Immunological characterization of the cytochrome o terminal oxidase from Escherichia coli

The cytochrome o terminal oxidase from Escherichia coli was immunochemically purified and monospecific antiserum toward cytochrome o was obtained. This antiserum is able to precipitate 100% of the ubiquinol-1 oxidase activity in Triton X-100 extracts of membranes from an E. coli strain in which cytochrome o is the only terminal oxidase. Cytochrome o was analyzed and quantitated using crossed immunoelectrophoresis, rocket immunoelectrophoresis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that cytochrome o is composed of four subunits of approximate equimolar stoichiometry with molecular weights of 51,000, 28,500, 18,000, and 12,700. The low temperature (77 K) reduced - oxidized spectrum of the immunoprecipitate shows two peaks at 555 and 562 nm, indicating b-type cytochromes. With the anti-cytochrome o and antiserum toward the cytochrome d terminal oxidase complex which was previously obtained, it is possible to immunochemically assay for all the cytochromes in the cytoplasmic membrane of aerobically grown E. coli. Preliminary results indicate that the biosynthesis of cytochrome o is repressed when cytochrome d is induced by lowering the dissolved oxygen concentration during cell growth.     

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.     

Succinate dehydrogenase in Rhodopseudomonas sphaeroides: subunit composition and immunocross-reactivity with other related bacteria.

Antibodies were raised against the succinate dehydrogenase (SDH) present in the chromatophores of phototrophically grown Rhodopseudomonas sphaeroides. Crossed immunoelectrophoresis experiments indicated that the SDH present in the cytoplasmic membranes of heterotrophically grown R. sphaeroides is probably the same enzyme observed in the chromatophores. The enzyme was extracted by Triton X-100 in a form which consisted of only two subunits (molecular weight, 68,000 and 30,000) and was not associated with a cytochrome b. The antibodies directed against SDH from R. sphaeroides showed no immunocross-reactivity with SDH from phylogenetically related bacterial species, including Rhodopseudomonas capsulata, Paracoccus denitrificans, Rhodopseudomonas palustris, Rhodospirillum rubrum, and Rhodospirillum fulvum.     

Immunological investigation of the distribution of cytochromes related to the two terminal oxidases of Escherichia coli in other gram-negative bacteria.

Monospecific antibodies were raised against the two terminal oxidase complexes of the aerobic respiratory chain of Escherichia coli. These are the cytochrome d and cytochrome o complexes. The antibodies were used to check for the occurrence of cross-reactive antigens in membrane preparations from a variety of gram-negative bacteria by rocket immunoelectrophoresis and immunoblotting techniques. With these criteria, proteins closely related to the cytochrome d complex of E. coli appeared to be widely distributed. Among the strains containing cytochrome d-related material were Serratia marcescens, Photobacterium phosphoreum, Salmonella typhimurium, Klebsiella pneumoniae, and Azotobacter vinelandii. The data suggest that the d-type terminal oxidase in many of these strains is associated in a complex with b-type and a1-type cytochromes, as has been found to be the case in E. coli. K. pneumoniae and S. typhimurium were also shown to have material cross-reactive to the E. coli cytochrome o complex.     

Relation between electromyogram and force in fatigue

The relationship between the surface electromyogram (SEMG) and force was examined during maximal voluntary contraction (MVC). Isometric MVC of elbow flexors were studied in 18 subjects who performed 27 trials, each consisting of six MVCs lasting 45 s at intervals of 30 s. There was a decrease in the median frequency (Fm) of the SEMG and of the compound action potentials (CAP) during MVC. The CAPs demonstrated that the fall in Fm was associated with a proportional increase in signal power, whereas CAP amplitude did not decrease, indicating intact neuromuscular transmission. The SEMG root-mean-square amplitude remained fairly constant, progressively deviating from force with time of contraction (r = 0.40). When SEMG amplitude was corrected for the Fm change, it tracked force more closely (r = 0.68), indicating a fall in motoneuron drive during MVC. The corrected SEMG was used to calculate the change in the generalized firing rate of motoneurons. The firing rate decreased 60% in the first and sixth contractions, tracked force closely, and corresponded to the firing rate fall seen in late adaptation of motoneurons (r = 0.90, P less than 0.001).     

Evidence for androgen independence of male mounting behavior in white-crowned sparrows (Zonotrichia leucophrys gambelii)

Previous experiments have shown that expression of mounting behavior in sexually inexperienced, adult male white-crowned sparrows does not require elevated plasma levels of androgen; adult males maintained on nonstimulatory short days mount sexually receptive females. The experiments reported here demonstrate that (1) sexually inexperienced, prepubertal males maintained on nonstimulatory short days show very low mounting rates in response to female sexual displays; (2) these males exhibit high mounting rates when exposed to stimulatory long days but androgen treatment on short days is ineffective in stimulating high mounting rates; and (3) prepubertal castration has no effect on the expression of mounting behavior by photostimulated adult males. Thus, there is no evidence that mounting behavior of adult male white-crowned sparrows depends on androgen.     

Molecular evolution of pteridophytes and their relationship to seed plants: Evidence from complete 18S rRNA gene sequences

Complete 18S ribosomal RNA sequence data from representatives of all extant pteridophyte lineages together with RNA sequences from different seed plants were used to infer a molecular phylogeny of vascular plants that included all major land plant lineages. The molecular data indicate that lycopsids are monophyletic and are the earliest diverging group within the vascular land plants, whereasPsilotum nudum is more closely related to the seed plants than to other pteridophyte lineages. The phylogenetic trees based on maximum likelihood, parsimony and distance analyses show substantial agreement with the evolutionary relationships of land plants as interpreted from the fossil record.     

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.