Heart failure with preserved ejection fraction diminishes peripheral hemodynamics and accelerates exercise-induced neuromuscular fatigue.

This study investigated the impact of HFpEF on neuromuscular fatigue and peripheral hemodynamics during small muscle mass exercise not limited by cardiac output. Eight HFpEF patients (NYHA II-III, ejection-fraction: 61 ± 2%) and eight healthy controls performed dynamic knee extension exercise (80% peak workload) to task failure and maximal intermittent quadriceps contractions (8 × 15 s). Controls repeated knee extension at the same absolute intensity as HFpEF. Leg blood flow (QL) was quantified using Doppler ultrasound. Pre/postexercise changes in quadriceps twitch torque (ΔQtw; peripheral fatigue), voluntary activation (ΔVA; central fatigue), and corticospinal excitability were quantified. At the same relative intensity, HFpEF (24 ± 5 W) and controls (42 ± 6 W) had a similar time-to-task failure (∼10 min), ΔQtw (∼50%), and ΔVA (∼6%). This resulted in a greater exercise-induced change in neuromuscular function per unit work in HFpEF, which was significantly correlated with a slower QL response time. Knee extension exercise at the same absolute intensity resulted in an ∼40% lower QL and greater ΔQtw and ΔVA in HFpEF than in controls. Corticospinal excitability remained unaltered during exercise in both groups. Finally, despite a similar ΔVA, ΔQtw was larger in HFpEF versus controls during isometric exercise. In conclusion, HFpEF patients are characterized by a similar development of central and peripheral fatigue as healthy controls when tested at the same relative intensity during exercise not limited by cardiac output. However, HFpEF patients have a greater susceptibility to neuromuscular fatigue during exercise at a given absolute intensity, and this impairs functional capacity. The patients' compromised QL response to exercise likely accounts, at least partly, for the patients' attenuated fatigue resistance.NEW & NOTEWORTHY The susceptibility to neuromuscular fatigue during exercise is substantially exaggerated in individuals with heart failure with a preserved ejection fraction. The faster rate of fatigue development is associated with the compromised peripheral hemodynamic response characterizing these patients during exercise. Given the role of neuromuscular fatigue as a factor limiting exercise, this impairment likely accounts for a significant portion of the exercise intolerance typical for this population.

Transplantation of pig stem cells into rat brain: proliferation during the first 8 weeks.

Previous work indicated that pig umbilical cord matrix (pUCM) cells are a type of primitive stem cell and that these cells could be recovered after central or peripheral injection into rats that did not receive immune suppression therapy. To determine the safety and proliferation potential of pUCM cells after brain transplantation, approximately 150 pUCM cells were transplanted into the brains of rats that previously received a striatal injection of the neurotoxin 6-hydroxydopamine (6-OHDA). The pUCM cells were previously engineered to express enhanced green fluorescent protein (eGFP); in this way, the graft cells were identified. The rats did not receive immune suppression therapy. There were no postsurgical complications and the animals thrived following transplantation. At 2, 4, 6, and 8 weeks after transplantation, two rats were sacrificed and the morphology, size and number of graft cells, and the percentage of tyrosine hydroxylase (TH)-positive graft cells were determined. The size distribution of the grafted pUCM cells was unimodal and normal, and the average size increased significantly over the 2- to 8-week survival period. The number of pUCM cells increased from approximately 5400 cells at the 2-week survival period post-transplantation to approximately 20,000 cells at the 8-week survival period. There was an increase in the percentage of TH-positive pUCM cells from approximately 1% at the 2-week survival period to approximately 6% at the 8-week survival period. There was no evidence of a significant host immune response at any time; for example, no accumulation of CD-4, CD-8, CD-11b, CD-161 cells in the transplantation site. These results suggest that pUCM cells engraft and proliferate without requiring immune suppression. These findings also suggest that a subset of pUCM cells can differentiate into TH-positive cells within 8 weeks after transplantation into the 6-OHDA lesioned rat brain.

