Low Molecular Weight Fragments Research Articles

Optimization of novel reversible Bruton's tyrosine kinase inhibitors identified using Tethering-fragment-based screens.

Since the approval of ibrutinib for the treatment of B-cell malignancies in 2012, numerous clinical trials have been reported using covalent inhibitors to target Bruton's tyrosine kinase (BTK) for oncology indications. However, a formidable challenge for the pharmaceutical industry has been the identification of reversible, selective, potent molecules for inhibition of BTK. Herein, we report application of Tethering-fragment-based screens to identify low molecular weight fragments which were further optimized to improve on-target potency and ADME properties leading to the discovery of reversible, selective, potent BTK inhibitors suitable for pre-clinical proof-of-concept studies.

Compositional characterization study on high -molecular -mass polymeric polyphenols in red wines by chemical degradation.

High-molecular-mass polymeric polyphenols (HPPCs) is the major group of polyphenols in red wine. However, owing to the diversity and structural complexity, its compositional characterization remains a difficulty. In this study, purified high-molecular-mass polymeric polyphenols (PHPPCs) was isolated by solid phase extraction combined with semi-preparative HPLC from red wine. Different chemical degradation methods (NaOH, HCl and benzyl mercaptan degradation) were used to decompose the PHPPCs into low molecular weight fragments. The chemical structures of the degradation fragments were verified by HPLC/UPLC-DAD and HPLC-FT-ICR-MS. The results showed that three proanthocyanidins, five proanthocyanidins benzylthioether, quercetin and fifteen anthocyanins were detected by degradation in the presence of benzyl mercaptan. Nine bound phenolic acids were found by NaOH degradation and twelve bound amino acids were proved by HCl. Among these different fragments detected,delphinidin-3-O-glucoside pyruvic adduct, four acylated anthocyanins, four hydroxyphenyl pyranoanthocyanins, quercetin, protocatechuic acid, vanillic acid, and cinnamic acid were first found in the PHPPCs. For the first time amino acids as constitutive units of polymeric polyphenols in red wines were verified. Based on the degradation fragments, the possible compositional characterization of HPPCs in red wine was postulated.

Assessment of RNAlater® as a Potential Method to Preserve Bovine Muscle Proteins Compared with Dry Ice in a Proteomic Study.

RNAlater® is regarded as a potential preservation method for proteins, while its effect on bovine muscle proteins has rarely been evaluated. Bovine muscle protein samples (n = 12) collected from three tender (Warner–Bratzler shear force: 30.02–31.74 N) and three tough (Warner–Bratzler shear force: 54.12–66.25 N) Longissimus thoracis et lumborum (LTL) samples, preserved using two different sampling preservation methods (RNAlater® and dry ice), at two post mortem time points (day 0 and day 14), were characterized using one-dimensional electrophoresis. Fourteen bands with molecular weights ranging from 15 to 250 kDa were verified, both in the dry ice and RNAlater® storage groups, at each time point, using image analysis. A shift from high to low molecular weight fragments, between day 0 and day 14, indicated proteolysis of the muscle proteins during post mortem storage. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses and database searching resulted in the identification of 10 proteins in four bands. Protein profiles of muscle preserved in RNAlater® were similar to those of muscle frozen on dry ice storage, both at day 0 and day 14. The results demonstrate that RNAlater® could be a simple and efficient way to preserve bovine muscle proteins for bovine muscle proteomic studies.

Counting on Fragment Based Drug Design Approach for Drug Discovery.

Fragment based drug design (FBDD) is a structure guided ligand design approach used in the process of drug discovery. It involves identification of low molecular weight fragments as hits followed by determination of their binding mode using X-ray crystallography and/or NMR spectroscopy. X-ray protein crystallography is one of the most sensitive biophysical methods used for screening and is least prone to false positives. It also provides detailed structural information of the protein-fragment complex at the atomic level. The retrieved binding information facilitates the optimization of fragments into drug like molecules. These identified molecules bind efficiently with the target proteins and form high quality binding interactions. Fragment-based screening using X-ray crystallography is, therefore, an efficient method for identifying binding hotspots on proteins that can be further exploited by chemists and biologists for the discovery of new drugs. The recent advancements in FBDD technique are illustrated in this review along with recently published success stories of FBDD technique in drug discovery.

