Rabbit muscle phosphofructokinase. Modification of molecular and regulatory kinetic properties with the affinity label 5'-p-(fluorosulfonyl)benzoyl adenosine.

The affinity label 5'-p-(fluorosulfonyl)benzoyl adenosine modifies rabbit muscle phosphofructokinase to the extent of one group/subunit. Modification appears to occur at a binding site specific for AMP, cyclic AMP, and ADP, i.e. those adenine nucleotides which are activators under conditions where regulatory kinetic behavior is obtained. The consequences of the modification are consistent with the model proposed previously for correlation between the pK of specific ionizable groups, regulatory kinetic behavior, ligand binding, and the reversible cold inactivation of the enzyme (Frieden, C., Gilbert. H. R., and Bock, P. E. (1976) J. Biol. Chem. 251, 5644-5647). Thus, the modification shifts the apparent pK of the essential ionizable groups from 6.9 to 6.4 at 25 degrees C, with the result that regulatory kinetic behavior at pH 6.9 and 25 degrees C is lost. Furthermore, the apparent affinity of a site (other than the active site) for ATP, as measured by ATP-dependent quenching of intrinsic protein fluorescence at pH 6.9 and 25 degrees C, is decreased by the modification. Regulatory kinetic behavior for both substrates is obtained with the modified enzyme at a lower pH, consistent with the downward shift in the pK of the ionizable groups, but sensitivity to cAMP activation is abolished by the modification. The loss of regulatory kinetic behavior upon modification of sulfhydryl groups does not appear to be the same as that due to modification by the affinity label.     

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.      

Cardiovascular effects of training for a marathon run in unfit middle aged men.

The effects of a 30 week exercise programme on serum lipid values, blood pressure, and cardiac function were assessed in a group of sedentary men aged 35-50 training for their first marathon. Mean serum cholesterol concentration (n = 33) fell by 12% from 6.54 (SE 0.18) to 5.76 (0.15) mmol/l (mean fall 0.78 mmol/l; 95% confidence interval 0.52 to 1.04 mmol/l), serum triglyceride concentration (n = 33) by 22% from 1.56 (0.17) to 1.21 (0.09) mmol/l (mean fall 0.34 mmol/l; 95% confidence interval 0.12 to 0.56 mmol/l), and mean blood pressure (n = 27) by 10% from 102 (2) to 92 (2) mm Hg (mean fall 10 mm Hg; 95% confidence interval 7 to 13 mm Hg). These changes were not explained by changes in body composition. Peak exercise left ventricular end diastolic volume (n = 16) increased with training; as a result of this and an increased exercise left ventricular ejection fraction peak exercise cardiac output increased from 19.9 (1.2) to 23.1 (3.0) l/min (mean rise 3.2 l/min; 95% confidence interval 1.5 to 5.0 l/min). Maximum oxygen consumption increased from 33.9 (1.6) to 39.0 (1.3) ml/kg/min (mean rise 5.0 ml/kg/min; 95% confidence interval 1.8 to 8.2 ml/kg/min). This study showed favourable effects on coronary risk factors and cardiac function and supports the place of regular exercise in coronary prevention programmes.     

Development of ionic conductances in neurons of the inferior olive in the rat: an in vitro study

Neurons of the inferior olive of the rat were studied at different stages of their postnatal (PN) development by using the current clamp technique in slices maintained in vitro. Antidromic and synaptic activation of inferior olivary neurons could be achieved in preparations as young as PN day 2. Neurons at this age already exhibited a variety of ionic conductances which included fast sodium-dependent spikes, high-threshold and low-threshold calcium spikes, potassium-dependent currents, Ca-dependent after-hyperpolarizing potentials (AHPS), and both instantaneous and time-dependent inward rectification at hyperpolarized levels of membrane potential. The two types of Ca-dependent responses recorded in olivary neurons during the first postnatal week were graded with the magnitude of the depolarization imposed on the cells. Furthermore, the high-threshold Ca spikes were only clearly observed during this early period when K conductances were depressed by the injection of caesium into the cells or by bath application of 4-aminopyridine. In contrast, the high-threshold Ca spikes could be obtained without suppression of K currents and were all-or-none in character in some neurons after PN day 8 and in all neurons after PN day 11. The observations suggest that the balance between K and Ca currents changes throughout maturation and is largely in favour of the K current until about the end of the first PN week. At all ages studied, the low-threshold Ca spikes were much less sensitive to the Ca channel blocker cadmium than were the high-threshold Ca spikes. Finally, spontaneous, regular oscillations of the membrane potential were observed for the first time at PN day 16 and were only commonly observed after PN day 19, suggesting a late development of electrotonic coupling between olivary neurons.     

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.     

