Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

The blood pressure decrease-induced sympathetic discharge following atrial natriuretic factor administration may offset its natriuretic action

Conscious SHR and WKY rats were infused during 7 days with ANF (Arg 101-Tyr 126), 100 ng/hr/rat, by means of miniosmotic pumps and their basal blood pressure (BP), changes in sodium excretion and urinary catecholamines compared with those at the last day of the infusion. The SHR initial BP of 181 +/- 3 mmHg gradually declined to 137 +/- 5 mmHg. No significant change in blood pressure was observed in the ANF-infused WKY group. However, WKY rats exhibited an increased sodium excretion and urinary dopamine/norepinephrine ratio when compared to sham-infused rats. No such differences were observed in SHR. It is suggested that an ANF-induced withdrawal of the renal sympathetic tone permits the manifestation of its natriuretic action in WKY rats. When, however, a BP decrease predominates, as in SHR, this decrease results in a reflex sympathetic discharge with a renal sympathetic activity over-riding the ANF induced natriuresis seen in WKY rats. Secondary sympathetic responses to the ANF-induced BP decrease have to be thus taken into account when a dissociation between the hypotensive and natriuretic action of ANF is observed in vivo.     

Effect of prolonged high salt intake on atrial natriuretic factor's kinetics in rats

Plasma atrial natriuretic factor (ANF) paradoxically decreases after 5 weeks (but not after 3 weeks) of 8% NaCl intake in normotensive rats. As this phenomenon remains unaccounted for by changes in ANF production, we studied the disappearance of [125I]ANF(99-126) from the circulation as an alternative explanation of plasma ANF decline. Following 5 weeks (but not 3 weeks) of an 8% NaCl diet, plasma concentrations of [125I]ANF were significantly decreased and metabolic clearance rate and volume of distribution were increased compared to control rats fed a 0.8% NaCl diet. By studying [125I]ANF tissue uptake we noted significantly greater peptide uptake after 5 weeks (but not after 3 weeks) of high salt consumption in several tissues. We hypothesize that prolonged (at least 5 weeks) 8% NaCl ingestion increases the density and/or affinity of ANF binding sites. These changes may be responsible for the previously observed decline in plasma ANF concentrations after a prolonged high salt intake.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Application of multivariate statistical analysis to estimate +Gz tolerance based on the changes of hemodynamic parameters during lower body negative pressure (LBNP)

The application of lower body negative pressure (LBNP) is very useful method for simulation of +Gz stress and for evaluation of orthostatic reaction. The different physiological changes that occur during LBNP test and +Gz acceleration test are similar. Lategola and Trent found that supine LBNP exposure at the level of -50 mmHg may be equivalent to +2Gz in producing the changes of heart rate (HR). Polese and coworkers compared hemodynamic changes occurring during upright and supine LBNP at the levels to -70 mmHg with identical measurements made during accelerations to +2Gz, +3Gz, and +4Gz in the same subjects. They noted for example that HR changes during upright LBNP exceeded HR supine levels. Peak values of HR during +3Gz and +4Gz significantly exceeded HR levels during both kinds of LBNP, but HR values at +2Gz were equivalent to those at -40 mmHg of upright and -70 mmHg of supine LBNP. So, the present study was undertaken to evaluate adaptating responses to LBNP stimulus at the level of -60 mmHg, regulatory mechanisms of the circulatory system (central and peripheral) and to look for the possibility of +Gz tolerance prediction based on the changes of some hemodynamic parameters during LBNP.     

Influence of endurance exercise performance on hemodynamic and hormonal responses to lower body negative pressure (LBNP) and +Gz tolerance in the aspect of individual sensitivity to motion sickness

A possible relationship between endurance exercise training, susceptibility to motion sickness, and orthostatic tolerance was investigated. Male subjects underwent acceleration tolerance tests, lower body negative pressure, and Coriolis tests. During the experimental protocol, hemodynamic parameters were measured including heart rate, stroke volume, blood pressure, and cardiac output, and blood was drawn and analyzed for various hormones. Specific results are presented and discussed.     