Increased Hyaluronan and TSG-6 in Association with Neuropathologic Changes of Alzheimer's Disease.

Little is known about the extracellular matrix (ECM) during progression of AD pathology. Brain ECM is abundant in hyaluronan (HA), a non-sulfated glycosaminoglycan synthesized by HA synthases (HAS) 1-3 in a high molecular weight (MW) form that is degraded into lower MW fragments. We hypothesized that pathologic severity of AD is associated with increases in HA and HA-associated ECM molecules. To test this hypothesis, we assessed HA accumulation and size; HA synthases (HAS) 1-3; and the HA-stabilizing hyaladherin, TSG-6 in parietal cortex samples from autopsied research subjects with not AD (CERAD = 0, Braak = 0- II, n = 12-21), intermediate AD (CERAD = 2, Braak = III-IV, n = 13-18), and high AD (CERAD = 3, Braak = V-VI, n = 32-40) neuropathologic change. By histochemistry, HA was associated with deposits of amyloid and tau, and was also found diffusely in brain parenchyma, with overall HA quantity (measured by ELSA) significantly greater in brains with high AD neuropathology. Mean HA MW was similar among the samples. HAS2 and TSG-6 mRNA expression, and TSG-6 protein levels were significantly increased in high AD and both molecules were present in vasculature, NeuN-positive neurons, and Iba1-positive microglia. These results did not change when accounting for gender, advanced age (≥ 90 years versus <90 years), or the clinical diagnosis of dementia. Collectively, our results indicate a positive correlation between HA accumulation and AD neuropathology, and suggest a possible role for HA synthesis and metabolism in AD progression.

Whole exome sequencing analysis of urine trans-renal tumour DNA in metastatic colorectal cancer patients

BackgroundThe analysis of circulating free tumour DNA (ctDNA) in blood, commonly referred as liquid biopsy, is being used to characterise patients with solid cancers. Tumour-specific genetic variants can also be present in DNA isolated from other body fluids, such as urine. Unlike blood, urine sampling is non-invasive, can be self-performed, and allows recurrent longitudinal monitoring. The features of tumour DNA that clears from the glomerular filtration barrier, named trans-renal tumour DNA (trtDNA), are largely unexplored.Patients and methodsSpecimens were collected from 24 patients with KRAS or BRAF mutant metastatic colorectal cancer (mCRC). Driver mutations were assessed by droplet digital PCR (ddPCR) in ctDNA from plasma and trtDNA from urine. Whole exome sequencing (WES) was performed in DNA isolated from tissue, plasma and urine.ResultsOut of the 24 CRC cases, only four had sufficient DNA to allow WES analyses in urine and plasma. We found that tumour alterations primarily reside in low molecular weight fragments (less than 112 bp). In patients whose trtDNA was more than 2.69% of the urine derived DNA, cancer-specific molecular alterations, mutational signatures and copy number profiles identified in urine DNA are comparable with those detected in plasma ctDNA.ConclusionsWith current technologies, WES analysis of trtDNA is feasible in a small fraction of mCRC patients. Tumour-related genetic information is mainly present in low molecular weight DNA fragments. Although the limited amounts of trtDNA poses analytical challenges, enrichment of low molecular weight DNAs and optimised computational tools can improve the detection of tumour-specific genetic information in urine.

