Clostridium tetani was discovered and identified as a Gram-positive anaerobic bacterium of the genus Clostridium, the possibility of turning its toxin into a valuable biological carrier to ameliorate neurodegenerative processes was inconceivable. However, the non-toxic carboxy-terminal fragment of the tetanus toxin heavy chain (fragment C) can be retrogradely transported to the central nervous system; therefore, fragment C has been used as a valuable biological carrier of neurotrophic factors to ameliorate neurodegenerative processes. More recently, the neuroprotective properties of fragment C have also been described in vitro and in vivo, involving the activation of Akt kinase and extracellular signal-regulated kinase (ERK) signaling cascades through neurotrophin tyrosine kinase (Trk) receptors. Although the precise mechanism of the molecular internalization of fragment C in neuronal cells remains unknown, fragment C could be internalized and translocated into the neuronal cytosol through a clathrin-mediated pathway dependent on proteins, such as dynamin and AP-2. In this review, the origins, molecular properties and possible signaling pathways of fragment C are reviewed to understand the biochemical characteristics of its intracellular and synaptic transport.When  Clostridial neurotoxin family comprises tetanus neurotoxin and seven distinct botulinum neurotoxins, which cause the diseases tetanus and botulism [Clostridial botulinum and Clostridial tetani produce the most potent toxins, botulinum and tetanus, due to their remarkable neurospecificity and their catalytic cleavage at low concentrations of neuronal substrates, which can oscillate between 10−12 and 10−13 M. The main difference between these toxins is in the intensity and duration of muscle paralysis. In fact, severe tetanus is characterized by violent and persistent spasms of the head, trunk and limb muscles [The botulism ,2. Regarin vitro and in vivo [The infectious nature of tetanus toxin was well documented before 1906, and the necessity of producing a tetanus antitoxin was decisive during the First World War . The pro in vivo ,5, and t in vivo . Additio in vivo . Supportdirect inhibition of motoneurons by impulses in Group I a afferent fibers of antagonistic muscles;inhibition by impulses in the Group I b afferent fibers from muscles of the same limb;inhibition of extensor motoneurons by impulses in Groups II and III muscle afferent fibers and in cutaneous afferent fibers; andinhibition of motoneurons following the activation of Renshaw cells by volleys in axon collaterals.These five forms of spinal inhibition were diminished and eventually abolished 8–10 h after injecting the toxin either peripherally into a mixed nerve trunk or directly into the spinal cord, which indicated that the toxin diffused slowly across the spinal cord, taking several hours to move 1 mm. From these results, it was also concluded that the toxin moved much faster longitudinally in nerve trunks and within the spinal cord .The nature of the action of tetanus toxin has been widely described in different animal models –15, explTetanus toxin is a single peptide of approximately 150 kDa, which consists of 1315 amino-acid residues. The toxin forms a two-chain activated molecule composed of a heavy chain (HC) and a light chain (LC) linked by a disulfide bond. The catalytic domain of the toxin resides in the LC, while the translocation and receptor-binding domains are present in HC –27 Figu. TetanusThe mechanism of cell internalization and catalytic activity of tetanus toxin can be summarized in few steps ,25 Figu:C-terminal region of HC;cell binding, mainly mediated by the ganglioside-recognition domain in the internalization into neuronal cells. Different internalization mechanisms have been described: the mechanism that tetanus toxin possibly follows to internalize into neurons may be a clathrin-mediated pathway, which is dependent on the proteins dynamin, AP-2 and AP180 . Other pmembrane translocation from mature endosomes into the neuronal cytoplasm. After incorporation of the toxin within endosomes, a structural change of the toxin is induced by the acidification of the endosomal environment; thus, a membrane-spanning pore forms. At this step, the oligomerization of four toxin amphipathic alpha-helices is required for channel formation; andtarget recognition and catalytic cleavage of neuronal substrates. Once the toxin reaches the cytoplasm, it specifically cleaves neuronal proteins integral to vesicular trafficking and neurotransmitter release. In particular, the synaptic vesicle protein synaptobrevin (VAMP) is the target of tetanus toxin. This protein belongs to a family of proteins that facilitate exocytosis in neurons known as SNARE proteins. The other members of this family are syntaxin and SNAP-25, which are the main molecular targets of botulinum toxin. SNARE proteins are formed by coiled-coil interactions of the alpha-helices of its members, which is required for membrane fusion –33.Clostridium botulinum, which is considered to be a potent blocker of synaptic transmission in peripheral cholinergic nervous system synapses. There are seven serologically distinct botulinum isoforms (denoted A–G), which exhibit strong amino-acid sequence similarity. The mature toxin is characterized by three main structural domains, an N-terminal light chain Zn2+-metalloprotease and the heavy chain that contains the N-terminal, of approximately 50-kDa, translocation domain, and the C-terminal, of approximately 50-kDa, receptor-binding domain. This receptor-binding domain comprises two subdomains, a β-sheet jelly-roll fold and a β-tree foil-fold carboxy subdomain. The seven botulinum toxin serotypes cleave specific residues on one of three SNARE proteins: botulinum serotypes B, D, F and G cleave VAMP, botulinum serotypes A and E cleave SNAP25, and botulinum serotype C cleaves SNAP25 and syntaxin 1a [Botulinum toxins are produced by the anaerobic bacterium taxin 1a ,28.N-terminal of HC, which allows for the translocation of the toxin through the lipid membrane. However, in the absence of fragment C, the toxin retains little ability to paralyze neuromuscular transmission [As previously mentioned, tetanus toxin can form channels in lipid membranes at low pH in the endosomal compartment of nerve cells. These channels are actually pores, which are involved in the transport of the toxin to the cytosol . The abismission . The lipsmission . Furthersmission .What is the molecular structure of fragment C that makes it essential for toxin behavior?The early step in tetanus-toxin internalization is cell binding. Lipid rafts are microdomains of the plasma membrane enriched in sphingolipids (gangliosides are members of this group of lipids), cholesterol and glycosylphosphatidylinositol (GPI)-anchored proteins. Lipid rafts behave as specialized domains for tetanus-toxin binding and internalization into neurons. In particular, gangliosides constitute the main part of mammalian plasma membranes. Their oligosaccharide residues and their ubiquity at the outer leaflet of the membrane allow them to function as bioactive signal transducers. The affinity of fragment C for gangliosides, which resides in the last 34 residues, has been widely tested in different animal models and in different tissues. For example, greater interaction was observed with long-chain gangliosides from rat brain than with similar gangliosides in rabbit kidney . FurtherN-acetylneuraminic acid (NeuAc) on one arm and a NeuAc dimer on the other  and GT1b , but not GM1 , are necessary for the binding process ,41. Speche other . The strAdditionally, the receptor-binding domain of tetanus and botulinum A and B toxins is structurally similar, containing two subdomains: an amino-terminal lectin-like jelly-roll subdomain and a carboxyl-terminal beta-trefoil subdomain linked by a single chain . Each ofBased on studies that included a combination of computational methods, these four carbohydrate-binding sites are localized in two different sites on the surface of fragment C: Site-1, a common site for all clostridial neurotoxins through which fragment C binds lactose and part of GT1b ; and the other well-characterized site Site-2, through which fragment C binds sialyllactose, lactose, disialyllactose (DiSia), a tripeptide Tyr-Glu-Trp (YEW) and the Gal-NAc part of GT1b. Site-2 has been shown to be vital for the toxicity of the toxin . FurtherIn particular, DiSia binding characterizes ganglioside binding to fragment C and could be the binding site for the ganglioside sugar moiety GD3 ,50. If lMore recent studies have suggested that gangliosides are functional dual receptors for tetanus toxin and they are necessary for high-affinity binding to neuronal and non-neuronal cells. In particular, these studies showed that HC of the toxin bound gangliosides via two carbohydrate-binding sites, the lactose binding site called W-pocket and the sialic acid binding site called R-pocket. Both W- and R-pockets are the binding sites for GM1a and GD3 gangliosides .N-glycosylated 15-kD receptor protein has also been described as a surface glycoprotein that interacts with tetanus toxin in neuronal cell-lines and motor neurons. The C-terminal subdomain of fragment C of tetanus toxin is sufficient and necessary for cell binding and interaction with the 15-kD putative receptor, highlighting the importance of this domain of fragment C for the neurospecific interaction of the toxin [NRT and TrkB [1226, suggesting that a protein as well as a ganglioside can bind to this domain [Although the affinity of fragment C for gangliosides has been widely characterized, another hypothesis could suggest that a high-affinity protein receptor can be involved in tetanus toxin internalization. Schiavo and co-workers showed that the he toxin . The samhe toxin . In addiand TrkB . Swaminas domain .C-terminus of the light chain enters the cytosol, where SNARE cleveage takes place and this is the last portion translocated that exit the channel. The disulfide bridge between light and heavy chains is crucial for botulinum toxicity and is required for chaperone function, acting as a principal determinant for cargo translocation and release [Similarly to tetanus toxin, botulinum toxin Types A, B, C and F bind gangliosides GT1b, GD1b and GD1a, whereas botulinum Type E binds GT1b and GT1a, botulinum Type D binds phosphatidylethanolamine (PE) and botulinum Type G recognizes all the gangliosides with an approximately similar affinity . Further release .One of the unique characteristics of tetanus toxin is that it can be transported retrogradely to the central nervous system from the circulatory system. The first question was whether fragment C itself could be transported by neurons similar to the native toxin while avoiding its toxicity. The trans-synaptic transport of fragment C was intensively studied in one of the best-characterized systems, the primary visual pathway ,58, conflacZ-fragment C. This protein has been tested in vitro and in vivo to determine its activity in the hypoglossal system, and the detection of the labeled motor neurons was dependent on time post-injection [One of the most well-known hybrid proteins that has been used for this purpose is the hybrid protein encoded by njection –62. Sincnjection . The pronjection .in vitro and in vivo [The advances in the understanding of these hybrid proteins have paved the way for new therapeutic approaches using fragment C as a carrier of neurotrophic factors to ameliorate the disease process of motor neuron diseases, neuropathies and pain. Moreover, fragment C represents a potential non-viral vector for delivering exogenous biomolecules, such as proteins and DNA, to neurons  in vivo . Among t in vivo .in vitro and in vivo. For example, GDNF-fragment C promotes neuronal survival and neurite outgrowth in animal models of Parkinson’s disease [Additionally, BDNF-fragment C and GDNF-fragment C hybrid proteins exert neuroprotective effects  disease  and amyo disease ,68, and  disease .in vitro in a dose-dependent manner [L and fragment C improved cell survival and decreased apoptosis in the glutamate-mediated excitotoxicity of SH-SY5Y