Genes in sweeping competition

Analysis of DNA variation is a powerful tool for detecting adaptation at the genomic level. The contribution of adaptive evolution is evident from examples of rapidly evolving genes, which represent the likely targets for strong selection. More subtle adaptation is also an integral component of routine maintenance of gene performance, continuously applied to every gene. Adaptive changes in the population are accomplished through selective sweeps, i.e. complete or partial fixation of beneficial alleles. The evidence is accumulating that selective sweeps are quite frequent events which, together with associated genetic hitchhiking, represent dominant forces that influence molecular evolution by shaping the variability pattern in the genome. Received 5 May 2000; revised 22 August 2000; accepted 24 August 2000     

Theory-ladenness of evidence: a case study from history of chemistry

This paper attempts to argue for the theory-ladenness of evidence. It does so by employing and analysing an episode from the history of eighteenth century chemistry. It delineates attempts by Joseph Priestley and Antoine Lavoisier to construct entirely different kinds of evidence for and against a particular hypothesis from a set of agreed upon observations or (raw) data. Based on an augmented version of a distinction, drawn by J. Bogen and J. Woodward, between data and phenomena it is shown that the role of theoretical auxiliary assumptions is very important in constructing evidence for (or against) a theory from observation or (raw) data. In revolutionary situations, rival groups hold radically different theories and theoretical auxiliary assumptions. These are employed to construct very different evidence from the agreed upon set of observations or (raw) data. Hence, theory resolution becomes difficult. It is argued that evidence construction is a multi-layered exercise and can be disputed at any level. What counts as unproblematic observation or (raw) data at one level may become problematic at another level. The contingency of these constructions and the (un)problematic nature of evidence are shown to be partially dependent upon the scientific knowledge that the scientific community possesses.     

Enzyme–substrate relationships in the ubiquitin system: approaches for identifying substrates of ubiquitin ligases

Protein ubiquitylation is an important post-translational modification, regulating aspects of virtually every biochemical pathway in eukaryotic cells. Hundreds of enzymes participate in the conjugation and deconjugation of ubiquitin, as well as the recognition, signaling functions, and degradation of ubiquitylated proteins. Regulation of ubiquitylation is most commonly at the level of recognition of substrates by E3 ubiquitin ligases. Characterization of the network of E3–substrate relationships is a major goal and challenge in the field, as this expected to yield fundamental biological insights and opportunities for drug development. There has been remarkable success in identifying substrates for some E3 ligases, in many instances using the standard protein–protein interaction techniques (e.g., two-hybrid screens and co-immunoprecipitations paired with mass spectrometry). However, some E3s have remained refractory to characterization, while others have simply not yet been studied due to the sheer number and diversity of E3s. This review will discuss the range of tools and techniques that can be used for substrate profiling of E3 ligases.     

What is mechanistic evidence,and why do we need it for evidence-based policy?

It has recently been argued that successful evidence-based policy should rely on two kinds of evidence: statistical and mechanistic. The former is held to be evidence that a policy brings about the desired outcome, and the latter concerns how it does so. Although agreeing with the spirit of this proposal, we argue that the underlying conception of mechanistic evidence as evidence that is different in kind from correlational, difference-making or statistical evidence, does not correctly capture the role that information about mechanisms should play in evidence-based policy. We offer an alternative account of mechanistic evidence as information concerning the causal pathway connecting the policy intervention to its outcome. Not only can this be analyzed as evidence of difference-making, it is also to be found at any level and is obtainable by a broad range of methods, both experimental and observational. Using behavioral policy as an illustration, we draw the implications of this revised understanding of mechanistic evidence for debates concerning policy extrapolation, evidence hierarchies, and evidence integration.     

Modulation of E-cadherin function and dysfunction by N-glycosylation

Several mechanisms have been proposed to explain the E-cadherin dysfunction in cancer, including genetic and epigenetic alterations. Nevertheless, a significant number of human carcinomas have been seen that show E-cadherin dysfunction that cannot be explained at the genetic/epigenetic level. A substantial body of evidence has appeared recently that supports the view that other mechanisms operating at the post-translational level may also affect E-cadherin function. The present review addresses molecular aspects related to E-cadherin N-glycosylation and evidence is presented showing that the modification of N-linked glycans on E-cadherin can affect the adhesive function of this adhesion molecule. The role of glycosyltransferases involved in the remodeling of N-glycans on E-cadherin, including N-acetylglucosaminyltransferase III (GnT-III), N-acetylglucosaminyltransferase V (GnT-V), and the α1,6 fucosyltransferase (FUT8) enzyme, is also discussed. Finally, this review discusses an alternative functional regulatory mechanism for E-cadherin operating at the post-translational level, N-glycosylation, that may underlie the E-cadherin dysfunction in some carcinomas.     

