Distribution patterns of postmortem damage in human mitochondrial DNA

The distribution of postmortem damage in mitochondrial DNA retrieved from 37 ancient human DNA samples was analyzed by cloning and was compared with a selection of published animal data. A relative rate of damage (rho(v)) was calculated for nucleotide positions within the human hypervariable region 1 (HVR1) and cytochrome oxidase subunit III genes. A comparison of damaged sites within and between the regions reveals that damage hotspots exist and that, in the HVR1, these correlate with sites known to have high in vivo mutation rates. Conversely, HVR1 subregions with known structural function, such as MT5, have lower in vivo mutation rates and lower postmortem-damage rates. The postmortem data also identify a possible functional subregion of the HVR1, termed "low-diversity 1," through the lack of sequence damage. The amount of postmortem damage observed in mitochondrial coding regions was significantly lower than in the HVR1, and, although hotspots were noted, these did not correlate with codon position. Finally, a simple method for the identification of incorrect archaeological haplogroup designations is introduced, on the basis of the observed spectrum of postmortem damage.     

Effect of disturbance on assemblages: an example using porifera

Extensive sponge assemblages are found in a number of habitats at Lough Hyne Marine Nature Reserve. These habitats are unusual in experiencing a range of environmental conditions, even though they are only separated by small geographic distances (1-500 m), reducing the possibility of confounding effects between study sites (e.g., silica concentrations and temperature). Sponge assemblages were examined on ephemeral (rocks), stable (cliffs), and artificial (slate panels) hard substrata from high- and low-energy environments that were used to represent two measures of disturbance (flow rate and habitat stability). Sponge assemblages varied considerably between habitat types such that only 26% (25 species) of species reported were common to both rock and cliff habitats. Seven species (of a total of 96 species) were found in the least-developed assemblages (slate panels) and were common to all habitats. Sponge assemblages on rocks and panels varied little between high- and low-energy environments, whereas assemblages inhabiting cliffs varied considerably. Assemblage composition was visualized using Bray-Curtis similarity analysis and Multi-Dimensional Scaling, which enabled differences and similarities between sponge assemblages to be visualized. Cliffs from high- and low-energy sites had different assemblage compositions compared to large rocks, small rocks, and panels, all of which had similar assemblages irrespective of environmental conditions. Differences in assemblages were partially attributed to sponge morphology (shape), as certain morphologies (e.g., arborescent species) were excluded from 2-D rock habitats. Other mechanisms were also considered responsible for the sponge assemblages associated with different habitats.     

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.     

Nitric oxide-dependent generation of reactive species in sickle cell disease. Actin tyrosine induces defective cytoskeletal polymerization

The intermittent vascular occlusion occurring in sickle cell disease (SCD) leads to ischemia-reperfusion injury and activation of inflammatory processes including enhanced production of reactive oxygen species and increased expression of inducible nitric-oxide synthase (NOS2). Appreciating that impaired nitric oxide-dependent vascular function and the concomitant formation of oxidizing and nitrating species occur in concert with increased rates of tissue reactive oxygen species production, liver and kidney NOS2 expression, tissue 3-nitrotyrosine (NO(2)Tyr) formation and apoptosis were evaluated in human SCD tissues and a murine model of SCD. Liver and kidney NOS2 expression and NO(2)Tyr immunoreactivity were significantly increased in SCD mice and humans, but not in nondiseased tissues. TdT-mediated nick end-label (TUNEL) staining showed apoptotic cells in regions expressing elevated levels of NOS2 and NO(2)Tyr in all SCD tissues. Gas chromatography mass spectrometry analysis revealed increased plasma protein NO(2)Tyr content and increased levels of hepatic and renal protein NO(2)Tyr derivatives in SCD (21.4 +/- 2.6 and 37.5 +/- 7.8 ng/mg) versus wild type mice (8.2 +/- 2.2 and 10 +/- 1.2 ng/mg), respectively. Western blot analysis and immunoprecipitation of SCD mouse liver and kidney proteins revealed one principal NO(2)Tyr-containing protein of 42 kDa, compared with controls. Enzymatic in-gel digestion and MALDI-TOF mass spectrometry identified this nitrated protein as actin. Electrospray ionization and fragment analysis by tandem mass spectrometry revealed that 3 of 15 actin tyrosine residues are nitrated (Tyr(91), Tyr(198), and Tyr(240)) at positions that significantly modify actin assembly. Confocal microscopy of SCD human and mouse tissues revealed that nitration led to morphologically distinct disorganization of filamentous actin. In aggregate, we have observed that the hemoglobin point mutation of sickle cell disease that mediates hemoglobin polymerization defects is translated, via inflammatory oxidant reactions, into defective cytoskeletal polymerization.     

NMDA receptor antagonism blocks experience-dependent expansion of hippocampal "place fields".

