Renal accumulation of amikacin,tobramycin and gentamycin in the rat

Summary Free and total concentrations of amikacin, tobramycin and gentamycin were measured separately in the rat kidney after equal weight by weight doses. The accumulation of aminoglycosides followed the order amikacin < tobramycin < gentamycin. The ratio between free and total aminoglycosides was similar (about 0.6) in all 3 aminoglycosides and independent on the length of administration.     

Annexin A5: shifting from a diagnostic towards a therapeutic realm

The surge in apoptosis research and the discovery of the phosphatidylserine binding properties of annexin A5 have propelled a tremendous interest in cell death detection technologies. In the past years, annexin A5 has evolved from an efficient assay for detection of apoptotic cells in vitro to an in vivo molecular imaging technology with potential clinical use. A second key discovery, the specific internalization properties of annexin A5, has opened the opportunity to use annexin A5 for therapeutic applications. Annexin A5-mediated internalization creates a novel therapeutic platform for targeted drug delivery and cell entry to treat various diseases, including cancer and cardiovascular disease. Received 29 June 2007; received after revision 19 July 2007; accepted 15 August 2007     

Mechanisms of sperm-egg interactions emerging from gene-manipulated animals

Untangling the molecular nature of sperm-egg interactions is fundamental if we are to understand fertilization. These phenomena have been studied for many years using biochemical approaches such as antibodies and ligands that interact with sperm or with eggs and their vestments. However, when homologous genetic recombination techniques were applied, most of the phenotypic factors of the gene-manipulated animals believed “essential” for fertilization were found to be dispensable. Of course, all biological systems contain redundancies and compensatory mechanisms, but as a whole the old model of fertilization clearly requires significant modification. In this review, we use the results of gene manipulation experiments in animals to propose the basis for a new vision. Received 26 January 2007; received after revision 7 March 2007; accepted 17 April 2007     

‘Heated’ Debates in Apoptosis

Hippocrates’ assertion that ‘what the lance does not heal, fire will’ underscores the fact that for thousands of years heat has been used to treat a variety of diseases, including cancer. Indeed, spontaneous tumor remission has been observed in patients following feverish infection [1], and expression of activated oncogenes, such as Ras, can render tumor cells sensitive to heat compared with normal cells [2, 3]. In the past, a primary drawback to the use of heat as a clinical therapy was the inability to selectively focus heat to tumors in situ. Of late, however, several approaches have been devised to deliver heat more precisely, including the use of heated nanoparticles, making hyperthermia a more clinically tractable treatment option [4, 5]. Despite these practical advances, the mechanisms responsible for heat shock-induced cell death remain controversial and ill-defined. In this Visions and Reflections we discuss recent findings surrounding the initiation of heat shock-induced apoptosis, and propose future areas of research. Received 17 March 2007; received after revision 25 April 2007; accepted 22 May 2007     

Cholera toxin structure, gene regulation and pathophysiological and immunological aspects

Many notions regarding the function, structure and regulation of cholera toxin expression have remained essentially unaltered in the last 15 years. At the same time, recent findings have generated additional perspectives. For example, the cholera toxin genes are now known to be carried by a non-lytic bacteriophage, a previously unsuspected condition. Understanding of how the expression of cholera toxin genes is controlled by the bacterium at the molecular level has advanced significantly and relationships with cell-density-associated (quorum-sensing) responses have recently been discovered. Regarding the cell intoxication process, the mode of entry and intracellular transport of cholera toxin are becoming clearer. In the immunological field, the strong oral immunogenicity of the non-toxic B subunit of cholera toxin (CTB) has been exploited in the development of a now widely licensed oral cholera vaccine. Additionally, CTB has been shown to induce tolerance against co-administered (linked) foreign antigens in some autoimmune and allergic diseases. Received 25 October 2007; accepted 12 December 2007     

The Penicillium chrysogenum antifungal protein PAF, a promising tool for the development of new antifungal therapies and fungal cell biology studies

In recent years the interest in antimicrobial proteins and peptides and their mode of action has been rapidly increasing due to their potential to prevent and combat microbial infections in all areas of life. A detailed knowledge about the function of such proteins is the most important requirement to consider them for future application. Our research in recent years has been focused on the low molecular weight, cysteine-rich and cationic antifungal protein PAF from Penicillium chrysogenum, which inhibits the growth of opportunistic zoo-pathogens including Aspergillus fumigatus, numerous plant-pathogenic fungi and the model organism Aspergillus nidulans. So far, the experimental results indicate that PAF elicits hyperpolarization of the plasma membrane and the activation of ion channels, followed by an increase in reactive oxygen species in the cell and the induction of an apoptosis-like phenotype. Detailed knowledge about the molecular mechanism of action of antifungal proteins such as PAF contributes to the development of new antimicrobial strategies that are urgently needed. Received 09 August 2007; received after revision 17 September 2007; accepted 19 September 2007     

Polyphenolic phytochemicals – just antioxidants or much more?

