Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production

Inhibition of mitochondrial respiratory chain complex I by rotenone had been found to induce cell death in a variety of cells. However, the mechanism is still elusive. Because reactive oxygen species (ROS) play an important role in apoptosis and inhibition of mitochondrial respiratory chain complex I by rotenone was thought to be able to elevate mitochondrial ROS production, we investigated the relationship between rotenone-induced apoptosis and mitochondrial reactive oxygen species. Rotenone was able to induce mitochondrial complex I substrate-supported mitochondrial ROS production both in isolated mitochondria from HL-60 cells as well as in cultured cells. Rotenone-induced apoptosis was confirmed by DNA fragmentation, cytochrome c release, and caspase 3 activity. A quantitative correlation between rotenone-induced apoptosis and rotenone-induced mitochondrial ROS production was identified. Rotenone-induced apoptosis was inhibited by treatment with antioxidants (glutathione, N-acetylcysteine, and vitamin C). The role of rotenone-induced mitochondrial ROS in apoptosis was also confirmed by the finding that HT1080 cells overexpressing magnesium superoxide dismutase were more resistant to rotenone-induced apoptosis than control cells. These results suggest that rotenone is able to induce apoptosis via enhancing the amount of mitochondrial reactive oxygen species production.     

Differential effects of isomeric incorporation of fluorophenylalanines into PvuII endonuclease

Incorporation of fluorine into proteins has long served as a means of probing structure and function, yet there are few studies that examine the impact of fluorine substitution, particularly at locations distant from the active sites of enzymes. The flexibility of isomeric fluorine incorporation at Phe is used to explore subtle substitution effects on enzyme activity and conformation. The unnatural amino acids o-, m-, and p-fluorophenylalanines were incorporated biosynthetically into the representative PvuII restriction endonuclease. Interestingly, m-fluoro-Phe-PvuII endonuclease exhibits very similar conformational stability to that of the native enzyme, but it exhibits a reproducible, 2-fold higher average specific activity. Given the level of incorporation and the distribution of species, the species of modified enzyme responsible for this increase in specific activity is most likely even faster. Further, moving the fluorine atom from the meta- to the para-position of Phe results in a 4-fold decrease in specific activity and a decrease in conformational stability of 1.5 kcal/mol. Since none of the Phe residues in PvuII endonuclease lies near the DNA recognition or catalytic sites, this differential behavior alludes to the impact of subtle changes in enzyme conformation on endonuclease activity and suggests novel ways to influence catalytic behavior.     

Activation of pyk2/related focal adhesion tyrosine kinase and focal adhesion kinase in cardiac remodeling

Cellular remodeling during progression of dilation involves focal adhesion contact reorganization. However, the signaling mechanisms and structural consequences leading to impaired cardiomyocyte adhesion are poorly defined. These events were studied in tropomodulin-overexpressing transgenic mice that develop dilated cardiomyopathy associated with chronic elevation of intracellular calcium. Analysis of tropomodulin-overexpressing transgenic hearts by immunoblot and confocal microscopy revealed activation and redistribution of signaling molecules known to regulate adhesion. Calcium-dependent pyk2/related focal adhesion tyrosine kinase (RAFTK) showed changes in expression and phosphorylation state, similar to changes observed for a related downstream target molecule of pyk2/RAFTK termed focal adhesion kinase. Paxillin, the target substrate molecule for focal adhesion kinase phosphorylation, was redistributed in tropomodulin-overexpressing transgenic hearts with enhanced paxillin phosphorylation and cleavage. Certain aspects of the in vivo signaling phenotype including increased paxillin phosphorylation could be recapitulated in vitro using neonatal rat cardiomyocytes infected with recombinant adenovirus to overexpress tropomodulin. In addition, increasing intracellular calcium levels with ionomycin induced pyk2/RAFTK phosphorylation, and adenovirally mediated expression of wild-type pyk2/RAFTK resulted in increased phospho-pyk2/RAFTK levels and concomitant paxillin phosphorylation. Collectively, these results delineate a cardiomyocyte signaling pathway associated with dilation that has potential relevance for cardiac remodeling, focal adhesion reorganization, and loss of contractility.     

