BBA - General Subjects (v.1790, #10)

Integrating evolutionary and molecular genetics of aging by Thomas Flatt; Paul S. Schmidt (951-962).
Aging or senescence is an age-dependent decline in physiological function, demographically manifest as decreased survival and fecundity with increasing age. Since aging is disadvantageous it should not evolve by natural selection. So why do organisms age and die? In the 1940s and 1950s evolutionary geneticists resolved this paradox by positing that aging evolves because selection is inefficient at maintaining function late in life. By the 1980s and 1990s this evolutionary theory of aging had received firm empirical support, but little was known about the mechanisms of aging. Around the same time biologists began to apply the tools of molecular genetics to aging and successfully identified mutations that affect longevity. Today, the molecular genetics of aging is a burgeoning field, but progress in evolutionary genetics of aging has largely stalled. Here we argue that some of the most exciting and unresolved questions about aging require an integration of molecular and evolutionary approaches. Is aging a universal process? Why do species age at different rates? Are the mechanisms of aging conserved or lineage-specific? Are longevity genes identified in the laboratory under selection in natural populations? What is the genetic basis of plasticity in aging in response to environmental cues and is this plasticity adaptive? What are the mechanisms underlying trade-offs between early fitness traits and life span? To answer these questions evolutionary biologists must adopt the tools of molecular biology, while molecular biologists must put their experiments into an evolutionary framework. The time is ripe for a synthesis of molecular biogerontology and the evolutionary biology of aging.
Keywords: Aging; Senescence; Longevity; Evolution; Genetics; Genetic variation;

Genome instability, cancer and aging by Alexander Y. Maslov; Jan Vijg (963-969).
DNA damage-driven genome instability underlies the diversity of life forms generated by the evolutionary process but is detrimental to the somatic cells of individual organisms. The cellular response to DNA damage can be roughly divided in two parts. First, when damage is severe, programmed cell death may occur or, alternatively, temporary or permanent cell cycle arrest. This protects against cancer but can have negative effects on the long term, e.g., by depleting stem cell reservoirs. Second, damage can be repaired through one or more of the many sophisticated genome maintenance pathways. However, erroneous DNA repair and incomplete restoration of chromatin after damage is resolved, produce mutations and epimutations, respectively, both of which have been shown to accumulate with age. An increased burden of mutations and/or epimutations in aged tissues increases cancer risk and adversely affects gene transcriptional regulation, leading to progressive decline in organ function. Cellular degeneration and uncontrolled cell proliferation are both major hallmarks of aging. Despite the fact that one seems to exclude the other, they both may be driven by a common mechanism. Here, we review age-related changes in the mammalian genome and their possible functional consequences, with special emphasis on genome instability in stem/progenitor cells.
Keywords: Aging; Cancer; DNA damage; Genome instability; Stem cell; Epimutation; Senescence; Apoptosis;

The impact of noncoding RNA on the biochemical and molecular mechanisms of aging by David J. Bates; Ruqiang Liang; Na Li; Eugenia Wang (970-979).
As the molecular mechanisms associated with aging become more understood, it is apparent that the normal processes involved in the development and metabolism of an organism are subject to changes that upset its crucial homeostatic balance, which in turn sets in motion the weakening and disease-prone process of senescence. This imbalance is the result of a variety of effectors, such as environmental insults, endogenous toxins, and genetic mishaps. In addition, it is highly probable that posttranscriptional regulatory events play a large role in the changes associated with aging. The emerging knowledge of posttranscriptional regulation is redefining our understanding of the complexities of cellular systems biology and genetics. The implications of the impact that small regulatory RNAs have on the many facets of developmental and molecular biology should be included as part of our current understanding of the biochemistry involved in these processes. These molecular regulators—along with other epigenetic events—restrict the flow of genetic expression, thus affording the cell an adjustable and tempered homeostatic balance control. Recent findings in the fields of organismal development, cancer, and aging indicate that small noncoding RNA plays a greater role than previously believed in orchestrating the changes associated with these processes. Furthermore, any misappropriations of these regulatory resources could lead to age-related diseases, and are therefore promising targets for prophylactics and therapeutics to combat maladies associated with aging. Here we report a brief overview of noncoding RNA as well as the potential roles of microRNAs in biochemical equilibriums where imbalance contributes to the many phenotypes of aging.
Keywords: MicroRNA; Oxidative stress; Inflammation; Intermediate metabolism; Cancer;

Protein aggregation as a paradigm of aging by Ariel B. Lindner; Alice Demarez (980-996).
The process of physiological decline leading to death of the individual is driven by the deteriorating capacity to withstand extrinsic and intrinsic hazards, resulting in damage accumulation with age. The dynamic changes with time of the network governing the outcome of misfolded proteins, exemplifying as intrinsic hazards, is considered here as a paradigm of aging. The main features of the network, namely, the non-linear increase of damage and the presence of amplifying feedback loops within the system are presented through a survey of the different components of the network and related cellular processes in aging and disease.
Keywords: Chaperone; Protein misfolding; Protein aggregation; Asymmetry; Proteostasis; Aging network;

For the past couple of decades, aging science has been rapidly evolving, and powerful genetic tools have identified a variety of evolutionarily conserved regulators and signaling pathways for the control of aging and longevity in model organisms. Nonetheless, a big challenge still remains to construct a comprehensive concept that could integrate many distinct layers of biological events into a systemic, hierarchical view of aging. The “heterochromatin island” hypothesis was originally proposed 10 years ago to explain deterministic and stochastic aspects of cellular and organismal aging, which drove the author to the study of evolutionarily conserved Sir2 proteins. Since a surprising discovery of their NAD-dependent deacetylase activity, Sir2 proteins, now called “sirtuins,” have been emerging as a critical epigenetic regulator for aging. In this review, I will follow the process of conceptual development from the heterochromatin island hypothesis to a novel, comprehensive concept of a systemic regulatory network for mammalian aging, named “NAD World,” summarizing recent studies on the mammalian NAD-dependent deacetylase Sirt1 and nicotinamide phosphoribosyltransferase (Nampt)-mediated systemic NAD biosynthesis. This new concept of the NAD World provides critical insights into a systemic regulatory mechanism that fundamentally connects metabolism and aging and also conveys the ideas of functional hierarchy and frailty for the regulation of aging in mammals.
Keywords: Aging; Metabolism; Sirt1; Nampt; Systemic NAD biosynthesis; Heterochromatin island; NAD World; Pancreatic beta cell; Neuron; Robustness; Frailty; System dynamics;

Is the oxidative stress theory of aging dead? by Viviana I. Pérez; Alex Bokov; Holly Van Remmen; James Mele; Qitao Ran; Yuji Ikeno; Arlan Richardson (1005-1014).
Currently, the oxidative stress (or free radical) theory of aging is the most popular explanation of how aging occurs at the molecular level. While data from studies in invertebrates (e.g., C. elegans and Drosophila) and rodents show a correlation between increased lifespan and resistance to oxidative stress (and in some cases reduced oxidative damage to macromolecules), direct evidence showing that alterations in oxidative damage/stress play a role in aging are limited to a few studies with transgenic Drosophila that overexpress antioxidant enzymes. Over the past eight years, our laboratory has conducted an exhaustive study on the effect of under- or overexpressing a large number and wide variety of genes coding for antioxidant enzymes. In this review, we present the survival data from these studies together. Because only one (the deletion of the Sod1 gene) of the 18 genetic manipulations we studied had an effect on lifespan, our data calls into serious question the hypothesis that alterations in oxidative damage/stress play a role in the longevity of mice.
Keywords: Antioxidant defense; Oxidative stress; Oxidative damage; Knockout mice; Transgenic mice; Longevity;