neuronal cells [Similarly, the hybrid protein that contains cardiotrophin-1 and fragment C promoted motor neuron survival t manner , and theal cells .NTR as the first membrane marker of the retrograde endocytic pathway used by fragment C of tetanus toxin [NTR is a transmembrane receptor for neurotrophic factors of the neurotrophin family, which comprises NGF, BDNF and neurotrophin-3 and -4/5. In addition to p75NTR, neurotrophins signal, via the tropomyosin-related kinase (Trk) family of receptors, tyrosine kinases Trk A, B and C. The two receptor systems can function synergistically, antagonistically or independently of each other in different cell types [NTR, TrkB and BDNF, which is strongly dependent on the activities of the small GTPases Rab5 and Rab7 [NTR is transported toward the soma without undergoing proteolytic cleavage. In particular, an impairment of Rab7 activity inhibits the trafficking of fragment C-, p75NTR- and TrkB-containing carriers. However, Rab7 impairment in PC12 cells led to an increase in TrkA and extracellular-regulated kinases (ERK-1/2) phosphorylation and the stimulation of NGF [Furthermore, fragment C of tetanus toxin and NGF share the same retrograde transport organelles, suggesting neurotrophin receptor p75us toxin . p75NTR ll types . The retand Rab7 , which sn of NGF .Considering the potential use of fragment C as a non-viral vector and its likely sharing a common molecular pathway as the one described for the neurotrophic factor BDNF , there iNTR [NTR [NTR to clathrin-coated pits to be delivered into the soma of motor neurons, which highlights the regulation of p75NTR signaling in response to neurotrophins [NTR can also interact with pro-neurotrophins and, therefore, it serves as a signaling component of the receptor complex for growth-inhibitory molecules of CNS, such as Nogo, prompting Nogo receptor-mediated signaling [The molecular pathway involving the activation of the Trk receptors is closely shared by neurotrophic factors and fragment C. The neurotrophin family has been shown to regulate survival, development and functional aspects of neurons in the central and peripheral nervous systems through the activation of one or more of the three members of the receptor tyrosine kinases  in cooperation with p75NTR . NGF cantrophins ,77. In aignaling ,79.Aguilera and co-workers described an increase in serotonin synthesis in the central nervous system induced by tetanus toxin, suggesting that the toxin-affected serotonergic innervation in the perinatal rat brain triggered the translocation of calcium-phosphatidylserine-dependent protein kinase C (PKC) . In partFurthermore, the activation of intracellular pathways related to the PLCγ-1 phosphorylation and activation of PKC isoforms and the kinases Akt (at Ser 473 and Thr 308) and ERK-1/2 (at Thr 202/Tyr 204) was induced by fragment C in rat brain synaptosomes and cultured cortical neurons. This signal pathway activation was dependent on time and concentration, suggesting that fragment C could exert neuroprotective effects, activating TrkA and TrkB receptors in a similar manner as do NGF and BDNF or neurotrophin-4/5 ,86.+)-triggered apoptosis, enhancing the survival pathways in rats with a dopaminergic lesion and improving different motor behaviors. In this study, fragment C induced Ser 112 and Ser 136 BAD phosphorylation, activated the transcription factor NF-κB, which prevents neuronal death, and induced a decrease in the release of cytochrome c and, consequently, a reduction in the activation of procaspase-3 and chromatin condensation [Fragment C also protected cerebellar granular cells against potassium deprivation-induced apoptotic death  and acteensation ,89  or naked DNA, encoding the recombinant molecule fragment C and BDNF (BDNF-TTC). In addition, a significant delay in the onset of symptoms and functional deficits, an improvement in the spinal motor neuron survival (down-regulation of caspase-1 and caspase-3 levels and a significant phosphorylation of serine/threonine protein kinase Akt) and a prolonged lifespan under both treatments was observed [G93A mice, reinforcing on the one hand the carrier properties of fragment C. The active state of BDNF in the recombinant molecule could suggest that BDNF could exert an autocrine and neuroprotective role together with TTC to a similar extent as TTC alone; however this effect could not be sufficient enough to prompt a synergistic effect. As a consequence, BDNF-TTC molecule could mainly use the same pathway that mimics a neurotrophic secretion route, prompting survival signals in the spinal cord of transgenic SOD1G93A mice [Consequently, the main advantage of using fragment C as a potential therapeutic agent in non-viral gene therapy is particularly interesting in amyotrophic lateral sclerosis (ALS). The failure of standard treatments in ALS could rely on the inappropriate route of administration and/or the poor bioavailability of molecules to the target cell . The subobserved ,93. Alth93A mice .Despite all these contributions to the understanding of the signaling pathway of fragment C, further studies should be performed to elucidate its neuroprotective effect.in vitro and in vivo studies have shown that fragment C can enhance cell survival by itself. This new property of fragment C has opened the door to the understanding of its possible, although not yet well-characterized, molecular pathways. Because the characterization of the protein-protein interaction at the molecular level is of high importance and the plasma membrane of each nervous cell contains a particular composition, a messenger, such as fragment C, can exert different effects depending on the signaling pathway it modulates, which is directly related to the type and distribution of different receptors in the plasma membrane. Thus, a messenger is not the molecule that carries the message; many results can be obtained when studying the same effector molecule under different experimental conditions.Fragment C is considered to be a useful and valuable tool to carry therapeutic molecules due to its efficient retroaxonal transport. This is the main reason why this particular property has been exploited as a therapeutic strategy in the central nervous system to ameliorate the disease process of well-known neurodegenerative diseases, such as Parkinson’s and ALS. However, recent Regarding fragment C, new studies will reveal the precise molecular mechanism by which it can induce its neuroprotective properties. This mechanism could shed light on using fragment C as an alternative therapeutic strategy for more neurodegenerative diseases in the near future.


During scleroderma (SSc) pathogenesis, fibroblasts acquire an activated phenotype characterized by enhanced production of extracellular matrix (ECM) and constitutive activation of several major signaling pathways including extracellular signal-related kinase (ERK1/2). Several studies have addressed the role of ERK1/2 in SSc fibrosis however the mechanism of its prolonged activation in SSc fibroblasts is still unknown. Protein phosphatase 2A (PP2A) is a key serine threonine phosphatase responsible for dephosphorylation of a wide array of signaling molecules. Recently published microarray data from cultured SSc fibroblasts suggests that the catalytic subunit (C-subunit) of PP2A is downregulated in SSc. In this study we examined the role and regulation of PP2A in SSc fibroblasts in the context of ERK1/2 phosphorylation and matrix production.We show for the first time that PP2A mRNA and protein expression are significantly reduced in SSc fibroblasts and correlate with an increase in ERK1/2 phosphorylation and collagen expression. Furthermore, transforming growth factor β (TGFβ), a major profibrotic cytokine implicated in SSc fibrosis, downregulates PP2A expression in healthy fibroblasts. PP2A-specific small interfering RNA (siRNA) was utilized to confirm the role of PP2A in ERK1/2 dephosphorylation in dermal fibroblasts. Accordingly, blockade of autocrine TGFβ signaling in SSc fibroblasts using soluble recombinant TGFβ receptor II (SRII) restored PP2A levels and decreased ERK1/2 phosphorylation and collagen expression. In addition, we observed that inhibition of ERK1/2 in SSc fibroblasts increased PP2A expression suggesting that ERK1/2 phosphorylation also contributes to maintaining low levels of PP2A, leading to an even further amplification of ERK1/2 phosphorylation.Taken together, these studies suggest that decreased PP2A levels in SSc is a result of constitutively activated autocrine TGFβ signaling and could contribute to enhanced phosphorylation of ERK1/2 and matrix production in SSc fibroblasts. Scleroderma (SSc) is an autoimmune connective tissue disease characterized by excess production and deposition of extracellular matrix proteins leading to fibrosis of the tissue. During this process, normal fibroblasts become 'activated' and acquire a fibrotic phenotype. Transforming growth factor β (TGFβ) is a major profibrotic cytokine that plays important roles in a variety of physiological processes including cell proliferation, differentiation and survival. Although the mechanism of SSc fibrosis is not fully understood, there is strong evidence to suggest that TGFβ is central to the development and maintenance of the SSc phenotype -3. Normaet al. recently reported that activation of the ERK1/2 pathway contributes to the enhanced fibrosis and contractile ability of scleroderma fibroblasts [vβ3 integrin, which contributes to the autocrine TGFβ signaling in scleroderma fibroblasts [During tissue injury, rapid release of TGFβ attracts inflammatory cells and fibroblasts to the site of injury, resulting in extracellular matrix production/remodeling and myofibroblast differentiation . In normroblasts . The ERKroblasts . HoweverProtein phosphatase 2A (PP2A) is a member of the PPP family and one of the most abundant serine-threonine phosphatases, accounting for a substantial part of the total phosphatase activity. PP2A plays an important role in signal transduction pathways, regulation of cell cycle and transcriptional and translational regulation . PP2A haBecause of the central role of TGFβ in the pathogenesis of SSc, we first examined the regulation of ERK1/2 phosphorylation by TGFβ treatment in healthy fibroblasts. To investigate the kinetics of ERK1/2 activation, time course experiments were performed. Near confluent cells were serum starved and then treated with TGFβ for increasing time periods ranging from 0-24 h. Using western blot analysis we observed that stimulation of cells with TGFβ resulted in rapid phosphorylation of ERK1/2 as early as 15 min and sustained ERK1/2 phosphorylation up to 24 h Figure . This suSince PP2A has been previously described as a major ERK1/2 phosphatase we next sought to determine whether TGFβ could also be involved in the regulation of PP2A expression in dermal fibroblasts. Confluent dermal fibroblasts were serum starved and then treated with TGFβ for different time periods. The mRNA levels of α and β isoforms of the PP2A catalytic subunit were analyzed by quantitative reverse transcription (qRT)-PCR. As shown in Figure To further confirm the role of PP2A in ERK1/2 phosphorylation in dermal fibroblasts, experiments were performed using okadaic acid (OA), a pharmacological inhibitor of PP2A activity, and PP2A-specific small interfering RNA (siRNA). Upon treatment of normal dermal fibroblasts with OA (2 nM) for 1 h, increased ERK1/2 phosphorylation was observed Figure . ConsistTo further study the relationship of PP2A and ERK1/2 phosphorylation in the pathological context, age, race and gender matched SSc and normal dermal fibroblasts obtained from patient biopsy were analyzed for PP2A expression and ERK1/2 activation. ERK1/2 phosphorylation was increased in SSc fibroblasts, consistent with data from previous reports  Figure . The mRNAutocrine TGFβ signaling has been reported to play a major role in the pathogenesis of SSc. and blockade of endogenous TGFβ signaling has been shown to attenuate the scleroderma fibrotic phenotype . RecombiThe activity of kinases and phosphatases is tightly regulated in the cell and often involve feedback