Polyphenolic phytochemicals are ubiquitous in plants, in which they function in various protective roles. A ‘recommended’ human diet contains significant quantities of polyphenolics, as they have long been assumed to be ‘antioxidants’ that scavenge excessive, damaging, free radicals arising from normal metabolic processes. There is recent evidence that polyphenolics also have ‘indirect’ antioxidant effects through induction of endogenous protective enzymes. There is also increasing evidence for many potential benefits through polyphenolic-mediated regulation of cellular processes such as inflammation. Inductive or signalling effects may occur at concentrations much lower than required for effective radical scavenging. Over the last 2 – 3 years, there have been many exciting new developments in the elucidation of the in vivo mechanisms of the health benefits of polyphenolics. We summarise the current knowledge of the intake, bio-availability and metabolism of polyphenolics, their antioxidant effects, regulatory effects on signalling pathways, neuro-protective effects and regulatory effects on energy metabolism and gut health. Received 14 May 2007; received after revision 27 June 2007; accepted 24 July 2007     

Sumoylation regulates diverse biological processes

Ten years after its discovery, the small ubiquitin-like protein modifier (SUMO) has emerged as a key regulator of proteins. While early studies indicated that sumoylation takes place mainly in the nucleus, an increasing number of non-nuclear substrates have recently been identified, suggesting a wider stage for sumoylation in the cell. Unlike ubiquitylation, which primarily targets a substrate for degradation, sumoylation regulates a substrate’s functions mainly by altering the intracellular localization, protein-protein interactions or other types of post-translational modifications. These changes in turn affect gene expression, genomic and chromosomal stability and integrity, and signal transduction. Sumoylation is counter-balanced by desumoylation, and well-balanced sumoylation is essential for normal cellular behaviors. Loss of the balance has been associated with a number of diseases. This paper reviews recent progress in the study of SUMO pathways, substrates, and cellular functions and highlights important findings that have accelerated advances in this study field and link sumoylation to human diseases. Received 19 March 2007; received after version 16 July 2007; accepted 1 August 2007     

NOD-like receptors: Ancient sentinels of the innate immune system

NOD-like receptors (NLRs) comprise a family of cytosolic proteins that have been implicated as ancient cellular sentinels mediating protective immune responses elicited by intracellular pathogens or endogenous danger signals. Genetic variants in NLR genes have been associated with complex chronic inflammatory barrier diseases (e.g. Crohn disease, bronchial asthma). In this review, we focus on the molecular pathophysiology of NLRs in the context of chronic inflammatory diseases and pinpoint recent advances in the evolutionary understanding of NLR biology. We propose that the field of NLRs may serve as a prototype for how a comprehensive understanding of an element of the immunological barrier will eventually lead to the development of targeted diagnostic, therapeutic and/or preventive strategies. Received 29 October 2007; received after revision 10 December 2007; accepted 19 December 2007     

Structure-function relationships of the polypyrimidine tract binding protein

The polypyrimidine tract binding protein (PTB) is a 58-kDa RNA binding protein involved in multiple aspects of mRNA metabolism including splicing regulation, polyadenylation, 3′end formation, internal ribosomal entry site-mediated translation, RNA localization and stability. PTB contains four RNA recognition motifs (RRMs) separated by three linkers. In this review we summarize structural information on PTB in solution that has been gathered during the past 7 years using NMR spectroscopy and small-angle X-ray scattering. The structures of all RRMs of PTB in their free state and in complex with short pyrimidine tracts, as well as a structural model of PTB RRM2 in complex with a peptide, revealed unusual structural features that provided new insights into the mechanisms of action of PTB in the different processes of RNA metabolism and in particular splicing regulation. Received 16 August 2007; received after revision 18 September 2007; accepted 2 October 2007     

Histone chaperones: 30 years from isolation to elucidation of the mechanisms of nucleosome assembly and disassembly