Mitochondrial DNA mutations and ageing by Laura C. Greaves; Doug M. Turnbull (1015-1020).
The mechanism by which we age has sparked a huge number of theories, and is an area of intense debate. As the elderly population rises, the importance of elucidating these mechanisms is becoming more apparent as age is the single biggest risk factor for a number of diseases such as cancer, diabetes and neurodegenerative disease. Mitochondrial DNA (MtDNA) mutations have been shown to accumulate in cells and tissues during the ageing process; however the question as to whether these mutations have a causal role in the ageing process remains an area of uncertainty. Here we review the current literature, and discuss the evidence for and against a causal role of mtDNA mutations in ageing and in the pathogenesis of age-related disease.
Keywords: Mitochondria; Mitochondrial DNA; Ageing; Mutation; Respiratory chain dysfunction;

Response to the increase of oxidative stress and mutation of mitochondrial DNA in aging by Yi-Shing Ma; Shi-Bei Wu; Wan-Yu Lee; Ju-San Cheng; Yau-Huei Wei (1021-1029).
In the aging process, mitochondrial function gradually declines with an increase of mutations in mitochondrial DNA (mtDNA) in tissue cells. Some of the aging-associated mtDNA mutations have been shown to result in not only inefficient generation of ATP but also increased production of reactive oxygen species (ROS) such as superoxide anions (O2) and hydrogen peroxide (H2O2) in the mitochondria of aging tissues. Extensive studies have revealed that such an increase of oxidative stress is a contributory factor for alterations in the expression and activities of antioxidant enzymes and increased oxidative damage to DNA, RNA, proteins, and lipids in tissues and cultured cells from elderly subjects. Recently, we observed that gene expression of several proteins and enzymes related to iron metabolism is altered and that aconitase is extremely susceptible to oxidative damage in senescent skin fibroblasts and in cybrids harboring aging-associated A8344G mutation of mtDNA. Of great importance is the perturbation at the protein and activity levels of several enzymes containing iron–sulfur clusters in skin fibroblasts of elderly subjects. Taken together, these findings suggest that cellular response to oxidative stress and oxidative damage elicited by mitochondrial dysfunction and/or mtDNA mutations plays an important role in human aging.
Keywords: Aging; Antioxidant enzyme; Iron–sulfur cluster; Mitochondrial DNA; Oxidative stress;

Hormesis, aging and longevity by Éric Le Bourg (1030-1039).
The beneficial effects of mild stress (or hormetic effects) on aging and longevity have been studied for several years. Mild stress appears to slightly increase longevity, delay behavioral aging, and increase resistance to some stresses. However, not all stresses have such beneficial effects and, for the time being, only a few studies have been done in mammals.
Keywords: Hormesis; Mild stress; Aging; Longevity; Resistance to stress; Caenorhabditis elegans; Drosophila melanogaster; Rodent; Human being; Therapy;

In 1935, Clive McCay and colleagues reported that decreasing the food intake of rats extends their life. This finding has been confirmed many times using rat and mouse models. The responsible dietary factor in rats is the reduced intake of energy; thus, this phenomenon is frequently referred to as caloric restriction. Although many hypotheses have been proposed during the past 74 years regarding the underlying mechanism, it is still not known. It is proposed that this lack of progress relates to the fact that most of these hypotheses have been based on a single underlying mechanism and that this is too narrow a focus. Rather, a broad framework is needed. Hormesis has been suggested as providing such a framework. Although it is likely that hormesis is involved in the actions of caloric restriction, it also is probably too narrowly focused. Based on currently available data, a provisional broad framework is presented depicting the complex of mechanisms that likely underlie the life-extending and other anti-aging actions of caloric restriction.
Keywords: Longevity; Hormesis; Stress; Signaling; Homeostasis; Mortality;

Klotho and aging by Makoto Kuro-o (1049-1058).
The klotho gene encodes a single-pass transmembrane protein that forms a complex with multiple fibroblast growth factor (FGF) receptors and functions as an obligatory co-receptor for FGF23, a bone-derived hormone that induces negative phosphate balance. Defects in either Klotho or Fgf23 gene expression cause not only phosphate retention but also a premature-aging syndrome in mice, unveiling a potential link between phosphate metabolism and aging. In addition, the extracellular domain of Klotho protein is clipped on the cell surface and secreted into blood stream, potentially functioning as an endocrine factor. The secreted Klotho protein has a putative sialidase activity that modifies glycans on the cell surface, which may explain the ability of secreted Klotho protein to regulate activity of multiple ion channels and growth factors including insulin, IGF-1, and Wnt. Secreted Klotho protein also protects cells and tissues from oxidative stress through a mechanism yet to be identified. Thus, the transmembrane and secreted forms of Klotho protein have distinct functions, which may collectively affect aging processes in mammals.
Keywords: Klotho; FGF23; Phosphate; Vitamin D; Sialidase; TRPV5;

PGC-1α in aging and anti-aging interventions by Rozalyn Anderson; Tomas Prolla (1059-1066).
Deregulation of mitochondrial function is a common feature in multiple aspects of aging. In addition to playing a role in aging-associated disease, decline in mitochondrial energy metabolism is likely to be important in the development of metabolic disease. Furthermore, altered mitochondrial function is a conserved feature in caloric restriction — a dietary intervention that delays aging in diverse species. The transcriptional co-activator PGC-1α is a critical regulator of mitochondrial energy metabolism and biogenesis. PGC-1α is uniquely poised as a potential target for correcting the effects of age on mitochondrial decline. We describe the cellular and tissue specific mechanisms of PGC-1α regulation and illustrate how these pathways may be involved in the aging process.
Keywords: Aging; Anti-aging; Caloric restriction; PGC-1α; Mitochondrial dysfunction; Aging associated disease; Resveratrol;

The TOR pathway comes of age by Monique N. Stanfel; Lara S. Shamieh; Matt Kaeberlein; Brian K. Kennedy (1067-1074).
Studies in a variety of model organisms indicate that nutrient signaling is tightly coupled to longevity. In nutrient replete conditions, organisms develop, grow, and age quickly. When nutrients become sparse as with dietary restriction, growth and development decline, stress response pathways become induced and organisms live longer. Considerable effort has been devoted to understanding the molecular events mediating lifespan extension by dietary restriction. One central focus has been on nutrient-responsive signal transduction pathways including insulin/IGF-1, AMP kinase, protein kinase A and the TOR pathway. Here we describe the increasingly prominent links between TOR signaling and aging in invertebrates. Longevity studies in mammals are not published to date. Instead, we highlight studies in mouse models, which indicate that dampening the TOR pathway leads to widespread protection from an array of age-related diseases.
Keywords: Aging; TOR; S6 kinase; Neurodegeneration; Metabolic syndrome; Cardiovascular disease; Dietary restriction;

Extreme-longevity mutations orchestrate silencing of multiple signaling pathways by Robert J. Shmookler Reis; Puneet Bharill; Cagdas Tazearslan; Srinivas Ayyadevara (1075-1083).
Long-lived mutants provide unique insights into the genetic factors that limit lifespan in wild-type animals. Most mutants and RNA interference targets found to extend life, typically by 1.5- to 2.5-fold, were discovered in C. elegans. Several longevity-assurance pathways are conserved across widely divergent taxa, indicating that mechanisms of lifespan regulation evolved several hundred million years ago. Strong mutations to the C. elegans gene encoding AGE-1/PI3KCS achieve unprecedented longevity by orchestrating the modulation (predominantly silencing) of multiple signaling pathways. This is evident in a profound attenuation of total kinase activity, leading to reduced phosphoprotein content. Mutations to the gene encoding the catalytic subunit of PI3K (phosphatidylinositol 3-kinase) have the potential to modulate all enzymes that depend on its product, PIP3, for membrane tethering or activation by other kinases. Remarkably, strong mutants inactivating PI3K also silence multiple signaling pathways at the transcript level, partially but not entirely mediated by the DAF-16/FOXO transcription factor. Mammals have a relatively large proportion of somatic cells, and survival depends on their replication, whereas somatic cell divisions in nematodes are limited to development and reproductive tissues. Thus, translation of longevity gains from nematodes to mammals requires disentangling the downstream consequences of signaling mutations, to avoid their deleterious consequences.
Keywords: Lifespan; Longevity; Caenorhabditis elegans; Insulin; IGF-1 (insulin-like growth factor 1);

