The Saccharomyces cerevisiae YOR163w gene encodes a diadenosine 5', 5"'-P1,P6-hexaphosphate (Ap6A) hydrolase member of the MutT motif (Nudix hydrolase) family

The YOR163w open reading frame on chromosome XV of the Saccharomyces cerevisiae genome encodes a member of the MutT motif (nudix hydrolase) family of enzymes of Mr 21,443. By cloning and expressing this gene in Escherichia coli and S. cerevisiae, we have shown the product to be a (di)adenosine polyphosphate hydrolase with a previously undescribed substrate specificity. Diadenosine 5',5"'-P1, P6-hexaphosphate is the preferred substrate, and hydrolysis in H218O shows that ADP and adenosine 5'-tetraphosphate are produced by attack at Pbeta and AMP and adenosine 5'-pentaphosphate are produced by attack at Palpha with a Km of 56 microM and kcat of 0.4 s-1. Diadenosine 5',5"'-P1,P5-pentaphosphate, adenosine 5'-pentaphosphate, and adenosine 5'-tetraphosphate are also substrates, but not diadenosine 5',5"'-P1,P4-tetraphosphate or other dinucleotides, mononucleotides, nucleotide sugars, or nucleotide alcohols. The enzyme, which was shown to be expressed in log phase yeast cells by immunoblotting, displays optimal activity at pH 6.9, 50 degrees C, and 4-10 mM Mg2+ (or 200 microM Mn2+). It has an absolute requirement for a reducing agent, such as dithiothreitol (1 mM), and is inhibited by Ca2+ with an IC50 of 3.3 mM and F- (noncompetitively) with a Ki of 80 microM. Its function may be to eliminate potentially toxic dinucleoside polyphosphates during sporulation.     

Conservation of alternative splicing and genomic organization of the myosin alkali light-chain (Mlc1) gene among Drosophila species

The Mlc1 gene of Drosophila melanogaster encodes two MLC1 isoforms via developmentally regulated alternative pre-mRNA splicing. In larval muscle and tubular and abdominal muscles of adults, all of the six exons are included in the spliced mRNA, whereas, in the fibrillar indirect flight muscle of adult, exon 5 is excluded from the mRNA. We show that this tissue-specific pattern of alternative splicing of the Mlc1 pre-mRNA is conserved in D. simulans, D. pseudoobscura, and D. virilis. Isolation and sequencing of the Mlc1 genes from these three other Drosophila species have revealed that the overall organization of the genes is identical and that the genes have maintained a very high level of sequence identity within the coding region. Pairwise amino acid identities are 94%-99%, and there are no charge changes among the proteins. Total nucleotide divergence within the coding region of the four genes supports the accepted genealogy of these species, but the data indicate a significantly higher rate of amino acid replacement in the branch leading to D. pseudoobscura. A comparison of nucleotide substitutions in the coding portions of exon 5 and exon 6, which encode the alternative carboxyl termini of the two MLC1 isoforms, suggests that exon 5 is subject to greater evolutionary constraints than is exon 6. In addition to the coding sequences, there is significant sequence conservation within the 5' and 3' noncoding DNA and two of the introns, including one that flanks exon 5. These regions are candidates for cis- regulatory elements. Our results suggest that evolutionary constraints are acting on both the coding and noncoding sequences of the Mlc1 gene to maintain proper expression and function of the two MLC1 polypeptides.      

