Interdoublet Sliding in Bovine Spermatozoa: Its Relationship to Flagellar Motility and the Action of Inhibitory Agents

Interdoublet sliding rates were assessed in bull sperm, utilizing a freeze–thaw procedure to allow axonemal disintegration. The sliding rate at 23°C increased with increasing MgATP concentrations up to 1 mMATP, to plateau at 8 μm/sec. The analyzed interdoublet shear in both live and demembranated (Triton X-100-extracted) bull sperm reactivated with 1 mMATP established maximal microtubule sliding rates at 6 μm/sec during flagellar beating. Therefore,in vitrosliding rates were sufficient to account for the beat in intact flagella. The effect of inhibitors of flagellar motility onin vitrosliding rates was evaluated. While 8 μMvanadate minimally reduced the sliding rate (to ≈ 4 μm/sec), only 0.5 μMvanadate was sufficient to terminate reactivated bull sperm motility. Nickel ion (0.66 mM) terminated all spontaneous motility, while only reducing microtubule sliding rates to ≈ 5.0 μm/sec. Exposing intact bull sperm to theophylline (1 mM), and incubating the subsequently demembranated sperm in cAMP (3 μM), improved flagellar motility, but had little impact on microtubule sliding rates as determined by axonemal disintegration. Furthermore, deactivating live sperm with 2 mMKCN and 4 mM2-deoxy- -glucose renders the subsequently reactivated sperm immotile (as long as exogenous cAMP is absent). Yet, this treatment only reduced the sliding rate by 38%. Paradoxically, 4 mMMgADP reduced the sliding rates most dramatically (86%), whereas demembranated sperm models retain a strong, coordinated beating pattern in the presence of MgADP. These results demonstrate that there is no direct relationship between interdoublet sliding rates and the capacity for coordinated flagellar beating.     

Evolution of eutherian cytochrome c oxidase subunit II: heterogeneous rates of protein evolution and altered interaction with cytochrome c

Cytochrome c oxidase subunit II (COII), encoded by the mitochondrial genome, exhibits one of the most heterogeneous rates of amino acid replacement among placental mammals. Moreover, it has been demonstrated that cytochrome c oxidase has undergone a structural change in higher primates which has altered its physical interaction with cytochrome c. We collected a large data set of COII sequences from several orders of mammals with emphasis on primates, rodents, and artiodactyls. Using phylogenetic hypotheses based on data independent of the COII gene, we demonstrated that an increased number of amino acid replacements are concentrated among higher primates. Incorporating approximate divergence dates derived from the fossil record, we find that most of the change occurred independently along the New World monkey lineage and in a rapid burst before apes and Old World monkeys diverged. There is some evidence that Old World monkeys have undergone a faster rate of nonsynonymous substitution than have apes. Rates of substitution at four-fold degenerate sites in primates are relatively homogeneous, indicating that the rate heterogeneity is restricted to nondegenerate sites. Excluding the rate acceleration mentioned above, primates, rodents, and artiodactyls have remarkably similar nonsynonymous replacement rates. A different pattern is observed for transversions at four-fold degenerate sites, for which rodents exhibit a higher rate of replacement than do primates and artiodactyls. Finally, we hypothesize specific amino acid replacements which may account for much of the structural difference in cytochrome c oxidase between higher primates and other mammals.      

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.     

Paired sequence difference in ribosomal RNAs: evolutionary and phylogenetic implications

Ribosomal RNAs have secondary structures that are maintained by internal Watson-Crick pairing. Through analysis of chordate, arthropod, and plant 5S ribosomal RNA sequences, we show that Darwinian selection operates on these nucleotide sequences to maintain functionally important secondary structure. Insect phylogenies based on nucleotide positions involved in pairing and the production of secondary structure are incongruent with those constructed on the basis of positions that are not. Furthermore, phylogeny reconstruction using these nonpairing bases is concordant with other, morphological data.      

