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The synaptic cleft of the neuromuscular junction (NMJ) consists of a highly specialized extracellular matrix (ECM) involved in synapse maturation, in the juxtaposition of pre- to post-synaptic areas, and in ensuring proper synaptic transmission. Key components of synaptic ECM, such as collagen IV, perlecan and biglycan, are binding partners of one of the most abundant ECM protein of skeletal muscle, collagen VI (ColVI), previously never linked to NMJ. Here, we demonstrate that ColVI is itself a component of this specialized ECM and that it is required for the structural and functional integrity of NMJs. In vivo, ColVI deficiency causes fragmentation of acetylcholine receptor (AChR) clusters, with abnormal expression of NMJ-enriched proteins and re-expression of fetal AChRγ subunit, both in Col6a1 null mice and in patients affected by Ullrich congenital muscular dystrophy (UCMD), the most severe form of ColVI-related myopathies. Ex vivo muscle preparations from ColVI null mice revealed altered neuromuscular transmission, with electrophysiological defects and decreased safety factor (i.e., the excess current generated in response to a nerve impulse over that required to reach the action potential threshold). Moreover, in vitro studies in differentiated C2C12 myotubes showed the ability of ColVI to induce AChR clustering and synaptic gene expression. These findings reveal a novel role for ColVI at the NMJ and point to the involvement of NMJ defects in the etiopathology of ColVI-related myopathies.

INTRODUCTION The cellular prion protein (PrP(C) ) is commonly recognized as the precursor of prions, the infectious agents of the fatal transmissible spongiform encephalopathies, or prion diseases. Despite extensive effort, the physiological role of PrP(C) is still ambiguous. Evidence has suggested that PrP(C) is involved in different cellular functions, including peripheral nerve integrity and skeletal muscle physiology. METHODS We analyzed the age-dependent influence of PrP(C) on treadmill test-based aerobic exercise capacity and on a series of morphological and metabolic parameters using wild-type and genetically modified mice of different ages expressing, or knockout (KO) for, PrP(C) . RESULTS We found that aged PrP-KO mice displayed a reduction in treadmill performance compared with PrP-expressing animals, which was associated with peripheral nerve demyelination and alterations of skeletal muscle fiber type. CONCLUSION PrP-KO mice have an age-dependent impairment of aerobic performance as a consequence of specific peripheral nerve and muscle alterations.

Alterations in the glial function of TDP-43 are becoming increasingly associated with the neurological symptoms observed in Amyotrophic Lateral Sclerosis (ALS), however, the physiological role of this protein in the glia or the mechanisms that may lead to neurodegeneration are unknown. To address these issues, we modulated the expression levels of TDP-43 in the Drosophila glia and found that the protein was required to regulate the subcellular wrapping of motoneuron axons, promote synaptic growth and the formation of glutamate receptor clusters at the neuromuscular junctions. Interestingly, we determined that the glutamate transporter EAAT1 mediated the regulatory functions of TDP-43 in the glia and demonstrated that genetic or pharmacological compensations of EAAT1 activity were sufficient to modulate glutamate receptor clustering and locomotive behaviors in flies. The data uncovers autonomous and non-autonomous functions of TDP-43 in the glia and suggests new experimentally based therapeutic strategies in ALS.

Insulin resistance and obesity are associated with a reduction of mitochondrial content in various tissues of mammals. Moreover, a reduced nitric oxide (NO) bioavailability impairs several cellular functions, including mitochondrial biogenesis and insulin-stimulated glucose uptake, two important mechanisms of body adaptation in response to physical exercise. Although these mechanisms have been thoroughly investigated in skeletal muscle and heart, few studies have focused on the effects of exercise on mitochondria and glucose metabolism in adipose tissue. In this study, we compared the in vivo effects of chronic exercise in subcutaneous adipose tissue of wild-type (WT) and endothelial NO synthase (eNOS) knockout (eNOS−/−) mice after a swim training period. We then investigated the in vitro effects of NO on mouse 3T3-L1 and human subcutaneous adipose tissue–derived adipocytes after a chronic treatment with an NO donor: diethylenetriamine-NO (DETA-NO). We observed that swim training increases mitochondrial biogenesis, mitochondrial DNA content, and glucose uptake in subcutaneous adipose tissue of WT but not eNOS−/− mice. Furthermore, we observed that DETA-NO promotes mitochondrial biogenesis and elongation, glucose uptake, and GLUT4 translocation in cultured murine and human adipocytes. These results point to the crucial role of the eNOS-derived NO in the metabolic adaptation of subcutaneous adipose tissue to exercise training.