A phylogenetic approach to the evolution of fish behaviour

Summary WHAT HAVE WE LEARNED?As I indicated in the introduction, this is a non-traditional review. I have not asked What generalizations can we draw about the evolution of fish behaviour based upon information gleaned from phylogenetically based studies? Instead, I have presented detailed discussions of those studies. The reason for this approach is quite simple: if all studies in such a wide area of investigation can be discussed at length in one relatively short paper, then the database is not large enough to warrant the move from information collection to information synthesis. The purpose of this review, then, has been to capture the enthusiasm of the phylogenetically orientated fish ethologists and to highlight their discoveries, in the hopes that this will stimulate further research. If successful, the next review of phylogeny and the evolution of fish behaviour will follow a more familiar pathway.Although the database does not allow us to draw generalizations about the evolution of specific behavioural characters in fishes, the studies to date have uncovered a number of more general evolutionary insights. First, phylogenetic conservatism is evident at all levels of analysis, from the muscle activity patterns that underlie behavioural characters (Lauder 1986; Westneat and Wainwright, 1989; Westneat 1991; Wainwright and Lauder, 1992) through foraging preferences (Winterbottom and McLennan, 1993) and egg deposition strategies (Johnston and Page, 1992) to parental care (Stiassney and Gerstner, 1992). This conservatism forms the backbone against which the appearance of novel behaviours (apomorphies) can be highlighted. Each species' behavioural repertoire is thus a unique combination of very old (plesiomorphic), relatively old (synapomorphic) and recently derived (autapomorphic) characters. Second, phylogenetic analysis has allowed us to investigate models of behavioural evolution that were constructed from a variety of microevolutionary fitness parameters. The macroevolutionary patterns have corroborated some parts of those models (transition from biparental to female-only care: Gross and Sargent, 1985; Stiassney and Gerstner, 1992; transition from fresh water to anadromy: Gross et al., 1988; Stearley, 1992) and highlighted other parts of the models that would benefit from a re-examination of the basic assumptions (transition from biparental or female-only to male-only care: Gross and Sargent, 1985; Stiassney and Gerstner, 1992). Third, expanding our evolutionary perspective to include clades of organisms has allowed researchers to formulate new theories of behavioural evolution incroporating information about the patterns of character origin and diversification as well as information about character maintenance (Ryan, 1900a; Ryan and Rand, 1990; Ryan et al., 1990a). And finally, examination of macroevolutionary correlations between the origin and diversification of behavioural characters has allowed us to make predictions about the forces influencing the evolution of those characters that can then be tested experimentally (McLennan et al., 1988; Basolo, 1990a,b, 1991; McLennan, 1991). The studies presented in this paper have spanned a wide theoretical arena. They have revealed a number of interesting insights about the evolution of behaviour, and in so doing, have demonstrated the hybrid vigour of a research programme based upon integrating phylogeny and experimental ethology, phylogeny and functional morphology, and phylogeny and behavioural ecology. The question to be answered now is:WHERE DO WE GO FROM HERE?If this fledgling research programme is to remain vigorous, we need to do two things. First, channels of communication must be re-opened between systematists and ethologists. Specifically, we need to encourage systematists to construct robust phylogenetic trees for groups of fish that either have already been well studied behaviourally and ecologically, or would be of interest to ethologists if a phylogeny existed (the belontiids, poeciliids, and rivulines come to mind, to name just a few). In the absence of such critical information, behavioural ecologists are faced with the option of investigating their ethological data based upon trees reconstructed from old classification schemes or phenograms, neither of which produces a robust phylogenetic hypothesis of genealogy. Researchers who have opted for this approach preface their investigations with the caveat that the analysis and conclusions are only preliminary because of the unsatisfactory nature of the phylogenetic hypotheses available to them. The importance of a preliminary analysis cannot be understimated for researchers who are frustrated by their inability to apply the phylogenetic approach to their burgeoning data sets. It is, however, critical to remember that a preliminary analysis can, at best, produce only tentative results. If the data themselves are both incomplete and ambiguous, this will compound the problems arising from the absence of a rigorous phylogenetic framework, which will produce a confusing picture of behavioural evolution. It is also important to realize that even the most robust phylogenetic tree is still only a hypothesis of genealogical relationships, a hypothesis that may change with the discovery of new data.Second, links must be forged between comparative ethology and behavioural ecology. All of the examples discussed in this paper uncovered a phylogenetic component in patterns of behavioural origin and diversification. The discovery of this phylogenetic influence, however, is only the first step in developing a comprehensive evolutionary picture because phylogenetic patterns can tell us very little about the processes underlying those patterns. In order to explore questions of process, we must incorporate information about the fitness parameters of behavioural characters into our evolutionary picture. For example, optimizing such parameters onto a phylogenetic tree may allow us to investigate whether there are any macroevolutionary correlations between the origin and divergence of a behaviour and a change in one (or more) of the fitness components. We must also incorporate information about the genetic, developmental and physiological control of behaviour into our comparative framework (Brooks and McLennan, 1991; Willis et al., 1991; Lauder et al., 1993). This is perhaps the most neglected aspect of comparative ethology and will thus be the most difficult to remedy. Details of the genetic and developmental systems underlying behaviour are known for only a handful of taxa and for only a handful of behaviours within those taxa. The physiological control of behaviour is better studied, but has yet to be placed within a phylogenetic context (but see e.g. Stearley, 1992, for an example of the insights that can be gained from such a study). The results of such a multilevel approach will be a more robust estimate of the relative roles for the effects of both phylogenetic heritage and environmental factors in the evolution of behaviour in fishes.     

The Contribution of Mitochondrial Respiratory Complexes to the Production of Reactive Oxygen Species

This work was focused on distinguishing the contribution of mitochondrial redox complexesto the production of reactive oxygen species (ROS) during cellular respiration. We were ableto accurately measure, for the first time, the basal production of ROS under uncoupled conditionsby using a very sensitive method, based on the fluorescent probe dichlorodihydrofluoresceindiacetate. The method also enabled the detection of the ROS generated by the oxidation ofthe endogenous substrates in the mitochondrial preparations and could be applied to bothmitochondria and live cells. Contrary to the commonly accepted view that complex III(ubiquinol:cytochrome c reductase) is the major contributor to mitochondrial ROS production, wefound that complex I (NADH-ubiquinone reductase) and complex II (succinate-ubiquinonereductase) are the predominant generators of ROS during prolonged respiration under uncoupledconditions. Complex II, in particular, appears to contribute to the basal production of ROSin cells.     

