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.     

Heterogeneous binding of high mobility group chromosomal proteins to nuclei

A dramatic difference is observed in the intracellular distribution of the high mobility group (HMG) proteins when chicken embryo fibroblasts are fractionated into nucleus and cytoplasm by either mass enucleation of cytochalasin-B-treated cells or by differential centrifugation of mechanically disrupted cells. Nuclei (karyoplasts) obtained by cytochalasin B treatment of cells contain more than 90 percent of the HMG 1, while enucleated cytoplasts contain the remainder. A similar distribution between karyoplasts and cytoplasts is observed for the H1 histones and the nucleosomal core histones as anticipated. The presence of these proteins, in low amounts, in the cytoplast preparation can be accounted for by the small percentage of unenucleated cells present. In contrast, the nuclei isolated from mechanically disrupted cells contain only 30-40 percent of the total HMGs 1 and 2, the remainder being recovered in the cytosol fraction. No histone is observed in the cytosol fraction. Unike the higher molecular weight HMGs, most of the HMGs 14 and 17 sediment with the nuclei after cell lysis by mechanical disruption. The distribution of HMGs is unaffected by incubating cells with cytochalasin B and mechanically fractionating rather than enucleating them. Therefore, the dramatic difference in HMG 1 distribution observed using the two fractionation techniques cannot be explained by a cytochalasin-B-induced redistribution. On reextraction and sedimentation of isolated nuclei obtained by mechanical cell disruption, only 8 percent of the HMG 1 is released to the supernate. Thus, the majority of the HMG 1 originally isolated with these nuclei, representing 35 percent of the total HMG 1, is stably bound, as is all the HMGs 14 and 17. The remaining 65 percent of the HMGs 1 and 2 is unstably bound and leaks to the cytosol fraction under the conditions of mechanical disruption. It is suggested that the unstably bound HMGs form a protein pool capable of equilibrating between cytoplasm and stably bound HMGs.     

Molecular evolution of rodent insulins

Several trees of amino acid sequences of rodent insulins were derived with the maximum-parsimony procedure. Possible orthologous and paralogous relationships were investigated. Except for a recent gene duplication in the ancestor of rat and mouse, there are no strong arguments for other paralogous relationships. Therefore, a tree in agreement with other biological data is the most reasonable one. According to this tree, the capacity to form zinc-binding hexamers was lost once in the ancestor of the hystricomorph rodents, followed by moderately increased evolutionary rates in the lineages to African porcupine and chinchilla but highly increased rates in at least three independent lines to other taxa of this suborder: guinea pig, cuis, and Octodontoidea (coypu and casiragua).      

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.