mechanisms, which help maintain the levels of cellular phosphorylation. A study performed in human lung fibroblasts suggests that silencing of ERK1/2 is associated with a decrease in PP2A activity . In ordeFibrosis is the hallmark of SSc fibroblasts and dysregulation of various signaling pathways have been implicated in increased collagen production in this disease. In SSc fibroblasts we observed an inverse correlation between PP2A levels and collagen expression Figure . To furtin vivo, has not been described in SSc fibroblasts. Previous reports indicated that an autocrine TGFβ signaling pathway contributes to the SSc phenotype [In this study we have demonstrated that TGFβ is a negative regulator of PP2A. We found decreased expression of both the α and β isoform of the catalytic subunit of PP2A after TGFβ stimulation. To our knowledge this is the first report to demonstrate that TGFβ is a negative regulator of PP2A gene expression. In accordance with previously published studies our data also confirms that TGFβ treatment activates prolonged ERK1/2 phosphorylation. This study provides new evidence for the contribution of PP2A to the pathogenesis of SSc. Analysis of fibroblasts cultured from SSc skin biopsies shows decreased protein levels of the PP2A catalytic subunit, with downregulation of both α and β isoforms at the mRNA levels, reproducing the effects of TGFβ in normal dermal fibroblasts. These data validate a previous gene array study, which showed decreased levels of the β isoform in cultured SSc fibroblasts. However, downregulation of the α isoform, which is the most abundant isoform henotype . We hypohenotype ,26,27. Iin vivo and its role in fibrosis. However there are several limitations to this approach considering the vast number of subunits and splice variants present for this molecule as well as the numerous substrates and methods of posttranslational regulation. Several experimental mouse models have been generated including the PP2AC knockout mouse and transgenic models of various other PP2A subunits [Reversible protein phosphorylation plays a central role in the regulation of vast majority of the biological processes. This process is tightly controlled by the protein kinases and phosphatases that together regulate the levels of cellular phosphoproteins. The balance between the activities of kinases and phosphatases is often disrupted during pathological conditions including neurodegenerative diseases and cancer ,28. Perssubunits . The PP2subunits . HoweverIn conclusion, this study describes a novel role for TGFβ in the regulation of PP2A gene expression. While our study focused on ERK1/2, PP2A dephosphorylates numerous signaling molecules, many of them with a potential role in fibrosis, and it is likely that such global downregulation of PP2A activity would modulate additional cellular pathways. We also show that SSc fibroblasts have decreased levels of PP2A and that this could be restored by blockade of autocrine TGFβ signaling, suggesting that negative regulation of the PP2A catalytic subunit gene expression may be a physiological mechanism by which sustained ERK1/2 phosphorylation occurs in SSc. This study highlights an unanticipated regulatory function for TGFβ in modulating PP2A activity and provides support for an essential role of PP2A in the pathogenesis of SSc. Further studies are necessary to gain insight into the role of PP2A and ERK1/2 activation in the modulation of ECM components in SSc fibroblasts.The following antibodies were used: anti-PP2A , anti-phospho-ERK1/2, anti-ERK1/2, anti-Akt , anti-phospho-Akt, Ser 473 , monoclonal β actin , anti-type 1 collagen .Recombinant human TGFβ1 was obtained from R&D Systems . OA was purchased from Sigma Aldrich. Tissue culture reagents, Dulbecco's modified Eagle medium (DMEM) and 100× antibiotic antimycotic solution (penicillin streptomycin and amphotericin B) were obtained from Gibco BRL  and fetal bovine serum was purchased from HyClone . Enhanced chemiluminescence reagent and bovine serum albumin (BSA) protein assay reagent were obtained from Pierce . TriReagent was purchased from the Molecular Research Center . Primers were purchased from Operon . SMARTpool siRNA against PP2A C-subunit was purchased from Dharmacon RNA Technologies  and Hiperfect siRNA transfection reagent from Qiagen .Human dermal fibroblast cultures were established from biopsy specimens obtained from the dorsal forearms of SSc patients with diffuse cutaneous disease and from age, race and gender matched healthy donors, upon informed consent and in compliance with the Institutional Review Board. Dermal fibroblasts were cultured from the biopsy specimens as described previously . Normal Total RNA was isolated from dermal fibroblasts using TriReagent (Molecular Research Center) according to the manufacturer's instructions. RNA (2 μg) was reverse transcribed in a 20-μl reaction using random primers and Transcriptor First Strand synthesis kit PCR was carried out using IQ SYBR Green mixture  on an iCycler PCR machine (Bio-Rad) using 1 μl of cDNA in triplicate with β actin as the internal control. The primers used are as follows. PP2A C-subunit α isoform: forward, 5'-GCACTTGATCGCCTACAAGA-3' and reverse, 5'-GAAATATCTTGCCCAAAGGTGT-3'. PP2A C-subunit β isoform: forward, 5'-TTCTTGTAGCATTAAAGGTGCGT-3' and reverse, 5'-CATTCCCATACTTCGCAGACA-3'.Whole cell protein extracts were prepared according to the manufacturer's recommendations (Pierce). Immunoblotting was performed as previously described .SMARTpool siRNA directed against human PP2A catalytic subunit was purchased from Dharmacon RNA Technologies. Negative-control siRNA was purchased from Qiagen  and Hiperfect transfection reagent (Qiagen) was used for transfection of dermal fibroblasts according to the manufacturer's recommendations.GHS was involved in the development/experimental design of the project, performed the majority of the experiments, performed data acquisition and analysis, and wrote the manuscript. AMB isolated SSc and normal fibroblasts from patient biopsies used for experiments, and was involved with experimental design. SSN performed some inhibitor experiments and was involved with experimental design. FH provided biopsies from patients and was involved with experimental design. MT was involved with the conception, experimental design and supervision of the study. All authors have read and approved the final manuscript.


Some of the latest research reported considerable abiotic CO2 absorption by alkaline soil, but the rate of CO2 absorption has been questioned by peer communities. To investigate the issue of carbon cycle in Central Asian desert ecosystems with alkaline soils, we have measured the NEE using eddy covariance (EC) method at two alkaline sites during growing season in Kazakhstan. The diurnal course of mean monthly NEE followed a clear sinusoidal pattern during growing season at both sites. Both sites showed significant net carbon uptake during daytime on sunny days with high photosynthetically active radiation (PAR) but net carbon loss at nighttime and on cloudy and rainy days. NEE has strong dependency on PAR and the response of NEE to precipitation resulted in an initial and significant carbon release to the atmosphere, similar to other ecosystems. These findings indicate that biotic processes dominated the carbon processes, and the contribution of abiotic carbon process to net ecosystem CO2 exchange may be trivial in alkaline soil desert ecosystems over Central Asia.Central Asia is covered by vast desert ecosystems, and the majority of these ecosystems have alkaline soils. Their contribution to global net ecosystem CO Although more than 950 site-years of eddy covariance (EC) data have been collected in the international network of FLUXNET  in combination with xerophytic dwarf semi-shrub, halophytic shrub, and psammophytic grasses . The fraction of vegetation coverage varied from 0 to 90%, with average value about 30–40% .The Aral Sea site  is located northeast of the Aral Sea. During the past half-century, the surface area of Aral Sea has significantly decreased by 75% , and grasses (with relatively large proportion in vegetation component) are distributed. The soil in the KZ-Bal site is takyr-like saline solonchak (Starodubtsev and Truskavetskiy http://ftp://sidads.colorado.edu/pub/DATASETS/NOAA/G02174/).The Balkhash Lake site  is located between the Balkhash Lake (200 km away) and the Kapchagay Reservoir (100 km away). The nearest town is Bakbakty, a town along the Ili River, 17 km north of the site. The KZ-Bal site is located at the transect between oasis croplands and original desert habitats. Around 3–5 km west of the site, irrigated crops, well-grown reed, and grasses are distributed. In the south of the site, a small village is resided. Both east and north regions of the KZ-Bal site are original desert ecosystems where desert semi-shrubs, shrubs  three-dimensional sonic anemometer thermometer  was utilized. A fast response (10 Hz) open path gas analyzer  was used to measure the mole densities of CO2 and H2O. Both instruments are mounted at 2.0 m above ground. The dominant wind direction at both sites was on average northeast . The data are stored in the format of GHG. Each GHG file is an archive containing the raw high-speed data (.data) and information on the study site (.metadata), both in readable text format.Raw data of the three wind components, the speed of sound, and COAncillary meteorological and soil measurements of relevance included photosynthetically active radiation (PAR) flux density , air temperature and humidity , downward and upward shortwave and longwave radiation  at 2.0 m above ground, and precipitation . Soil temperature , soil moisture content , and soil heat flux  were measured at 0.20, 0.40, 0.60, 0.80 m depth below the ground. These data were recorded by the data logger at 10 Hz and saved as half-hourly averages.Up to date, available data covered the period between 30 April and 18 August 2012 at the KZ-Ara site and between 23 May and 6 Sep 2012 at the KZ-Ara site. These data will be used for the analysis in this study.2 fluxes) according to Mauder and Foken 2 flux data were 15.7% (836 in 5328) and 30.1% (1544 in 5136) at the KZ-Ara and KZ-Bal sites, respectively.Data processing and gap filling was carried out in three steps. First, GHG files were imported into EddyPro software (version 4.0.0) to calculate out 30 min blocks of flux data. Tilt correction, turbulent fluctuation blocking, time lag compensation, spike detection and removal, and other statistical tests and spectral corrections were applied with the standard functionality of the “Express Model” option in the software. EddyPro also outputs quality flags for all flux variables  artificial neural network (ANN)  using a training data set without any “bad” value. The input data are first classified into five nodes based on Self-Organising Feature Map, so that each node represents an individual region of the input space. At each node, a linear regression is implemented between input variables and the interested output flux variable. Finally, the flux time series with gaps is estimated based on a piecewise linear approximation of the training data set  in a year was below 0°C and mean temperature exceeding 10°C was from May to September  could vary site by site  was independent of u* in a broad range between 0 and 0.9 m s−1 as shown by a relatively horizontally linear regression between normalized nighttime NEE and u*   and wind direction distribution  on nighttime respiration is apparently small  Fig. C. The deion Fig. D. At theion Fig. D where oμmol m−2 s−1 at the KZ-Ara and KZ-Bal sites, respectively. The significant difference in maximum carbon uptake rates between the two sites and the difference in the month of peak carbon uptake rates were possibly related to the vegetation compositions and climatic conditions. At the KZ-Ara site, the vegetation around the site was all desert plants, shrubs, or grasses and no human disturbance applied. The temperature at the KZ-Ara site is higher than that at the KZ-Bal site, which may cause earlier phenology for plants' primary production. At the KZ-Bal site, the observed