Low dietary fish-oil threshold for myocardial membrane n-3 PUFA enrichment independent of n-6 PUFA intake in rats

Long chain n-3 PUFA docosahexaenoic acid (DHA) is important for heart and brain function. Investigations of biologically plausible mechanisms using animal models associate cardioprotection with DHA incorporation into myocardial membranes that are largely derived from supra-physiological fish oil (FO) intake. We measured the incorporation of DHA into myocardial membranes of rats from low dietary FO intake within human dietary range and quantitatively assessed the influence of dietary n-6 PUFA. With rats fed diets containing 0.16%–5% FO, equal to 0.12%–8.7% energy (%en) as eicosapentaenoic acid (EPA) and DHA (EPA+DHA), and either 1.5%en or 7.5%en n-6 PUFA (linoleic acid) for four weeks, dietary n-6:n-3 PUFA ratios ranged from 74 to 0.3. Myocardial DHA concentration increased in a log-linear fashion with a dietary threshold of 0.019%en as EPA+DHA and half maximal dietary [EPA+DHA] equal to 0.29%en (95% CI, 0.23–0.35). Dietary linoleic acid intake did not influence myocardial DHA. Myocardial membranes are sensitive to absolute dietary intake of long chain n-3 PUFA at low %en in the rat, equivalent to a human intake of one meal of fatty fish per week or less. The dietary ratio of n-6:n-3 PUFA has no influence on long chain n-3 PUFA cellular incorporation from dietary fish oil.     

Role of interleukin-7 in degenerative and inflammatory joint diseases

IL-7 is known foremost for its immunostimulatory capacities, including potent T cell-dependent catabolic effects on bone. In joint diseases like rheumatoid arthritis and osteoarthritis, IL-7, via immune activation, can induce joint destruction. Now it has been demonstrated that increased IL-7 levels are produced by human articular chondrocytes of older individuals and osteoarthritis patients. IL-7 stimulates production of proteases by IL-7 receptor-expressing chondrocytes and enhances cartilage matrix degradation. This indicates that IL-7, indirectly via immune activation, but also by a direct action on cartilage, contributes to joint destruction in rheumatic diseases.     

The Concept of Co-option: Why Evolution Often Looks Miraculous

Darwin believed that evolution generally occurred through a series of small, gradual changes. This proposal was counter-intuitive to many people because it seemed likely that “transitional” forms would not survive. Darwin, and later Cuènot, recognized that this problem was easily solved if characters that had evolved for one reason changed their function at a later time with little to no concurrent structural modification, at least initially. In other words, traits that had evolved under one set of conditions were co-opted to serve a different function under a second set of conditions. This meant that organisms carried with them in the structures of their genes, proteins, morphological, physiological, and behavioral characters the potential for rapid evolutionary change, so rapid, indeed, that the process looked miraculous and Lamarckian. In this paper, I discuss some of the paradigm examples of co-option, from genes to behavior.

Duration of ischaemia determines matrix metalloproteinase-2 activation in the reperfused rabbit heart

It has been hypothesised that activation of matrix metalloproteinase-2 (MMP-2) contributes to reversible myocardial dysfunction (stunning) following short-term ischaemia and reperfusion. Gelatin zymography was used to measure release of both pro-MMP-2 (72 kDa) and MMP-2 (62 kDa), into the coronary effluent from isolated, perfused rabbit hearts during 90 min aerobic perfusion (control), or low-flow ischaemia (15 or 60 min at 1 mL/min), followed by 60 min reperfusion. In controls, pro-MMP-2 was detected in the coronary effluent throughout the first 30 min of aerobic perfusion, but MMP-2 was not detected. In contrast, MMP-2 was detected in the coronary effluent during reperfusion after both 15 and 60 min ischaemia. However, while left ventricular systolic function was impaired after both 15 min and 60 min ischaemia, a significant increase in the release of MMP-2 was only detected in hearts following 60 min ischaemia. The dissociation between mechanical function and MMP-2 levels suggest that MMP-2 does not contribute to myocardial stunning in this model, but may contribute to myocardial dysfunction following prolonged ischaemia.     

Structure and substrate-binding mechanism of human Ap4A hydrolase

Asymmetric diadenosine 5',5'-P(1),P(4)-tetraphosphate (Ap(4)A) hydrolases play a major role in maintaining homeostasis by cleaving the metabolite diadenosine tetraphosphate (Ap(4)A) back into ATP and AMP. The NMR solution structures of the 17-kDa human asymmetric Ap(4)A hydrolase have been solved in both the presence and absence of the product ATP. The adenine moiety of the nucleotide predominantly binds in a ring stacking arrangement equivalent to that observed in the x-ray structure of the homologue from Caenorhabditis elegans. The binding site is, however, markedly divergent to that observed in the plant/pathogenic bacteria class of enzymes, opening avenues for the exploration of specific therapeutics. Binding of ATP induces substantial conformational and dynamic changes that were not observed in the C. elegans structure. In contrast to the C. elegans homologue, important side chains that play a major role in substrate binding do not have to reorient to accommodate the ligand. This may have important implications in the mechanism of substrate recognition in this class of enzymes.