Influence of peroxynitrite on energy metabolism and cardiac function in a rat ischemia-reperfusion model

Ischemia-reperfusion generates peroxynitrite (ONOO-), which interacts with many of the systems altered by ischemia-reperfusion. This study examines the influence of endogenously produced ONOO- on cardiac metabolism and function. Nitro-L-arginine (an inhibitor of ONOO- biosynthesis) and urate (a scavenger of ONOO-) were utilized to investigate potential pathophysiological roles for ONOO- in a rat Langendorff heart model perfused with glucose-containing saline at constant pressure and exposed to 30 min of ischemia followed by 60 min of reperfusion. In this model, ischemia-reperfusion decreased contractile function (e.g., left ventricular developed pressure), cardiac work (rate-pressure product), efficiency of O2 utilization, membrane-bound creatine kinase activity, and NMR-detectable ATP and creatine phosphate without significantly altering the recovery of coronary flow, heart rate, lactate release, and muscle pH. Treatment with urate and nitro-L-arginine produced a substantial recovery of left ventricular developed pressure, rate-pressure product, efficiency of O2 utilization, creatine kinase activity, and NMR-detectable creatine phosphate and a partial recovery of ATP. The pattern of effects observed in this study and in previously published work with similar models suggests that ONOO- may alter key steps in the efficiency of mitochondrial high-energy phosphate generation.     

Nuclear CaMKII inhibits neuronal differentiation of PC12 cells without affecting MAPK or CREB activation

Ca²⁺/calmodulin-regulatedprotein kinase II (CaMKII) mediates many cellular events. The fourCaMKII isoforms have numerous splice variants, three of which containnuclear localization signals. Little is known about the role of nuclearlocalized CaMKII in neuronal development. To study this process, PC12cells were transfected to produce CaMKII targeted to either thecytoplasm or the nucleus and then treated with nerve growth factor(NGF). NGF triggers a signaling cascade (MAPK) that results in thedifferentiation of PC12 cells into a neuronal phenotype, marked byneurite outgrowth. The present study found that cells expressingnuclear targeted CaMKII failed to grow neurites, whereas cellsexpressing cytoplasmic CaMKII readily produced neurites. Inhibition ofneuronal differentiation by nuclear CaMKII was independent of MAPKsignaling, as sustained Erk phosphorylation was not affected.Phosphorylation of CREB was also unaffected. Thus nuclear CaMKIImodifies neuronal differentiation by a mechanism independent of MAPKand CREB activation.

     

DNA strand scission by a Cu(I).adenylated polymeric template: preliminary mechanistic and recycling studies

Cleavage of a model phosphate ester and supercoiled plasmid DNA by a Cu(I).adenylated polymer template and preliminary mechanistic investigations are reported. A novel paradigm for the design of recyclable nucleolytic reagents allowing multiple use of this catalyst is also demonstrated     

Arresting and releasing Staphylococcal alpha-hemolysin at intermediate stages of pore formation by engineered disulfide bonds

alpha-Hemolysin (alphaHL) is secreted by Staphylococcus aureus as a water-soluble monomer that assembles into a heptamer to form a transmembrane pore on a target membrane. The crystal structures of the LukF water-soluble monomer and the membrane-bound alpha-hemolysin heptamer show that large conformational changes occur during assembly. However, the mechanism of assembly and pore formation is still unclear, primarily because of the difficulty in obtaining structural information on assembly intermediates. Our goal is to use disulfide bonds to selectively arrest and release alphaHL from intermediate stages of the assembly process and to use these mutants to test mechanistic hypotheses. To accomplish this, we created four double cysteine mutants, D108C/K154C (alphaHL-A), M113C/K147C (alphaHL-B), H48C/ N121C (alphaHL-C), I5C/G130C (alphaHL-D), in which disulfide bonds may form between the pre-stem domain and the beta-sandwich domain to prevent pre-stem rearrangement and membrane insertion. Among the four mutants, alphaHL-A is remarkably stable, is produced at a level at least 10-fold greater than that of the wild-type protein, is monomeric in aqueous solution, and has hemolytic activity that can be regulated by the presence or absence of reducing agents. Cross-linking analysis showed that alphaHL-A assembles on a membrane into an oligomer, which is likely to be a heptamer, in the absence of a reducing agent, suggesting that oxidized alphaHL-A is halted at a heptameric prepore state. Therefore, conformational rearrangements at positions 108 and 154 are critical for the completion of alphaHL assembly but are not essential for membrane binding or for formation of an oligomeric prepore intermediate.     

pH Dependence of structural stability of interleukin-2 and granulocyte colony-stimulating factor

After a cytokine binds to its receptor on the cell surface (pH approximately 7), the complex is internalized into acidic endosomal compartments (pH approximately 5-6), where partially unfolded intermediates can form. The nature of these structural transitions was studied for wild-type interleukin-2 (IL-2) and wild-type granulocyte colony-stimulating factor (G-CSF). A noncoincidence of denaturation transitions in the secondary and tertiary structure of IL-2 and tertiary structural perturbations in G-CSF suggest the presence of an intermediate state for each, a common feature of this structural family of four-helical bundle proteins. Unexpectedly, both IL-2 and G-CSF display monotonic increases in stability as the pH is decreased from 7 to 4. We hypothesize that such cytokines with cell-based clearance mechanisms in vivo may have evolved to help stabilize endosomal complexes for sorting to lysosomal degradation. We show that mutants of both IL-2 and G-CSF have differential stabilities to their wild-type counterparts as a function of pH, and that these differences may explain the differences in ligand trafficking and depletion. Further understanding of the structural changes accompanying unfolding may help guide cytokine design with respect to ligand binding, endocytic trafficking, and, consequently, therapeutic efficacy.     