Significance of the variation in haemolymph copper-protein ratio in the crabScylla serrata (Forskal) during different hours of the day

Summary InScylla serrata, haemolymph copper is bound to protein and it can be more reliably determined by the 2,2 biquinoline method than by other spectrophotometric methods. Ionic or free copper is absent from the haemolymph. The lack of significant time-of-day variation in copper concentration and the occurrence of variation in total protein concentration and copper-protein ratio, indicate fluctuations in copper-free proteins, which may either be periodically sequestered or released by the tissues during different hours of the day.     

DING proteins: numerous functions, elusive genes, a potential for health

DING proteins, named after their conserved N-terminus, form an overlooked protein family whose members were generally discovered through serendipity. It is characterized by an unusually high sequence conservation, even between distantly related species, and by an outstanding diversity of activities and ligands. They all share a demonstrated capacity to bind phosphate with high affinity or at least a predicted phosphate-binding site. However, DING protein genes are conspicuously absent from databases. The many novel family members identified in recent years have confirmed that DING proteins are ubiquitous not only in animals and plants but probably also in prokaryotes. At the functional level, there is increasing evidence that they participate in many health-related processes such as cancers as well as bacterial (Pseudomonas) and viral (HIV) infections, by mechanisms that are now beginning to be understood. They thus represent potent targets for the development of novel therapeutic approaches, especially against HIV. The few genomic sequences that are now available are starting to give some clues on why DING protein genes and mRNAs are well conserved and difficult to clone. This could open a new era of research, of both fundamental and applied importance.     

NDE1 and NDEL1 from genes to (mal)functions: parallel but distinct roles impacting on neurodevelopmental disorders and psychiatric illness

NDE1 (Nuclear Distribution Element 1, also known as NudE) and NDEL1 (NDE-Like 1, also known as NudEL) are the mammalian homologues of the fungus nudE gene, with important and at least partially overlapping roles for brain development. While a large number of studies describe the various properties and functions of these proteins, many do not directly compare the similarities and differences between NDE1 and NDEL1. Although sharing a high degree structural similarity and multiple common cellular roles, each protein presents several distinct features that justify their parallel but also unique functions. Notably both proteins have key binding partners in dynein, LIS1 and DISC1, which impact on neurodevelopmental and psychiatric illnesses. Both are implicated in schizophrenia through genetic and functional evidence, with NDE1 also strongly implicated in microcephaly, as well as other neurodevelopmental and psychiatric conditions through copy number variation, while NDEL1 possesses an oligopeptidase activity with a unique potential as a biomarker in schizophrenia. In this review, we aim to give a comprehensive overview of the various cellular roles of these proteins in a “bottom-up” manner, from their biochemistry and protein–protein interactions on the molecular level, up to the consequences for neuronal differentiation, and ultimately to their importance for correct cortical development, with direct consequences for the pathophysiology of neurodevelopmental and mental illness.     

Über den Auslesewert chromosomaler Strukturtypen vonDrosophila subobscura unter experimentellen Bedingungen

Summary In artificial populations ofDrosophila subobscura, arising from the mating of strains of different origin and different chromosomal structure, it was demonstrated that also in the X-chromosome an adaptive equilibrium is reached between two different structural types, although heterosis only acts in the females. There is a significant excess of females in the hybrid-generation in both reciprocal matings, especially at low temperature. The sexratio of the pure strains is 1:1. The excess may be caused by a selective advantage of the structurally heterozygous females in competition with the hemizygous males.     

Age-dependent changes in hippocampal synaptic transmission and plasticity in the PLB1Triple Alzheimer mouse

