Septal deafferentation produces continuous rhythmic slow activity (theta) in the rat hippocampus

Hippocampal EEG was recorded in behaving rats in which the brain stem afferents to the septal region were previously damaged. In these animals rhythmic slow activity (RSA or theta) was continuously present, including drinking and immobility. The average frequency of RSA, however, was significantly higher during running (8-9 Hz) than during drinking or awake motionless state (6-8 Hz). In normal rats irregular sharp waves, rather than RSA were present during drinking and immobility. The results suggest that brain stem afferents are necessary to suppress the rhythmic firing of septal "pacemaker" cells.     

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.      

A Unitary Anesthetic Binding Site at High Resolution

Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABA_A receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABA_A receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show that apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABA_A receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels.Most general anesthetics alter the activity of ligand-gated ion channels, and electrophysiology, photolabeling, and transgenic animal experiments imply that this effect contributes to the mechanism of anesthesia (1–9). Although the molecular mechanism for this effect is not yet clear, photolabeling studies indicate that anesthetics bind within the transmembrane regions of Cys-loop ligand-gated ion channels such as the nicotinic acetylcholine and the γ-aminobutyric acid (GABA)² type A receptors (2, 9–11). Practical difficulties associated with overexpression, purification, and crystallization of ion channels have thus far stymied investigation of the structural and energetic bases underlying anesthetic recognition. However, general anesthetics also bind specifically to sites in soluble proteins, including firefly luciferase, human serum albumin (HSA), and horse spleen apoferritin (HSAF) (12–14), and x-ray crystal structures have been determined for complexes of these proteins with several general anesthetics (14–16). In particular, HSAF is an attractive model for studying anesthetic-protein interactions because it has the highest affinity for anesthetics of any protein studied to date, has a unique anesthetic binding site, and is a multimer of 4-helix bundles, much like the putative anesthetic binding regions in ligand-gated channels. In addition, apoferritin is commercially available and crystallizes readily. Most importantly, however, the affinity of HSAF for a broad range of general anesthetics is highly correlated with anesthetic potency, confirming the utility and relevance of this model system (17).Ferritin is a 24-mer iron-binding protein. It sequesters free iron ions, thereby helping to maintain non-toxic levels of iron in the cell and functioning as a cellular iron reservoir (18, 19). Each subunit has a molecular mass of ∼20 kDa and adopts a 4-helix bundle fold. The 24-mer forms a hollow, roughly spherical particle with 432 symmetry. Two ferritin isoforms are found in mammals, heavy (H) and light (L), and 24-mers can contain all H chains, all L chains, or mixtures of varying stoichiometry; the biological significance of the H/L ratio is not yet clear (20).In addition to the large central cavity, the apoferritin 24-mer contains additional, smaller cavities at the dimer interfaces; these smaller cavities are of an appropriate size to accommodate anesthetics. X-ray crystallography has confirmed that this interfacial cavity is the binding site for the inhalational anesthetics halothane and isoflurane, and isothermal titration calorimetry (ITC) measurements have shown that this interfacial site has a relatively high affinity for these anesthetics (K_a values ∼10⁵ m⁻¹) (14).General anesthetics fall into at least two broad classes, inhalational and injectable. Whereas both classes of drugs can induce the amnesia, immobility, and hypnosis associated with anesthesia, molecules in the two classes differ substantially in their chemical and physical properties. Prior to this work, only one crystal structure has been available for an injectable general anesthetic complexed with a protein-propofol, bound to HSA (16). This structure revealed that the propofol binding sites on this protein do not, by and large, overlap with the binding sites for inhalational anesthetics. This raises the question of whether the two types of drug invariably bind to separate sets of targets, or whether they could possibly transduce their effects by binding to a single protein site. To address this question we assessed whether propofol binds to the apoferritin site that had been previously identified as the binding site for inhalational anesthetics. Using x-ray crystallography, calorimetry, and molecular modeling, we show that the two types of anesthetics do indeed share a common binding site. We also investigated structure-binding relationships for a homologous series of propofol-like compounds and found that, remarkably, the energetics of binding to apoferritin precisely match the compound''s abilities to potentiate GABA effects at GABA_A receptors, suggesting that similar structural and physicochemical factors mediate anesthetic recognition by both apoferritin and ligand-gated ion channels. This argues for the possibility that anesthetic binding might trigger structural and dynamic alterations in GABA_A receptors similar to those observed in apoferritin, and that these changes underlie anesthetic effects.     

Crystal structure of a Josephin-ubiquitin complex: evolutionary restraints on ataxin-3 deubiquitinating activity

The Josephin domain is a conserved cysteine protease domain found in four human deubiquitinating enzymes: ataxin-3, the ataxin-3-like protein (ATXN3L), Josephin-1, and Josephin-2. Josephin domains from these four proteins were purified and assayed for their ability to cleave ubiquitin substrates. Reaction rates differed markedly both among the different proteins and for different substrates with a given protein. The ATXN3L Josephin domain is a significantly more efficient enzyme than the ataxin-3 domain despite their sharing 85% sequence identity. To understand the structural basis of this difference, the 2.6 Å x-ray crystal structure of the ATXN3L Josephin domain in complex with ubiquitin was determined. Although ataxin-3 and ATXN3L adopt similar folds, they bind ubiquitin in different, overlapping sites. Mutations were made in ataxin-3 at selected positions, introducing the corresponding ATXN3L residue. Only three such mutations are sufficient to increase the catalytic activity of the ataxin-3 domain to levels comparable with that of ATXN3L, suggesting that ataxin-3 has been subject to evolutionary restraints that keep its deubiquitinating activity in check.     

Evoked potentials during the elaboration of a conditioned reflex to hippocampal stimulation in rats

In experiments on 9 rats, the study of evoked potentials (EPs) of the CA1 field of the dorsal hippocampus to stimulation of its symmetrical part serving as a signal of drinking conditioned reflex (CR) showed that during reflex elaboration, the amplitude of the main EP components significantly decreased; CR did not appear when the population spike (PS) was absent in the hippocampal response. PS always accompanying CR was not specific only of it, it was also recorded at other behavioural reactions. Changes of fascia dentata EPs in the process of CR elaboration to stimulation of its symmetrical part consisted in decrease of the initial negative wave and increase of the positive one. The obtained data point to a significant reconstruction of excitatory and inhibitory inputs to the hippocampus and fascia dentata under the influence of conditioned activity.