Some three decades have passed since the discovery of nucleosomes in 1974 and the first isolation of a histone chaperone in 1978. While various types of histone chaperones have been isolated and functionally analyzed, the elementary processes of nucleosome assembly and disassembly have been less well characterized. Recently, the tertiary structure of a hetero-trimeric complex composed of the histone chaperone CIA/ASF1 and the histone H3-H4 dimer was determined, and this complex was proposed to be an intermediate in nucleosome assembly and disassembly reactions. In addition, CIA alone was biochemically shown to dissociate the histone (H3-H4)₂ tetramer into two histone H3-H4 dimers. This activity suggested that CIA regulates the semi-conservative replication of nucleosomes. Here, we provide an overview of prominent histone chaperones with the goal of elucidating the mechanisms that preserve and modify epigenetic information. We also discuss the reactions involved in nucleosome assembly and disassembly. Received 5 July 2007; received after revision 8 September 2007; accepted 13 September 2007 M. Eitoku, L. Satoa: These authors contributed equally to this work.     

Pannexins and gap junction protein diversity

Gap junctions (GJs) are composed of proteins that form a channel connecting the cytoplasm of adjacent cells. Connexins were initially considered to be the only proteins capable of GJ formation. Another family of GJ proteins (innexins) were first found in invertebrates and were proposed to be renamed pannexins after their orthologs were discovered in vertebrates. The lack of both connexins and pannexins in the genomes of some metazoans suggests that other, still undiscovered GJ proteins exist. In vertebrates, connexins and pannexins co-exist. Here we discuss whether vertebrate pannexins have a nonredundant role in animal physiology. Pannexin channels appear to be suited for ATP and calcium signaling and play a role in the maintenance of calcium homeostasis by mechanisms implicating both GJ and nonjunctional function. Suggested roles in the ischemic death of neurons, schizophrenia, inflammation and tumor suppression have drawn much attention to exploring the molecular properties and cellular functions of pannexins. Received 22 April 2007; received after revision 9 September 2007; accepted 19 September 2007     

Cofactor-induced and mutational activity enhancement of coagulation factor VIIa

Coagulation factor VIIa (FVIIa) is an atypical member of the trypsin family of serine proteases. It fails to attain spontaneously its catalytically competent conformation and requires its protein cofactor tissue factor (TF) to accomplish this. Over a number of years, this unique behaviour of FVIIa has prompted investigations of the TF-induced activation mechanism and the zymogenicity determinants in factor VIIa. Factor VIIa has gained additional interest in the past decade because of its development into a clinically useful haemostatic agent. Here, we present an overview of the current knowledge about the TF-induced allosteric activation of FVIIa and the various molecular approaches to enhance the intrinsic activity and efficacy of FVIIa. Received 18 October 2007; received after revision 12 November 2007; accepted 14 November 2007     

Molecular mimicry in neurological disease: what is the evidence?

Autoimmune diseases are a leading cause of disability and are increasing in incidence in industrialized countries. How people develop autoimmune diseases is not completely understood, but is related to an interaction between genetic background, environmental agents, autoantigens and the immune response. Molecular mimicry continues to be an important hypothesis that explains how an infection with an environmental agent results in autoimmune disease of the nervous system and other target organs. Although molecular mimicry has yet to be unequivocally proven, in the past several years there has been a sharpening of its definition with better experimental data implicating it as a cause of neurological disease in humans. Received 9 July 2007; received after revision 15 November 2007; accepted 27 November 2007     

Histone deacetylases: Focus on the nervous system

Neurodegenerative disease strikes millions worldwide and there is mounting evidence suggesting that underlying the onset and progression of these debilitating diseases is inappropriate neuronal apoptosis. Recent reports have implicated a family of proteins known as histone deacetylases (HDACs) in various neuronal processes including the neuronal death program. Initial headway in this field has been made largely through the use of broad-spectrum HDAC inhibitors. In fact, pharmacological inhibition of HDAC activity has been shown to protect neurons in several models of neurodegeneration. The observation that HDAC inhibitors can have opposing effects in different paradigms of neurodegeneration suggests that individual members of the HDAC protein family may play distinct roles that could depend on the specific cell type under study. The purpose of this review is to detail work involving the use of HDAC inhibitors within the context of neurodegeneration and examine the roles of individual HDAC members in the nervous system with specific focus on neuronal cell death. Received 25 January 2007; received after revision 3 April 2007; accepted 26 April 2007     

Modulation of protein biophysical properties by chemical glycosylation: biochemical insights and biomedical implications