Several genetically engineered models exist that mimic aspects of the pathological and cognitive hallmarks of Alzheimer’s disease (AD). Here we report on a novel mouse model generated by targeted knock-in of transgenes containing mutated human amyloid precursor protein (APP) and microtubule-associated protein tau genes, inserted into the HPRT locus and controlled by the CaMKIIα regulatory element. These mice were crossed with an asymptomatic presenilin1_A246E overexpressing line to generate PLB1_Triple mice. Gene expression analysis and in situ hybridization confirmed stable, forebrain-specific, and gene-dose-dependent transgene expression. Brain tissue harvested from homozygous, heterozygous, and wild-type cohorts aged between 3 and 24 months was analyzed immunohistochemically and electrophysiologically. Homozygous PLB1_Triple offspring presented with mostly intracellular cortical and hippocampal human APP/amyloid, first detected reliably at 6 months. Human tau was already uncovered at 3 months (phospho-tau at 6 months) and labeling intensifying progressively with age. Gene-dose dependence was confirmed in age-matched heterozygous females that accumulated less tau and amyloid protein. General excitability of hippocampal neurones was not altered in slices from PLB1_Triple mice up to 12 months, but 2-year-old homozygous PLB1_Triple mice had smaller synaptically evoked postsynaptic potentials compared with wild types. Synaptic plasticity (paired-pulse depression/facilitation and long-term potentiation) of synaptic CA1 pyramidal cell responses was deficient from 6 months of age. Long-term depression was not affected at any age or in any genotype. Therefore, despite comparatively subtle gene expression and protein build-up, PLB1_Triple mice develop age-dependent progressive phenotypes, suggesting that aggressive protein accumulation is not necessary to reconstruct endophenotypes of AD.     

Evidence of undiscovered cell regulatory mechanisms: phosphoproteins and protein kinases in mitochondria

The finding that mitochondria contain substrates for protein kinases lead to the discovery that protein kinases are located in the mitochondria of certain tissues and species. These include pyruvate dyhydrogenase kinase, branched-chain α-ketoacid dehydrogenase kinase, protein kinase A, protein kinase Cδ, stress-activated kinase and A-Raf as well as unidentified kinases. Recent evidence suggests that mitochondrial protein kinases may be involved in physiological processes such as apoptosis and steroidogenesis. Additionally, the novel finding of low-molecular-weight GTP-binding proteins in mitochondria suggests the possibility that these may interact with mitochondrial protein kinases to regulate the activity of mitochondrial effector proteins. The fact that there are components of cellular regulatory systems in mitochondria indicates the exciting possibility of undiscovered systems regulating mitochondrial physiology. Received 19 June 2001; received after revision 7 August 2001; accepted 8 August 2001     

Redox-dependent thiol modifications: implications for the release of extracellular vesicles

Extracellular vesicles (EVs), including microvesicles and exosomes, are emerging as important regulators of homeostasis and pathophysiology. During pro-inflammatory and pro-oxidant conditions, EV release is induced. As EVs released under such conditions often exert pro-inflammatory and procoagulant effects, they may actively promote the pathogenesis of chronic diseases. There is evidence that thiol group-containing antioxidants can prevent EV induction by pro-inflammatory and oxidative stimuli, likely by protecting protein thiols of the EV-secreting cells from oxidation. As the redox state of protein thiols greatly impacts three-dimensional protein structure and, consequently, function, redox modifications of protein thiols may directly modulate EV release in response to changes in the cell’s redox environment. In this review article, we discuss targets of redox-dependent thiol modifications that are known or expected to be involved in the regulation of EV release, namely redox-sensitive calcium channels, N-ethylmaleimide sensitive factor, protein disulfide isomerase, phospholipid flippases, actin filaments, calpains and cell surface-exposed thiols. Thiol protection is proposed as a strategy for preventing detrimental changes in EV signaling in response to inflammation and oxidative stress. Identification of the thiol-containing proteins that modulate EV release in pro-oxidant environments could provide a rationale for broad application of thiol group-containing antioxidants in chronic inflammatory diseases.     

Diacylglycerol kinases in nuclear lipid-dependent signal transduction pathways

Several independent groups have shown that lipid-dependent signal transduction systems operate in the nucleus and that they are regulated independently from their membrane and cytosolic counterparts. A sizable body of evidence suggests that nuclear lipid signaling controls critical biological functions such as cell proliferation and differentiation. Diacylglycerol is a fundamental lipid second messenger which is produced in the nucleus. The levels of nuclear diacylglycerol fluctuate during the cell cycle progression, suggesting that such a molecule has important regulatory roles. Most likely, nuclear diacylglycerol serves as a chemoattractant for some isoforms of protein kinase C that migrate to the nucleus in response to a variety of agonists. The nucleus also contains diacylglycerol kinases, i.e. the enzymes that, by converting diacylglycerol into phosphatidic acid, terminate diacylglycerol-dependent events. A number of diacylglycerol kinases encoded by separate genes are present in the mammalian genome. This review aims at highlighting the different isotypes of diacylglycerol kinases identified at the nuclear level as well as at discussing their potential function and regulation. Received 4 December 2001; received after revision 28 January 2002; accepted 31 January 2002     

Muscleblind participates in RNA toxicity of expanded CAG and CUG repeats in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