Effects on Embryo Development Time and Survival of Intercrossing Three Geographically Separate Populations of Southeast Alaska coho salmon, Oncorhynchus kisutch

We investigated adaptive differences among three geographically separate populations of coho salmon (Oncorhynchus kisutch) by forming first generation intercrosses (hybrid lines) and comparing them to parental types (control lines). Broodstock for the experiment came from the Gastineau Hatchery in Juneau, Hidden Falls Hatchery on Baranof Island, and Neets Bay Hatchery near Ketchikan, Alaska. All were isolated hatchery populations separated by 220–400 km and derived 15–20 years previously from single local wild populations. For each population, gametes were taken from 50 mature salmon of each sex and combined to form nine lines (three control and six hybrid); each line had 50 full-sibling families which were assigned to separate cells of an incubator at Gastineau Hatchery. Embryo survival and development times were measured as indicators of locally adapted fitness traits. Two of the control lines had higher survival rates than hybrid lines formed between either of their parental populations and other populations. Differences (p < 0.05) were found between development times for control and hybrid groups, which varied by as many as 20 days between families and as many as 30 days between control lines. The intermediate expression of development time of intercrossed lines is consistent with additive genetic variation of development time between the ancestral populations of coho salmon and indicates that important genetic divergence exists between the populations. A loss of local adaptation through a change in seasonal timing of completion of embryonic development would occur in intercrosses between the populations.     

HDAC6 and Ubp-M BUZ domains recognize specific C-terminal sequences of proteins

The BUZ/Znf-UBP domain is a protein module found in the cytoplasmic deacetylase HDAC6, E3 ubiquitin ligase BRAP2/IMP, and a subfamily of ubiquitin-specific proteases. Although several BUZ domains have been shown to bind ubiquitin with high affinity by recognizing its C-terminal sequence (RLRGG-COOH), it is currently unknown whether the interaction is sequence-specific or whether the BUZ domains are capable of binding to proteins other than ubiquitin. In this work, the BUZ domains of HDAC6 and Ubp-M were subjected to screening against a one-bead-one-compound (OBOC) peptide library that exhibited random peptide sequences with free C-termini. Sequence analysis of the selected binding peptides as well as alanine scanning studies revealed that the BUZ domains require a C-terminal Gly-Gly motif for binding. At the more N-terminal positions, the two BUZ domains have distinct sequence specificities, allowing them to bind to different peptides and/or proteins. A database search of the human proteome on the basis of the BUZ domain specificities identified 11 and 24 potential partner proteins for Ubp-M and HDAC6 BUZ domains, respectively. Peptides corresponding to the C-terminal sequences of four of the predicted binding partners (FBXO11, histone H4, PTOV1, and FAT10) were synthesized and tested for binding to the BUZ domains by fluorescence polarization. All four peptides bound to the HDAC6 BUZ domain with low micromolar K(D) values and less tightly to the Ubp-M BUZ domain. Finally, in vitro pull-down assays showed that the Ubp-M BUZ domain was capable of binding to the histone H3-histone H4 tetramer protein complex. Our results suggest that BUZ domains are sequence-specific protein-binding modules, with each BUZ domain potentially binding to a different subset of proteins.     

Phenotypic plasticity and population viability: the importance of environmental predictability

Phenotypic plasticity plays a key role in modulating how environmental variation influences population dynamics, but we have only rudimentary understanding of how plasticity interacts with the magnitude and predictability of environmental variation to affect population dynamics and persistence. We developed a stochastic individual-based model, in which phenotypes could respond to a temporally fluctuating environmental cue and fitness depended on the match between the phenotype and a randomly fluctuating trait optimum, to assess the absolute fitness and population dynamic consequences of plasticity under different levels of environmental stochasticity and cue reliability. When cue and optimum were tightly correlated, plasticity buffered absolute fitness from environmental variability, and population size remained high and relatively invariant. In contrast, when this correlation weakened and environmental variability was high, strong plasticity reduced population size, and populations with excessively strong plasticity had substantially greater extinction probability. Given that environments might become more variable and unpredictable in the future owing to anthropogenic influences, reaction norms that evolved under historic selective regimes could imperil populations in novel or changing environmental contexts. We suggest that demographic models (e.g. population viability analyses) would benefit from a more explicit consideration of how phenotypic plasticity influences population responses to environmental change.