Cytokine production in motor neurons by poliovirus replicon vector gene delivery.

Poliovirus replicon vectors transiently express foreign proteins selectively in motor neurons of the anterior horn of the spinal cord. Here we intraspinally inoculated mice transgenic for the poliovirus receptor (PVR) with replicons encoding murine tumor necrosis factor alpha (mTNF-alpha). We detected high-level expression of mTNF-alpha in the spinal cords of these animals at 8-12 h post inoculation; this returned to background by 72 h. The mice exhibited ataxia and tail atony, whereas animals given a replicon encoding green fluorescent protein (GFP) exhibited no neurological symptoms. Histology of spinal cords from mice given the replicon encoding mTNF-alpha revealed neuronal chromatolysis, reactive astrogliosis, decreased expression of myelin basic protein, and demyelination. These animals recovered with only slight residual damage. This study shows that replicon vectors have potential for targeted delivery of therapeutic proteins to the central nervous system and provide a new approach for treatment of spinal cord trauma and neurological disease.

Targeted foreign gene expression in spinal cord neurons using poliovirus replicons.

A hallmark of poliovirus is the propensity to infect and replicate in spinal cord neurons of the central nervous system. Previously, we characterized a poliovirus self-replicating RNA genome (replicon), which encodes firefly luciferase in place of the capsid genes. This replicon is encapsidated into an authentic poliovirion by providing the poliovirus capsid protein in trans. The amount of enzymatically active luciferase in cells infected with this replicon correlated with the infectious dose. To begin to characterize the in vivo infectious potential of replicons, we have inoculated mice transgenic for the human receptor for poliovirus (PVR), either intracranially or intraspinally, with the replicon encoding luciferase. Wild-type poliovirus delivered to PVR mice via intracranial or intraspinal routes resulted in paralysis and death. Replicon preparations were shown by a sensitive biological assay to be free of infectious poliovirus. Neither intracranial nor intraspinal inoculation of the replicon encoding luciferase resulted in any obvious paralysis or disease symptoms. Following intraspinal inoculation with replicons encoding luciferase, luciferase enzyme activity was detected at 4 h post-inoculation, with peak activity at approximately 8 h post-inoculation; by 48 - 72 h, the luciferase activity had returned to background levels. Luciferase activity was detected in spinal cord predominantly near the site of inoculation, although activity was detected anterior and posterior to the site of inoculation, indicating that replicons undergo limited movement within the CNS presumably via the cerebrospinal fluid. In stark contrast to poliovirus though, inoculation of replicons into the spinal cords of PVR mice did not result in noticeable pathogenesis. Using immunofluorescence with antibodies to double-stain for replicons and neurons, we determined that replicons exclusively infect the neurons of the spinal cord, with the expression of the luciferase and replicon proteins confined to the cytoplasm of the infected cells. Replicons, then, possess the identical capacity for infection of spinal cord neurons in vivo as poliovirus. The lack of discernible neuronal destruction following replicon inoculation into the spinal cord suggests that some of the pathogenesis observed during a poliovirus infection might not be due entirely to primary infection of neurons. Finally, the results of this study point to future use of replicons as a means to target recombinant protein expression to neurons in the spinal cord.

Responses to escalating predatory threat in garter and ribbon snakes (Thamnophis).

Several models suggest that animals perform a stable sequence of responses during increasing threat from a potential predator. To test the comparative utility of such models, 45 garter and ribbon snakes from four species (genus Thamnophis) were given an antipredator behavior test in which the snakes were exposed to a series of five levels of escalating threat: (a) threat is out of view, (b) threat looms over arena, (c) threat is nonmoving human hand near snake's head, (d) threat is moving human hand, and (e) threat is experimenter's tapping the snake's body. Five different antipredator behaviors were measured. It was found that both qualitative and quantitative changes in behavior occurred between threat levels. However, species differences were also found in the occurrence and amount of every behavior recorded, which indicates that even closely related species may differ in behaviors exhibited in different phases of a predatory encounter.