We have utilized Caenorhabditis elegans as a model to investigate the toxicity and underlying mechanism of untranslated CAG repeats in comparison to CUG repeats. Our results indicate that CAG repeats can be toxic at the RNA level in a length-dependent manner, similar to that of CUG repeats. Both CAG and CUG repeats of toxic length form nuclear foci and co-localize with C. elegans muscleblind (CeMBL), implying that CeMBL may play a role in repeat RNA toxicity. Consistently, the phenotypes of worms expressing toxic CAG and CUG repeats, including shortened life span and reduced motility rate, were partially reversed by CeMbl over-expression. These results provide the first experimental evidence to show that the RNA toxicity induced by expanded CAG and CUG repeats can be mediated, at least in part, through the functional alteration of muscleblind in worms.     

An ancient cytokine,astakine, mediates circadian regulation of invertebrate hematopoiesis

Invertebrate circulating hemocytes are key players in the innate immune defense and their continuous renewal from hematopoietic tissues is tightly regulated in crustaceans by astakine, a new family of cytokines sharing a prokineticin (PROK) domain. In vertebrates, brain PROKs function as transmitters of circadian rhythms and we present evidence that hemocyte release from hematopoietic tissues in crayfish is under circadian regulation, a direct result of rhythmic expression of astakine. We demonstrate that the observed variation in astakine expression has an impact on innate immunity assessed as susceptibility to a pathogenic Pseudomonas species. These findings enlighten the importance of comparing immune responses at fixed times not to neglect circadian regulation of innate immunity. Moreover, our results entail an evolutionary conserved function for prokineticins as mediators of circadian rhythm, and for the first time show a role for this domain in circadian regulation of hematopoiesis that may have implications also in vertebrates.     

Identification of factor XIII-A as a marker of alternative macrophage activation

Factor XIII subunit A of blood coagulation (FXIII-A) is known to be synthesized but not secreted by the monocyte/macrophage cell line. On the basis of its intracellular localization and substrate profile, FXIII-A is thought to be involved in certain intracellular processes. Our present study was designed to monitor the changes in FXIII-A gene expression and protein production in long-term culture of human monocytes during their differentiation into macrophages in the presence of activating agents (interleukin-4, interferon-γ, Mycobacterium bovis BCG) inducing classical and alternative activation pathways. By using quantitative RT-PCR and fluorescent image analysis at the single-cell level we demonstrated that the expression of FXIII-A both at the mRNA as well as at the protein level is inversely regulated during the two activation programmes. Here we conclude that FXIII-A expression is an intracellular marker for alternatively activated macrophages, while its absence in monocyte-derived macrophages indicates their classically activated state.Received 2 June 2005; received after revision 12 July 2005; accepted 22 July 2005     

Deamidation and isoaspartate formation in proteins: unwanted alterations or surreptitious signals?

Formation of -linked Asp-Xaa peptide bonds – isoaspartyl (isoAsp) sites – arise in proteins via succinimide–linked deamidation of asparagine or dehydration of aspartate, reactions which represent a major source of spontaneous protein damage under physiological conditions. Accumulation of atypical isoaspartyl sites is minimized in vivo by the activity of protein L-isoaspartyl O–methyltransferase (PIMT), which regenerates a normal peptide bond. Loss of PIMT has harmful consequences, especially in neurons; thus, formation of isoAsp sites and their subsequent correction by PIMT is widely believed to constitute an important pathway of protein damage and repair. Recent evidence is mounting, however, that deamidation and isoaspartate formation may, in some instances, constitute a novel mechanism for intentional modification of protein structure. Herein we describe the mechanism of Asx rearrangement, summarize the evidence that PIMT serves an important repair function, and then focus on emerging evidence that deamidation and isoAsp formation may sometimes have a useful function.Received 16 October 2002; received after revision 11 December 2002; accepted 12 December 2002     

Glycolipid transfer protein and intracellular traffic of glucosylceramide

Summary Glycolipid transfer protein (GL-TP), a nonglycosylated protein with a molecular weight of 22,000 K, has been purified from pig brain. The protein transfers, by a carrier mechanism, glycolipids with a -glucosyl or -galactosyl residue directly linked to either ceramide or diacylglycerol. GL-TP appears to be present in most animal cells, and evidence has been obtained which indicates that it is a cytoplasmic protein. Little is known about the function of GL-TP. Current evidence indicates that glycosphingolipid glycosylation occurs at the luminal side of the Golgi apparatus, except for the glucosylation of ceramide, which has been shown to occur at the cytoplasmic side of the Golgi or endoplasmic membrane. It appears most likely that GL-TP participates in the intracellular traffic of glucosylceramide.