Invertebrate models of age-related muscle degeneration by Hrvoje Augustin; Linda Partridge (1084-1094).
Functional and structural deterioration of muscles is an inevitable consequence of ageing in a wide variety of animal species. What underlies these changes is a complex network of interactions between the muscle-intrinsic and muscle-extrinsic factors, making it very difficult to distinguish between the cause and the consequence. Many of the genes, structures, and processes implicated in mammalian skeletal muscle ageing are preserved in invertebrate species Drosophila melanogaster and Caenorhabditis elegans. The absence in these organisms of mechanisms that promote muscle regeneration, and substantially different hormonal environment, warrant caution when extrapolating experimental data from studies conducted in invertebrates to mammalian species. The simplicity and accessibility of these models, however, offer ample opportunities for studying age-related myopathologies as well as investigating drugs and therapies to alleviate them.
Keywords: Muscle; Ageing; Drosophila melanogaster; Caenorhabditis elegans; Motor unit; Excitation–contraction coupling; Neuromuscular junction;

Lens aging: Effects of crystallins by K. Krishna Sharma; Puttur Santhoshkumar (1095-1108).
The primary function of the eye lens is to focus light on the retina. The major proteins in the lens—α, β, and γ-crystallins—are constantly subjected to age-related changes such as oxidation, deamidation, truncation, glycation, and methylation. Such age-related modifications are cumulative and affect crystallin structure and function. With time, the modified crystallins aggregate, causing the lens to increasingly scatter light on the retina instead of focusing light on it and causing the lens to lose its transparency gradually and become opaque. Age-related lens opacity, or cataract, is the major cause of blindness worldwide. We review deamidation, and glycation that occur in the lenses during aging keeping in mind the structural and functional changes that these modifications bring about in the proteins. In addition, we review proteolysis and discuss recent observations on how crystallin fragments generated in vivo, through their anti-chaperone activity may cause crystallin aggregation in aging lenses. We also review hyperbaric oxygen treatment induced guinea pig and ‘humanized’ ascorbate transporting mouse models as suitable options for studies on age-related changes in lens proteins.
Keywords: Lens crystallin; Aging; Lens opacity; Chaperone; Deamidation; Glycation; Oxidation; Peptide;

The role of advanced glycation end products in retinal ageing and disease by Josephine V. Glenn; Alan W. Stitt (1109-1116).
The retina is exposed to a lifetime of potentially damaging environmental and physiological factors that make the component cells exquisitely sensitive to age-related processes. Retinal ageing is complex and a raft of abnormalities can accumulate in all layers of the retina. Some of this pathology serves as a sinister preamble to serious conditions such as age-related macular degeneration (AMD) which remains the leading cause of irreversible blindness in the Western world.The formation of advanced glycation end products (AGEs) is a natural function of ageing but accumulation of these adducts also represents a key pathophysiological event in a range of important human diseases. AGEs act as mediators of neurodegeneration, induce irreversible changes in the extracellular matrix, vascular dysfunction and pro-inflammatory signalling. Since many cells and tissues of the eye are profoundly influenced by such processes, it is fitting that advanced glycation is now receiving considerable attention as a possible pathogenic factor in visual disorders.This review presents the current evidence for a pathogenic role for AGEs and activation of the receptor for AGEs (RAGE) in initiation and progression of retinal disease. It draws upon the clinical and experimental literature and highlights the opportunities for further research that would definitively establish these adducts as important instigators of retinal disease. The therapeutic potential for novel agents that can ameliorate AGE formation of attenuate RAGE signalling in the retina is also discussed.
Keywords: Ageing; Advanced glycation end product; RPE; AMD;

Role of visceral adipose tissue in aging by Derek M. Huffman; Nir Barzilai (1117-1123).
Visceral fat (VF) accretion is a hallmark of aging in humans. Epidemiologic studies have implicated abdominal obesity as a major risk factor for insulin resistance, type 2 diabetes, cardiovascular disease, metabolic syndrome and death.Studies utilizing novel rodent models of visceral obesity and surgical strategies in humans have been undertaken to determine if subcutaneous (SC) abdominal or VF are causally linked to age-related diseases.Specific depletion or expansion of the VF depot using genetic or surgical tools in rodents has been shown to have direct effects on disease risk. In contrast, surgically removing large quantities of SC fat does not consistently improve metabolic parameters in humans or rodents, while benefits were observed with SC fat expansion in mice, suggesting that SC fat accrual is not an important contributor to metabolic decline. There is also compelling evidence in humans that abdominal obesity is a stronger risk factor for mortality risk than general obesity. Likewise, we have shown that surgical removal of VF improves mean and maximum lifespan in rats, providing the first causal evidence that VF depletion may be an important underlying cause of improved lifespan with caloric restriction.This review provides both corollary and causal evidence for the importance of accounting for body fat distribution, and specifically VF, when assessing disease and mortality risk. Given the hazards of VF accumulation on health, treatment strategies aimed at selectively depleting VF should be considered as a viable tool to effectively reduce disease risk in humans.
Keywords: Visceral fat; Longevity; Obesity; Metabolic syndrome; Restriction;

Late-onset Alzheimer disease is the most common form of dementia and is strongly associated with age. Today, around 24 million people suffer from dementia and with aging of industrial populations this number will significantly increase throughout the next decades. An effective therapy that successfully decelerates or prevents the progressive neurodegeneration does not exist. Histopathologically Alzheimer disease is characterized by extensive extracellular amyloid β (Aβ) plaques, intracellular neurofibrillary tangles (NFTs), synaptic loss and neuronal cell death in distinct brain regions. The molecular correlation of Aβ or NFTs and development of late-onset Alzheimer disease needs further clarification. This review focuses on structural and functional alterations of the brain during aging, age-associated imbalances of defences against oxidative stress and age-related alterations of the metabolism of Aβ, via a comparison of observations in healthy aged individuals and cognitively impaired or AD patients. Although our understanding of brain region-specific neuronal aging is still incomplete, the early structural and molecular changes in the transition from cognitive health to impairment are subtle and the actual factors triggering the severe brain atrophy during LOAD remain ambiguous.
Keywords: Alzheimer disease; Aging; Amyloid β;

Population aging is progressing rapidly in many industrialized countries. The United States population aged 65 and over is expected to double in size within the next 25 years. In sedentary people eating Western diets aging is associated with the development of serious chronic diseases, including type 2 diabetes mellitus, cancer and cardiovascular diseases. About 80% of adults over 65 years of age have at least one chronic disease, and 50% have at least two chronic diseases. These chronic diseases are the most important cause of illness and mortality burden, and they have become the leading driver of healthcare costs, constituting an important burden for our society. Data from epidemiological studies and clinical trials indicate that many age-associated chronic diseases can be prevented, and even reversed, with the implementation of healthy lifestyle interventions. Several recent studies suggest that more drastic interventions (i.e. calorie restriction without malnutrition and moderate protein restriction with adequate nutrition) may have additional beneficial effects on several metabolic and hormonal factors that are implicated in the biology of aging itself. Additional studies are needed to understand the complex interactions of factors that regulate aging and age-associated chronic disease.
Keywords: Aging; Chronic disease; Calorie restriction; Physical exercise; Prevention;

HAX-1: A multifunctional protein with emerging roles in human disease by Bengt Fadeel; Ewa Grzybowska (1139-1148).
HS-1-associated protein X-1 (HAX-1) was identified more than 10 years ago as a novel protein with ubiquitous tissue expression and a predominantly mitochondrial localization at the subcellular level. Recent studies have shown that homozygous mutations in the HAX1 gene are associated with autosomal recessive forms of severe congenital neutropenia (also known as Kostmann disease), and results from studies in mice and men are beginning to unravel a prominent role for HAX-1 in apoptosis signaling not only in the hematopoietic compartment, but also in the central nervous system. Moreover, several different cellular and viral binding partners of HAX-1 have been identified thus pointing toward a complex and multifunctional role of this protein. HAX-1 has also been shown to bind to the 3′ untranslated regions of certain mRNAs and could therefore contribute to the regulation of transport and/or stability of such transcripts. The present review discusses the emerging and divergent roles of HAX-1, including its involvement in cell migration, apoptosis signaling, and mRNA surveillance. The importance of HAX-1 in human disease is also highlighted and outstanding questions that remain to be addressed are identified.
Keywords: Apoptosis; Cancer; Cell migration; Neutropenia; Splice variant;