Molecular homology and DNA hybridization.

We reviewed the concept of homology, which can broadly be defined as a correspondence between characteristics that is caused by continuity of information (Van Valen 1982). The concept applies widely in molecular biology when correspondence is taken to mean a genetic relationship resulting from a unique heritable modification of a feature at some previous point in time. Such correspondence can be established for features within a single organism as well as between organisms, making paralogy a valid form of molecular homology under this definition. Molecular homology can be recognized at a variety of organizational levels, which are interdependent. For example, the recognition of homology at the site level involves a statement of homology at the sequence level, and vice versa. This hierarchy, the potential for nonhomologous identity at the site level, and such processes as sequence transposition combine to yield a molecular equivalent to complex structural homology at the anatomical level. As a result, statements of homology between heritable units can involve a valid sense of percent homology. We analyzed DNA hybridization with respect to the problems of recognizing homology and using it in phylogenetic inference. Under a model requiring continuous divergence among compared sequences, DNA hybridization distances embed evolutionary hierarchy, and groups inferred using pairwise methods of tree reconstruction are based on underlying patterns of apomorphic homology. Thus, symple-siomorphic homology will not confound DNA hybridization phylogenies. However, nonhomologous identities that act like apomorphic homologies can lead to inaccurate reconstructions. The main difference between methods of phylogenetic analysis of DNA sequences is that parsimony methods permit hypotheses of nonhomology, whereas distance methods do not.

Indexes to the reassociation and stability of solution DNA hybrids.

The computation, assumptions, and properties of DNA-hybrid stability and reassociation indexes were reviewed. Different methods of computing the same index typically yielded similar values. However, because dissociation curves change from asymmetric to symmetric as increasingly divergent DNAs are compared, adequate determination of mode required fitting a complex function. Delta Tm, delta mode, and delta T50H correlated well up to ca. 12, and all were found to be useful indexes of genomic similarity in that range. They also exhibited similar levels of error, even though T50H comprises a percent reassociation component with relatively large variance. At greater distances, the delta Tm scale became markedly compressed because of the boundary imposed by the temperature of hybrid formation (incubation temperature). Though not compressed or technically limited by it, delta mode and delta T50H could not be extrapolated with certainty below the incubation temperature. Among theoretical problems discussed: Tm and mode index an increasingly small percentage of the genome as the extent of reassociation decreases, and they may compare different genomic segments as DNAs become highly diverged. T50H relies upon the assumptions that all sequences evolve at a constant rate and that reassociation behavior is the same among all sequences regardless of their extent of divergence. Tm and T50H may be biased by self-hybridization of repetitive elements or cross-hybridization of paralogous sequences. Delta mode is free of such biases as long as the genomes under comparison are not too diverged. No index was found to be best in all circumstances.

DNA evolutionary rates in nine-primaried passerine birds.

Differences in single-copy nuclear-DNA sequences among 13 species of passerine birds were measured using DNA-DNA hybridization. A matrix of pairwise dissimilarity values (delta mode distances) was constructed from analysis of fitted thermal dissociation curves. A least-squares method of phylogenetic estimation was used to construct two topologies from the distance matrix, one constraining branch lengths of sister taxa to be equal and the other permitting such lengths to vary. These topologies were identical in the pattern of branching of taxa, and the difference in their sums of squares was not statistically significant, suggesting that rates of DNA evolution in sister groups of nine-primaried oscines are equal. A nonparametric test for nonrandom variation in distances of sister groups to outgroup taxa revealed no statistically significant deviation from random variation that would be expected as a result of measurement error. However, the level of measurement error was such that rates of DNA evolution in sister taxa could vary by as much as 10% without being detected with the statistical methods used here.