NEE may be impacted by surrounding oasis crops and reed and grasses grown alongside the acequia.The mean diurnal NEE in each month followed a clear sinusoidal dynamic during the growing season Fig. . Mean diμmol m−2 s−1 in May to −1.1 μmol m−2 s−1 in August. The daytime mean NEE decreased from May to August. In contrast, mean monthly nighttime NEE ranged from 1.65 μmol m−2 s−1 in May and June to 1.0 μmol m−2 s−1 in August  = Aexp(−PAR/B) + C  represents nighttime respiration. Monthly variations of parameter C indicate seasonal changes in maximum CO2 uptake. A comparatively good match between the measured data and the resulted model output was derived for the months with the highest net CO2 exchange (parameter C in the fitting equation), that is, May  at the KZ-Ara site and Jul  at the KZ-Bal site  NEE  of near surface air or soil temperature . On cloudy or rainy days, daily NEE at both sites tended to be positive, that is, net carbon release to the atmosphere. For instance, consecutive rainfall on the days 23–24 June caused noticeable carbon loss at the KZ-Ara site. Similarly, five consecutive days of rainfall from 30 May to 3 June led to consecutively significant and considerable carbon release (0.5–3.5 gC m−2 day−1) into the atmosphere at the KZ-Bal site. On other cloudy days, for example, the day on 27 May at the KZ-Ara site with low PAR of 250 μmol m−2 s−1 showed a net carbon loss.Figure ite Fig. A and daiite Fig. B. Corres2 exchange from desert areas has received much attention. Underlying reasons may come from the vast extent of arid and semi-arid ecosystems in terrestrial ecosystems   during the growing season was strongly related to the fact that the site was actually impacted by human interference  was questionable (Schlesinger et al. −2 year−1 boundary (Fig. Global terrestrial ecosystem showed a quasi-Gaussian probability distribution with the mean NEE of −183 gC mion Fig. , which iary Fig. . However2 exchange at the two desert ecosystems in Kazakhstan where soil was featured as alkaline and high soil pH value. Latest reports based on continuously measured soil respiration in alkaline soil exhibited significant net carbon loss at both daytime and nighttime (Ma et al. 2 absorption by alkaline soil (Serrano-Ortiz et al. 2 uptake by alkaline soils may not be noticeable and its contribution to net ecosystem CO2 exchange should be fairly limited.Both sites of desert ecosystems with alkaline soils in Kazakhstan acted as obvious net carbon release at nighttime and on cloudy and rainy days, but net carbon uptake at daytime on sunny days Figs  and 9. C2 exchange based on the measurements of EC in two sites of desert ecosystems with alkaline soils in Central Asia. The results found that the diurnal courses of NEE in each month followed clear sinusoidal patterns during growing season. Negative values of mean NEE were found at daytime on sunny days, indicating a net carbon uptake. In contrast, positive values of mean NEE were observed on cloudy or rainy days and at nighttime, which implied a net carbon source. Furthermore, strong dependency of NEE on PAR and the response of NEE to precipitation indicated that desert ecosystems with alkaline soils were still dominated by biotic factors, similar to other ecosystems, and abiotic CO2 absorption by alkaline soils may be trivial in terms of magnitude and aptitude.The present study provides the first insight on the diurnal, daytime, nighttime, and daily rates of net ecosystem CO


CD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPCCD36 are not well defined. We have employed mass spectrometry-based tyrosine, serine, and threonine phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPCCD36 as well as by the strong physiological agonist thrombin. oxPCCD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide candidates in the study of platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCγ2 was identified in platelets activated by oxPCCD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPCCD36. Our results provide multiple insights into the mechanism of platelet activation and specifically in platelet regulation by oxPCCD36.Specific oxidized phospholipids (oxPC Dyslipidemia is associated with oxidative stress and platelet hyper-reactivity, a condition that increases the risk of thrombotic complications in cardiovascular pathologies In vitro, CD36 mediates platelet activation by a group of specific oxidized phospholipids (oxPCCD36) derived from the oxidation of 1-palmitoyl-2-arachidonyl-phosphatidylcholine (PAPC) and 1-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC). Electrostatic interaction of the negatively charged carboxylate at sn-2 position of oxPCCD36 with lysine CD36, amyloid-like structures, lysophosphatidylcholine, oxysterols, PAF receptor activating-oxidized lipids) that can lead to the activation of several independent signaling mechanisms We previously showed that the scavenger receptor CD36 is the major receptor on platelets that promotes platelet reactivity in hyperlipidemic conditions in response to oxidized phospholipids Phosphoproteomic studies are particularly useful in recognizing platelet signaling mechanisms, since genomic approaches are limited in anucleated cells. Currently, phosphoproteomics based on mass spectrometry provide the most comprehensive analysis for identification and quantitation of phosphorylation sites from diverse biological samples 2 (PGI2) sodium salt, Sepharose 2B, BSA, DMSO, and U73122 were purchased from Sigma-Aldrich . Human α-thrombin was from Enzyme Research Laboratories  and ADP from Chrono-log . PE-conjugated anti–P-selectin antibody was obtained from Becton Dickinson  and mouse anti-CD36 (clone FA6-152) was purchased from Beckman Coulter . PP2, PP3, PD98059, and BAY61-3606 were obtained from EMD Millipore . PLPC  was from Avanti Polar Lipids . KODA-PC (9-keto-12-oxo-10-dodecenoic acid ester of 2-lyso-phosphocholine) was synthesized as described previously Prostaglandin IAll experiments done with human blood were approved by the Cleveland Clinic's Institutional Review Board. Written informed consent was obtained in accordance with the Declaration of Helsinki.2 was added after blood collection into ACD to a final concentration 0.1 µg/mL. Platelet-rich plasma (PRP) was separated by centrifugation at 150 g . Platelets were pelleted from PRP by centrifugation at 930 g , resuspended in Tyrode's buffer , and further purified by gel filtration using a Sepharose 2B column. The concentration of platelets was determined using Cellometer M10  and adjusted to concentrations indicated with Tyrode's buffer. CaCl2 and MgCl2 were added to a final concentration of 2 mM and 1 mM, respectively, for phosphoproteomic and flow cytometry experiments.Human venous blood was drawn from healthy donors into acid-citrate-dextrose solution . PGI8/mL) were incubated in Tyrode's buffer with 50 µM KODA-PC or PLPC (as control) for 30 min at 37°C. Platelets were then centrifuged at 3,700 g  and lysed in 8 M urea, 50 mM Tris-HCl (pH 7.4), 1 mM Na3VO4, and 1 mM NaF with sonication. Phosphoproteome changes induced by the agonist from a single biological experiment were assessed based on trypsin digestion of the protein lysate, phosphopeptide enrichment, mass spectrometry analysis, chromatography alignment, quantitation, and bioinformatics and enrichment analysis as described in http://proteomecentral.proteomexchange.org) via the PRIDE partner repository Human platelets isolated by gel filtration (2.7×108/mL) and treated with indicated inhibitors and agonists were pelleted at 960 g . The platelets were then lysed for 30 min on ice with 50 mM Tris-HCl (pH 7.4), 1 % IGEPAL CA-630, 0.25 % sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin, 1 mM Na3VO4, and 1 mM NaF. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with rabbit anti-human phospho-PLCγ2 Tyr759, phospho-PLCγ2 Tyr1217, phospho-SYK Tyr323, phospho-SYK Tyr525/526 (C87C1), or phospho-Src family Tyr416 . The membranes were stripped and then probed for normalization with rabbit anti-human SYK (C-20)  or PLCγ2 .Platelets isolated by gel filtration (2×107/mL) were incubated in Tyrode's buffer with the indicated inhibitors for 30 min and then with agonists for another 30 min. Platelets were incubated with PE-conjugated anti–P-selectin antibody and then fixed with 2 % formaldehyde in PBS. Data was acquired using a FACSCalibur instrument  and analyzed using FlowJo . Data are presented as mean ±SD. Significant changes were determined using one-way ANOVA with Bonferroni post-test. Significance was accepted at the level of P<0.05.Human platelets isolated by gel filtration  domain . Since recognition of phosphoinositides is a major function of PH-domains, this finding probably reflects a major role for PI-3-kinase pathway in platelets activated by thrombin as compared to KODA-PC.KODA-PC induced significant increased or decreased phosphorylation (≥1.5 fold change) of 162 sites in 115 proteins. KODA-PC induced changes are reported here for the first time. Thrombin induced significant (de)phosphorylation of 334 sites of 181 proteins. Direct comparison of phosphorylation sites found in both sets of experiments indicate that many phosphorylation events induced by KODA-PC and thrombin are similar in their trend, although the fold increase was generally higher for thrombin . There wConsensus motifs extracted from either phosphorylation or dephosphorylation events induced by each agonist indicated the presence of known kinase activities and binding targets  [29], XXC) present in peroxiredoxins with antioxidant properties eriments  confirmeComparison of our mass spectrometry results  without assigning the specific member(s) of the family. Western blotting analysis detected an increase in phosphorylation of several bands corresponding to members of the SFK . Figure S3. Motif-X analysis of sites that were (de)phosphorylated after platelet activation by KODA-PC and thrombin. Figure S4. Selected GO-terms enriched from the list of genes identified by phosphoproteomic in platelets activated by KODA-PC and thrombin. Figure S5. PLC inhibitor blocks platelet P-selectin expression induced by different ligands. Figure S6. ERK kinase is not required for P-selectin expression induced by KODA-PC. Figure S7. KODA-PC induces Src-family kinases (SFK) phosphorylation. Figure S8. SYK and PLCγ2 phosphorylation induced by KODA-PC at different time points in the presence of CD36 blocking antibody and chemical inhibitors to SFK and SYK. Figure S9. Surface level expression of CD41 and CD61 is not affected by KODA-PC, thrombin, chemical inhibitors PP2 and BAY61-3606, or blocking antibody to CD36. Figure S10. Phosphoproteome network regulating integrin in platelets activated by KODA-PC.(PDF)Click here for additional data file.File S2Table S1. Identification and quantitation of protein phosphorylation sites from platelets activated with KODA and thrombin using enrichment of phosphopeptides. Table S2. Number of copies and rank of phosphorylated proteins detected in this study. Table S3. Complete list of KEGG pathways enriched in the list of phosphoproteins modulated by KODA and thrombin in platelets. Table S4. Complete list of GO-terms enriched in the list of phosphoproteins modulated by KODA and thrombin in platelets. Table S5. Prediction of kinases responsible for KODA- and Thrombin-induced protein phosphorylation. Table S6. List of shared phosphoryaltion sites described in Table S1 and Burkhart, J.M. et al. Blood. 120: e73-82, 2012.(XLSX)Click here for additional data file.Materials and Methods S1Detailed description of trypsin digestion of the protein lysate, phosphopeptide enrichment, mass spectrometry analysis, chromatography alignment, quantitation, and bioinformatics and enrichment analysis employed to study the phosphoproteome changes induced by the agonists.(PDF)Click here for additional data file.Annotated Spectra S1(ZIP)Click here for additional data file.Annotated Spectra S2(ZIP)Click here for additional data file.Annotated Spectra S3(ZIP)Click here for additional data file.Annotated Spectra S4(ZIP)Click here for additional data file.Annotated Spectra S5(ZIP)Click here for additional data file.