Scorpions are among the oldest venomous living organisms and the family Buthidae is the largest and most medically relevant one. Scorpion venoms include many toxic peptides, but recently, a metalloprotease from Tityus serrulatus called antarease was reported to be capable of cleaving VAMP2, a protein involved in the neuroparalytic syndromes of tetanus and botulism. We have produced antarease and an inactive metalloprotease mutant in a recombinant form and analyzed their enzymatic activity on recombinant VAMP2 in vitro and on mammalian and insect neuromuscular junction. The purified recombinant antarease paralyzed the neuromuscular junctions of mice and of Drosophila melanogaster whilst the mutant was inactive. We were unable to demonstrate any cleavage of VAMP2 under conditions which leads to VAMP proteolysis by botulinum neurotoxin type B. Antarease caused a reduced release probability, mainly due to defects upstream of the synaptic vesicles fusion process. Paired pulse experiments indicate that antarease might proteolytically inactivate a voltage-gated calcium channel.

Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion, growth, proliferation and differentiation. Recently, covalent and selective functionalization of glass and titanium surfaces with an adhesive peptide (HVP) mapped on [351–359] sequence of human Vitronectin allowed to selectively increase osteoblast attachment and adhesion strength in in vitro assays, and to promote osseointegration in in vivo studies. For the first time to our knowledge, in this study we investigated the resistance of adhesion sequences to proteolytic digestion: HVP was completely cleaved after 5 h. In order to overcome the enzymatic degradation of the native peptide under physiological conditions we synthetized three analogues of HVP sequence. A retro-inverted peptide D-2HVP, composed of D amino acids, was completely stable in serum-containing medium. In addition, glass surfaces functionalized with D-2HVP increased human osteoblast adhesion as compared to the native peptide and maintained deposition of calcium. Interestingly, D-2HVP increased expression of IBSP, VTN and SPP1 genes as compared to HVP functionalized surfaces. Total internal reflection fluorescence microscope analysis showed cells with numerous filopodia spread on D-2HVP-functionalized surfaces. Therefore, the D-2HVP sequence is proposed as new osteoblast adhesive peptide with increased bioactivity and high proteolytic resistance., Graphical abstract Image 1, Highlights • We first investigated the resistance of adhesive peptides to proteolytic digestion. • We synthesized peptides completely stable to enzymatic digestion. • A grafted retro-inverted sequence increased h-osteoblast adhesion and gene expression. • A grafted retro-inverted peptide induced cell focal adhesions and filopodia.

The neuromuscular junction is exposed to different types of insult, including mechanical trauma, toxins and autoimmune antibodies and, accordingly, has retained through evolution a remarkable ability to regenerate. Regeneration is driven by multiple signals that are exchanged among the cellular components of the junction. These signals are largely unknown. Miller Fisher syndrome is a variant of Guillain–Barré syndrome caused by autoimmune antibodies specific for epitopes of peripheral axon terminals. Using an animal model of Miller Fisher syndrome, we recently reported that a monoclonal anti-polysialoganglioside GQ1b antibody plus complement damages nerve terminals with production of mitochondrial hydrogen peroxide, which activates Schwann cells. Several additional signaling molecules are likely to be involved in the activation of the regeneration program in these cells. Using an in vitro cellular model consisting of co-cultured primary neurons and Schwann cells, we found that ATP is released by neurons injured by the anti-GQ1b antibody plus complement. Neuron-derived ATP acts as an alarm messenger for Schwann cells, where it induces the activation of intracellular pathways, including calcium signaling, cAMP and CREB, which, in turn, produce signals that promote nerve regeneration. These results contribute to defining the cross-talk taking place at the neuromuscular junction when it is attacked by anti-gangliosides autoantibodies plus complement, which is crucial for nerve regeneration and is also likely to be important in other peripheral neuropathies., Summary: ATP released by degenerating neurons participates in neuron-Schwann cell communication in an in vitro model of Miller Fisher syndrome and activates Schwann cell pro-regenerative properties.