Alpha-lipoic acid as a dietary supplement: Molecular mechanisms and therapeutic potential by Kate Petersen Shay; Régis F. Moreau; Eric J. Smith; Anthony R. Smith; Tory M. Hagen (1149-1160).
Alpha-lipoic acid (LA) has become a common ingredient in multivitamin formulas, anti-aging supplements, and even pet food. It is well-defined as a therapy for preventing diabetic polyneuropathies, and scavenges free radicals, chelates metals, and restores intracellular glutathione levels which otherwise decline with age. How do the biochemical properties of LA relate to its biological effects? Herein, we review the molecular mechanisms of LA discovered using cell and animal models, and the effects of LA on human subjects. Though LA has long been touted as an antioxidant, it has also been shown to improve glucose and ascorbate handling, increase eNOS activity, activate Phase II detoxification via the transcription factor Nrf2, and lower expression of MMP-9 and VCAM-1 through repression of NF-kappa B. LA and its reduced form, dihydrolipoic acid, may use their chemical properties as a redox couple to alter protein conformations by forming mixed disulfides. Beneficial effects are achieved with low micromolar levels of LA, suggesting that some of its therapeutic potential extends beyond the strict definition of an antioxidant. Current trials are investigating whether these beneficial properties of LA make it an appropriate treatment not just for diabetes, but also for the prevention of vascular disease, hypertension, and inflammation.
Keywords: Alpha-lipoic acid; Antioxidant; Aging; Glutathione; Diabetes; Dietary supplement;

L-cysteine supplementation attenuates local inflammation and restores gut homeostasis in a porcine model of colitis by C.J. Kim; J. Kovacs-Nolan; C. Yang; T. Archbold; M.Z. Fan; Y. Mine (1161-1169).
Inflammatory bowel disease (IBD), a chronic inflammation of the gastrointestinal tract, is characterized by a deregulation of the mucosal immune system and resistance of activated T cells to apoptosis. Current therapeutics show limited efficacy and potential toxicity; therefore there is a need for novel approaches for the treatment of IBD. L-cysteine was examined for its ability to reduce colitis symptoms and modulate local gene expression in a DSS-induced porcine model of colitis.Colitis was induced via intra-gastric infusion of dextran sodium sulfate (DSS), followed by the administration of L-cysteine or saline. Clinical signs, morphological measurements, histology and gut permeability were assessed for the prognosis of colitis. Local tissue production of cytokines and gene expression in the colon were analyzed by ELISA and real-time RT-PCR.L-cysteine supplementation attenuated DSS-induced weight loss and intestinal permeability, reduced local chemokine expression and neutrophil influx, and markedly improved colon histology. Furthermore, cysteine significantly reduced the expression of pro-inflammatory cytokines, including TNF-α, IL-6, IL-12p40, IL-1β, and resulted in increased expression of the apoptosis initiator caspase-8 and decreased expression of the pro-survival genes cFLIP and Bcl-xL.These results suggest that L-cysteine administration aids in restoring gut immune homeostasis by attenuating inflammatory responses and restoring susceptibility of activated immune cells to apoptosis, and that cysteine supplementation may be a novel therapeutic strategy for the treatment of IBD.
Keywords: Inflammatory bowel disease (IBD); Dextran sodium sulfate (DSS); L-cysteine; Inflammation; Cytokine; Apoptosis;

Reduction of gene expression by a hairpin-loop structured oligodeoxynucleotide: Alternative to siRNA and antisense by Terry Kwok; Jochen Heinrich; Jiunshan Jung-Shiu; Michelle G. Meier; Srikanth Mathur; Karin Moelling (1170-1178).
We previously described the inhibition of HIV-1 replication by a 54-mer hairpin-loop structured oligodeoxynucleotide (ODN) A, which binds the polypurine tract (PPT) on HIV-1 RNA. ODN A was shown to lead to reduced viral RNA in virions or early during infection.Here we demonstrated that ODN A was able to cause hydrolysis of viral RNA not only by retroviral RT-associated RNase H but also cellular RNase H1 and RNase H2 in vitro. Furthermore, ODN A reduced gene expression in a dose-dependent manner in a cell-based reporter assay where a PPT sequence was inserted in the 5′ untranslated region of the reporter gene. The efficacy of ODN A was higher than that of its siRNA and antisense counterparts. By knocking down cellular RNases H, we showed that RNase H1 contributed to the gene silencing by ODN A but the possibility of a partial contribution of RNase H-independent mechanisms could not be ruled out.Our findings highlight the potential application of hairpin-loop structured ODNs for reduction of gene expression in mammalian cells and underscore the possibility of using ODN A to trigger the hydrolysis of HIV RNA in infected cells by cellular RNases H.
Keywords: Gene silencing; Reverse transcriptase-RNase H; Polypurine tract; siRNA; Antisense;

We have shown that protein kinase Cδ (PKCδ) inhibition results in increased endothelial cell (EC) permeability and decreased RhoA activity; which correlated with diminished stress fibers (SF) and focal adhesions (FA). We have also shown co-precipitation of p190RhoGAP (p190) with PKCδ. Here, we investigated if PKCδ regulates p190 and whether PKCδ-mediated changes in SF and FA or permeability were dependent upon p190.Protein–protein interaction and activity analyses were performed using co-precipitation assays. Analysis of p190 phosphorylation was performed using in vitro kinase assays. SF and FA were analyzed by immunofluorescence analyses. EC monolayer permeability was measured using electrical cell impedance sensor (ECIS) technique.Inhibition of PKCδ increased p190 activity, while PKCδ overexpression diminished p190 activity. PKCδ bound to and phosphorylated both p190FF and p190GTPase domains. p190 protein overexpression diminished SF and FA formation and RhoA activity. Disruption of SF and FA or increased permeability induced upon PKCδ inhibition, were not attenuated in EC in which the p190 isoforms were suppressed individually or concurrently.Our findings suggest that while PKCδ can regulate p190 activity, possibly at the FF and/or GTPase domains, the effect of PKCδ inhibition on SF and FA and barrier dysfunction occurs through a pathway independent of p190.
Keywords: PKC; p190RhoGAP; Endothelium; Permeability; Focal adhesions; Stress fibers;

Modulation of thioredoxin reductase-2 expression in EAhy926 cells: Implications for endothelial selenoprotein hierarchy by Michael S. Crane; Alexander F. Howie; John R. Arthur; Fergus Nicol; Lynne K. Crosley; Geoffrey J. Beckett (1191-1197).
We examined the expression of the mitochondrial selenoenzyme TrxR2 in the endothelial cell line EAhy926 under conditions known to modify its cytoplasmic counterpart TrxR1.Cells were cultured with varying concentrations of selenite, sulforaphane or the Ca2+ ionophore A23187 for 72-h, prior to assay of TrxR concentration and activity. Further cultures underwent prolonged (7-day) Se-depletion before selenoprotein measurement.In Se-deficient cultures, neither Se, A23187 or sulforaphane affected TrxR2 concentration, while these treatments induced TrxR1 concentration (p  < 0.05). When co-incubated, optimal concentrations of Se (40 nM) and sulforaphane (4 μM) only modestly increased TrxR2 protein (∼ 1.3-fold), compared with TrxR1 (∼ 4-fold). In Se-deficient cells, TrxR activity was unaffected by sulforaphane or A23187. Prolonged Se-depletion caused a comparatively small reduction in TrxR2 (66% TrxR2 retained) against TrxR1 and glutathione peroxidase-1 activity (38% and 17% retained, respectively).The relative resistance of TrxR2 to Se-deprivation and induction by sulforaphane and A23187 suggests TrxR2 lies near the top of the selenoprotein hierarchy in EAhy926 cells and exhibits near maximum expression under a range of culture conditions. In Se deficiency an inactive (possibly truncated) TrxR1 is produced in response to stimulus by sulforaphane and A23187.These observations underpin a likely critical antioxidant role for TrxR2 and TrxR1 in the endothelium.
Keywords: Mitochondrial thioredoxin reductase; Endothelium; Selenium; Sulforaphane; Selenoprotein hierarchy;