In agreement with theories of sequence learning, hippocampal place representations expand asymmetrically during repeated route following. This behaviorally induced, experience-dependent expression of neuronal plasticity was blocked by the NMDA(R) antagonist CPP, suggesting that it may result from the temporal asymmetry and associative properties of LTP. NMDA(R) antagonism, however, had no effect on the range of the progressive shift of firing phase of hippocampal cells, relative to the theta rhythm, as the rat traverses the cell's "place field." Thus, when place fields normally expand with experience, the relationship between firing phase and position is altered, as predicted by models that account for "phase precession" on the basis of asymmetry of synaptic connection strengths. These effects of CPP mimic changes that occur during normal aging, suggesting mechanisms by which sequence learning deficits may arise in aged animals.     

Viscoelastic properties of pressure overload hypertrophied myocardium: effect of serine protease treatment

To determine whether and to what extent one component of the extracellular matrix, fibrillar collagen, contributes causally to abnormalities in viscoelasticity, collagen was acutely degraded by activation of endogenous matrix metalloproteinases (MMPs) with the serine protease plasmin. Papillary muscles were isolated from normal cats and cats with right ventricular pressure overload hypertrophy (POH) induced by pulmonary artery banding. Plasmin treatment caused MMP activation, collagen degradation, decreased the elastic stiffness constant, and decreased the viscosity constant in both normal and POH muscles. Thus, whereas many mechanisms may contribute to the abnormalities in myocardial viscoelasticity in the POH myocardium, changes in fibrillar collagen appear to play a predominant role.     

Conserved residues in the putative catalytic triad of human bile acid Coenzyme A:amino acid N-acyltransferase

Human bile acid-CoA:amino acid N-acyltransferase (hBAT), an enzyme catalyzing the conjugation of bile acids with the amino acids glycine or taurine has significant sequence homology with dienelactone hydrolases and other alpha/beta hydrolases. These enzymes have a conserved catalytic triad that maps onto the mammalian BATs at residues Cys-235, Asp-328, and His-362 of the human sequence, albeit that the hydrolases contain a serine instead of a cysteine. In the present study, the function of the putative catalytic triad of hBAT was examined by chemical modification with the cysteine alkylating reagent N-ethylmaleimide (NEM) and by site-directed mutagenesis of the triad residues followed by enzymology studies of mutant and wild-type hBATs. Treatment with NEM caused inactivation of wild-type hBAT. However, preincubation of wild-type hBAT with the substrate cholyl-CoA before NEM treatment prevented loss of N-acyltransferase activity. Substitution of His-362 or Asp-328 with alanine results in inactivation of hBAT. Although substitution of Cys-235 with serine generated an hBAT mutant with lower N-acyltransferase activity, it substantially increased the bile acid-CoA thioesterase activity compared with wild type. In summary, data from this study support the existence of an essential catalytic triad within hBAT consisting of Cys-235, His-362, and Asp-328 with Cys-235 serving as the probable nucleophile and thus the site of covalent attachment of the bile acid molecule.     

Molecular cloning and expression of rat liver bile acid CoA ligase

Bile acid CoA ligase (BAL) is responsible for catalyzing the first step in the conjugation of bile acids with amino acids. Sequencing of putative rat liver BAL cDNAs identified a cDNA (rBAL-1) possessing a 51 nucleotide 5'-untranslated region, an open reading frame of 2,070 bases encoding a 690 aa protein with a molecular mass of 75,960 Da, and a 138 nucleotide 3'-nontranslated region followed by a poly(A) tail. Identity of the cDNA was established by: 1) the rBAL-1 open reading frame encoded peptides obtained by chemical sequencing of the purified rBAL protein; 2) expressed rBAL-1 protein comigrated with purified rBAL during SDS-polyacrylamide gel electrophoresis; and 3) rBAL-1 expressed in insect Sf9 cells had enzymatic properties that were comparable to the enzyme isolated from rat liver. Evidence for a relationship between fatty acid and bile acid metabolism is suggested by specific inhibition of rBAL-1 by cis-unsaturated fatty acids and its high homology to a human very long chain fatty acid CoA ligase. In summary, these results indicate that the cDNA for rat liver BAL has been isolated and expression of the rBAL cDNA in insect Sf9 cells results in a catalytically active enzyme capable of utilizing several different bile acids as substrates.     

Heregulin promotes expression and subcellular redistribution of ADP-ribosylation factor 3

To identify genes whose expression is modulated by heregulin-beta1 (HRG), a regulatory polypeptide for mammary epithelial cells, we performed differential display screening of MCF-7 cell mRNA. One cDNA clone upregulated by HRG was identical to human ADP-ribosylation factor 3 (ARF3), a guanine nucleotide-binding protein functioning in vesicular trafficking, phospholipase D activation and intracellular transport. HRG treatment increased expression of ARF3 mRNA and protein. Also, HRG triggered a rapid redistribution of ARF3, first to cell membranes and then to the nuclear compartment, where ARF3 colocalized with acetylated histone H3 in discrete regions. In addition, the ARF3 protein was developmentally regulated in the mammary gland with the highest levels in virgin and post-weaning glands. Together, these findings suggest for the first time that stimulation of ARF3 expression, subcellular redistribution and interaction with acetylated histone H3 may play a role in the action of HRG in mammary epithelial cells.