An analysis of SNAP-25 isoform sequences indicates that there is a highly conserved arginine residue (198 in vertebrates, 206 in the genus Drosophila) within the C-terminal region, which is cleaved by botulinum neurotoxin A, with consequent blockade of neuroexocytosis. The possibility that it may play an important role in the function of the neuroexocytosis machinery was tested at neuromuscular junctions of Drosophila melanogaster larvae expressing SNAP-25 in which Arg206 had been replaced by alanine. Electrophysiological recordings of spontaneous and evoked neurotransmitter release under different conditions as well as testing for the assembly of the SNARE complex indicate that this residue, which is at the P1′ position of the botulinum neurotoxin A cleavage site, plays an essential role in neuroexocytosis. Computer graphic modelling suggests that this arginine residue mediates protein–protein contacts within a rosette of SNARE complexes that assembles to mediate the fusion of synaptic vesicles with the presynaptic plasma membrane.

Although it is widely accepted that mitochondria in living cells can efficiently uptake Ca(2+) during stimulation because of their vicinity to microdomains of high [Ca(2+)], the direct proof of Ca(2+) hot spots' existence is still lacking. Thanks to a GFP-based Ca(2+) probe localized on the cytosolic surface of the outer mitochondrial membrane, we demonstrate that, upon Ca(2+) mobilization, the [Ca(2+)] in small regions of the mitochondrial surface reaches levels 5- to 10-fold higher than in the bulk cytosol. We also show that the [Ca(2+)] to which mitochondria are exposed during capacitative Ca(2+) influx is similar between near plasma membrane mitochondria and organelles deeply located in the cytoplasm, whereas it is 2- to 3-fold higher in subplasma membrane mitochondria upon activation of voltage-gated Ca(2+) channels. These results demonstrate that mitochondria are exposed to Ca(2+) hot spots close to the ER but are excluded from the regions where capacitative Ca(2+) influx occurs.

Human Wolf-Hirschhorn syndrome (WHS) is a multigenic disorder resulting from a hemizygous deletion on chromosome 4. LETM1 is the best candidate gene for seizures, the strongest haploinsufficiency phenotype of WHS patients. Here, we identify the Drosophila gene CG4589 as the ortholog of LETM1 and name the gene DmLETM1. Using RNA interference approaches in both Drosophila melanogaster cultured cells and the adult fly, we have assayed the effects of down-regulating the LETM1 gene on mitochondrial function. We also show that DmLETM1 complements growth and mitochondrial K(+)/H(+) exchange (KHE) activity in yeast deficient for LETM1. Genetic studies allowing the conditional inactivation of LETM1 function in specific tissues demonstrate that the depletion of DmLETM1 results in roughening of the adult eye, mitochondrial swelling and developmental lethality in third-instar larvae, possibly the result of deregulated mitophagy. Neuronal specific down-regulation of DmLETM1 results in impairment of locomotor behavior in the fly and reduced synaptic neurotransmitter release. Taken together our results demonstrate the function of DmLETM1 as a mitochondrial osmoregulator through its KHE activity and uncover a pathophysiological WHS phenotype in the model organism D. melanogaster.

The cellular prion protein (PrP(C) ) is commonly recognized as the precursor of prions, the infectious agents of the fatal transmissible spongiform encephalopathies, or prion diseases. Despite extensive effort, the physiological role of PrP(C) is still ambiguous. Evidence has suggested that PrP(C) is involved in different cellular functions, including peripheral nerve integrity and skeletal muscle physiology.We analyzed the age-dependent influence of PrP(C) on treadmill test-based aerobic exercise capacity and on a series of morphological and metabolic parameters using wild-type and genetically modified mice of different ages expressing, or knockout (KO) for, PrP(C) .We found that aged PrP-KO mice displayed a reduction in treadmill performance compared with PrP-expressing animals, which was associated with peripheral nerve demyelination and alterations of skeletal muscle fiber type.PrP-KO mice have an age-dependent impairment of aerobic performance as a consequence of specific peripheral nerve and muscle alterations.

