Role of thiocyanate, bromide and hypobromous acid in hydrogen peroxide-induced apoptosis

We have previously reported that H₂O₂-induced apoptosis in HL-60 human leukemia cells takes place in the presence of chloride, requires myeloperoxidase (MPO), and occurs through oxidative reactions involving hypochlorous acid and chloramines. We now report that when chloride is replaced by the pseudohalide thiocyanate, there is little or no H₂O₂-induced apoptosis. Furthermore, thiocyanate inhibits H₂O₂-induced apoptosis when chloride is present at physiological concentrations, and this occurs at thiocyanate concentrations that are present in human serum and saliva. In contrast, bromide can substitute for chloride in H₂O₂-induced apoptosis, but results in a lower percent of the cells induced into apoptosis. Hypobromous acid is likely a short-lived intermediate in this H₂O₂/MPO/bromide apoptosis, and reagent hypobromous acid and bromamines induce apoptosis in HL-60 cells. We conclude that the physiologic concentrations of thiocyanate found in human plasma could modulate the cytototoxicity of H₂O₂ and its resulting highly toxic MPO-generated hypochlorous acid by competing with chloride for MPO. Furthermore, the oxidative products of the reaction of thiocyanate with MPO are relatively innocuous for human leukemic cells in culture. In contrast, bromide can support H₂O₂/MPO/halide apoptosis, but is less potent than chloride and it has no effect in the presence of physiological levels of chloride.     

Overexpression of manganese superoxide dismutase promotes the survival of prostate cancer cells exposed to hyperthermia

It has been hypothesized that exposure of cells to hyperthermia results in an increased flux of reactive oxygen species (ROS), primarily superoxide anion radicals, and that increasing antioxidant enzyme levels will result in protection of cells from the toxicity of these ROS. In this study, the prostate cancer cell line, PC-3, and its manganese superoxide dismutase (MnSOD)-overexpressing clones were subjected to hyperthermia (43°C, 1 h). Increased expression of MnSOD increased the mitochondrial membrane potential (MMP). Hyperthermic exposure of PC-3 cells resulted in increased ROS production, as determined by aconitase inactivation, lipid peroxidation, and H₂O₂ formation with a reduction in cell survival. In contrast, PC-3 cells overexpressing MnSOD had less ROS production, less lipid peroxidation, and greater cell survival compared to PC-3 Wt cells. Since MnSOD removes superoxide, these results suggest that superoxide free radical or its reaction products are responsible for part of the cytotoxicity associated with hyperthermia and that MnSOD can reduce cellular injury and thereby enhance heat tolerance.     

Extreme difference in rate of mitochondrial and nuclear DNA evolution in a large ectotherm, Galápagos tortoises

We sequenced approximately 4.5 kb of mtDNA from 161 individuals representing 11 named taxa of giant Galápagos tortoises (Geochelone nigra) and about 4 kb of non-coding nuclear DNA from fewer individuals of these same 11 taxa. In comparing mtDNA and nucDNA divergences, only silent substitutions (introns, ITS, mtDNA control region, and synonymous substitutions in coding sequences) were considered. mtDNA divergence was about 30 times greater than that for nucDNA. This rate discrepancy for mtDNA and nucDNA is the greatest yet documented and is particularly surprising for large ectothermic animals that are thought to have relatively low rates of mtDNA evolution. This observation may be due to the somewhat unusual reproductive biology and biogeographic history of these organisms. The implication is that the ratio of effective population size of nucDNA/mtDNA is much greater than the usually assumed four. The nearly neutral theory of molecular evolution predicts this would lead to a greater difference between rates of evolution.     

Induction of flexibility through protein-protein interactions

The dimerization/docking (D/D) domain of the cyclic AMP-dependent protein kinase (PKA) holoenzyme mediates important protein-protein interactions that direct the subcellular localization of the enzyme. A kinase anchoring proteins (AKAPs) provide the molecular scaffold for the localization of PKA. The recent solution structures of two D/D AKAP complexes revealed that the AKAP binds to a surface-exposed, hydrophobic groove on the D/D. In the present study, we present an analysis of the changes in hydrogen/deuterium exchange protection and internal motions of the backbone of the D/D when free and bound to the prototype anchoring protein, Ht31(pep). We observe that formation of the complex results in significant, but small, increases in H/D exchange protection factors as well as increases in backbone flexibility, throughout the D/D, and in particular, in the hydrophobic binding groove. This unusual observation of increased backbone flexibility and marginal H/D exchange protection, despite high affinity protein-ligand interactions, may be a general effect observed for the stabilization of hydrophobic ligand/hydrophobic pocket interactions.     

Dynamics of DNA molecules in a membrane channel probed by active control techniques

The dynamics of single-stranded DNA in an alpha-Hemolysin protein pore was studied at the single-molecule level. The escape time for DNA molecules initially drawn into the pore was measured in the absence of an externally applied electric field. These measurements revealed two well-separated timescales, one of which is surprisingly long (on the order of milliseconds). We characterized the long timescale as being associated with the binding and unbinding of DNA from the pore. We have also found that a transmembrane potential as small as 20 mV strongly biased the escape of DNA from the pore. These experiments have been made possible due to the development of a feedback control system, allowing the rapid modulation of the applied force on individual DNA molecules while inside the pore.     