The glycoproteins on the cell surface are altered during apoptosis and play an important role in phagocytic clearance of apoptotic cells.We classified Jurkat cells treated with etoposide as viable and early apoptotic cells, late apoptotic cells or secondary necrotic cells based on propidium iodide staining and scattered grams and estimated the expression levels of glycoproteins on the cell surface.The cell surface expression levels of intercellular adhesion molecules (ICAM)-2 and -3 on the apoptotic cells were markedly lower, while those of calnexin, calreticulin, and lysosome-associated membrane proteins (LAMP)-1 and -2 were significantly higher compared to non-apoptotic cells. These decreases in ICAM-2 and -3 on the apoptotic cell surface were reduced in the presence of metalloproteinase inhibitors and caspase inhibitors, respectively. Confocal microscopic analysis revealed that calnexin and calreticulin were assembled around fragmented nuclei of blebbed apoptotic cells.These results suggest that alteration of glycoproteins on the cell surface during apoptosis is associated with shedding and intracellular translocation of glycoproteins.
Keywords: Apoptosis; ICAM; Calnexin; LAMP; Metalloproteinase; Intracellular translocation;

Mouse carnitine–acylcarnitine translocase (CACT) is transcriptionally regulated by PPARα and PPARδ in liver cells by Anke Gutgesell; Gaiping Wen; Bettina König; Alexander Koch; Julia Spielmann; Gabriele I. Stangl; Klaus Eder; Robert Ringseis (1206-1216).
Hepatic PPARα acts as the primary mediator of the adaptive response to fasting by upregulation of a number of genes involved in fatty acid catabolism. Whether carnitine–acylcarnitine translocase (CACT), which mediates the import of acylcarnitines into the mitochondrial matrix for subsequent β-oxidation of fatty acid moieties, is also regulated by PPARα in the liver has not yet been investigated.Herein, we observed that hepatic mRNA abundance of CACT was increased by both, fasting and treatment with PPARα agonist WY-14,643 in wild-type mice but not PPARα-knockout mice (P  < 0.05). Cell culture experiments revealed that CACT mRNA abundance was higher in liver cells treated with either WY-14,643 or PPARδ agonist GW0742, but not with PPARγ agonist troglitazone (TGZ) than in control cells (P  < 0.05). In addition, reporter assays revealed activation of mouse CACT promoter by WY-14,643 and GW0742, but not TGZ. Moreover, deletion and mutation analyses of CACT promoter and 5′-UTR revealed one functional PPRE in the 5′-UTR of mouse CACT. CACT is upregulated by PPARα and PPARδ, probably by binding to a functional PPRE at position + 45 to + 57 relative to the transcription start site. The upregulation of CACT by PPARα and PPARδ, which are both important for the regulation of fatty acid oxidation in tissues during fasting, may increase the import of acylcarnitine into the mitochondrial matrix during fasting.
Keywords: Carnitine–acylcarnitine translocase (CACT); Peroxisome proliferator-activated receptor α (PPARα); Liver; Fasting;

Analyses of non-leucine-rich repeat (non-LRR) regions intervening between LRRs in proteins by Norio Matsushima; Tomoko Mikami; Takanori Tanaka; Hiroki Miyashita; Keiko Yamada; Yoshio Kuroki (1217-1237).
Many proteins have LRR (leucine-rich repeat) units interrupted by non-LRRs which we call IR (non-LRR island region).We identified proteins containing LRR@IRs (LRRs having IR) by using a new method and then analyzed their natures and distributions.LRR@IR proteins were found in over two hundred proteins from prokaryotes and from eukaryotes. These are divided into twenty-one different protein families. The IRs occur one to four times in LRR regions and range in length from 5 to 11,265 residues. The IR lengths in Fungi adenylate cyclases (acys) range from 5 to 116 residues; there are 22 LRR repeats. The IRs in Leishmania proteophosphoglycans (ppgs) vary from 105 to 11,265 residues. These results indicate that the IRs evolved rapidly. A group of LRR@IR proteins—LRRC17, chondroadherin-like protein, ppgs, and four Pseudomonas proteins—have a super motif consisting of an LRR block and its adjacent LRR@IR region. This indicates that the entire super motif experienced duplication. The sequence analysis of IRs offers functional similarity in some LRR@IR protein families.This study suggests that various IRs and super motifs provide a great variety of structures and functions for LRRs.
Keywords: Leucine-rich repeat; Island region; Fungi adenylate cyclase; Leishmania proteophosphoglycans; Intrinsically disordered region; Rapid evolution; Super motif; Duplication;

Oligomerization of Bacillus subtilis DesR is required for fine tuning regulation of membrane fluidity by Sebastián R. Najle; María E. Inda; Diego de Mendoza; Larisa E. Cybulski (1238-1243).
The DesK-DesR two-component system regulates the order of membrane lipids in the bacterium Bacillus subtilis by controlling the expression of the des gene coding for the delta 5-acyl-lipid desaturase. To activate des transcription, the membrane-bound histidine kinase DesK phosphorylates the response regulator DesR. This covalent modification of the regulatory domain of dimeric DesR promotes, in a cooperative fashion, the hierarchical occupation of two adjacent, non-identical, DesR-P binding sites, so that there is a shift in the equilibrium toward the tetrameric active form of the response regulator. However, the mechanism of regulation of DesR activity by phosphorylation and oligomerization is not well understood.We employed deletion analysis and reporter fusions to study the role of the N-terminal domain on DesR activity. In addition, electromobility shift assays were used to analyze the binding capacity of the transcription factor to deletion mutants of the des promoter.We show that DesR lacking the N-terminal domain is still able to bind to the des promoter. We also demonstrate that if the RA site is moved closer to the − 35 region of Pdes, the adjacent site RB is dispensable for activation.Our results indicate that the unphosphorylated regulatory domain of DesR obstructs the access of the recognition helix of DesR to its DNA target. In addition, we present evidence showing that RB is physiologically relevant to control the activation of the des gene when the levels of DesR-P reach a critical threshold.
Keywords: Transcriptional regulation; Two-component system; Desaturase; Membrane fluidity;

The assembly of Ser/Thr-linked O-glycans of mucins with core 2 structures is initiated by polypeptide GalNAc-transferase (ppGalNAc-T), followed by the action of core 1 β3-Gal-transferase (C1GalT) and core 2 β6-GlcNAc-transferase (C2GnT). β4-Gal-transferase (β4GalT) extends core 2 and forms the backbone structure for biologically important epitopes. O-glycan structures are often abnormal in chronic diseases. The goal of this work is to determine if the activity and specificity of these enzymes are directed by the sequences and glycosylation of substrates.We studied the specificities of four enzymes that synthesize extended O-glycan core 2 using as acceptor substrates synthetic mucin derived peptides and glycopeptides, substituted with GalNAc or O-glycan core structures 1, 2, 3, 4 and 6.Specific Thr residues were found to be preferred sites for the addition of GalNAc, and Pro in the + 3 position was found to especially enhance primary glycosylation. An inverse relationship was found between the size of adjacent glycans and the rate of GalNAc addition. All four enzymes could distinguish between substrates having different amino acid sequences and O-glycosylated sites. A short glycopeptide Galβ1–3GalNAcα-TAGV was identified as an efficient C2GnT substrate.The activities of four enzymes assembling the extended core 2 structure are affected by the amino acid sequence and presence of carbohydrates on nearby residues in acceptor glycopeptides. In particular, the sequences and O-glycosylation patterns direct the addition of the first and second sugar residues by ppGalNAc-T and C1GalT which act in a site directed fashion.Knowledge of site directed processing enhances our understanding of the control of O-glycosylation in normal cells and in disease.
Keywords: Polypeptide GalNAc-transferase; Core 1 β3-Gal-transferase; Core 2 β6-GlcNAc-transferase; β4-Gal-transferase; O-glycopeptide; Specificity;

