Cdk12 maintains the integrity of adult axons by suppressing actin remodeling.

The role of cyclin-dependent kinases (CDKs) that are ubiquitously expressed in the adult nervous system remains unclear. Cdk12 is enriched in terminally differentiated neurons where its conical role in the cell cycle progression is redundant. We find that in adult neurons Cdk12 acts a negative regulator of actin formation, mitochondrial dynamics and neuronal physiology. Cdk12 maintains the size of the axon at sites proximal to the cell body through the transcription of homeostatic enzymes in the 1-carbon by folate pathway which utilize the amino acid homocysteine. Loss of Cdk12 leads to elevated homocysteine and in turn leads to uncontrolled F-actin formation and axonal swelling. Actin remodeling further induces Drp1-dependent fission of mitochondria and the breakdown of axon-soma filtration barrier allowing soma restricted cargos to enter the axon. We demonstrate that Cdk12 is also an essential gene for long-term neuronal survival and loss of this gene causes age-dependent neurodegeneration. Hyperhomocysteinemia, actin changes, and mitochondrial fragmentation are associated with several neurodegenerative conditions such as Alzheimer's disease and we provide a candidate molecular pathway to link together such pathological events.

Monophyly of terrestrial adephagan beetles as indicated by three nuclear genes (Coleoptera: Carabidae and Trachypachidae).

The beetle suborder Adephaga is traditionally divided into two sections on the basis of habitat, terrestrial Geadephaga and aquatic Hydradephaga. Monophyly of both groups is uncertain, and the relationship of the two groups has implications for inferring habitat transitions within Adephaga. Here we examine phylogenetic relationships of these groups using evidence provided by DNA sequences from all four suborders of beetles, including 60 species of Adephaga, four Archostemata, three Myxophaga, and ten Polyphaga. We studied 18S ribosomal DNA and 28S ribosomal DNA, aligned with consideration of secondary structure, as well as the nuclear protein-coding gene wingless. Independent and combined Bayesian, likelihood, and parsimony analyses of all three genes supported placement of Trachypachidae in a monophyletic Geadephaga, although for analyses of 28S rDNA and some parsimony analyses only if Coleoptera is constrained to be monophyletic. Most analyses showed limited support for the monophyly of Hydradephaga. Outside of Adephaga, there is support from the ribosomal genes for a sister group relationship between Adephaga and Polyphaga. Within the small number of sampled Polyphaga, analyses of 18S rDNA, wingless, and the combined matrix supports monophyly of Polyphaga exclusive of Scirtoidea. Unconstrained analyses of the evolution of habitat suggest that Adephaga was ancestrally aquatic with one transition to terrestrial. However, in analyses constrained to disallow changes from aquatic to terrestrial habitat, the phylogenies imply two origins of aquatic habit within Adephaga.

The glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, and pyruvate kinase are components of the K(ATP) channel macromolecular complex and regulate its function.

The regulation of ATP-sensitive potassium (K(ATP)) channel activity is complex and a multitude of factors determine their open probability. Physiologically and pathophysiologically, the most important of these are intracellular nucleotides, with a long-recognized role for glycolytically derived ATP in regulating channel activity. To identify novel regulatory subunits of the K(ATP) channel complex, we performed a two-hybrid protein-protein interaction screen, using as bait the mouse Kir6.2 C terminus. Screening a rat heart cDNA library, we identified two potential interacting proteins to be the glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triose-phosphate isomerase. The veracity of interaction was verified by co-immunoprecipitation techniques in transfected mammalian cells. We additionally demonstrated that pyruvate kinase also interacts with Kir6.2 subunits. The physiological relevance of these interactions is illustrated by the demonstration that native Kir6.2 protein similarly interact with GAPDH and pyruvate kinase in rat heart membrane fractions and that Kir6.2 protein co-localize with these glycolytic enzymes in rat ventricular myocytes. The functional relevance of our findings is demonstrated by the ability of GAPDH or pyruvate kinase substrates to directly block the K(ATP) channel under patch clamp recording conditions. Taken together, our data provide direct evidence for the concept that key enzymes involved in glycolytic ATP production are part of a multisubunit K(ATP) channel protein complex. Our data are consistent with the concept that the activity of these enzymes (possibly by ATP formation in the immediate intracellular microenvironment of this macromolecular K(ATP) channel complex) causes channel closure.

Differential effects of linoleic Acid metabolites on cardiac sodium current.

9,10-Epoxy-12-octadecenoic acid (EOA), a metabolite of linoleic acid, causes cardiac arrest in dogs. Other metabolites of linoleic acid also have toxic effects. This study investigates the mechanism of action of four of these compounds on cardiac Na(+) current (I(Na)). The whole-cell patch-clamp technique was used to investigate the effects of EOA, 9,10-dihydroxy-12-octadecenoic acid (DHOA), and their corresponding methyl esters (9,10-epoxy-12-octadecenoic methyl ester, EOM; and 9,10-dihydroxy-12-octadecenoic methyl ester, DHOM) on I(Na) in isolated adult rat ventricular myocytes. Extracellular application of each compound elicited a concentration-dependent inhibition of I(Na). The dose-response curve yielded 50% inhibition concentrations of 301 +/- 117 microM for DHOA, 41 +/- 6 microM for DHOM, 34 +/- 5 microM for EOA, and 160 +/- 41 microM for EOM. Although there was no effect on activation, 50 microM DHOM, EOA, and EOM significantly hyperpolarized the steady-state inactivation curve by approximately -6 mV. Furthermore, EOM significantly increased the slope of the steady-state inactivation curve. These compounds also seemed to stabilize the inactivated state because the time for recovery from inactivation was significantly slowed from a control value of 12.9 +/- 0.5 ms to 30.5 +/- 3.3, 31.4 +/- 1.4, and 20.5 +/- 1.0 ms by 50 microM DHOM, EOA, and EOM, respectively. These compounds have multiple actions on Na(+) channels and that despite their structural similarities their actions differ from each other. The steady-state block of I(Na) suggests that either the pore is being blocked or the channels are prevented from gating to the open state. In addition, these compounds stabilize the inactivated state and promote increased population of a slower inactivated state.