QTL analysis of an intervarietal set of substitution lines in Brassica napus: (i) Seed oil content and fatty acid composition

Backcross breeding with marker-assisted selection was used to construct an intervarietal set of part chromosome substitution lines in Brassica napus, formed from a cross between two winter varieties of oilseed rape: Tapidor and Victor. A total of 22 lines from this substitution library were examined over a 3-year period, in a total of nine field trials, for seed oil fatty acid composition and seed oil content. Trialing of the substitution lines gave evidence for the existence of 13 quantitative trait loci (QTL). All 13 QTL affected fatty acid composition of the seed, and were distributed among linkage groups 1, 3, 6, 7, 8, 11, 13, 14, 18, and 19. Seven of these QTL, on linkage groups 3, 6, 8, 13, 14, 18, and 19, also affected total seed oil content. The positions of these QTL are compared to those in the published literature and with respect to erucic acid QTL previously identified in a backcross population of the same cross. The substitution line approach gives increased precision and sensitivity for QTL mapping compared to other methods.     

Folding of intracellular retinol and retinoic acid binding proteins

The folding mechanisms of cellular retinol binding protein II (CRBP II), cellular retinoic acid binding protein I (CRABP I), and cellular retinoic acid binding protein II (CRABP II) were examined. These beta-sheet proteins have very similar structures and higher sequence homologies than most proteins in this diverse family. They have similar stabilities and show completely reversible folding at equilibrium with urea as a denaturant. The unfolding kinetics of these proteins were monitored during folding and unfolding by circular dichroism (CD) and fluorescence. During unfolding, CRABP II showed no intermediates, CRABP I had an intermediate with nativelike secondary structure, and CRBP II had an intermediate that lacked secondary structure. The refolding kinetics of these proteins were more similar. Each protein showed a burst-phase change in intensity by both CD and fluorescence, followed by a single observed phase by both CD and fluorescence and one or two additional refolding phases by fluorescence. The fluorescence spectral properties of the intermediate states were similar and suggested a gradual increase in the amount of native tertiary structure present for each step in a sequential path. However, the rates of folding differed by as much as 3 orders of magnitude and were slower than those expected from the contact order and topology of these proteins. As such, proteins with the same final structure may not follow the same route to the native state.     

Cell survival and proliferation are modified by insulin-like growth factor 2 between days 9 and 10 of mouse gestation

The size of mammalian species involves the interaction of multiple genetic modifiers that control the timing and extent of growth mechanisms. Disruption of the paternal allele of the imprinted embryonic gene coding for insulin-like growth factor 2 (IGF2, Igf2(+m/-p)), results in viable mice that are 60% the weight of wild-type littermates. Differences in weight are first detected at embryonic day (E) 11, and the growth deficit is maintained throughout life. We report the mechanisms that account for this unusual phenotype. In order to quantify growth, we used novel methods to generate single cell suspensions of post-implantation mouse embryos. We were then able to quantify cell number, cell proliferation and cell death between E8.5 and E11.5 using flow cytometry. Determination of total embryo cell number also allowed us to time litters by a method other than by plugging. Wild-type and Igf2(+m/-p) embryos accumulated similar total cell numbers up to E9.25, but cell number began to diverge by around E9.5, with significant differences by E11 (75% of wild type). A relative increase in pyknotic nuclei, sub-GI cytometry counts and caspase activity, all indicative of cell death, occurred in Igf2(+m/-p) embryos at E9.25, reverting to wild-type levels by E9.75. This was followed at E9.75 by a significant reduction in the proportion of cells in S phase, quantified by S-phase cytometry counts and BrdU labelling. No significant differences in cell size were detected. We conclude that the majority of the cell number differences between wild-type and Igf2(+m/-p) mice can be accounted for by modification of cell survival and proliferation during the period (E9 to E10) of post-implantation development.     

Gill-derived glands in glandulocaudine fishes (teleostei: Characidae: Glandulocaudinae)

The Glandulocaudinae is a subfamily of neotropical characid fishes from Central and South America. A unifying feature of the subfamily is the caudal gland, found almost exclusively in males. The gland consists of tissue on the base of the caudal fin covered in part by hypertrophied scales. Scale movement as the caudal fin is flexed appears to facilitate the release of chemical compounds from the glandular tissue. We describe here a different structure, found in the gill cavity of mature males in 12 of 17 glandulocaudine genera examined. Termed a gill gland, it develops as a male secondary sex character and appears morphologically suited to release chemical signals. The gland forms by the growth of tissue over and around 4-13 anterior gill filaments on the first gill arch, forming chambers with ventral openings. Within the gland chambers, gill secondary lamellae usually shorten and may disappear. When secondary lamellae persist, simple columnar epithelial cells develop between them. In the absence of secondary lamellae, the gland chambers are lined with a simple cuboidal or columnar epithelium. Gland size and the degree of gill modification vary among species. Gill glands appear absent in five glandulocaudine genera, suggesting character reversals based on current phylogenetic hypotheses and systematic classification. Gill gland morphology suggests that this structure releases chemical compounds into the gill current. The presence of gill glands only in mature males suggests a function in reproduction and/or male aggression. Together with studies of the caudal gland, this research suggests that chemical signals may play important roles in glandulocaudine reproduction.