bFGF induces changes in hyaluronan synthase and hyaluronidase isoform expression and modulates the migration capacity of fibrosarcoma cells by Aikaterini Berdiaki; Dragana Nikitovic; Aristeidis Tsatsakis; Pavlos Katonis; Nikos K. Karamanos; George N. Tzanakakis (1258-1265).
Hyaluronan (HA) a glycosaminoglycan, is capable of transmitting extracellular matrix derived signals to regulate cellular functions. In this study, we investigated whether the changes in HT1080 and B6FS fibrosarcoma cell lines HA metabolism induced by basic fibroblast growth factor (bFGF) are correlated to their migration.Real-time PCR, in vitro wound healing assay, siRNA transfection, enzyme digestions, western blotting and immunofluorescence were utilized.bFGF inhibited the degradation of HA by decreasing hyaluronidase-2 expression in HT1080 cells (p  = 0.0028), increased HA-synthase-1 and -2 expression as we previously found and enhanced high molecular weight HA deposition in the pericellular matrix. Increased endogenous HA production (p  = 0.0022) and treatment with exogenous high molecular weight HA (p  = 0.0268) correlated with a significant decrease of HT1080 cell migration capacity. Transfection with siHAS2 and siHAS1 showed that mainly HAS1 synthesized high molecular weight HA regulates HT1080 cell motility. Induced degradation of the HA content by hyaluronidase treatment and addition of low molecular weight HA, resulted in a significant stimulation of HT1080 cells' motility (p  < 0.01). In contrast, no effects on B6FS fibrosarcoma cell motility were observed.bFGF regulates, in a cell-specific manner the migration capability of fibrosarcoma cells by modulating their HA metabolism.HA metabolism is suggested to be a potential therapeutic target in fibrosarcoma.
Keywords: Fibrosarcoma; Hyaluronan; Basic fibroblast growth factor; Hyaluronan synthase; Hyaluronidase;

Identification and characterization of retinoid-active short-chain dehydrogenases/reductases in Drosophila melanogaster by Olga V. Belyaeva; Seung-Ah Lee; Oleg V. Kolupaev; Natalia Y. Kedishvili (1266-1273).
In chordates, retinoid metabolism is an important target of short-chain dehydrogenases/reductases (SDRs). It is not known whether SDRs play a role in retinoid metabolism of protostomes, such as Drosophila melanogaster. Drosophila genome was searched for genes encoding proteins with ∼ 50% identity to human retinol dehydrogenase 12 (RDH12). The corresponding proteins were expressed in Sf9 cells and biochemically characterized. Their phylogenetic relationships were analyzed using PHYLIP software.A total of six Drosophila SDR genes were identified. Five of these genes are clustered on chromosome 2 and one is located on chromosome X. The deduced proteins are 300 to 406 amino acids long and are associated with microsomal membranes. They recognize all-trans-retinaldehyde and all-trans-3-hydroxyretinaldehyde as substrates and prefer NADPH as a cofactor. Phylogenetically, Drosophila SDRs belong to the same branch of the SDR superfamily as human RDH12, indicating a common ancestry early in bilaterian evolution, before a protostome–deuterostome split.Similarities in the substrate and cofactor specificities of Drosophila versus human SDRs suggest conservation of their function in retinoid metabolism throughout protostome and deuterostome phyla.The discovery of Drosophila retinaldehyde reductases sheds new light on the conversion of β-carotene and zeaxantine to visual pigment and provides a better understanding of the evolutionary roots of retinoid-active SDRs.
Keywords: Retinaldehyde; Retinol; Dehydrogenase; Reductase; Drosophila; Visual pigment;

Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca2+ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca2+/Mg2+ binding to human scramblases and conformational changes taking place in them remains unknown.In the present study, we analyzed the Ca2+ and Mg2+ binding to the calcium binding motifs of hPLSCR1–4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry.The results in this study show that (i) affinities of the peptides are in the order hPLSCR1   > hPLSCR3 > hPLSCR2 > hPLSCR4 for Ca2+ and in the order hPLSCR1 > hPLSCR2 > hPLSCR3 > hPLSCR4 for Mg2+, (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca2+ and Mg2+ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families.Based on the above results, we hypothesize that the Ca2+ binding motif of hPLSCR1 is a novel type of Ca2+ binding motif.Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function.
Keywords: Scramblase; Peptides; Phospholipids; Calcium binding; Tryptophan fluorescence; Isothermal titration calorimetry;

The twin phenomena of aggregation and degradation are classically associated with protein storage. However, although aggregation has been thought to be a possible consequence of protein degradation, it has never before been proposed to be a cause of degradation.Proteins stored under physiological conditions and electrophoresed on SDS-PAGE were examined zymographically for the presence of detergent-resistant high molecular weight (HMW) forms, and association of such HMW forms with time-correlated, seeding-dependent gelatinolytic activity, under various conditions.Eight different proteins aggregate naturally during storage at near-neutral pH, with concomitant development of ‘gelatinolytic’ activity diminished greatly by storage at low temperatures, extremes of pH, arginine, imidazole, BSA, azide, EDTA, DTT, PMSF (but not AEBSF), and diisopropyl fluorophosphate (DFP), suggesting involvement of surface serine residues in a novel aggregate-borne proteolytic activity.Naturally-formed aggregates of proteins appear to use surface serines to perform peptide bond hydrolysis, explaining degradation of proteins during storage, and indicating why aggregates are cytotoxic.The study suggests that a bi-directional cause–effect relationship operates between protein aggregation, and protein degradation, providing clues to the design of better conditions for long-term protein storage.
Keywords: Protein aggregation; Protein degradation; Proteolysis; Gelatinolysis; Aggregate-borne gelatinolytic activity;

Identification and biochemical analysis of a homolog of a sulfate transporter from a vanadium-rich ascidian Ascidia sydneiensis samea by Tatsuya Ueki; Nobuaki Furuno; Qiang Xu; Yuya Nitta; Kan Kanamori; Hitoshi Michibata (1295-1300).
Several species of ascidians accumulate extremely high levels of vanadium ions in the vacuoles of their blood cells (vanadocytes). The vacuoles of vanadocytes also contain many protons and sulfate ions. To maintain the concentration of sulfate ions, an active transporter must exist in the blood cells, but no such transporter has been reported in vanadium-accumulating ascidians.We determined the concentration of vanadium and sulfate ions in the blood cells (except for the giant cells) of Ascidia sydneiensis samea. We cloned cDNA for an Slc13-type sulfate transporter, AsSUL1, expressed in the vanadocytes of A. sydneiensis samea. The synthetic mRNA of AsSUL1 was introduced into Xenopus oocytes, and its ability to transport sulfate ions was analyzed.The concentrations of vanadium and sulfate ions in the blood cells (except for the giant cells) were 38 mM and 86 mM, respectively. The concentration of sulfate ions in the blood plasma was 25 mM. The transport activity of AsSUL1 was dependent on sodium ions, and its maximum velocity and apparent affinity were 2500 pmol/oocyte/h and 1.75 mM, respectively.This could account for active uptake of sulfate ions from blood plasma where sulfate concentration is 25 mM, as determined in this study.
Keywords: Vanadium; Ascidian; Sulfate; Vacuole; Transporter;