An acute and highly reproducible motor axon terminal degeneration followed by complete regeneration is induced by some animal presynaptic neurotoxins, representing an appropriate and controlled system to dissect the molecular mechanisms underlying degeneration and regeneration of peripheral nerve terminals. We have previously shown that nerve terminals exposed to spider or snake presynaptic neurotoxins degenerate as a result of calcium overload and mitochondrial failure. Here we show that toxin-treated primary neurons release signaling molecules derived from mitochondria: hydrogen peroxide, mitochondrial DNA, and cytochrome c. These molecules activate isolated primary Schwann cells, Schwann cells cocultured with neurons and at neuromuscular junction in vivo through the MAPK pathway. We propose that this inter- and intracellular signaling is involved in triggering the regeneration of peripheral nerve terminals affected by other forms of neurodegenerative diseases.

The features of implant devices and the reactions of bone-derived cells to foreign surfaces determine implant success during osseointegration. In an attempt to better understand the mechanisms underlying osteoblasts attachment and spreading, in this study adhesive peptides containing the fibronectin sequence motif for integrin binding (Arg-Gly-Asp, RGD) or mapping the human vitronectin protein (HVP) were grafted on glass and titanium surfaces with or without chemically induced controlled immobilization. As shown by total internal reflection fluorescence microscopy, human osteoblasts develop adhesion patches only on specifically immobilized peptides. Indeed, cells quickly develop focal adhesions on RGD-grafted surfaces, while HVP peptide promotes filopodia, structures involved in cellular spreading. As indicated by immunocytochemistry and quantitative polymerase chain reaction, focal adhesions kinase activation is delayed on HVP peptides with respect to RGD while an osteogenic phenotypic response appears within 24 h on osteoblasts cultured on both peptides. Cellular pathways underlying osteoblasts attachment are, however, different. As demonstrated by adhesion blocking assays, integrins are mainly involved in osteoblast adhesion to RGD peptide, while HVP selects osteoblasts for attachment through proteoglycan-mediated interactions. Thus an interfacial layer of an endosseous device grafted with specifically immobilized HVP peptide not only selects the attachment and supports differentiation of osteoblasts but also promotes cellular migration.

The SNARE proteins VAMP/synaptobrevin, SNAP-25 and Syntaxin are core components of the apparatus that mediates neurotransmitter release. They form a heterotrimeric complex and an undetermined number of SNARE complexes assemble to form a super-complex. Here, we present a radial model of this nanomachine, derived from experiments performed with botulinum neurotoxins, which led to the identification of one arginine in SNAP-25 and one aspartate in Syntaxin (R206 and D253 in Drosophila melanogaster). These residues are highly conserved and predicted to play a major role in the protein-protein interactions among SNARE complexes by forming an ionic couple. Accordingly, we generated transgenic Drosophila lines expressing SNAREs mutated in these residues and performed an electrophysiological analysis of their neuromuscular junctions. Our results indicate that SNAP-25-R206 and Syntaxin-D253 play a major role in neuroexocytosis and support a radial assembly of several SNARE complexes interacting via the ionic couple formed by these two residues.