Arenaviruses include lethal human pathogens which pose serious public health threats. So far, no FDA approved vaccines are available against arenavirus infections, and therapeutic options are limited, making the identification of novel drug targets for the development of efficacious therapeutics an urgent need. Arenaviruses are comprised of two RNA genome segments and four proteins, the polymerase L, the envelope glycoprotein GP, the matrix protein Z, and the nucleoprotein NP. A crucial step in the arenavirus life-cycle is the biosynthesis and maturation of the GP precursor (GPC) by cellular signal peptidases and the cellular enzyme Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P) yielding a tripartite mature GP complex formed by GP1/GP2 and a stable signal peptide (SSP). GPC cleavage by SKI-1/S1P is crucial for fusion competence and incorporation of mature GP into nascent budding virion particles. In a first part of our review, we cover basic aspects and newer developments in the biosynthesis of arenavirus GP and its molecular interaction with SKI-1/S1P. A second part will then highlight the potential of SKI-1/S1P-mediated processing of arenavirus GPC as a novel target for therapeutic intervention to combat human pathogenic arenaviruses.  Arenaviridae. The “sandy” appearance of the viral particles in electron microscopy, due to the presence of host ribosomes, is at the origin of the family name, which has its roots in the Latin word “arena”, meaning sand.Arenaviruses are world-wide distributed negative strand RNA viruses that include a number of important human pathogens. The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) was discovered by Armstrong and Lille upon passage of infectious material derived from a fatal case of encephalitis during the 1933 outbreak in St Louis (USA) , while TCurrently, 22 species of arenaviruses, belonging to one singe genus, are recognized . Based oSeveral arenaviruses are causative agents of severe viral hemorrhagic fevers (VHF) in humans which are among the most devastating human diseases . Among tvia vertical transmission from acutely infected mothers in utero or perinatally resulting in severe brain malformation including hydrocephalus, chorioretinitis and mental retardation . In. In67]. In vivo studies suggest that blocking SKI-1/S1P activity may be an alternative approach to lower plasma cholesterol [woodrat mutation, which shows partial loss of enzyme function, leads to hypersensitivity to dextran sodium sulfate-induced colitis [The biological activities of SKI-1/S1P are numerous and affect essential cellular functions. Among the best characterized SKI-1/S1P substrates are the Sterol Regulating Element Binding Proteins (SREBPs), transcription factors activated via limited proteolysis to control cell lipid homeostasis ,73. In vlesterol . The SRElesterol  CREBH, Llesterol . Very felesterol , pro-bralesterol  and repulesterol . SKI-1/Slesterol ,81, and lesterol , but thelesterol . Even in colitis , and cau colitis .In contrast to cellular substrates of SKI-1/S1P that are processed in the median Golgi, LASV GPC is cleaved in the ER/cis-Golgi and LCMV GPC in a late Golgi compartment ,71,86. TA characteristic common to most arenavirus GPCs  Figure  is the sYISRRLL↓ sequence mimicking the cleavage site of LASV GPC is the best substrate to assess in vitro SKI-1/S1P enzymatic activity [Specific GPC residues surrounding the cleavage site were shown to influence maturation. An F259A point mutation at P7 position of LCMV GPC greatly impairs processing . Analysiactivity ,78,90. The N-terminus of LASV GP2 contains two stretches of highly conserved hydrophobic amino acids. Alanine scanning of the N-terminal 260-266 and 276-298 sequences revealed that maturation of the envelope GPC does not occur in presence of single point mutations at positions 260, 261, 262, 280, 284, 285, 286, 292, and 293, despite GPC folding and reaching the plasma membrane . Indeed,A large body of evidence indicate a crucial role of the cytoplasmic domains of viral envelope GPs in assembly, fusogenicity, and infectivity of enveloped viruses, including Newcastle disease virus  Measles in vivo were not feasible [in vitro IC50 in the nanomolar range [in vivo, significantly lowering plasma cholesterol levels [via direct interference with the SKI-1/S1P-mediated GPC cleavage [in vitro on synthetic peptides mimicking the cleavage site of the LASV GPC: processing of the LASV GPC derived Ac-IYISRRLL-MCA peptide is inhibited by PF-429242 with an IC50 of 130 nM [in vitro [Similarly to other enveloped viruses, whose GPs are processed by PCs, such as HIV-1 and influenza virus, arenaviruses strictly rely on SKI-1/S1P for productive infection. In SKI-1/S1P-deficient cells, uncleaved GPCs are correctly transported to the plasma membrane but fail to be incorporated into budding particles, which remain “naked”, and lack infectivity ,51,65. Tfeasible . Rationaar range . The coml levels . PF-4292cleavage . The on-f 130 nM . The smaf 130 nM . Combinain vitro , suggestin vivo is further illustrated in mice homozygous for the hypomorphic Mbtps1 wrt allele (woodrat mutation), carrying a defective but still partially active SKI-1/S1P are resistant to LCMV infection [A hallmark of persistent infection is the down-regulation of GPC expression to almost undetectable levels with respect to the viral NP . During nfection . in vitro without affecting the SREBP-2 pathway. As mentioned above, residues surrounding GPC processing site affect substrate-enzyme recognition. In particular, an aromatic amino acid at P7 was found to be crucial for processing of both LASV [Given the importance of arenavirus envelope GPC processing for productive infection and its dependency on a protease involved in several crucial host functions, efforts have been made to understand how viral substrate and the enzyme interact, with the goal to eventually develop substrate-specific drugs. Biochemical characterization of SKI-1/S1P-GPC interaction revealed that auto-processing at the B/B’ sites of SKI-1/S1P is crucial for effective GPC maturation but not cellular SREBP-2 activation . The exaoth LASV  and LCMVoth LASV , making In vitro inhibition of SKI-1/S1P-mediated processing of GPC greatly limits virus cell-to-cell propagation, thus representing a promising antiviral strategy. In vivo evaluation of SKI-1/S1P inhibitors as antivirals, e.g. PF-429242, would be of great importance to understand the efficacy of interfering with normal GPC maturation on arenavirus infections. During drug treatment, dietary supplementation of cholesterol and other lipids may be taken into account to compensate for plasma cholesterol reduction, resulting from SREBPs inhibition. Novel substrate specific SKI-1/S1P inhibitors may be of great value to minimize side effects. The envelope glycoprotein processing is a key step in arenavirus life-cycle. via its late or RING domain with Tsg101, a member of the ESCRT machinery [per se budding properties and does not require any other viral protein to accomplish its function. However, the ability to produce infectious particles resides on the assembly of the four different viral proteins in a synchronized fashion. Thus, Z is required to interact with the SSP/GP1/GP2 complex that decorates the surface of the nascent particle. Confocal and biochemical analyses showed a direct LCMV and LASV GP1/GP2-Z interaction, dependent on the stable signal peptide of the GP complex [Viral infection induces a global reprogramming of cellular processes, including alterations the organization of intracellular membranes and organelles ,106. Assachinery ,36. Myriachinery . Z posse complex . Cells t complex . Sub-cel complex . Z myris complex . However complex . Further complex . Inhibit


Left ventricular noncompaction (LVNC) is a myocardial disorder characterized by excessive left ventricular (LV) trabeculae. Current methods for quantification of LV trabeculae have limitations. The aim of this study is to describe a novel technique for quantifying LV trabeculation using cardiovascular magnetic resonance (CMR) and fractal geometry. Observing that trabeculae appear complex and irregular, we hypothesize that measuring the fractal dimension (FD) of the endocardial border provides a quantitative parameter that can be used to distinguish normal from abnormal trabecular patterns.n=30; healthy whites, n=75; healthy blacks, n=30.Fractal analysis is a method of quantifying complex geometric patterns in biological structures. The resulting FD is a unitless measure index of how completely the object fills space. FD increases with increased structural complexity. LV FD was measured using a box-counting method on CMR short-axis cine stacks. Three groups were studied: LVNC (defined by Jenni criteria), In healthy volunteers FD varied in a characteristic pattern from base to apex along the LV. This pattern was altered in LVNC where apical FD were abnormally elevated. In healthy volunteers, blacks had higher FD than whites in the apical third of the LV  (mean±s.e.m.). Comparing LVNC with healthy volunteers, maximal apical FD was higher in LVNC . The fractal method was more accurate and reproducible  than two other CMR criteria for LVNC (Petersen and Jacquier).FD is higher in LVNC patients compared to healthy volunteers and is higher in healthy blacks than in whites. Fractal analysis provides a quantitative measure of trabeculation and has high reproducibility and accuracy for LVNC diagnosis when compared to current CMR criteria. Left ventricular noncompaction of the myocardium (LVNC) is a disorder of endomyocardial morphogenesis characterized by excessive left ventricular (LV) trabeculations and deep intertrabecular recesses.LVNC occurs in association with many congenital cardiac disorders, but it is unclear whether isolated LVNC is a separate disease entity or a morphological trait shared by phenotypically distinct cardiomyopathies. Familial disease is estimated to occur in 18% to 64% of adults  with isoSeveral attempts to quantify the extent of ventricular trabeculation as a means of diagnosing LVNC have been made using echocardiography but these criteria ,6,7 poorFine trabeculae are a feature of the normal LV and their extent may vary among healthy subjects of different racial backgrounds ,14. ExisA better approach may be to measure the complexity of the endocardial border, producing a continuous variable that can be used to both diagnose LVNC and assess its severity. One method to achieve this is to perform a fractal analysis of the endocardial border. Fractal analysis is a method of quantifying the complex geometric patterns which are encountered in biological, natural or mathematical structures ,17. The In LVNC, the excessive trabeculae create a highly irregular endocardial border. Fractal analysis of these borders in LVNC should generate a higher FD when compared to normal hearts.In this proof of principle study we have assessed the utility of measuring FD as a method for discriminating patients with LVNC from ethnically diverse healthy subjects. The specific aims were to assess: i) the FD in LVNC patients and healthy white and black volunteers and ii) the diagnostic accuracy and reproducibility of FD for identification of LVNC compared to current standard imaging criteria.n = 30); healthy white (n = 75) and healthy black (n = 30) volunteers. Ethnicity was self-defined. Healthy normal volunteers had a body-mass index > 18.5 kg/m2 and < 30.0 kg/m2 and were not involved in high-level competitive sports.Three adult populations (aged 18–85 years) were studied between January 2010 and February 2012: LVNC cases  were recruited from a dedicated cardiomyopathy clinic at The Heart Hospital, UCLH. Inclusion criteria for LVNC cases were: i) the fulfillment of Jenni for LVNC ,18 and ifor LVNC . To ensuClinical scans  were performed on all subjects using a 1.5-T magnet . CMR short-axis volumetric studies  were acqLV volumes, ejection fraction and LV mass were determined in all study participants using CMRtools . Two CMR methods for identifying LVNC