Xylanases (EC hydrolyze xylan, one of the most abundant plant polysaccharides found in nature, and have many potential applications in biotechnology.Molecular dynamics simulations were used to investigate the effects of temperature between 298 to 338 K and xylobiose binding on residues located in the substrate-binding cleft of the family 11 xylanase from Bacillus circulans (BcX).In the absence of xylobiose the BcX exhibits temperature dependent movement of the thumb region which adopts an open conformation exposing the active site at the optimum catalytic temperature (328 K). In the presence of substrate, the thumb region restricts access to the active site at all temperatures, and this conformation is maintained by substrate/protein hydrogen bonds involving active site residues, including hydrogen bonds between Tyr69 and the 2′ hydroxyl group of the substrate. Substrate access to the active site is regulated by temperature dependent motions that are restricted to the thumb region, and the BcX/substrate complex is stabilized by extensive intermolecular hydrogen bonding with residues in the active site.These results call for a revision of both the “hinge-bending” model for the activity of group 11 xylanases, and the role of Tyr69 in the catalytic mechanism.
Keywords: Enzyme–substrate interaction; Xylanase; Thermostability;

Proton motive force dissipation precludes interaction of microcin J25 with RNA polymerase, but enhances reactive oxygen species overproduction by Fernando G. Dupuy; María V. Niklison Chirou; Beatriz Fernández de Arcuri; Carlos J. Minahk; Roberto D. Morero (1307-1313).
Microcin J25 targets the RNA polymerase as well as bacterial membranes. Because there is scarce information on the relationship between the uptake and the activity, a fluorescent microcin J25-derivative was used to further characterize its mechanism of action.MccJ25 I13K was labeled with FITC and its uptake by sensitive cells was assessed by fluorescence measurements from supernatants of MccJ25-Escherichia coli suspensions. The interaction of the peptide with bacterial membranes was investigated by fluorescence resonance energy transfer. Oxygen consumption was measured with Clark-type electrode. RNA synthesis was evaluated in vivo by incorporation of [3H]uridine. ROS production was assayed by measuring the fluorescence emission of the ROS-sensitive probe 5(and 6)-carboxy-2’,7’-dichlorodihydrofluorescein diacetate.The protonophore 2,4-dinitrophenol decreased 80% of the MccJ25 uptake and prevented inhibition of transcriptional activity, the antibiotic intracellular target. On the other hand, peptide binding to bacterial membranes was not affected and antibacterial activity remained nearly unchanged. Proton gradient dissipation by protonophore accelerated cell oxygen consumption rates and enhanced MccJ25-related reactive oxygen species overproduction.The deleterious reactive oxygen species would be produced as a consequence of the minor fraction of MccJ25 that interacts with the bacterial plasma membrane from the periplasmic side. These results show the first evidence of the mechanism underlying ROS production in sensitive bacteria.
Keywords: Microcin; Reactive oxygen species; 2,4 DNP; Peptide uptake; E. coli;

Syndecan-1 and syndecan-4 are involved in RANTES/CCL5-induced migration and invasion of human hepatoma cells by Faten Charni; Veronique Friand; Oualid Haddad; Hanna Hlawaty; Loïc Martin; Roger Vassy; Olivier Oudar; Liliane Gattegno; Nathalie Charnaux; Angela Sutton (1314-1326).
We previously demonstrated that the CC-chemokine Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES)/CCL5 exerts pro-tumoral effects on human hepatoma Huh7 cells through its G protein-coupled receptor, CCR1. Glycosaminoglycans play major roles in these biological events.In the present study, we explored 1/ the signalling pathways underlying RANTES/CCL5-mediated hepatoma cell migration or invasion by the use of specific pharmacological inhibitors, 2/ the role of RANTES/CCL5 oligomerization in these effects by using a dimeric RANTES/CCL5, 3/ the possible involvement of two membrane heparan sulfate proteoglycans, syndecan-1 (SDC-1) and syndecan-4 (SDC-4) in RANTES/CCL5-induced cell chemotaxis and spreading by pre-incubating cells with specific antibodies or by reducing SDC-1 or -4 expression by RNA interference.The present data suggest that focal adhesion kinase phosphorylation, phosphoinositide 3-kinase-, mitogen-activated protein kinase- and Rho kinase activations are involved in RANTES/CCL5 pro-tumoral effects on Huh7 cells. Interference with oligomerization of the chemokine reduced RANTES/CCL5-mediated cell chemotaxis. This study also indicates that SDC-1 and -4 may be required for HepG2, Hep3B and Huh7 human hepatoma cell migration, invasion or spreading induced by the chemokine. These results also further demonstrate the involvement of glycosaminoglycans as the glycosaminoglycan-binding deficient RANTES/CCL5 variant, in which arginine 47 was replaced by lysine, was devoid of effect.The modulation of RANTES/CCL5-mediated cellular effects by targeting the chemokine-syndecan interaction could represent a new therapeutic approach for hepatocellular carcinoma.
Keywords: Chemokine; Syndecan; Hepatocellular carcinoma; RANTES/CCL5; Chemotaxis; Spreading;

Characterization of vanadium-binding sites of the vanadium-binding protein Vanabin2 by site-directed mutagenesis by Tatsuya Ueki; Norifumi Kawakami; Masaaki Toshishige; Koichi Matsuo; Kunihiko Gekko; Hitoshi Michibata (1327-1333).
Vanabins are a unique protein family of vanadium-binding proteins with nine disulfide bonds. Possible binding sites for VO2+ in Vanabin2 from a vanadium-rich ascidian Ascidia sydneiensis samea have been detected by nuclear magnetic resonance study, but the metal selectivity and metal-binding ability of each site was not examined.In order to reveal functional contribution of each binding site, we prepared several mutants of Vanabin2 by in vitro site-directed mutagenesis and analyzed their metal selectivity and affinity by immobilized metal-ion affinity chromatography and Hummel Dreyer method.Mutation at K10/R60 (site 1) markedly reduced the affinity for VO2+. Mutation at K24/K38/R41/R42 (site 2) decreased the maximum binding number, but only slightly increased the overall affinity for VO2+. Secondary structure of both mutants was the same as that of the wild type as assessed by circular dichroism spectroscopy. Mutation in disulfide bonds near the site 1 did not affect its high affinity binding capacity, while those near the site 2 decreased the overall affinity for VO2+.These results suggested that the site 1 is a high affinity binding site for VO2+, while the site 2 composes a moderate affinity site for multiple VO2+.
Keywords: Ascidian; Vanadium; Metal-binding protein;

Characterization of ordered aggregates of cerato-platanin and their involvement in fungus–host interactions by Luigia Pazzagli; Camilla Zoppi; Lara Carresi; Bruno Tiribilli; Francesca Sbrana; Silvia Schiff; Thelma A. Pertinhez; Aniello Scala; Gianni Cappugi (1334-1344).
The “cerato-platanin family” consists of fungal-secreted proteins that are involved in various stages of the host–fungus interaction and act as phytotoxins, elicitors of defense responses and allergens. Cerato-platanin (CP) is a moderately hydrophobic protein secreted and localized in the cell wall of Ceratocystis platani, the causal agent of a severe disease of Platanus. These properties make CP like the hydrophobins: these are self-assembling proteins that form a surface coating which is involved in the formation of aerial hyphae and in adherence to surfaces.CP aggregation was monitored by ThT, circular dichroism, and AFM. The eliciting activity of CP aggregates was assayed on leaves and cells.The CP self-assembles forming amyloid-like aggregates via a nucleated growth mechanism which is joined up with a cleavage of the N-terminus. The ovoidal shape and the lack of a clear transition toward an all-β structure distinguish these aggregates from typical amyloid fibrils. Moreover, CP aggregates interact with hydrophobic surfaces and enhance the hypersensitive response of Platanus.CP forms “ordered aggregates” for which the soluble prefibrillar structures are the end point of the aggregation process, and do not evolve to insoluble fibrils. An involvement in host–microbe interaction is also suggested.
Keywords: Amyloid-like assemblage; Cerato-platanin family; Fungal protein; Host–microbe interaction; Hydrophobin; Protein aggregation;