The β2 auxiliary subunit of voltage-gated sodium channels (VGSCs) appears at early stages of brain development. It is abundantly expressed in the mammalian central nervous system where it forms complexes with different channel isoforms, including Na(v)1.2. From the structural point of view, β2 is a transmembrane protein: at its extracellular N-terminus an Ig-like type C2 domain mediates the binding to the pore-forming alpha subunit with disulfide bonds and the interactions with the extracellular matrix. Given this structural versatility, β2 has been suggested to play multiple functions ranging from channel targeting to the plasma membrane and gating modulation to control of cell adhesion. We report that, when expressed in Chinese Hamster Ovary cells CHO-K1, the subunit accumulates at the perimetral region of adhesion and particularly in large lamellipodia-like membrane processes where it induces formation of filopodia-like structures. When overexpressed in developing embryonic rat hippocampal neurons in vitro, β2 specifically promotes formation of filopodia-like processes in dendrites leading to expansion of the arborization tree, while axonal branching remains unaltered. In contrast to this striking and highly specific effect on dendritic morphology, the targeting of functional sodium channels to the plasma membrane, including the preferential localization of Na(v)1.2 at the axon, and their gating properties are only minimally affected. From these and previously reported observations it is suggested that β2, among its multiple functions, may contribute to promote dendritic outgrowth and to regulate neuronal wiring at specific stages of neuronal development.

s / Pancreatology 12 (2012) 502–597 505 Results: During the course of the study, the average level of IL-6 was significantly (p

Mitochondrial Ca2+-uptake is important for the regulation of aerobic ATP production and is involved in apoptosis. Muscle fibers contract in response to transient elevation in intracellular calcium concentration. At rest, the Ca2+ stored inside the sarcoplasmic reticulum (SR) is predominantly bound to calsequestrin (CASQ). Since muscle fibers lacking calsequestrin (CASQ) have a reduced SR Ca2+ content and present alterations in mitochondrial morphology that may affect function, we investigated mitochondrial Ca2+-handling using a ratiometric FRET-based calcium indicator (mtD3cpv Cameleon) targeted to the mitochondrial matrix. Experiments were conducted in enzymatically dissociated FDB muscle fibers from wild type (WT) and CASQ-null mice, lacking both CASQ isoforms either in the presence (1 mM) or absence of extracellular calcium. Small but significant differences in free mitochondrial Ca2+-concentration ([Ca2+]mito) were observed between quiescent WT and CASQ-null fibers. The free [Ca2+]mito during steady state electrical stimulation at 1 Hz showed a rapid increase with a 10% - 90% rise time of 18.4±0.4 ms. The decline in [Ca2+]mito during and after stimulation trains was governed by 3 temporally distinct processes with rate constants of approximately 40 s−1, 1.6 s−1 and 0.2 s−1 (at 26 oC). During the sustained contractions in WT fibers, frequency-dependent increases in free [Ca2+]mito occurred, which were smaller in the absence than in the presence of external Ca2+. In CASQ-null fibers the increase in free [Ca2+]mito was less pronounced, and in the absence of extracellular Ca2+, the increase in free [Ca2+]mito was virtually absent. These results provide direct evidence for rapid Ca2+ uptake by the mitochondria and suggest that mitochondrial Ca2+ uptake is sensitive to the amount of Ca2+ available inside the sarcoplasmic reticulum as well as in the extracellular spaces.

Mutations in the EFHC1 gene have been linked to juvenile myoclonic epilepsy. To understand EFHC1 function in vivo, we generated knockout Drosophila for the fly homolog Defhc1.1. We found that the neuromuscular junction synapse of Defhc1.1 mutants displays an increased number of satellite boutons resulting in increased spontaneous neurotransmitter release. Defhc1.1 binds to microtubules in vitro and overlaps in vivo with axonal and synaptic microtubules. Elimination of Defhc1.1 from synaptic terminals reduces the number of microtubule loops, suggesting that Defhc1.1 is a negative regulator of microtubule dynamics. In fact, pharmacological treatment of Defhc1.1 mutants with vinblastine, an inhibitor of microtubule dynamics, suppresses the satellite bouton phenotype. Furthermore, Defhc1.1 mutants display overgrowth of the dendritic arbor and Defhc1.1 overexpression reduces dendrite elaboration. These results suggest that Defhc1.1 functions as an inhibitor of neurite growth by finely tuning the microtubule cytoskeleton dynamics and that EFHC1-dependent juvenile myoclonic epilepsy may result from augmented spontaneous neurotransmitter release due to overgrowth of neuronal processes.