Glycosylation constitutes one of the most important posttranslational modifications employed by biological systems to modulate protein biophysical properties. Due to the direct biochemical and biomedical implications of achieving control over protein stability and function by chemical means, there has been great interest in recent years towards the development of chemical strategies for protein glycosylation. Since current knowledge about glycoprotein biophysics has been mainly derived from the study of naturally glycosylated proteins, chemical glycosylation provides novel insights into its mechanistic understanding by affording control over glycosylation parameters. This review presents a survey of the effects that natural and chemical glycosylation have on the fundamental biophysical properties of proteins (structure, dynamics, stability, and function). This is complemented by a mechanistic discussion of how glycans achieve such effects and discussion of the implications of employing chemical glycosylation as a tool to exert control over protein biophysical properties within biochemical and biomedical applications. Received 15 December 2006; received after revision 28 March 2007; accepted 25 April 2007     

Molecular genetics of RecQ helicase disorders

The RecQ helicases belong to the Superfamily II group of DNA helicases, and are defined by amino acid motifs that show sequence similarity to the catalytic domain of Escherichia coli RecQ. RecQ helicases have crucial roles in the maintenance of genome stability. In humans, there are five RecQ helicases and deficiencies in three of them cause genetic disorders characterised by cancer predisposition, premature aging and/or developmental abnormalities. RecQ helicase-deficient cells exhibit aberrant genetic recombination and/or DNA replication, which result in chromosomal instability and a decreased potential for proliferation. Here, we review the current knowledge of the molecular genetics of RecQ helicases, focusing on the human RecQ helicase disorders and mouse models of these conditions. Received 9 March 2007; received after revision 26 April 2007; accepted 2 May 2007     

Defining a neuron: neuronal ELAV proteins

Neuronal cells strongly depend on the control exerted by RNA-binding proteins (RBPs) on gene expression for the establishment and maintenance of their phenotype. Neuronal ELAV (nELAV) proteins are RBPs able to influence virtually every aspect of the postsynthesis fate of bound mRNAs, from polyadenylation, alternative splicing and nuclear export to cytoplasmic localization, stability and translation. They enhance gene expression through the last two, best documented activities, increasing mRNA half-life and promoting protein synthesis by a still-unknown molecular mechanism. Developmentally, nELAV proteins have been shown to act as inducers of the transition between neural stem/progenitor cells and differentiation-committed cells, also assisting these neuroblasts in the completion of their maturation program. In brain physiology, they are also the first RBPs demonstrated to have a pivotal role in memory, where they probably control mRNA availability for translation in subcellular domains, thereby providing a biochemical means for selective increase in synaptic strength. Received 15 January 2007; received after revision 10 August 2007; accepted 6 September 2007     

Interaction of aging-associated signaling cascades: Inhibition of NF-κB signaling by longevity factors FoxOs and SIRT1

Research on aging in model organisms has revealed different molecular mechanisms involved in the regulation of the lifespan. Studies on Saccharomyces cerevisiae have highlighted the role of the Sir2 family of genes, human Sirtuin homologs, as the longevity factors. In Caenorhabditis elegans, the daf-16 gene, a mammalian homolog of FoxO genes, was shown to function as a longevity gene. A wide array of studies has provided evidence for a role of the activation of innate immunity during aging process in mammals. This process has been called inflamm-aging. The master regulator of innate immunity is the NF-κB system. In this review, we focus on the several interactions of aging-associated signaling cascades regulated either by Sirtuins and FoxOs or NF-κB signaling pathways. We provide evidence that signaling via the longevity factors of FoxOs and SIRT1 can inhibit NF-κB signaling and simultaneously protect against inflamm-aging process. Received 4 October 2007; received after revision 7 November 2007; accepted 9 November 2007     

Alternative exon usage selectively determines both tissue distribution and subcellular localization of the acyl-CoA thioesterase 7 gene products

Acyl-CoA thioesterases (ACOTs) catalyze the hydrolysis of acyl-CoAs to free fatty acids and coenzyme A. Recent studies have demonstrated that one gene named Acot7, reported to be mainly expressed in brain and testis, is transcribed in several different isoforms by alternative usage of first exons. Strongly decreased levels of ACOT7 activity and protein in both mitochondria and cytosol was reported in patients diagnosed with fatty acid oxidation defects, linking ACOT7 function to regulation of fatty acid oxidation in other tissues. In this study, we have identified five possible first exons in mouse Acot7 (Acot7a–e) and show that all five first exons are transcribed in a tissue-specific manner. Taken together, these data show that the Acot7 gene is expressed as multiple isoforms in a tissue-specific manner, and that expression in tissues other than brain and testis is likely to play important roles in fatty acid metabolism. Received 5 February 2007: received after revision 3 April 2007; accepted 19 April 2007