Sequence alignment of 18S ribosomal RNA and the basal relationships of Adephagan beetles: evidence for monophyly of aquatic families and the placement of Trachypachidae.

Current hypotheses regarding family relationships in the suborder Adephaga (Coleoptera) are conflicting. Here we report full-length 18S ribosomal RNA sequences of 39 adephagans and 13 outgroup taxa. Data analysis focused on the impact of sequence alignment on tree topology, using two principally different approaches. Tree alignments, which seek to minimize indels and substitutions on the tree in a single step, as implemented in an approximate procedure by the computer program POY, were contrasted with a more traditional procedure based on alignments followed by phylogenetic inference based on parsimony, likelihood, and distance analyses. Despite substantial differences between the procedures, phylogenetic conclusions regarding basal relationships within Adephaga and relationships between the four suborders of Coleoptera were broadly similar. The analysis weakly supports monophyly of Adephaga, with Polyphaga usually as its sister, and the two small suborders Myxophaga and Archostemata basal to them. In some analyses, however, Polyphaga was reconstructed as having arisen from within Hydradephaga. Adephaga generally split into two monophyletic groups, corresponding to the terrestrial Geadephaga and the aquatic Hydradephaga, as initially proposed by Crowson in 1955, consistent with a single colonization of the aquatic environment by adephagan ancestors and contradicting the recent proposition of three independent invasions. A monophyletic Hydradephaga is consistently, though not strongly, supported under most analyses, and a parametric bootstrapping test significantly rejects an hypothesis of nonmonophyly. The enigmatic Trachypachidae, which exhibit many similarities to aquatic forms but whose species are entirely terrestrial, were usually recovered as a basal lineage within Geadephaga. Strong evidence opposes the view that terrestrial trachypachids are related to the dytiscoid water beetles.

Molecular phylogeny of glutathione-S-transferases.

The glutathione-S-transferase (GST) protein superfamily is currently composed of nearly 100 sequences. This study documents a greater phylogenetic diversity of GSTs than previously realized. Parsimony and distance phylogenetic methods of GST amino acid sequences yielded virtually the same results. There appear to be at least 25 groups (families) of GST-like proteins, as different from one another as are the currently recognized classes. This diversity will require the design of a new nomenclature for this large protein superfamily. There is one well-supported large clade containing the mammalian mu, pi, and alpha classes as well as GSTs from molluscs, helminths, nematodes, and arthropods.

NEXUS: an extensible file format for systematic information.

NEXUS is a file format designed to contain systematic data for use by computer programs. The goals of the format are to allow future expansion, to include diverse kinds of information, to be independent of particular computer operating systems, and to be easily processed by a program. To this end, the format is modular, with a file consisting of separate blocks, each containing one particular kind of information, and consisting of standardized commands. Public blocks (those containing information utilized by several programs) house information about taxa, morphological and molecular characters, distances, genetic codes, assumptions, sets, trees, etc.; private blocks contain information of relevance to single programs. A detailed description of commands in public blocks is given. Guidelines are provided for reading and writing NEXUS files and for extending the format.

Interactive analysis of phylogeny and character evolution using the computer program MacClade.

Computer programs for phylogenetic analysis have been important tools in systematics and evolutionary biology, but most have been designed primarily for the reconstruction of phylogenetic trees and not the interpretation of patterns of character evolution. Described here is the computer program MacClade, designed for interactive analysis of character evolution and phylogeny. For a given tree and a matrix of character data, MacClade displays its reconstruction of character evolution by shading the branches of the tree to indicate ancestral states. Trees can be manipulated for instance by picking up and moving branches. Assumptions underlying the reconstruction of character evolution can be varied extensively. With these manipulations and MacClade's graphical feedback, one can explore the relationships among phylogenetic trees, character data, assumptions and interpretations of character evolution. MacClade has extensive facilities for editing data, displaying various summaries of character evolution in charts and diagrams, and printing.

Innovation, ideology and innocence.

Innovative solutions are required to deal with the inadequacies of existing medical education programmes, in both the developed and developing countries, if we are to promote the evolution of more acceptable, more efficient and more effective health care. The newly formed Network of Community-oriented Educational Institutions for Health Sciences is attempting to promote a series of educational and organisational innovations, aimed at the preparation of health professionals who will be better equipped and better motivated to meet the real health needs of the populations they are to serve. Such innovations encounter many obstacles, of which ideologically-based inflexibility of thought is the most important. Ideologies are here defined, in Barnett's words, as "patterns of belief ... quasi-conceptual, quasi-affective sets assumed to be true", having the power to prohibit "rigorous or experimental examination of novel experience." Such systems of thought have 'negative, restrictive and pathological effects' on the development of innovative solutions to complex problems. A particularly destructive and inhibiting stereotype has flowed from an ideological commitment to the achievement of 'international excellence'. A state of what in this paper is called 'innocence' is regarded as mandatory for the implementation of studies based on a commitment to the empirical approach, to the null hypothesis and above all to the evaluation of the process and outcome of educational and other interventions in the health care system.