Evidence for "twisted plane" undulations in golden hamster sperm tails

Motile spermatozoa from the golden hamster have been arrested by rapid freezing and then fixed with glutaraldehyde at low temperature after substitution with ethylene glycol. As far as can be judged, the flagellar waveforms thus stabilized are similar to those seen in living sperm; in contrast, fixation in glutaraldehyde, without prior freezing, induces agonal changes in flagellar conformation. The characteristics waveform after freeze substitution contains three bends. Approx. half of these flagella are entirely planar. The rest are three dimensional, with the third bend displaced in a regular way from the plane containing the proximal two bends. From the geometry of these flagella, it is concluded that the plane of action of a given bending cycle undergoes a clockwise twist (from a forward viewpoint) as the cycle is succeeded by new bending cycles. This "twisted plane" undulation is quite different from helical movement. The twisting seems to occur abruptly, between cycles, as if each bending cycle has a preferred plane of action. The mechanism underlying the twisting is uncertain. However, on the basis of the angular displacements between the preferred planes, and the findings from electron microscopy, the following idea is presented as a working hypothesis: that, if the most proximal plane of bending is topographically determined by peripheral doublet 1, then successive distal planes of action are influenced predominantly by doublets 2, 3, etc., in clockwise sequence. The merits and weaknesses of this hypothesis are discussed.     

An Invasive Grass Increases Live Fuel Proportion and Reduces Fire Spread in a Simulated Grassland

Fire is a globally important ecosystem process, and invasive grass species generally increase fire spread by increasing the fuel load and continuity of native grassland fuelbeds. We suggest that invasive grasses that are photosynthetically active, while the native plant community is dormant reduce fire spread by introducing high-moisture, live vegetation gaps in the fuelbed. We describe the invasion pattern of a high-moisture, cool-season grass, tall fescue (Schedonorus phoenix (Scop.) Holub), in tallgrass prairie, and use spatially explicit fire behavior models to simulate fire spread under several combinations of fuel load, invasion, and fire weather scenarios. Reduced fuel load and increased extent of tall fescue invasion reduced fire spread, but high wind speed and low relative humidity can partially mitigate these effects. We attribute reduced fire spread to asynchrony in the growing seasons of the exotic, cool-season grass, tall fescue, and the native, warm-season tallgrass prairie community in this model system. Reduced fire spread under low fuel load scenarios indicate that fuel load is an important factor in fire spread, especially in invaded fuel beds. These results present a novel connection between fire behavior and asynchronous phenology between invasive grasses and native plant communities in pyrogenic ecosystems.     

Biological and Structural Characterization of Glycosylation on Ephrin-A1, a Preferred Ligand for EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase is overexpressed in a number of malignancies and is activated by ephrin ligands, most commonly by ephrin-A1. The crystal structure of the ligand-receptor complex revealed a glycosylation on the Asn-26 of ephrin-A1. Here we report for the first time the significance of the glycosylation in the biology of EphA2 and ephrin-A1. Ephrin-A1 was enzymatically deglycosylated, and its activity was evaluated in several assays using glioblastoma (GBM) cells and recombinant EphA2. We found that deglycosylated ephrin-A1 does not efficiently induce EphA2 receptor internalization and degradation, and does not activate the downstream signaling pathways involved in cell migration and proliferation. Data obtained by surface plasmon resonance confirms that deglycosylated ephrin-A1 does not bind EphA2 with high affinity. Mutations in the glycosylation site on ephrin-A1 result in protein aggregation and mislocalization. Analysis of Eph/ephrin crystal structures reveals an interaction between the ligand''s carbohydrates and two residues of EphA2: Asp-78 and Lys-136. These findings suggest that the glycosylation on ephrin-A1 plays a critical role in the binding and activation of the EphA2 receptor.