T5 spinal cord transection increases susceptibility to reperfusion-induced ventricular tachycardia by enhancing sympathetic activity in conscious rats

We recently documented that paraplegia (T(5) spinal cord transection) alters cardiac electrophysiology and increases the susceptibility to ventricular tachyarrhythmias induced by programmed electrical stimulation. However, coronary artery occlusion is the leading cause of death in industrially developed countries and will be the major cause of death in the world by the year 2020. The majority of these deaths result from tachyarrhythmias that culminate in ventricular fibrillation. beta-Adrenergic receptor antagonists have been shown to reduce the incidence of sudden cardiac death. Therefore, we tested the hypothesis that chronic T(5) spinal cord transection increases the susceptibility to clinically relevant ischemia-reperfusion-induced sustained ventricular tachycardia due to enhanced sympathetic activity. Intact and chronic (4 wk after transection) T(5) spinal cord-transected (T(5)X) male rats were instrumented to record arterial pressure, body temperature, and ECG. In addition, a snare was placed around the left main coronary artery. The susceptibility to sustained ventricular tachycardia produced by 2.5 min of occlusion and reperfusion of the left main coronary artery was determined in conscious rats by pulling on the snare. Reperfusion culminated in sustained ventricular tachycardia in 100% of T(5)X rats (susceptible T(5)X, 10 of 10) and 0% of intact rats [susceptible intact, 0 of 10 (P < 0.05, T(5)X vs. intact)]. Beta-adrenergic receptor blockade prevented reperfusion-induced sustained ventricular tachycardia in T(5)X rats [susceptible T(5)X 0 of 8, 0% (P < 0.05)]. Thus paraplegia increases the susceptibility to reperfusion-induced sustained ventricular tachycardia due to enhanced sympathetic activity.     

Incorporation and utilization of [3-13C]lactate and [1,2-13C]acetate by rat skeletal muscle.

Skeletal muscle can utilize many different substrates, and traditional methodologies allow only indirect discrimination between oxidative and nonoxidative uptake of substrate, possibly with contamination by metabolism of other internal organs. Our goal was to apply 1H- and 13C-nuclear magnetic resonance spectroscopy to monitor the patterns of [3-13C]lactate and [1,2-13C]acetate (model of simple carbohydrates and fats, respectively) utilization in resting vs. contracting muscle extracts of the isolated perfused rat hindquarter. Total metabolite concentrations were measured by using NADH-linked fluorometric assays. Fractional oxidation of [3-13C]lactate was unchanged by contraction despite vascular endogenous lactate accumulation. Although label accumulated in several citric acid cycle (CAC) intermediates, contraction did not increase the concentration of CAC intermediates in any muscle extracts. We conclude that 1) the isolated rat hindquarter is a viable, well-controlled model for measuring skeletal muscle 13C-labeled substrate utilization; 2) lactate is readily oxidized even during contractile activity; 3) entry and exit from the CAC, via oxidative and nonoxidative pathways, is a component of normal muscle metabolism and function; and 4) there are possible differences between gastrocnemius and soleus muscles in utilization of nonoxidative pathways.     

Evolutionary optimization of an anatomical suction cup: Lip collagen content and its correlation with flow and substrate in Neotropical suckermouth catfishes (Loricarioidei)

In riverine ecosystems, downstream drag caused by fast-flowing water poses a significant challenge to rheophilic organisms. In neotropical rivers, many members of a diverse radiation of suckermouth catfishes (Loricarioidei) resist drag in part by using modified lips that form an oral suction cup composed of thick flesh. Histological composition and morphology of this cup are interspecifically highly variable. Through an examination of 23 loricarioid species, we determined that the tissue most responsible for lip fleshiness is collagen. We hypothesized that lip collagen content is interspecifically correlated with substrate and flow so that fishes living on rocky substrates in high-flow environments have the largest, most collagenous lips. By mapping the amount and distribution of lip collagen onto a phylogeny and conducting ANOVA tests, we found support for this hypothesis. Moreover, these traits evolved multiple times in correlation with substrate and flow, suggesting they are an effective means for improving suction-based attachment. We hypothesize that collagen functions to reinforce oral suction cups, reducing the likelihood of slipping, buckling, and failure under high-flow, high-drag conditions. Macroevolutionary patterns among loricarioid catfishes suggest that for maximum performance, biomimetic suction cups should vary in material density according to drag and substrate requirements.     