After isolating NT-S100A8 from pancreatic cancer (PC) tissue of diabetic patients, we verified whether this peptide alters PC cell growth and invasion and/or insulin release and [Ca(2+)](i) oscillations of insulin secreting cells and/or insulin signaling. BxPC3, Capan1, MiaPaCa2, Panc1 (PC cell lines) cell growth, and invasion were assessed in the absence or presence of 50, 200, and 500 nM NT-S100A8. In NT-S100A8 stimulated beta-TC6 (insulinoma cell line) culture medium, insulin and [Ca(2+)](i) were measured at 2, 3, 5, 10, 15, 30, and 60 min, and [Ca(2+)](i) oscillations were monitored (epifluorescence) for 3 min. Five hundred nanomolars NT-S100A8 stimulated BxPC3 cell growth only and dose dependently reduced MiaPaCa2 and Panc1 invasion. Five hundred nanomolars NT-S100A8 induced a rapid insulin release and enhanced beta-TC6 [Ca(2+)](i) oscillations after both one (F = 6.05, P < 0.01) and 2 min (F = 7.42, P < 0.01). In the presence of NT-S100A8, [Ca(2+)] in beta-TC6 culture medium significantly decreased with respect to control cells (F = 6.3, P < 0.01). NT-S100A8 did not counteract insulin induced phosphorylation of the insulin receptor. Akt and I kappa B-alpha, but it independently activated Akt and NF-kappa B signaling in PC cells. In conclusion, NT-S100A8 exerts a mild effect on PC cell growth, while it reduces PC cell invasion, possibly by Akt and NF-kappa B signaling, NT-S100A8 enhances [Ca(2+)](i) oscillations and insulin release, probably by inducing Ca(2+) influx from the extracellular space, but it does not interfere with insulin signaling.

The present study is aimed to design a prototype of hybrid silicon-muscle cell junction, analog to an artificial neuromuscular junction prototype and relevant to the development of advanced neuro-prostheses and bionic systems. The device achieves focal Electric Capacitive Stimulation (ECS) by coupling of single cells and semiconductors, without electrochemical reaction with the substrate. A voltage change applied to a stimulation spot beneath an electrogenic cell leads to a capacitive current (charge accumulation) that opens voltage-gated ion channels in the membrane and generates an action potential. The myo-electronic junction was employed to chronically stimulate muscle cells via ECS and to induce cytosolic calcium transients in myotubes, fibers isolated from mouse FDB (fast [Ca 2+ ] i transients) and surprisingly also in undifferentiated myoblasts (slow [Ca 2+ ] i waves). The hybrid junction elicited, via chronic ECS, a differential reprogramming of single muscle cells by inducing early muscle contraction maturation and plasticity effects, such as NFAT-C3 nuclear translocation. In addition, in the presence of agrin, chronic ECS induced a modulation of AchR clustering which simulates in vitro synaptogenesis. This methodology can coordinate the myogenic differentiation, thus offering direct but non-invasive single cell/wiring, providing a platform for regenerative medicine strategies.

Electrolyte-oxide-semiconductor capacitors (EOSCs) are a class of microtransducers for extracellular electrical stimulation that have been successfully employed to activate voltage-dependent sodium channels at the neuronal soma to generate action potentials in vitro. In the present work, we report on their use to control Ca2+ signalling in cultured mammalian cells, including neurons. Evidence is provided that EOSC stimulation with voltage waveforms in the microsecond or nanosecond range activates two distinct Ca2+ pathways, either by triggering Ca2+ entry through the plasma membrane or its release from intracellular stores. Ca2+ signals were activated in non-neuronal and neuronal cell lines, CHO-K1 and SH-SY5Y. On this basis, stimulation was tailored to rat and bovine neurons to mimic physiological somatic Ca2+ transients evoked by glutamate. Being minimally invasive and easy to use, the new method represents a versatile complement to standard electrophysiology and imaging techniques for the investigation of Ca2+ signalling in dissociated primary neurons and cell lines.