were assessed in this study.For the identification of LVNC using the Petersen method , all segFor the Jacquier method , the epiFractal analysis was performed on the end-diastolic frames of each short-axis slice in the LV stack using an in-house macro designed for public domain software, ImageJ  versiona. To ensFractal analysis of the endocardial border was performed using the standard box-counting method available in the FracLac plug-in . In this method, a grid of known spacing  was placed over the endocardial border image  and the number of boxes that contained non-zero pixels was counted  curve (AUC) was calculated using the method described by Hanley et al. [c + [specificityc-1]), with c representing ranges over all possible criterion values.A two-sided p value < 0.05 was considered significant. Statistical analysis was performed in SPSS for Windows version 20.0 . Descriptive data are expressed as mean ± standard error of mean (s.e.m.) except where otherwise stated. Distribution of data was assessed on histograms and using Shapiro-Wilk test. Patient group characteristics were non-normally distributed and compared by the Kruskal-Wallis and Pearson’s Chi Square non parametric tests as appropriate. Correlation between continuous variables was assessed with Pearson’s Correlation Coefficient. FD was compared between the three study populations using Student’s y et al. . Optimaln = 20 for each of LVNC cases, healthy white and healthy black volunteers) by two experienced and blinded readers (reader G.C. for intra-observer and reader A.B. for inter-observer ratings). Intra-observer readings were performed with one-month temporal separation between repeat analyses. Paired measurements from fractal analysis were evaluated using the Bland-Altman method [2/n-1) . Intraclass correlation coefficient (ICC) was used to compare reproducibility between the various methods. Variability of binary outcome data for each method using established cut-off values was assessed using Fleiss’ Kappa test for concordance. For all methods, sensitivity, specificity, positive and negative predictive values with exact 95% CI are reported [Intra and inter-observer reproducibility of fractal analysis, perimetry, Petersen and Jacquier techniques were assessed on 60 subjects  Figures . Global In the basal third of the LV, there was no difference in maximal FD between LVNC patients (1.325 ± 0.007) and healthy black  or white  volunteers. In the middle third, maximal FD in LVNC (1.348 ± 0.011) was higher than in healthy black and white populations . The greatest difference in maximal FD between LVNC and healthy populations was registered in the apical third (p < 0.00001). Maximal apical FD was: LVNC, 1.392 ± 0.010; healthy white, 1.235 ± 0.004; healthy black, 1.253 ± 0.005 . In the LVNC group but not in the healthy volunteers, a correlation existed between FD and LV size . FD remained elevated even in those LVNC patients without dilatation (59% of LVNC cohort) when compared to healthy volunteers (p < 0.0001). No significant correlation was demonstrated between FD and ejection fraction in the three populations .No significant correlation was demonstrated between FD and body-surface area in the three populations  measured using the Jacquier technique, were significantly higher than in healthy volunteers Table .2  and favourable coefficients of variation (5% for both intra and inter-observer readings). Comparison between methods showed better reproducibility for global LV FD, maximal apical FD and total LV perimetry when compared to the Petersen and Jacquier methods Table .The main findings of this study were that: i) FD was significantly higher in LVNC patients compared to healthy volunteers, ii) FD was higher in healthy blacks than in whites, and iii) fractal analysis was accurate and reproducible when compared to current techniques. The fractal method permits better identification of patients with LVNC.Although classically used to describe ‘pure fractals’ , fractal analysis can also be performed on real-world objects . In natuFractal analysis has already found widespread application in the medical imaging field for the analysis of lungs , vasculaThe elevated FD noted at the mid-ventricular level in both LVNC and healthy volunteers Figure  is explaWhile the apex of healthy hearts is known to be lined by numerous trabeculae ,36, thesThe healthy LV is more trabeculated in blacks than in whites as evidenced by higher global LV FD and maximal apical FD in this study. This ethnic difference, suggested before ,14,38 bun = 367) Kawel [In a large multi-ethnic cohort free from hypertension and cardiac disease at baseline (7) Kawel  reportedAccurate diagnosis of LVNC is required for the appropriate management of confirmed cases and it avoids the unjustified diagnosis of cardiomyopathy in healthy subjects ,40. ApplWe found a positive correlation between FD and LV size within the LVNC population. This raises the question about whether the higher FD recorded in LVNC cases could simply have been the result of larger cardiac size. To address this, we showed how FD remained elevated even amongst those LVNC cases with non-dilated ventricles. Furthermore, the box-counting approach is scaled so each FD is calculated relative to the size of the ROI (size of the LV). This correlation between FD and LV size is interesting, and merits further study involving a larger more extensively-phenotyped population.The main limitation of this study is the lack of an LVNC reference-standard, so a composite diagnostic criterion was used to define our LVNC cases. This was a single-centre study and thus the sensitivity of the developed methodology to the use of different scanners, different manufacturers and to varying scan parameters has not been tested. The last apical slice of short-axis stack, containing a few milliliters of blood and highly prone to partial voluming, was omitted from analysis (in line with the approaches of Petersen et al.  and KaweThe LV FD could permit the stratification of hearts imaged by CMR across a spectrum of normal to abnormal trabecular patterning but ethnically-appropriate, population-based, normative reference ranges for trabeculation in health will be needed for this.In this study, we describe a novel reproducible approach to measuring LV trabecular complexity using fractal analysis applied to CMR. FD is higher in LVNC patients compared to healthy volunteers and is higher in healthy blacks than in whites. In subjects with a high pre-test probability for disease, the biological signal measured by fractal analysis distinguishes LVNC from health with high diagnostic accuracy.AUC: Area under curve; CI: Confidence interval; CMR: Cardiovascular magnetic resonance; FD: Fractal dimension; ICC: Intraclass correlation coefficient; LV: Left ventricle/ventricular; LVNC: Left ventricular noncompaction; NC/C: Noncompacted to compacted ratio; RC: Repeatability coefficient; ROC: Receiver operating characteristics; ROI: Region of interest; s.d.: Standard deviation; s.e.m: Standard error of mean.The authors declare that they have no competing interests.All authors have contributed significantly to the submitted work: JCM. and PME conceived and directed the project. JCM, VM, PME and GC wrote the article. JCM, VM, PME, ASF and GC developed the fractal technique for use with CMR. GC, CC, ASF, DS, SA and JCM performed the CMR imaging in humans. GC and JCM analyzed the human data. GC and AB performed the reproducibility experiments. PME, VM, TM, RW and WJM provided expert advice and critical review of the manuscript. All authors read and approved the final manuscript.Detailed clinical characteristics of the 30 LVNC cases.Click here for file


Protein kinases provide a platform for the integration of signal transduction networks. A key feature of transmitting these cellular signals is the ability of protein kinases to activate one another by phosphorylation. A number of kinases are predicted by sequence homology to be incapable of phosphoryl group transfer due to degradation of their catalytic motifs. These are termed pseudokinases and because of the assumed lack of phosphoryltransfer activity their biological role in cellular transduction has been mysterious. Recent structure–function studies have uncovered the molecular determinants for protein kinase inactivity and have shed light to the biological functions and evolution of this enigmatic subset of the human kinome. Pseudokinases act as signal transducers by bringing together components of signalling networks, as well as allosteric activators of active protein kinases. These et al., 2006). However, these data contradict reports from genetic studies suggesting that kinase catalytic activity per se may not be required for IPP complex function  with Ser324  is involved in signalling from the transmembrane integrin receptor to the actin cytoskeleton, and as such regulates many cell-adhesion-dependent processes . ILK is l., 2010 . It is pl., 2010 •• has unh Ser324 c and e. h Ser324 a and e. h Ser324 e, similah Ser324 d. While  binding ••. Thus,HER3/ErbB3 is a member of the human epidermal growth family (HER) of tyrosine kinase receptors that also includes HER1/ErbB1, HER2/ErbB2 and HER4/ErbB4. Of the four members, HER3 is classified as a pseudokinase because it lacks two of the eleven residues important for catalysis c 37••,3•, suggesVRK3 is a human vaccinia related kinase and lacks catalytic activity owing to the substitution of six out of eleven active site residues a and b. Toxoplasma gondii have been reported recently [et al. reported that ROP2 does not interact with ATP [2+ ion is present in the ROP2/8 active site  family, ROP2 and ROP8 from the intracellular parasite recently ••,45. Ofrecently ••,45. Onwith ATP ••. Non-cive site h, and thive site a. Hence,ive site h, which The link between nucleotide pocket occupancy of a kinase/pseudokinase domain and its conformation has not been fully appreciated, despite potential functional implications and examples in other classes of enzymes that bind nucleotides . Pseudokinases like STRADα and HER3 bind ATP with low nanomolar affinity ••,22•. SRecently, conformational regulation of a kinase domain through nucleotide pocket occupancy has also been described for catalytically active kinases. For instance in the case of IRE1 (Inositol-requiring enzyme 1), its phosphoryl transfer activity is functionally dispensable . Insteadet al., 2006 raised a monoclonal antibody that recognises the pseudokinase domain of guanylyl cyclase receptor 2C (GC-C) and showed that the immunoreactivity was compromised upon ATP binding, suggesting large conformational shifts between the ATP bound/unbound forms of this domain [Another prominent example of a pseudokinase influenced by ATP binding is the family of receptor guanylyl cyclases (RGC), that produce the second messenger cGMP in response to the binding of several natriuretic factors (reviewed in ). These s domain . Fine epErbB genes coupled by the allosteric mechanism of activation rather than phosphorylation, may be the cause for the loss of catalytic activity of ErbB3. It is possible that other pseudokinases have also evolved in a similar way. Given the proximity to the EGF receptor tyrosine kinases in the human kinome, EphB6 and EphB10 may be regulating the active members of the Eph receptor tyrosine kinase family by using an as yet to be determined, analogous trans-activation mechanism to ErbB3.Interestingly, there are a number of membrane receptors that contain a predicted cytoplasmic pseudokinase domain, including members of the ephrin receptor EphB6 and EphB10 as well as members of the epidermal growth factor receptor family ErbB3/Her3. As already discussed above the four members of the EGF receptor kinases (ErbB1–4) are envisaged to trans-activate by forming so-called asymmetric heterodimers, upon ligand binding rather than phosphorylation ••,38. Th2+ levels [Another example where a pseudokinase forms complexes with active kinases are members of the kinase suppressor of Ras 1 and 2 (KSR1/2), that are essential