Differences in genome-wide repeat sequence instability conferred by proofreading and mismatch repair defects

Mutation rates are used to calibrate molecular clocks and to link genetic variants with human disease. However, mutation rates are not uniform across each eukaryotic genome. Rates for insertion/deletion (indel) mutations have been found to vary widely when examined in vitro and at specific loci in vivo. Here, we report the genome-wide rates of formation and repair of indels made during replication of yeast nuclear DNA. Using over 6000 indels accumulated in four mismatch repair (MMR) defective strains, and statistical corrections for false negatives, we find that indel rates increase by 100 000-fold with increasing homonucleotide run length, representing the greatest effect on replication fidelity of any known genomic parameter. Nonetheless, long genomic homopolymer runs are overrepresented relative to random chance, implying positive selection. Proofreading defects in the replicative polymerases selectively increase indel rates in short repetitive tracts, likely reflecting the distance over which Pols δ and ϵ interact with duplex DNA upstream of the polymerase active site. In contrast, MMR defects hugely increase indel mutagenesis in long repetitive sequences. Because repetitive sequences are not uniformly distributed among genomic functional elements, the quantitatively different consequences on genome-wide repeat sequence instability conferred by defects in proofreading and MMR have important biological implications.     

Microanatomy of the paired‐fin pads of ostariophysan fishes (Teleostei: Ostariophysi)

Members of the teleost superorder Ostariophysi dominate freshwater habitats on all continents except Antarctica and Australia. Obligate benthic and rheophilic taxa from four different orders of the Ostariophysi (Gonorynchiformes, Cypriniformes, Characiformes, and Siluriformes) frequently exhibit thickened pads of skin along the ventral surface of the anteriormost ray or rays of horizontally orientated paired (pectoral and pelvic) fins. Such paired‐fin pads, though convergent, are externally homogenous across ostariophysan groups (particularly nonsiluriform taxa) and have been considered previously to be the result of epidermal modification. Histological examination of the pectoral and/or pelvic fins of 44 species of ostariophysans (including members of the Gonorynchiforms, Cypriniformes, Characiformes, and Siluriformes) revealed a tremendous and previously unrecognized diversity in the cellular arrangement of the skin layers (epidermis and subdermis) contributing to the paired‐fin pads. Three types of paired‐fin pads (Types 1–3) are identified in nonsiluriform ostariophysan fishes, based on differences in the cellular arrangement of the epidermis and subdermis. The paired‐fin pads of siluriforms may or may not exhibit a deep series of ridges and grooves across the surface. Two distinct patterns of unculus producing cells are identified in the epidermis of the paired‐fin pads of siluriforms, one of which is characterized by distinct bands of keratinization throughout the epidermis and is described in Amphilius platychir (Amphiliidae) for the first time. General histological comparisons between the paired fins of benthic and rheophilic ostariophysan and nonostariophysan percomorph fishes are provided, and the possible function(s) of the paired‐fin pads of ostariophysan fish are discussed. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

Energy-minimized structures and MO levels of catalysts related to [RuO(hpsd)(bpy)] that competently hydroxylate benzene (hpsd(2-)=(2-hydroxyphenyl)salicyldiminato)