The association between lameness,ovarian cysts and fertility in lactating dairy cows

The objective of this observational study was to evaluate the association between lameness, ovarian cysts, and fertility in lactating dairy cows. Data analysis of historical records from a 3000 Holstein farm was conducted. Sixty-five cows that became lame within 30 days postpartum were used as cases, and 130 nonlame cows served as controls. The outcome variables were incidence of ovarian cysts (OC, %), conception rate at first service (CRFS, %), overall pregnancy rate (PR, %), and calving to first service interval (CFSI, day), Incidence of OC and CRFS were analyzed by logistic regression, PR by survival analysis and CFSI by ANOVA. Lame cows had a lower CRFS (17.5% versus 42.6%) and higher incidence of OC (25.0% versus 11.1%) than controls (P0.05). There was a multicollinearity relationship between lameness and ovarian cysts. The results show that cows that became lame within the first 30 days postpartum were associated with a higher incidence of ovarian cysts, a lower likelihood of pregnancy, and lower fertility than control cows. Because this is an observational study it is not possible to conclude a cause-effect relationship.     

Reversing the substrate specificities of phenylalanine and tyrosine hydroxylase: aspartate 425 of tyrosine hydroxylase is essential for L-DOPA formation

The catalytic domains of the pterin-dependent enzymes phenylalanine hydroxylase and tyrosine hydroxylase are homologous, yet differ in their substrate specificities. To probe the structural basis for the differences in specificity, seven residues in the active site of phenylalanine hydroxylase whose side chains are dissimilar in the two enzymes were mutated to the corresponding residues in tyrosine hydroxylase. Analysis of the effects of the mutations on the isolated catalytic domain of phenylalanine hydroxylase identified three residues that contribute to the ability to hydroxylate tyrosine, His264, Tyr277, and Val379. These mutations were incorporated into full-length phenylalanine hydroxylase and the complementary mutations into tyrosine hydroxylase. The steady-state kinetic parameters of the mutated enzymes showed that the identity of the residue in tyrosine hydroxylase at the position corresponding to position 379 of phenylalanine hydroxylase is critical for dihydroxyphenylalanine formation. The relative specificity of tyrosine hydroxylase for phenylalanine versus tyrosine, as measured by the (V/K(phe))/(V/K(tyr)) value, increased by 80000-fold in the D425V enzyme. However, mutation of the corresponding valine 379 of phenylalanine hydroxylase to aspartate was not sufficient to allow phenylalanine hydroxylase to form dihydroxyphenylalanine at rates comparable to that of tyrosine hydroxylase. The double mutant V379D/H264Q PheH was the most active at tyrosine hydroxylation, showing a 3000-fold decrease in the (V/K(phe))/(V/K(tyr)) value.     

Quantitative methanol-burning lung model for validating gas-exchange measurements over wide ranges of FIO2

Themethanol-burning lung model has been used as a technique for generatinga predictable ratio of carbon dioxide production (CO₂) to oxygen consumption(O₂) or respiratoryquotient (RQ). Although an accurate RQ can be generated, quantitativelypredictable and adjustableO₂ andCO₂ cannot be generated. Wedescribe a new burner device in which the combustion rate of methanolis always equal to the infusion rate of fuel over an extended range ofO₂ concentrations. This permitsthe assembly of a methanol-burning lung model that is usable withO₂ concentrations up to 100% and provides continuously adjustable and quantitativeO₂ (69-1,525 ml/min)and CO₂ (46-1,016ml/min) at a RQ of 0.667.

     

Genetic diversity in Puerto Rico and its implications for the peopling of the Island and the West Indies

Puerto Rico and the surrounding islands rest on the eastern fringe of the Caribbean's Greater Antilles, located less than 100 miles northwest of the Lesser Antilles. Puerto Ricans are genetic descendants of pre‐Columbian peoples, as well as peoples of European and African descent through 500 years of migration to the island. To infer these patterns of pre‐Columbian and historic peopling of the Caribbean, we characterized genetic diversity in 326 individuals from the southeastern region of Puerto Rico and the island municipality of Vieques. We sequenced the mitochondrial DNA (mtDNA) control region of all of the samples and the complete mitogenomes of 12 of them to infer their putative place of origin. In addition, we genotyped 121 male samples for 25 Y‐chromosome single nucleotide polymorphism and 17 STR loci. Approximately 60% of the participants had indigenous mtDNA haplotypes (mostly from haplogroups A2 and C1), while 25% had African and 15% European haplotypes. Three A2 sublineages were unique to the Greater Antilles, one of which was similar to Mesoamerican types, while C1b haplogroups showed links to South America, suggesting that people reached the island from the two distinct continental source areas. However, none of the male participants had indigenous Y‐chromosomes, with 85% of them instead being European/Mediterranean and 15% sub‐Saharan African in origin. West Eurasian Y‐chromosome short tandem repeat haplotypes were quite diverse and showed similarities to those observed in southern Europe, North Africa and the Middle East. These results attest to the distinct, yet equally complex, pasts for the male and female ancestors of modern day Puerto Ricans. Am J Phys Anthropol 155:352–368, 2014. © 2014 Wiley Periodicals, Inc.