish meal over the first 35 d of supplementation compared with cows supplemented with corn gluten meal (P < 0.05). Endometrial docosahexaenoic acid did not differ (P = 0.12), whereas eicosapentaenoic acid was greater (P < 0.05) in cows supplemented with fish meal than in cows supplemented with corn gluten meal. These results indicate that dietary fish meal alters plasma and endometrial n-3 fatty acid composition in beef cows.

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Lipid microdomains are ordered regions on the plasma membrane of cells, rich in cholesterol and sphingolipids, ranging in size from 10 to 200 nm in diameter. These lipid-ordered domains may serve as platforms to facilitate colocalization of intracellular signaling proteins during agonist-induced signal transduction. It is hypothesized that fish oil will disrupt the lipid microdomains, increasing spatial distribution of these lipid-ordered domains and lateral mobility of the prostaglandin (PG) F2α (FP) receptors in bovine luteal cells. The objectives of this study were to examine the effects of fish oil on (1) the spatial distribution of lipid microdomains, (2) lateral mobility of FP receptors, and (3) lateral mobility of FP receptors in the presence of PGF2α on the plasma membrane of bovine luteal cells in vitro. Bovine ovaries were obtained from a local abattoir and corpora lutea were digested using collagenase. In experiment 1, lipid microdomains were labeled using cholera toxin subunit B Alexa Fluor 555. Domains were detected as distinct patches on the plasma membrane of mixed luteal cells. Fish oil treatment decreased fluorescent intensity in a dose-dependent manner (P < 0.01). In experiment 2, single particle tracking was used to examine the effects of fish oil treatment on lateral mobility of FP receptors. Fish oil treatment increased microdiffusion and macrodiffusion coefficients of FP receptors as compared to control cells (P < 0.05). In addition, compartment diameters of domains were larger, and residence times were reduced for receptors in fish oil–treated cells (P < 0.05). In experiment 3, single particle tracking was used to determine the effects of PGF2α on lateral mobility of FP receptors and influence of fish oil treatment. Lateral mobility of receptors was decreased within 5 min following the addition of ligand for control cells (P < 0.05). However, lateral mobility of receptors was unaffected by addition of ligand for fish oil–treated cells (P > 0.10). The data presented provide strong evidence that fish oil causes a disruption in lipid microdomains and affects lateral mobility of FP receptors in the absence and presence of PGF2α.

Metalloproteinases are members of a family of proteinases that remodel the extracellular matrix throughout the body. To test the hypothesis that metalloproteinases are regulated by gonadotropin-induced changes during follicular growth, rats were injected with eCG (20 IU, s.c.), and ovaries and serum were collected at the time of eCG administration (0 h) and at 6, 12, 24, 36, or 48 h later for analysis of metalloproteinase mRNA expression, metalloproteinase activity, and steroidogenesis. Serum estradiol levels increased from 18.9 pg/ml at 0 h to 503.8 pg/ml at 48 h. Analysis of mRNA expression was performed for collagenase-3, 72-kDa gelatinase, and 92-kDa gelatinase (n = 3-4). For collagenase-3, eCG stimulated a 32-fold increase in collagenase-3 mRNA at 48 h after eCG injection as compared to that in ovaries collected at the time of eCG administration (i.e., 0-h control). The mRNA levels for 72-kDa gelatinase were 2.8-fold compared to 0 h at 36 h after eCG treatment and returned to control levels by 48 h after gonadotropin treatment. Levels of the 92-kDa mRNA expression peaked at 24 h (4. 2-fold compared to 0 h) and returned to control levels by 36 h. Gel zymography revealed 3 gelatinolytic bands corresponding to the gelatinases of approximately 72 kDa, 92 kDa, and 105 kDa. Analysis of metalloproteinase activity as the degradation of collagen or gelatin per ovary showed an increase in gelatinolytic and collagenolytic activity between 12 and 48 h after eCG treatment. In summary, these findings demonstrate that the gonadotropin induction of folliculogenesis results in changes in the metalloproteinases that may be responsible for extracellular matrix remodeling associated with follicular growth.

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