A protective role of the transient potential vanilloid receptor 1 (TRPV1) in intestinal inflamma- tion induced by dinitrobenzene sulphonic acid (DNBS) has been recently demonstrated. Curcumin, the major active component of turmeric, is also able to prevent and ameliorate the severity of the damage in DNBS-induced colitis. We evaluated the possibility that curcumin (45 mg kg )1 day p.o. for 2 days before and 5 days after the induction of colitis) was able to reduce DNBS-induced colitis in mice, by acting as a TRPV1 agonist. Macroscopic damage score, histologi- cal damage score and colonic myeloperoxidase (MPO) activity were significantly lower (by 71%, 65% and 73%, respectively; P < 0.01), in animals treated with curcumin compared with untreated animals. Capsa- zepine (30 mg kg )1 , i.p.), a TRPV1 receptor antagonist, completely abolished the protective effects of curcu- min. To extend these data in vitro, Xenopus oocytes expressing rat TRPV1 were examined. Capsaicin- evoked currents (3.3 lmol L )1 ) disappeared subse- quent either to removal of the agonist or subsequent to the addition of capsazepine. However, curcumin (30 lmol L )1 ) was ineffective both as regard direct modification of cell membrane currents and as regard interference with capsaicin-mediated effects. As sen- sitization of the TRPV1 receptor by mediators of inflammation in damaged tissues has been shown previously, our results suggest that in inflamed, but not in normal tissue, curcumin can interact with the TRPV1 receptor to mediate its protective action in DNBS-induced colitis.

Background In order to gain further insight on the crosstalk between pancreatic cancer (PDAC) and stromal cells, we investigated interactions occurring between TGFβ1 and the inflammatory proteins S100A8, S100A9 and NT-S100A8, a PDAC-associated S100A8 derived peptide, in cell signaling, intracellular calcium (Cai2+) and epithelial to mesenchymal transition (EMT). NF-κB, Akt and mTOR pathways, Cai2+ and EMT were studied in well (Capan1 and BxPC3) and poorly differentiated (Panc1 and MiaPaCa2) cell lines. Results NT-S100A8, one of the low molecular weight N-terminal peptides from S100A8 to be released by PDAC-derived proteases, shared many effects on NF-κB, Akt and mTOR signaling with S100A8, but mainly with TGFβ1. The chief effects of S100A8, S100A9 and NT-S100A8 were to inhibit NF-κB and stimulate mTOR; the molecules inhibited Akt in Smad4-expressing, while stimulated Akt in Smad4 negative cells. By restoring Smad4 expression in BxPC3 and silencing it in MiaPaCa2, S100A8 and NT-S100A8 were shown to inhibit NF-κB and Akt in the presence of an intact TGFβ1 canonical signaling pathway. TGFβ1 counteracted S100A8, S100A9 and NT-S100A8 effects in Smad4 expressing, not in Smad4 negative cells, while it synergized with NT-S100A8 in altering Cai2+ and stimulating PDAC cell growth. The effects of TGFβ1 on both EMT (increased Twist and decreased N-Cadherin expression) and Cai2+ were antagonized by S100A9, which formed heterodimers with TGFβ1 (MALDI-TOF/MS and co-immuno-precipitation). Conclusions The effects of S100A8 and S100A9 on PDAC cell signaling appear to be cell-type and context dependent. NT-S100A8 mimics the effects of TGFβ1 on cell signaling, and the formation of complexes between TGFβ1 with S100A9 appears to be the molecular mechanism underlying the reciprocal antagonism of these molecules on cell signaling, Cai2+ and EMT.

The cytosolic free Ca2+-concentrations transients elicited during muscular contraction are well characterized but little is known about the free [Ca2+] dynamics inside the sarcoplasmic reticulum (SR). FRET-based Ca2+ indicators (Cameleons) allowed us to investigate SR Ca2+-handling with high time resolution (9 ms or less). The impact of calsequestrin (CS) on SR [Ca2+] was studied in enzymatically dissociated FDB muscle fibers from wild type (WT), CS type 1(-/-) (KO) and double CS type 1/CS type 2(-/-) (DKO) mice 7 days after transfection with the cDNA coding for D1ER.[Ca2+] measurements were performed at rest and during repetitive stimulation at 1, 5, 20 and 60 Hz, and the YFP(535nm)/CFP(480nm) ratio was deemed as a measure of the free SR [Ca2+].The SR [Ca2+] at rest did not differ between WT (n=26), KO (n=25) and DKO (n=21) fibers while during electrical stimulation they were rather small in WT, reflecting powerful buffering of SR [Ca2+]. In KO and DKO fibers, a significant reduction in the SR [Ca2+] occurred, which increased in parallel with the stimulation frequency. At 60 Hz the SR became virtually depleted of Ca2+, both in KO and DKO fibers. Calcium reuptake during and after the trains of stimuli was governed by 3 rate constants of 50 s−1, 1-5 s−1 and 0.3 s−1 (at 26°C). In conclusion, CS-KO fibers represent a unique model to resolve the kinetics of SR release and reuptake.

