Cytotoxic effects of peroxynitrite, polymorphonuclear neutrophils, free-radical scavengers, inhibitors of myeloperoxidase, and inhibitors of nitric oxide synthase on bovine mammary secretory epithelial cells

OBJECTIVE: To determine cytotoxic effects of activated polymorphonuclear neutrophils (PMN) and peroxynitrite on bovine mammary secretory epithelial cells before and after addition of nitric oxide synthase inhibitors, myeloperoxidase (MPO) inhibitors, and free-radical scavengers. SAMPLE POPULATION: Polymorphonuclear neutrophils from 3 lactating cows. PROCEDURE: Cells from the bovine mammary epithelial cell line MAC-T were cultured. Monolayers were treated with activated bovine PMN, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), 3-morpholino-sydnonimine (SIN-1), 4-amino-benzoic acid hydrazide (ABAH), NG-monomethyl-L-arginine, histidine, and superoxide dismutase (SOD). At 24 hours, activity of lactate dehydrogenase in culture medium was used as a relative index of cell death. Tyrosine nitration of proteins in MAC-T cell lysates was determined by visual examination of immunoblots. RESULTS: Lipopolysaccharide, PMA, and < or = 0.1 mM SIN-1 were not toxic to MAC-T cells. Activated PMN, > or = 6 mg of histidine/ml, and 0.5 mM SIN-1 were toxic. Together, histidine and 500,000 activated PMN/ml also were toxic. NG-monomethyl-L-arginine did not have an effect, but ABAH decreased PMN-mediated cytotoxicity. Ten and 50 U of SOD/ml protected MAC-T cells from cytotoxic effects of 0.5 mM SIN-1. Compared with control samples, nitration of MAC-T tyrosine residues decreased after addition of 500,000 PMN/ml or > or = 6 mg of histidine/ml. Superoxide dismutase increased and SIN-1 decreased tyrosine nitration of MAC-T cell proteins in a dose-responsive manner. CONCLUSIONS AND CLINICAL RELEVANCE: Peroxynitrite, MPO, and histidine are toxic to mammary secretory epithelial cells. Superoxide dismutase and inhibition of MPO activity mitigate these effects. Nitration of MAC-T cell tyrosine residues may be positively associated with viability.     

Germination and net in vitro growth of peach, almond and peach-almond hybrid embryos in response to mannitol inclusion in the nutrient medium

Nine Prunus accessions were evaluated for germination and plantlet growth in an in vitro osmotic screening test using mannitol as an osmoticum. Embryos from diverse peaches, almonds and peach-almond hybrids were cultured in Woody Plant Medium, and in this same medium modified with the inclusion of 350 mM mannitol. Embryos were stratified in vitro for 60 days, induced to germinate for a two week period and then allowed to develop and grow for another 20 days prior to harvest. Fresh weights of both roots and shoots as well as percent germination were recorded at harvest. The main effects of nutrient medium, germplasm type (peach, peach-almond, almond) and specific Prunus accession were all highly significant (P = 0.01) with regard to fresh weight of roots and shoots. Embryo germination was affected significantly by the inclusion of mannitol in the nutrient medium and by the particular germplasm type (P=0.01 and P=0.05, respectively). Significant interactions of nutrient medium × Prunus accession and nutrient medium × germplasm type were also detected for both root and shoot fresh weights. Almond germplasm cultured in Woody Plant Medium with 350 mM mannitol produced significantly more roots and shoots than either peaches or peach-almond hybrids grown in the same medium. Peach-almond hybrid embryos were observed to germinate at a significantly higher frequency than peach embryos when averaged across both media. Results obtained in this study indicate a wide range of average plantlet fresh weight relative to the specific germplasm challenged in the osmotic screen. This revised version was published online in August 2006 with corrections to the Cover Date.     

