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.      

Diel drift of Chironomidae larvae in a pristine Idaho mountain stream

Simultaneous hourly net collections in a meadow and canyon reach of a mountain stream determined diel and spatial abundances of drifting Chironomidae larvae. Sixty-one taxa were identified to the lowest practical level, 52 in the meadow and 41 in the canyon. Orthocladiinae was the most abundant subfamily with 32 taxa and a 24 h mean density of 294 individuals 100 m⁻³ (meadow) and 26 taxa and a mean of 648 individuals 100 m⁻³ (canyon). Chironominae was the second most abundant subfamily. Nonchironomid invertebrates at both sites and total Chironomidae larvae (meadow) were predominantly night-drifting. Parakiefferiella and Psectrocladius were day-drifting (meadow) whereas 8 other chironomid taxa (meadow) and 2 taxa (canyon) were night-drifting. All others were aperiodic or too rare to test periodicity, Stempellinella cf brevis Edwards exhibited catastrophic drift in the canyon only. The different drift patterns between sites is attributed to greater loss of streambed habitat in the canyon compared to the meadow as streamflow decreased. Consequent crowding of chironomid larvae in the canyon caused catastrophic drift or interfered with drift periodicty. This study adds to knowledge of Chironomidae drift and shows influences on drift of hydrologic and geomorphic conditions.     

A comparative study of the development of Eimeria nieschulzi in vitro under aerobic and reducing conditions

Sporozoites of the rat coccidian, Eimeria nieschulzi Dieben, 1924 (Apicomplexa: Eimeriidae), were inoculated onto monolayers of normal rat kidney (NRK) fibroblasts and cultured either under aerobic (5% CO2/95% air) or reducing (desiccator jars modified into candle jars) conditions in RPMI-1640 supplemented with 5% fetal bovine serum, sodium bicarbonate, and antibiotics. Under aerobic conditions, first-generation meronts were observed at 2 days postinoculation (DPI) and, except for individual third-generation meronts that were seen at 5 and 6 DPI, no further development was noted. Under reducing conditions, however, first-generation meronts observed at 2-5 DPI underwent additional development to form second-generation meronts (3-5 DPI), third-generation meronts (3-7 DPI), and a small number of fourth-generation meronts (5-8 DPI). Both second- and third-generation meronts were abnormal, exhibiting gigantism although the merozoites produced appeared normal. The gradual degeneration of cell monolayers under reducing conditions prevented further observations beyond 8 DPI. These results suggest that atmospheric conditions play an important role in the development of E. nieschulzi and maintenance of reducing conditions may be one key to achieving enhanced development of some species of coccidia in vitro.     

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.     

Solubilization of native actin monomers from human erythrocyte membranes

Up to 50% of the actin in erythrocyte membranes can be solubilized at low ionic strength in a form capable of inhibiting DNAse I, in the presence of 0.4 mM ATP and 0.05 mM calcium. In the absence of calcium and ATP, actin is released but is apparently rapidly denatured. Solubilization of G-actin increases with temperature up to 37 degrees C. At higher temperatures, actin is released rapidly but quickly loses its ability to inhibit DNAse I.     

Metal-ion catalysis of aldose-ketose isomerizations in acidic solutions

A comparative study has been made of the ability of metal ions to catalyze the Lobry de Bruyn-Alberda van Ekenstein transformation. An analytical enzymic assay system, which is highly sensitive and selective, was developed to monitor the rates of isomerization of hexose and triose phosphates. The metal ions catalyze the isomerization of hexose phosphates by virtue of their Lewis acid character with the first transition series being most effective. The metal ions also appear to have no ability to direct the course of the reactions. Amino acids also catalyze the isomerization apparently by virtue of the free carboxylate anion.     

MPphiWR-2, a cryptic phage associated withMicromonospora purpurea ATCC 15835

Summary A previously undescribed cryptic phage was found associated withMicromonospora purpurea ATCC 15835.     

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.      

Comparative development of Cryptosporidium parvum (Apicomplexa) in 11 continuous host cell lines

Abstract Using standardized media, incubation, and parasite inoculating procedures, we compared development of Crytosporidium parvum between Madin-Darby bovine kidney (MDBK) cells and 10 additional host cell lines available through the American Type Culture Collection. Parasite development was assessed by counting parasite numbers atop monlayers in 25 random oil fields 68 h post-infection using Nomarski interference-contrast optics. Results revealed that the human ileocecal adenocarcinoma (HCT-8) cell line supported nearly twice the number of parasite developmental stages than MDBK cells or any of the other host cell types.     

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.