The cytosolic free Ca 2+ transients elicited by muscle fiber excitation are well characterized, but little is known about the free [Ca 2+ ] dynamics within the sarcoplasmic reticulum (SR). A targetable ratiometric FRET-based calcium indicator (D1ER Cameleon) allowed us to investigate SR Ca 2+ dynamics and analyze the impact of calsequestrin (CSQ) on SR [Ca 2+ ] in enzymatically dissociated flexor digitorum brevis muscle fibers from WT and CSQ-KO mice lacking isoform 1 (CSQ-KO) or both isoforms [CSQ-double KO (DKO)]. At rest, free SR [Ca 2+ ] did not differ between WT, CSQ-KO, and CSQ-DKO fibers. During sustained contractions, changes were rather small in WT, reflecting powerful buffering of CSQ, whereas in CSQ-KO fibers, significant drops in SR [Ca 2+ ] occurred. Their amplitude increased with stimulation frequency between 1 and 60 Hz. At 60 Hz, the SR became virtually depleted of Ca 2+ , both in CSQ-KO and CSQ-DKO fibers. In CSQ-KO fibers, cytosolic free calcium detected with Fura-2 declined during repetitive stimulation, indicating that SR calcium content was insufficient for sustained contractile activity. SR Ca 2+ reuptake during and after stimulation trains appeared to be governed by three temporally distinct processes with rate constants of 50, 1–5, and 0.3 s −1 (at 26 °C), reflecting activity of the SR Ca 2+ pump and interplay of luminal and cytosolic Ca 2+ buffers and pointing to store-operated calcium entry (SOCE). SOCE might play an essential role during muscle contractures responsible for the malignant hyperthermia-like syndrome in mice lacking CSQ.

Calsequestrin (CASQ) is an acidic, high capacity Ca2+-binding protein located within the terminal cisternae of the sarcoplasmic reticulum (SR). CASQ1 is the major SR Ca2+- buffer in fast muscle fibers, whereas CASQ2 predominates in cardiomyocytes and slow muscle fibers. Mice devoid of CASQ were generated by crossing mice lacking CASQ1 (Paolini et al J Physiol 2007, 583, 767) and mice lacking CASQ2 (Knollman et al J Clin Inv 2006, 116, 2510).When studied ex vivo, fast muscles (EDL) showed an earlier decline in tension during tetanic stimulation than WT fibers. This was not the case in slow muscle, soleus. Such decline of the developed tension was compatible with the decay of free cytosolic calcium during repeated stimulation observed using Fura-2 in single intact fast FDB muscle fibers.Single permeabilized fibers from EDL and tibialis anterior showed a reduction of the amount of calcium released by 30 mM caffeine, which is supposed to release about 80% of SR calcium (Murphy et al. J Physiol 2009, 587, 443), thus suggesting a depletion of SR. The depletion was confirmed by measurements of SR fee [Ca2+] using a FRET-based indicator (D1ER) genetically targeted to the SR. During contraction a massive/marked reduction in intraluminal free [Ca2+] was observed to a level close to full depletion.The results are consistent with the function of CASQ as a intraluminal SR buffer. CASQ1/2 null mice lack sufficient compensation for the loss in buffering power by other SR proteins. The diversity between slow and fast muscles might be attributed to the presence of parvalbumin as a relevant sink of calcium in cytosol of fast but not slow muscles (Celio et al Nature 1982, 297, 504).