Modification of sugar profiles in California adapted apricots (Prunus armeniaca L.) through breeding with Central Asian germplasm

Summary Central Asian apricot germplasm was used in hybridizations with California adapted apricots to increase Brix levels and improve fresh eating quality. Fruit from parental trees, the F₁ hybrid and two backcross families were evaluated for fruit quality traits and analyzed by HPLC for specific sugar content. The F₁ hybrid between Central Asian and California adapted apricots was intermediate to its parents in many of the evaluated characteristics and levels of specific sugars. When the F₁ hybrid was backcrossed to California adapted apricots ‘Lorna’ and ‘Robada,’ the resulting hybrids were diverse in Brix, juice acidity, fruit size and profiles of specific sugars. Glucose: fructose ratios higher that 3.3 were encountered in fruit from five of the 22 analyzed seedlings, and fructose: sorbitol ratio ranged from 0.67 to 6.46. Brix and total sugar content correlated significantly with each other and with both sucrose and glucose. No significant correlations existed between sorbitol and any of the other analyzed sugars, nor with Brix or total sugars. The results demonstrated the extent of sugar profile modification possible in California adapted apricots after just two generations of breeding with Central Asian apricot germplasm.     

Improved seed development and germination of stenospermic grapes by plant growth regulators

Plant growth regulators were applied to the foliage and immature fruit clusters of the stenospermic grape selection ‘C35-33’ at various periods before bloom to stimulate viable seed development. In the 1987 season five different plant growth regulators were used, but in 1988 the growth retardants Cycocel and XE-1019 were used exclusively. Chemical treatments applied 35 days after bud break increased significantly germination percentage. Experimental results indicate that the use of certain plant growth regulators may aid in increasing the efficiency of seedless grape breeding by providing an alternative to in-ovulo embryo culture.     

Inhibiting fungus on largemouth bass eggs with copper sulfate and its toxicity to fry and juveniles

This study determined the effectiveness of copper sulfate (CuSO₄) to inhibit fungal growth (caused by Saprolegnia spp.) on largemouth bass (LMB) eggs spawned on/in fiber mats in very high-alkalinity/-hardness waters; experiments also determined the toxicity of CuSO₄ to LMB fry and juveniles. An untreated control and three CuSO₄ concentrations (10, 20, and 40 mg/L) were tested under a flow-through scenario in the effectiveness experiment. Eggs were treated daily until hatching began. Fungal load at the time of hatch had a total area > 3.0 cm² in the untreated controls, total area 1.0 to <2.0 cm² in the 10 and 20 mg/L CuSO₄ treatments, and total area > 1.0 cm² in the 40 mg/L CuSO₄ treatments. Fungus samples were identified as Saprolegnia australis. The 24-hr median lethal concentration (LC50) values on the LMB yolk-sac and swim-up fry were 32.0 and 4.6 mg/L CuSO₄, respectively; the No Observed Effect Concentrations (NOEC) were 16.0 and 0.125 mg/L CuSO₄, respectively. Juvenile LMB were extremely tolerant to CuSO₄, and their 24-hr LC50 value was 185.5 mg/L; the NOEC was 64 mg/L. This study indicates that CuSO₄ can be an important resource for hatcheries to control egg fungus, especially in high-alkalinity/-hardness waters.     

Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas

Previous studies have demonstrated that allelic deletions of the short arm of chromosome 17 occur in over 75% of colorectal carcinomas. Twenty chromosome 17p markers were used to localize the common region of deletion in these tumors to a region contained within bands 17p12 to 17p13.3. This region contains the gene for the transformation-associated protein p53. Southern and Northern blot hybridization experiments provided no evidence for gross alterations of the p53 gene or surrounding sequences. As a more rigorous test of the possibility that p53 was a target of the deletions, the p53 coding regions from two tumors were analyzed; these two tumors, like most colorectal carcinomas, had allelic deletions of chromosome 17p and expressed considerable amounts of p53 messenger RNA from the remaining allele. The remaining p53 allele was mutated in both tumors, with an alanine substituted for valine at codon 143 of one tumor and a histidine substituted for arginine at codon 175 of the second tumor. Both mutations occurred in a highly conserved region of the p53 gene that was previously found to be mutated in murine p53 oncogenes. The data suggest that p53 gene mutations may be involved in colorectal neoplasia, perhaps through inactivation of a tumor suppressor function of the wild-type p53 gene.