for Ras induced activation of the RAF-MEK-ERK module in MAP kinase signalling. These predicted pseudokinases act as scaffolds bringing together the three components of the MAP kinase pathway , thus regulating signalling output and potentiation . The rol+ levels . The lacet al. demonstrate that KSR can function as an allosteric activator of RAF, without the requirement for KSR phosphoryltransfer activity [A prominent finding that suggests KSR may have functions beyond the scaffolding component, came from the recent revelation that RAF kinase domains form homodimers and heterodimers resulting in activated RAF kinase •• Figurd. This sactivity ••.Perhaps the most studied pseudokinase domain belongs to the members of Janus tyrosine kinase (JAK) family. The JAK isoforms include JAK1, JAK2, JAK3 and TYK2 all of which contain an N-terminal pseudokinase domain (JH2) followed by a kinase domain (JH1). This characteristic feature of containing two kinase domains gives JAKs their name, referring to the two-faced Roman god Janus. JAKs respond upon receptor binding to cytokines and phosphorylate the cytoplasmic region of these cytokine receptors, thus creating sites of interaction for downstream signalling molecules. The pseudokinase domain is required for JAK2 auto-inhibition and is essential for JAK2 cytokine activation . A gain-How did pseudokinases evolve? In general it is plausible to believe that kinases evolved from ATP binding enzymes, since nucleotide binding must have preceded catalytic function. Therefore it is not surprising that ligand (ATP) binding plays a key regulatory component for pseudokinase/kinase function. As a consequence there are two possibilities—either pseudokinases represent ‘would-be’ active kinases, or they represent kinases that have lost their catalytic activity.The first scenario could be true for proteins that are well conserved as pseudokinases throughout eukaryotic kinase evolution such as SCYL1-3 and GCN2. For instance, ATP-binding precursors of yeast SCY1 and GCN2 may have failed to ‘mature’ as active protein kinases capable of phosphoryl transfer. Instead, important non-catalytic functions were conserved throughout evolution for these pseudokinases. In GCN2 this could have been aided by loss of evolutionary pressure for kinase maturation, since another kinase domain is present in the same polypeptide chain.However, the reasoning above cannot account for a significant number of pseudokinases that do not have clear pseudokinase homologues in primitive species . Therefore these may have evolved via a different route. One possibility is that pseudokinases have evolved from active kinases once capable of phosphorylating and activating their substrates, but have lost their activity during evolution. The structures of STRADα, ILK and VRK3 show that despite being inactive, these pseudokinases are able to assume an active conformation with a highly organised active site poised for phosphoryl transfer. In addition, the structure of the LKB1 heterotrimer and ILK-α-parvin complex, reveal that STRAD and ILK can bind LKB1 and α-parvin respectively as pseudosubstrates ••,36••. The process of using previously catalytically competent domains as scaffolds is plausible from an evolutionary perspective as enzymes have evolved to bind their substrates. This is also observed in other classes of enzymes such as pseudophosphatases , and simThe phenomenon of catalytically inactive members is not restricted to enzyme families involved in protein phosphorylation alone, but extends to other processes of postranslational modifications such as ubiquitinylation. A class of ubiquitin E2 ligases called Uev (ubiquitin E2 variant) domains have the same fold as E2 enzymes but lack certain catalytic residues and are hence devoid of catalytic activity . SimilarIn recent years, a burst of studies have provided valuable insights into the biological functions and the mechanism of action of pseudokinases. An important revelation is that the initially predicted fraction of kinases that are truly ‘dead’, ∼10% of the human kinome, may have been an overestimate. Despite variations in the catalytic motifs of pseudokinases, nature has acquired alternative mechanisms to correct for the missing functions in phosphoryl transfer, by making use of the versatile kinase fold. Exhaustive structural studies of protein kinases have established that the kinase/pseudokinase domain is plastic, and attains many different conformations in response to binding of micromolecular and macromolecular ligands. Similarly, large conformational changes occur upon postranslational modifications such as phosphorylation. Therefore, pseudokinases can be envisaged as ‘elastic’ scaffolds, bringing together components of a particular signalling network, as well as being allosteric activators of protein kinases.• of special interest•• of outstanding interestPapers of particular interest, published within the annual period of review, have been highlighted as:


As found, the metals produced, although in a different way, dose- and time-dependent changes of all these parameters. Importantly, Cd2+ beginning from 10 [mu]M and already at short incubation time (3 h) significantly inhibited the FCCP-uncoupled cell respiration; besides, practically the complete inhibition of the respiration was reached after 3 h incubation with 50 [mu]M Hg2+ or 500 [mu]M Cd2+, whereas even after 48 h exposure with 500 [mu]M Cu2+, only a 50% inhibition of the respiration occurred. Against the Cd2+-induced cell injury, not only different antioxidants and mitochondrial permeability transition pore inhibitors were protective but also such mtETC effectors as FCCP and stigmatellin (complex III inhibitor). However, all mtETC effectors used did not protect against the Hg2+- or Cu2+-induced cell damage. Notably, stigmatellin was shown to be one of the strongest protectors against the Cd2+-induced cell damage, producing a 15–20% increase in the cell viability. The mechanisms of the mtETC involvement in the heavy-metal-induced mitochondrial membrane permeabilization and cell death are discussed.To clarify the role of mitochondrial electron transport chain (mtETC) in heavy-metal-induced neurotoxicity, we studied action of Cd IμM of Cd2+ induced a significant increase in the ROS formation after 30 min of incubation with the cells, whereas after 3 h already all Cd2+ concentrations under test stimulated strongly the intracellular ROS production  all concentrations of the metal studied enhanced the intracellular ROS generation  and the2+, beginning from 10 μM and already at short incubation time (3 h), produced significant inhibition of FCCP-uncoupled respiration of the cells  and starting from 24 h of treatment, the significant changes in the cellular respiration occurred  were strongly depressed, indicating a potent inhibitory effect on the respiratory chain. It should be remind that the participation of the mtETC could be checked by estimation of the uncoupled respiration rate, the inhibition of which is considered a marker of the disturbance of the respiratory chain components. Cdhe cells . The comcubation . As to Cion time  h, produin situ . This small but significant effect of stigmatellin was observed despite that this concentration of the complex III inhibitor per se stimulated the ROS production of the control cells, upon the average, on 25%.We obtained also that Ru-360 , RR (MCU inhibitor), CsA and BKA (MPT pore inhibitors), FCCP (protonophoric uncoupler), stigmatellin (complex III inhibitor), and different antioxidants partially prevented the Cd2+-induced necrosis  as a key feature that is in line with previous observations of Armstrong et al. [2+, which partially depressed by Cu2+, ethoxyformic anhydride , and N-ethylmaleimide, NEM, the Rieske ISP participates as well  [The results, revealed by us on the intact cells herein and before , areol, DTT, , 17, 53)ol, DTT, , 49. Furol, DTT, , 55. Not by Cd2+ . Besides Q0 site . In addicceptor) .2+, which is known to induce both necrosis and/or apoptosis, and even can produce antiapoptotic effect [2+ produced a rapid and transient ROS generation, mitochondrial dysfunction, and apoptosis; moreover, the Cd2+-triggered apoptosis was not inhibited by CsA and BKA while it was blocked by NAC pretreatment. In some other cells [2+-induced apoptosis was mitochondria- and ROS-dependent event that, in opposite, was inhibited by oligomycin [It should be mentioned, however, that there is some cell type specificity in toxic action of Cdc effect , 58. Forc effect , Cd2+ prer cells , 60, 61,igomycin , 61, andigomycin . Besidesigomycin –66. 2+ as a probe correlate well with data existed in literature when MPT and/or cell death are produced by different other prooxidants and effectors [2+ led to the MPT induction; moreover, they found that the reactive group of salicylate for inducing oxidative stress was the hydroxyl group which, by interacting with Fe-S cluster N2 of complex I, produced the ROS increase [2 [It seems important to say that findings obtained with Cdffectors , 67–72. ffectors , 68 as wffectors –77. Furtffectors –84, inclffectors . In partrease [2 . The imprease [2 , ceramidrease [2 –90, and rease [2 , 92 durirease [2 –100 and rease [2 , 102 increase [2 –107 are 2+ and/or Ca2+plus Pi. On the basis of own findings [2+-activated CsA-sensitive pore originally proposed by Halestrap (for reviews see [iC) had been integrated with an idea of Fontaine and Bernardi (for review see [2+(Me2+)-binding site(s), critical for the MPT induction, and depending on conditions and cell type, either one or both complexes could be involved in triggering of the MPT pore assembly; besides, the complex I of the respiratory chain likely constitutes the P-site while complex III—the S-site of the MPT pore [2+-binding sites but also main loci for ROS generation that was instrumental in oxidation of critical thiol groups and the MPT pore opening [2+-binding site(s) are most likely disposed: (i) on the way of reverse electron transfer from succinate to NAD+ (complex I); (ii) on cytochrome b somewhere near heme bL and close to stigmatellin binding site (complex III) [2+-dependent CsA-sensitive) MPT pore complex, while complex III is likely involved in the “unregulated”  MPT pore assembly and might concern to the external (inhibitory) Ca2+-binding site. Some of these views become more valid in the light of new interesting experimental data obtained by different investigators during the latest time [2+ binding sites and dithiols is so mysterious and intrigues problems, the decision of which, we hope, could bring new understanding of processes of aging and death.In general, all the above-mentioned data support our hypothesis about the possible involvement of the mtETC components, namely, a supercomplex formed by complex I (P-site) and complex III (S-site) in the mitochondrial membrane permeabilization mediated by the MPT pore  in whicview see ) concernMPT pore . Later, MPT pore . In part opening . The afolex III) . Our reclex III) , 109 extlex III) . Moreoveest time ; the com2+-induced injury, not only well-known antioxidants and the MPT pore and MCU inhibitors are effective but also different mtETC effectors. Among mtETC effectors under test, only stigmatellin produced the significant and sustained protection of PC12 cells. Moreover, stigmatellin was found to be one of the strongest protectors that exhibited its action not only on different types of the cells  of neurotoxic action of the heavy metals. Notably, the data obtained herein further support our hypothesis about the direct involvement of the mtETC components in the mitochondrial membrane permeabilization mediated by the MPT pore assembly [In conclusion, the results obtained in this study give new important information to understand the mechanism(s) underlying the heavy-metal-produced neurotoxic action, pointing to the critical involvement of mitochondrial dysfunction in the heavy-metal-induced cell death. We have shown that against the Cdd AS-30D ) but on d AS-30D , 49. Allassembly –18, 49.