Gene therapy: prospects for glycolipid storage diseases

Lysosomal storage diseases comprise a group of about 40 disorders, which in most cases are due to the deficiency of a lysosomal enzyme. Since lysosomal enzymes are involved in the degradation of various compounds, the diseases can be further subdivided according to which pathway is affected. Thus, enzyme deficiencies in the degradation pathway of glycosaminoglycans cause mucopolysaccharidosis, and deficiencies affecting glycopeptides cause glycoproteinosis. In glycolipid storage diseases enzymes are deficient that are involved in the degradation of sphingolipids. Mouse models are available for most of these diseases, and some of these mouse models have been used to study the applicability of in vivo gene therapy. We review the rationale for gene therapy in lysosomal disorders and present data, in particular, about trials in an animal model of metachromatic leukodystrophy. The data of these trials are compared with those obtained with animal models of other lysosomal diseases.     

Microspatial genetic heterogeneity and gene flow in stray cats (Felis catus L.): a comparison of coat colour and microsatellite loci

We analysed levels of genetic differentiation between nine local urban colonies of stray cats using eight coat colour and nine microsatellite loci. Both types of markers revealed a strong differentiation between colonies (FST = 0.15 and 0.09 for coat colour and microsatellite loci, respectively). Three coat colour loci showed extreme levels of genetic differentiation comparatively to other loci and are strongly suspected to be under divergent selective pressures. Microsatellite loci showed significant heterozygote deficiency within colonies (FIS = 0.14), suggesting that coat colour loci are not appropriate to investigate genetic structure at a fine scale because coat colour allele frequencies are based on Hardy-Weinberg equilibrium. The reported pattern conformed to that predicted from the social structuring of cat colonies: aggressive exclusion of immigrants, inbreeding and very low dispersal rate.     

How to perform measurements in a hovering animal's wake: physical modelling of the vortex wake of the hawkmoth, Manduca sexta

The vortex wake structure of the hawkmoth, Manduca sexta, was investigated using a vortex ring generator. Based on existing kinematic and morphological data, a piston and tube apparatus was constructed to produce circular vortex rings with the same size and disc loading as a hovering hawkmoth. Results show that the artificial rings were initially laminar, but developed turbulence owing to azimuthal wave instability. The initial impulse and circulation were accurately estimated for laminar rings using particle image velocimetry; after the transition to turbulence, initial circulation was generally underestimated. The underestimate for turbulent rings can be corrected if the transition time and velocity profile are accurately known, but this correction will not be feasible for experiments on real animals. It is therefore crucial that the circulation and impulse be estimated while the wake vortices are still laminar. The scaling of the ring Reynolds number suggests that flying animals of about the size of hawkmoths may be the largest animals whose wakes stay laminar for long enough to perform such measurements during hovering. Thus, at low advance ratios, they may be the largest animals for which wake circulation and impulse can be accurately measured.     

20-HETE inotropic effects involve the activation of a nonselective cationic current in airway smooth muscle

20-Hydroxyeicosatetraenoic acid (20-HETE) controls several mechanisms such as vasoactivity, mitogenicity, and ion transport in various tissues. Our goal was to quantify the effects of 20-HETE on the electrophysiological properties of airway smooth muscle (ASM). Isometric tension measurements, performed on guinea pig ASM, showed that 20-HETE induced a dose-dependent inotropic effect with an EC50 value of 1.5 microM. This inotropic response was insensitive to GF-109203X, a PKC inhibitor. The sustained contraction, requiring Ca2+ entry, was partially blocked by either 100 microM Gd3+ or 1 microM nifedipine, revealing the involvement of noncapacitative Ca2+ entry and L-type Ca2+ channels, respectively. Microelectrode measurements showed that 3 microM 20-HETE depolarized the membrane potential in guinea pig ASM by 13 +/- 2mV(n = 7), as did 30 microM 1-oleoyl-2-acetyl-sn-glycerol. Depolarizing effects were also observed in the absence of epithelium. Patch-clamp recordings demonstrated that 1 microM 20-HETE activated a nonselective cationic inward current that may be supported by the activation of transient receptor potential channels. The presence of canonical transient receptor potential mRNA was confirmed by RT-PCR in guinea pig ASM cells.     

Lack of genetic linkage evidence for a trans-acting factor having a large effect on plasma lipoprotein[a] levels in African Americans

The distribution of plasma lipoprotein[a] (Lp[a]) concentrations, a risk factor for cardiovascular disease, varies greatly among racial groups, with African Americans having values that are shifted toward higher levels than those of whites. The underlying cause of this heterogeneity is unknown, but a role for "trans-acting" factors has been hypothesized. This study used genetic linkage analysis to localize genetic factors influencing Lp[a] levels in African Americans that were absent in other populations; linkage results were analyzed separately in non-Hispanic whites, Hispanic whites, and African Americans. As expected, all three samples showed highly significant linkage at the approximate location of the lysophosphatidic acid locus. The white populations also independently had regions of significant linkage on chromosome 19 (LOD 3.80) and suggestive linkage on chromosomes 12 (LOD 1.60), 14 (LOD 2.56), and 19 (LOD 2.52).No linkage evidence was found to support the hypothesis of another single gene with large effects specifically segregating in African Americans that may account for their elevated Lp[a] levels.