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.     

Influence of a fibric acid type of hypolipidemic agent on the oxidative metabolism of arachidonic acid by liver microsomal cytochrome P-450

The regiospecificity of arachidonic acid oxygenation, catalyzed by rat liver microsomal fractions in the presence of NADPH, can be altered by animal pretreatment with a fibric acid type of hypolipidemic drug, ciprofibrate. While microsomal fractions isolated from either control or phenobarbital-treated animals oxygenate arachidonic acid to mainly epoxyeicosatrienoic acids (EETs), animal pretreatment with ciprofibrate results in an eightfold stimulation of omega and omega-1 oxidation, concomitant with a net decrease in the formation of both HETEs and EETs. The isomeric composition of the EETs and of the omega and omega-1 oxidation products formed is also dependent on the type of animal pretreatment. Associated decreases in the amounts of HETEs and the rate of hydrogen peroxide formation suggests a modification of the "uncoupler action" of arachidonic acid during the function of different cytochromes P-450.     

Arachidonic acid epoxygenase: structural characterization and quantification of epoxyeicosatrienoates in plasma.

Gas chromatographic/mass spectroscopic and chiral analysis showed the presence of enzymatically derived 8,9-, 11,12- and 14,15-EET in rat plasma (2.8:1:3.4 molar ratio, respectively; 10.2 +/- 0.4 ng total EET/ml plasma). Greater than 90% of the plasma EETs was esterified to the phospholipids of circulating lipoproteins. The lipoprotein fraction with the highest EET concentration was LDL (8.1 +/- 0.9 ng/mg of protein) followed by HDL and VLDL (3.5 +/- 0.1 and 1.9 +/- 0.3 ng/mg of protein, respectively). In light of the biological activities of the EETs, these results suggest a potential systemic function for the cytochrome P-450 epoxygenase.     

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.      

The Drosophila segment polarity gene patched interacts with decapentaplegic in wing development.

The decapentaplegic (dpp) gene of Drosophila melanogaster encodes a polypeptide of the transforming growth factor-beta family of secreted factors. It is required for the proper development of both embryonic and adult structures, and may act as a morphogen in the embryo. In wing imaginal discs, dpp is expressed and required in a stripe of cells near the anterior-posterior compartment boundary. Here we show that viable mutations in the segment polarity genes patched (ptc) and costal-2 (cos2) cause specific alterations in dpp expression within the anterior compartment of the wing imaginal disc. The interaction between ptc and dpp is particularly interesting; both genes are expressed with similar patterns at the anterior-posterior compartment boundary of the disc, and mis-expressed in a similar way in segment polarity mutant backgrounds like ptc and cos2. This mis-expression of dpp could be correlated with some of the features of the adult mutant phenotypes. We propose that ptc controls dpp expression in the imaginal discs, and that the restricted expression of dpp near the anterior-posterior compartment boundary is essential to maintain the wild-type morphology of the wing disc.     

Phagocytosis of Intravenously Administered Particles by Leukocytes Adhered to the Aortic Endothelium of the Rat

Phagocytosis has been used to characterize on a functional basis leukocytes adhered to the aortic endothelium of the rat. After intravenous administration of particles, phagocytosis was observed microscopically in esterase-positive leukocytes adhered to the endothelium in whole mounts of aorta. Polybead^R blue and red, 0.5 and 1 μm particle size, were inadequate because they were insufficiently colored to be identified individually at 400. Fluoresbrite^tm YG 0.25 and 0.50 μm at doses of 0.2 and 2 ± 0.3 m1/100 g, respectively, produced endothelial lesions. The same occurred with Monastral blue B^R (MbB) at 0.3 ml/100 g, red iron at 2 ± 16 mg/100 g and India ink at different concentrations depending on the supplier. At lower particle doses, lesions were not found. Deferoxamine mesylate 1.5 mg/100 g intravenous and allopurinol 5 mg/100 g intraperitoneal administered before the particles diminished the number and intensity of lesions. In none of the cases studied was the percentage of phagocytic cells greater than 50%. Clearance curves of MbB and Fluoresbrite^tm indicated rapid disappearance of particles from the blood. Results indicate that administration of particulate suspensions is not a good method for characterizing the phagocytic leukocytes adhering to the aortic endothelium because low doses produce rapid clearance of particles, thus impeding sufficient leukocyte loading, and higher doses produce endothelial lesions that often impair reliable counting of the adhering leukocytes.     

Arachidonic acid epoxidation: epoxyeicosatrienoic acids are endogenous constituents of rat liver

Epoxyeicosatrienoic acids have been isolated and purified from the livers of male rats. They were identified by gas chromatography-mass spectrometric techniques. These results expand the list of in vivo-produced eicosanoids. Their documented in vitro biological activities suggest a role for them in cell and tissue homeostasis.     

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.     

Genes involved in the activation of the bithorax complex ofDrosophila

Summary We have studied the genetic properties and developmental effects of several mutations, in eight different loci, which alter the specification of embryonic segments. All of the changes are related to the transformations caused by mutants of the bithorax complex.Several properties: the interactions of these mutants with different mutants of the bithorax complex, the interactions between themselves, the effect of changing dosages of their wildtype alleles and their response to ether induction of phenocopies permit one to distinguish between those mutations which affect activation of the bithorax genes in early embryogenesis and those which affect expression of the bithorax genes during development. This paper deals mainly with the Rg-bx locus and its interactions with other loci which affect segment specification.In particular two loci show genetic and developmental characteristics which seem to conform to those expected for a repressor coding gene (Polycomb) and for an inducer synthesizing gene (Rg-bx) active in a negative control system of the genes in the bithorax complex. A model for the interaction of the wildtype products of these genes to determine the segmental characteristics of both the thorax and abdomen is proposed.