Oncogenes as the center of cancer research focused attention 30 years ago on signal transduction especially tyrosine phosphorylation. Discovery that the Jun oncoprotein was a site-specific DNA binding protein demanded a connection of signaling abnormalities with transcriptional control . The cytLess than a year later Richard Jove and colleagues made a convincing first link between STAT3 and oncogenesis . Src-traSo while a STAT3 cancer connection has been extensively documented, what precisely does STAT3 do to promote or sustain cancer? Much work on STAT3 gene targets supports an anti-apoptotic role for STAT3 as well as a positive affect on cell growth.Now, low and behold, STAT3 appears to be involved with the earliest recognized metabolic abnormality of cancer cells, energy derivation through glycolysis. Valeria Poli and a host of colleagues  have pubMost of the new experiments  use miceHowever, we never obtained (attempts were made) to get a Y705F mutant of STAT3C and did not demonstrate therefore that the mutant required phosphorylation of Y705 just as does the wild type in order to function as a oncogene. Peter Shaw however has corrected our error. STAT3C indeed requires Y705 phosphorylation to be active . Shaw anC/C MEFs. And HIF1α increases transcription of all known glycolytic enzymes. STAT3c/c activity also increases mRNA for a set of proteins, including PDK-l (pyruvate dehydrogenase kinase 1) which blocks the shuttling of pyruvate into the citric acid cycle in mitochondria sending it into the glycolytic pathway with resulting increased lactic acid production. With the increased dependence on glycolysis STAT3C/C cells require high glucose to avoid apoptosis. A decreased mitochondrial oxidative phosphorylation attendant to presence of STAT3C/C likely depends on a lowered amount of mRNA for mitochondrial proteins, e.g. ATP synthase, for the G subunit of the mitochondrial FO complex, for fumarate hydratase.The Warburg type tumor metabolism in STAT3C/C cells depends on an increase in levels of HIF1 α that is at least partly due to increased STAT3 dependent transcription shown earlier by Niu et al.  with furThe authors discuss the recent findings of Andy Larner and David Levy ,17 that C/C) is a separate set of events from the serine 727 phosphorylation inside the mitochondria. Thus there is no contradiction between the two sets of findings but emphasizes STAT3 has multiple roles in cell transformation. The powerful message of the newest results is that STAT3 is involved in the time-honored characteristic of cancer cells, dependence on glycolytic energy derivation.A major point made in the Demaria et al. paper is that the metabolic effect brought about by phosphorylating tyrosine 705 activates STAT3 in the cell nucleus (as must occur at a low level in STAT3


ELOVL4), which leads to vision loss in young adults. ELOVL4 is an integral endoplasmic reticulum (ER) protein that mediates the elongation of very long chain (VLC) FAs. Mutations in ELOVL4 lead to truncation and mislocalization of the translated protein from the ER, the site of FA elongation. Little is known about the enzymatic elongation of VLC-FAs by ELOVL4. We over-expressed full-length mouse ELOVL4, an N-glycosylation-deficient mutant, an ER-retention mutant, and mutants of active site histidines to parse their individual roles in VLC-FA elongation. ELOVL4 elongated appropriate precursors to the corresponding VLC-FA species ≥28 carbons. Active site histidine mutants of ELOVL4 did not elongate appropriate precursors, establishing ELOVL4 as the elongase. Displacing ELOVL4 from the ER was sufficient to cause loss of condensation activity, while absence of N-glycosylation was irrelevant for enzyme function. This study shows that ELOVL4 enzymatic activity is governed by individual histidines in its active site and the ER microenvironment, both of which are essential for elongation of VLC-FAs.Autosomal dominant Stargardt-like macular dystrophy (STGD3) in humans results from mutations in elongation of very long chain FAs-like 4 ( ELOVL4) gene  retention signal. The truncated gene product is subsequently mislocalized to other cellular compartments, resulting in protein aggregation  FAs greater than 26 carbons in length  in humans was described by three independent research groups as resulting from mutations in the elongation of very long chain fatty acids-like 4 (L4) gene –3. Domin in humanregation –7. Bearin length , 9.trans-2,3-enoyl-CoA reductase (TER)  . ELOVL4 se (TER)  and HACDse (TER)  when coese (TER) .VLC-FAs greater than 26 carbons (>C26) are unique in that they are found only in tissues expressing ELOVL4 such as retina, brain, testes, and skin , with th1) ELOVL4 has an N-glycosylation consensus site at the N terminus (2) ELOVL4 contains an active site comprising a histidine-rich motif (HXXHH) proposed to chelate iron, which acts as an electron transfer moiety during O2-dependent redox reactions (3) a dilysine ER retention motif at the C terminus that localizes ELOVL4 to the ER, where KAR, TER, and HACD1–4 are also located. Lysine residues juxtaposed to the C terminus of transmembrane proteins provide a recognition signal for the ER retention machinery to allow retrieval and retention of ER-resident proteins  proteins . The depHence, in this study we sought to characterize the enzymatic activity of the ELOVL4 protein and the contribution of each of the protein motifs toward the synthesis of VLC-FAs. We hypothesized that ELOVL4 is indeed the elongase that mediates successive elongation steps generating FA chain lengths ≥28 carbons, and that these individual motifs are critical for enzyme function. Using a gain-of-function approach, we show conclusively that ELOVL4 is the elongase responsible for elongation of FAs to C28, C30, C32, and C36 FAs. We also show that this activity is dependent on the coordination of the histidine residues in the active site and requires localization to the ER, while N-glycosylation is dispensable for enzyme function.2 plates with DMEM-nutrient mixture F-12  supplemented with 10% heat-inactivated FBS  and antibiotics [100 units/ml each of penicillin and streptomycin (Invitrogen)]. HEK293T and HeLa cells were grown in DMEM medium supplemented with 10% FBS and antibiotics.ARPE19 cells were cultured in 10 cmElovl4 (WT and 5 bp deletion) were PCR amplified and cloned in frame with a triple hemagglutinin (HA) tag in 3 BSENXpKH vector (kindly provided by Dr. Scott M. Plafker). Active site mutants of WT ELOVL4 were constructed by mutating single histidines to glutamine or by mutating all three histidines to glutamines. N-glycosylation mutant was constructed by mutating the NDTV consensus site to NDAV and the lysine mutant was constructed by mutating the two lysines in the ER retention signal to arginines. All mutagenesis was performed using QuickChange II site directed mutagenesis kits . Adenovirus particles (Ad5) were generated as previously described  at 1:1,000 dilution to detect untagged ELOVL4 as reported in Agbaga et al. . In thisCell pellets were lysed in lysis buffer containing 20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, complete protease inhibitors (EDTA free) , 1 mM PMSF, and 1% Triton X-100, and processed as previously described . Equal aHeLa cells were grown on Labtek chamber slides and transiently transfected with ELOVL4 constructs. After 48 h, slides were rinsed and fixed as per Logan et al. . The sliARPE19 cells and HEK293T cells were transduced overnight with mouse ELOVL4 adenovirus and treated with FA precursors as described in Logan et al. , and 10 μM 2-[14C]malonyl-CoA at 37°C for 1 h. Individual reactions were initiated with the addition of 200 μg of microsomal protein. To measure condensing activity alone, NADPH/NADH was omitted from the reaction buffer. FAMEs were spotted on C-18 reverse phase silica high-performance TLC plates  and resolved using CHCl3:methanol:water  as the mobile phase. The plates were exposed to a multi-sensitive storage phosphor screen for 48 h and the radioactivity visualized using a Packard Cyclone Plus phosphor imager . Data were analyzed as per Logan et al.  were analyzed using a multivariate ANOVA followed by a post hoc Scheffe test using Statistica 2000 software . Data are represented as the mean ± SD. Significance is indicated by ELOVL4 has been shown to elongate 26:0 to 28:0 in culture ; howeverHere we demonstrate that, in addition to elongation of 26:0 to 28:0 , ELOVL4 ELOVL4 (Elovl4 cDNA were constructed (VLC-FA elongation is affected by mutations in exon 6 of ELOVL4 . Howeverstructed . To studAd5) of the HA-tagged constructs was generated by homologous recombination using RAPAd CMV adenoviral expression system . Recombinant adenovirus for untagged ELOVL4 was generated previously as described in Agbaga et al. , and active site mutants  in HEK293T cells and supplemented with or without [nontreated (NT)] 20:5n3 . UT and These experiments demonstrate conclusively that the enzymatic activity of ELOVL4 is mediated by the histidine residues in the active site.ELOVL4 is N-glycosylated, a posttranslational modification known to be important for the proper folding and stability of proteins, albeit the relevance of this glycosylation for enzyme activity is unclear. We over-expressed ΔNG mutant and WT in ARPE19 cells and supplemented with 20:5n3 . GFP-expMutations in ELOVL4 result in truncation and loss of the C terminus of the protein, the region encoding the dilysine ER-retention signal, and subsequent loss in enzyme activity –3, 9. WhDuring FA elongation, ELOVL4 mediates the first rate-limiting condensation reaction between malonyl-CoA and a fatty acyl-CoA to generate the 3-keto-acyl intermediate . Other eELOVL4 develop Stargardt macular dystrophy (STGD3) characterized by early onset retinal degeneration and loss of vision . Rhodopsin is dually glycosylated at N2 and N15 , and mutiability . Howeveriability . Thus, s1) access to the precursor is lost; 2) association with the other components of the elongation machinery, for example KAR and TER, might be disbanded; or 3) both. These findings suggest that the mere mislocalization of the STGD3 mutant would render it inactive. However, we have shown that even when the STGD3 mutant was forced to localize to the ER, it lacked condensation activity (STGD3 mutant protein has been shown to be mislocalized in cultured cells , 7, 51, activity . This suIn conclusion, we have demonstrated the prominent features of ELOVL4 for optimal enzyme activity during biosynthesis of VLC-FAs. More importantly, our analysis of ELOVL4 enzyme activity paves an avenue to study this unique class of FAs, the VLC-PUFAs, which have long been overlooked and undervalued.