Requirement of the SH4 and tyrosine-kinase domains but not the kinase activity of Lyn for its biosynthetic targeting to caveolin-positive Golgi membranes by Kikuko Ikeda; Yuji Nakayama; Mayuko Ishii; Yuuki Obata; Kousuke Kasahara; Yasunori Fukumoto; Naoto Yamaguchi (1345-1352).
The Src-family non-receptor-type tyrosine kinase Lyn, which is often associated with chemotherapeutic resistance in cancer, localizes not only to the plasma membrane but also Golgi membranes. Recently, we showed that Lyn, which is synthesized in the cytosol, is transported from the Golgi to the plasma membrane along the secretory pathway. However, it is still unclear how Golgi targeting of newly synthesized Lyn is regulated.Subcellular localization of Lyn and its mutants was determined by confocal microscopy.We show that the kinase domain, but not the SH3 and SH2 domains, of Lyn is required for the targeting of Lyn to the Golgi, whereas the N-terminal lipids of the Lyn SH4 domain are not sufficient for its Golgi targeting. Although intact Lyn, which colocalizes with caveolin-positive Golgi membranes, can traffic toward the plasma membrane, kinase domain-deleted Lyn is immobilized on caveolin-negative Golgi membranes.Besides the SH4 domain, the Lyn kinase domain is important for targeting of newly synthesized Lyn to the Golgi, especially caveolin-positive transport membranes. Our results provide a novel role of the Lyn catalytic domain in the Golgi targeting of newly synthesized Lyn in a manner independent of its kinase activity.
Keywords: Golgi caveolin; Kinase domain; Lipid modification; Lyn; Src-family tyrosine kinase; Targeting;

Differential effect of molecular size HA in mouse chondrocytes stimulated with PMA by Giuseppe M. Campo; Angela Avenoso; Salvatore Campo; Angela D'Ascola; Paola Traina; Alberto Calatroni (1353-1367).
Hyaluronan (HA) fragments elicit the expression of inflammatory mediators through a mechanism involving the CD44 receptor. This study investigated the effects of HA at different molecular weights on PMA-induced inflammation in mouse chondrocytes.mRNA and related protein levels were measured for CD44, PKCδ, PKCɛ, TNF-α, IL-1β, MMP-13, and iNOS in chondrocytes, untreated or PMA treated, with and without the addition of HA. The level of NF-kB activation was also assayed.CD44, PKCδ, and PKCɛ mRNA expression resulted higher than controls in chondrocytes treated with PMA. PMA also induced NF-kB up-regulation and increased TNF-α, IL-1β, MMP-13, and iNOS expression. HA treatment produced different effects: low MW HA up-regulated CD44 expression, increased PKCδ and PKCɛ levels, and enhanced inflammation in untreated chondrocytes; while in PMA-treated cells it increased CD44, PKCδ, PKCɛ, NF-kB, TNF-α, IL-1β, MMP-13, and iNOS expression and enhanced the effects of PMA; medium MW HA did not exert action; high MW HA had no effect on untreated chondrocytes; however, it reduced PKCδ, PKCɛ, NF-kB activation and inflammation in PMA-stimulated cells. Specific CD44 blocking antibody was utilised to confirm CD44 as the target of HA modulation.These data suggest that HA via CD44 may modulate inflammation via its different molecular mass.
Keywords: CD44; Hyaluronan; Chondrocytes; Interleukins; Inflammation; PMA;

An NMR method for the determination of protein binding interfaces using TEMPOL-induced chemical shift perturbations by Jun Moriya; Masayoshi Sakakura; Yuji Tokunaga; R. Scott Prosser; Ichio Shimada (1368-1376).
The determination of protein–protein interfaces is of crucial importance to understand protein function and to guide the design of compounds. To identify protein–protein interface by NMR spectroscopy, 13C NMR paramagnetic shifts induced by freely diffusing 4-hydroxy-2, 2, 6, 6-tetramethyl-piperidine-1-oxyl (TEMPOL) are promising, because TEMPOL affects distinct 13C NMR chemical shifts of the solvent accessible nuclei belonging to proteins of interest, while 13C nuclei within the interior of the proteins may be distinguished by a lack of such shifts.We measured the 13C NMR paramagnetic shifts induced by TEMPOL by recording 13C–13C TOCSY spectra for ubiquitin in the free state and the complex state with yeast ubiquitin hydrolase1 (YUH1).Upon complexation of ubiquitin with YUH1, 13C NMR paramagnetic shifts associated with the protein binding interface were reduced by 0.05 ppm or more. The identified interfacial atoms agreed with the prior X-ray crystallographic data.The TEMPOL-induced 13C chemical shift perturbation is useful to determine precise protein–protein interfaces.The present method is a useful method to determine protein–protein interface by NMR, because it has advantages in easy sample preparations, simple data analyses, and wide applicabilities.
Keywords: 13C NMR; Paramagnetic shift; Protein–protein interaction; Surface analysis; TEMPOL;

Anticoagulant activity of a sulfated polysaccharide from the green alga Arthrospira platensis by Hatem Majdoub; Mohamed Ben Mansour; Frédéric Chaubet; Mohamed S. Roudesli; Raoui M. Maaroufi (1377-1381).
The polysaccharide of culture medium from Arthrospira platensis was extracted by ultrafiltration, partially characterized and assayed for anticoagulant activity.The crude polysaccharidic fraction was fractionated by anion exchange chromatography on DEAE-cellulose, subjected to acetate cellulose electrophoresis and characterized by physicochemical procedures. The anticoagulant effect of the ultrafiltrated polysaccharide was checked by several coagulation tests.Anion exchange chromatography revealed in the whole ultrafiltrated polysaccharidic fraction the occurrence of a sulfated spirulan-like component designated PUF2. The average molecular weight of PUF2 was determined by size exclusion chromatography combined with multi-angle light scattering (SEC-MALS) and viscosimetry and was 199 kDa and the sulfate content was 20% weight/dry weight. The physicochemical characterization indicated the occurrence of rhamnose (49.7%), galacturonic and glucuronic acid (32% of total sugar). The anticoagulant effect of this sulfated polysaccharide was mainly due to the potentiation of thrombin inhibition by heparin cofactor II and was 4-times higher than that of the porcine dermatan sulfate whereas it had no effect on anti-Xa activity.An ultrafiltrated sulfated polysaccharide, likely a calcium spirulan was obtained from the culture medium of A. platensis and showed an anticoagulant activity mediated by heparin cofactor II.Old culture medium of A. platensis may represent an important source for the spirulan-like PUF2 which was endowed with potentially useful anticoagulant properties whereas its obtention by ultrafiltration may represent an extraction procedure of interest.
Keywords: Anticoagulant activity; Arthrospira platensis; Calcium spirulan; Heparin cofactor II; Thrombin; Ultrafiltrated polysaccharide;

Antiprion action of new cyclodextrin analogues by Karl McEvoy; Hilary E.M. McMahon (1382-1386).
Prion disorders are characterised by the accumulation of a misfolded isoform (PrPSc) of the host encoded prion protein (PrPC). This paper examines the antiprion potential of cyclodextrin (CD) analogues and it identifies sulphated-β-cyclodextrin, with a half-maximal inhibitory concentration (IC50) of 2.4 μM, as having 31-fold greater antiprion activity than that previously reported for β-cyclodextrin (βCD).Scrapie infected cells were treated with a range of βCD analogues. This enabled a CD structure to antiprion activity analysis to be carried out. The metachromatic activity of each of the cyclodextrins was determined, this test is employed to mimic complexation of glycosaminogylcans to a cell membrane.Sulphated-βCD had an IC50 of 2.4 μM and it was the only CD found to have metachromatic activity. Its activity was equivalent to that of heparin and heparin sulphate, this may account for sulphated-βCD's superior antiprion action.In solution heparin can form a helical structure with a hydrophobic interior, the hydrophobic interior of cyclic CDs is vital for CD molecule encapsulation. The controlled CD structure, however, restricts degradation by human enzymes; consequently sulphated-CDs could be ideal candidates in the search for prion therapeutics. Sulphated-CDs may open up avenues for the treatment of TSEs.
Keywords: Cyclodextrin; Prion; Scrapie;