Restricted range of ocular accommodation in barn owls (Aves:Tytonidae)

Summary In an examination of the focusing abilities of 15 species of owls, the North American barn owl, Tyto alba pratincola (Bonaparte 1838), was an outstanding accommodator, having a range of accommodation exceeding 10 diopters (Murphy and Howland 1983). Using comparable methods, we examined the accommodation of 4 specimens of the Australian barn owl, Tyto alba delicatula (Gould 1837). We failed to elicit accommodation greater than two diopters, and most stimuli failed to evoke any discernable accommodation at all. Furthermore, examination of other Australian tytonid owls, the grass owl, T. longimembris, the sooty owl, T. tenebricosa, and both the mainland and Tasmanian subspecies of the masked owl, T. novaehollandiae novaehollandiae and T. novaehollandiae castanops, also failed to reveal anything but very moderate accommodative ranges. We conclude that the outstanding accommodative ability of the American barn owl is truly an exception to the modest accommodative abilities of the tytonid owls generally.     

Structural basis for the variation of pH-dependent redox potentials of Pseudomonas cytochromes c-551

The redox potentials of many c-type cytochromes vary with pH over the physiological pH range. We have investigated the pH dependence of redox potential for the four homologous cytochromes c-551 from Pseudomonas aeruginosa, Pseudomonas stutzeri strain 221, Pseudomonas stutzeri strain 224, and Pseudomonas mendocina . The pH dependence is due to an ionizable group that ionizes with pKox in ferricytochrome c-551 but with a higher pK, pKred , in ferrocytochrome c-551. For P. aeruginosa cytochrome c-551 it has been shown that this ionizable group is one of the heme propionic acid substituents [Moore, G. R., Pettigrew , G. W., Pitt , R. C., & Williams, R. J. P. (1980) Biochim. Biophys. Acta 590, 261-271]but the values of pKox and pKred are significantly lower in this protein than in the other three cytochromes. NMR and chemical modification studies show that for the two P. stutzeri cytochromes c-551 and P. mendocina cytochrome c-551, this propionic acid substituent is again important for the pH dependence of the redox potential. However, a histidine occurring at position 47 in their sequences hydrogen bonds to the propionic acid and thereby raises its pK. In P. aeruginosa cytochrome c-551, His-47 is substituted by Arg-47. Hydrogen-bonding schemes involving His-47 and the propionic acid are proposed.     

Phylogenetic relations between microbats, megabats and primates (Mammalia: Chiroptera and Primates)

We examine the paraphylectic hypothesis of bat origins, both in the light of previous discussions, and in the light of new evidence from our analyses of neurological traits and wing morphology. Megabats share with primates a variety of complex details in the organization of neural pathways that have not been found in any other mammalian group, particularly not in microbats. The features previously used to link microbats and megabats have been examined and found to be questionable bases for support of a monophyletic origin. In particular, morphological analyses of the musculoskeletal adaptations associated with the flight apparatus are consistent with two separate origins of the mammalian wing. Taken together, these analyses suggest that megabats evolved from an early branch of the primate lineage. This branch was comprised of moderate-sized, phytophagous gliders, of which the other living descendants are the dermopterans. Microbats, in contrast, probably evolved much earlier from small, agile insectivores whose forelimbs had long metacarpals in relation to their phalanges.     

Crystallization and preliminary X-ray studies of Escherichia coli glycerol kinase

Escherichia coli glycerol kinase, a major regulatory enzyme which catalyzes the reversible MgATP-dependent phosphorylation of glycerol has been crystallized by the hanging drop vapor diffusion method at room temperature. Three different crystal forms have been obtained in the presence of glycerol and appear to be suitable for X-ray crystallographic studies. Vapor diffusion against 55% ammonium sulfate and 1% beta-octyl glucoside (pH 7.0) yields rhombohedral crystals with space group R32, a = b = 277.1 A, c = 78.7 A (hexagonal indexing) containing a dimer of Mr 112,000 in the asymmetric unit (Vm = 2.64 A3/dalton). Vapor diffusion against sodium chloride in the presence of 10% (w/v) polyethylene glycol (pH 6.5 to 7.0) yields two different crystal forms, both with space group P2(1). The first form has a = 88.1 A, b = 99.3 A, c = 114.6 A, beta = 119 degrees, the second form has a = 92.5 A, b = 117.6 A, c = 108.3 A, beta = 93.64 degrees. Addition of ADP enhances growth of the monoclinic forms. These forms appear to contain an entire tetramer of Mr 224,000 in the asymmetric unit and have Vm values of 2.28 and 2.65 A3/dalton, respectively. All forms diffract to better than 3.0 A resolution while the second monoclinic form diffracts to approximately 1.8 A.     

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.