Two series of complexes with formal oxidation state assignments of {Ru^V(O²⁻)} have been examined by molecular mechanics and molecular orbital methods at the level of PM3 calculations in order to assess the origin of differences in the activity of these complexes in the conversion of benzene to phenol by oxygen transfer. The first series includes complexes of general formula [RuO(hpsd)(XY)]ⁿ⁺ with hpsd²⁻ (also known in the literature as amp²⁻)=(2-hydroxyphenyl)salicyldiminato; XY=bpy(2,2^′-bipyridine) and other py-X, wherein the second pyridyl group of bpy is changed to X=-CH₂N(CH₃)₂ (stronger σ-donor X), -CH₂P(CH₃)₂ (better π-acceptor X), -CO₂ ⁻ (weak π-donor X), -CH₂S⁻ (strong π-donor X), and -CH₂C(CH₃)₂ ⁻ (very strong σ-donor X).A second series of complexes, [RuO(TDL)(bpy)]ⁿ⁺ was also studied with TDL=(tridentate ligand) of the parent hpsd²⁻ (or amp²⁻); cpsd²⁻=(N-(2-carboxyphenyl)salicylaldiminato); cppc⁻=(N-2-carboxyphenylpyridine-2-carboxaldiminato); and hppc⁻=(2-hydroxyphenyl)2-pyridylcarboxaldiminato (or app⁻). Experimentally, the activity order based upon the percentage yields of oxygenated products for [RuO(TDL)(bpy)]ⁿ⁺ is as follows for TDL’s=hpsd²⁻ (91%) > cppc⁻ (87%) > cpsd²⁻ (84%) > hppc⁻ (80%). The rates approach toward saturation in reactivity as a function of the fractional positive charge on the apical O center: cppc⁻ (0.233) > hpsd²⁻ (0.166) > cpsd²⁻ (0.105) > hppc⁻ (0.041). The reactivity order follows chelate ring strain influences of the TDL, with 5,6-membered chelate rings; hpsd²⁻ and cppc⁻ > 6,6; cpsd²⁻ > 5,5; hppc⁻.It was determined that the general structures of these complexes are best described as pentagonal pyramidal (rather than pseudo-octahedral) with the RuO unit apical, the three donors of hpsd²⁻, cpsd²⁻, cppc⁻ or hppc⁻, and the two donors of XY ligands adopting a waffled arrangement around the Ru center as the remaining donors of the pentagonal set. The donor most trans to the apical RuO is approximately at 140°, rather than 180°. Ligands such as hpsd²⁻ (amp²⁻) are not retained in a single planar array, but rather with one of the aromatic donors turned upward to shield the approach of the RuO unit from one side. The ligand series [RuO(hpsd)(XY)]⁺ averages angles between adjacent atoms of the pentagonal set of 75.4° instead of a theoretical 72.0°; angles between the apical RuO and adjacent donors average 111° but with wide deviations (±30°) depending upon the donors of the TDL.Small changes in the donor atom positions, and in the capability of the “trans” donor’s σ-donor strength, and whether it is a π-acceptor or a π-donor, modulate the degree of mixing of ligand orbitals and the LUMO/SOMO energy gap which influences reactivity. The presence of a π-acceptor ligand provides the most destabilization of Ru-O π bonding, and this appears to be the best way to increase the activity of these catalysts toward oxidation of C₆H₆ to C₆H₅OH. Also, implicated in the activity of the catalysts is the need for two non-innocent phenolate donors that raise the energy of orbitals on the apical O atom. This increases the oxenoid character of the terminal O, and makes the insertion into a C-H bond more favored.     

Mismatch repair-independent tandem repeat sequence instability resulting from ribonucleotide incorporation by DNA polymerase ε

During DNA synthesis in vitro using dNTP and rNTP concentrations present in vivo, yeast replicative DNA polymerases α, δ and ? (Pols α, δ and ?) stably incorporate rNTPs into DNA. rNTPs are also incorporated during replication in vivo, and they are repaired in an RNase H2-dependent manner. In strains encoding a mutator allele of Pol ? (pol2-M644G), failure to remove rNMPs from DNA due to deletion of the RNH201 gene encoding the catalytic subunit of RNase H2, results in deletion of 2-5 base pairs in short repetitive sequences. Deletion rates depend on the orientation of the reporter gene relative to a nearby replication origin, suggesting that mutations result from rNMPs incorporated during replication. Here we demonstrate that 2-5 base pair deletion mutagenesis also strongly increases in rnh201Δ strains encoding wild type DNA polymerases. As in the pol2-M644G strains, the deletions occur at repetitive sequences and are orientation-dependent, suggesting that mismatches involving misaligned strands arise that could be subject to mismatch repair. Unexpectedly however, 2-5 base pair deletion rates resulting from loss of RNH201 in the pol2-M644G strain are unaffected by concomitant loss of MSH3, MSH6, or both. It could be that the mismatch repair machinery is unable to repair mismatches resulting from unrepaired rNMPs incorporated into DNA by M644G Pol ?, but this possibility is belied by the observation that Msh2-Msh6 can bind to a ribonucleotide-containing mismatch. Alternatively, following incorporation of rNMPs by M644G Pol ? during replication, the conversion of unrepaired rNMPs into mutations may occur outside the context of replication, e.g., during the repair of nicks resulting from rNMPs in DNA. The results make interesting predictions that can be tested.     

Regulation of Spine Density and Morphology by IQGAP1 Protein Domains

IQGAP1 is a scaffolding protein that regulates spine number. We now show a differential role for IQGAP1 domains in spine morphogenesis, in which a region of the N-terminus that promotes Arp2/3-mediated actin polymerization and branching stimulates spine head formation while a region that binds to Cdc42 and Rac is required for stalk extension. Conversely, IQGAP1 rescues spine deficiency induced by expression of dominant negative Cdc42 by stimulating formation of stubby spines. Together, our observations place IQGAP1 as a crucial regulator of spine number and shape acting through the N-Wasp Arp2/3 complex, as well as upstream and downstream of Cdc42.