Enzymatic treatments as alternative to produce chitin fragments of low molecular weight from Alternaria alternata

ABSTRACTChitin and its oligosaccharides are involved in the plant defense response against fungal pathogens. In most studies, these molecules come from crustacean shell, and there are scarce studies on the use of fungal chitin. Usually the extraction of chitin is by alkaline treatments, which affect the acetylation degree (DA), and the obtaining of oligosaccharides of low molecular weight; so the use of enzymes is proposed as an alternative treatment to obtain chitin oligosaccharides from fungi. The objective of this work was to characterize the chitin fragments of Alternaria alternata extracted by alkaline and enzymatic treatments. The chitin extracted was ultrasonicated and ultrafiltrated to produce chitin fragments. Enzymatic treatments decreased the protein content to 29%, and the bands corresponding to β‐glucans decreased when β‐1,3‐glucanase was applied. Fragments smaller than 1 kDa, DA of 76.7%, and 35.4 μg glucosamine/mg dry weight were obtained by enzymatic treatments. The enzymatic treatments show promising results to extract chitin fragments from A. alternata without severely affect the DA of the molecule. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019, 136, 47339.

Heterodimeric Plasmonic Nanogaps for Biosensing.

We report the study of heterodimeric plasmonic nanogaps created between gold nanostar (AuNS) tips and gold nanospheres. The selective binding is realized by properly functionalizing the two nanostructures; in particular, the hot electrons injected at the nanostar tips trigger a regio-specific chemical link with the functionalized nanospheres. AuNSs were synthesized in a simple, one-step, surfactant-free, high-yield wet-chemistry method. The high aspect ratio of the sharp nanostar tip collects and concentrates intense electromagnetic fields in ultrasmall surfaces with small curvature radius. The extremities of these surface tips become plasmonic hot spots, allowing significant intensity enhancement of local fields and hot-electron injection. Electron energy-loss spectroscopy (EELS) was performed to spatially map local plasmonic modes of the nanostar. The presence of different kinds of modes at different position of these nanostars makes them one of the most efficient, unique, and smart plasmonic antennas. These modes are harnessed to mediate the formation of heterodimers (nanostar-nanosphere) through hot-electron-induced chemical modification of the tip. For an AuNS-nanosphere heterodimeric gap, the intensity enhancement factor in the hot-spot region was determined to be 106, which is an order of magnitude greater than the single nanostar tip. The intense local electric field within the nanogap results in ultra-high sensitivity for the presence of bioanalytes captured in that region. In case of a single BSA molecule (66.5 KDa), the sensitivity was evaluated to be about 1940 nm/RIU for a single AuNS, but was 5800 nm/RIU for the AuNS-nanosphere heterodimer. This indicates that this heterodimeric nanostructure can be used as an ultrasensitive plasmonic biosensor to detect single protein molecules or nucleic acid fragments of lower molecular weight with high specificity.

STD NMR as a Technique for Ligand Screening and Structural Studies.

STD NMR is a powerful ligand-based tool for screening small molecules and low molecular weight fragments for their interaction with a given macromolecule. Such information is invaluable both in the drug discovery sector and in understanding fundamental biological interactions. Recently, powerful methods have been developed to extract a greater wealth of information from the STD NMR experiment, including ligand binding epitopes, dissociation constant determination, and mapping of binding site properties. Herein we describe these STD NMR experiments, giving practical examples for each approach, and highlight the important parameters and common pitfalls that must be considered for a successful experiment.

Identification and characterization of the fucoidanase gene from Luteolibacter algae H18

Fucoidan is a hetero-sulfated polysaccharide found in brown algae and has received much attention as an ingredient in functional and health foods. The marine bacterial strain Luteolibacter algae H18 degrades fucoidan from Cladosiphon okamuranus. We purified the fucoidanase from a cell-free extract of L. algae H18, used it to decrease the molecular weight of deacetylated-fucoidan, determined the N-terminal amino acid sequence of the enzyme, and identified the gene involved in the degradation of fucoidan, fct114, in a draft genome sequence of strain H18. The gene product was heterologously produced in Escherichia coli and demonstrated to catalyze the degradation of deacetylated-fucoidan into lower molecular weight fragments. The mass of the gene product Fct114 is 112kDa (1026 amino acid residues). The general properties of the enzyme were investigated by measuring the amount of reducing ends produced from deacetylated-fucoidan during the reaction. The enzyme was inactive toward fucoidans from other brown seaweed species or toward polysaccharides such as alginic acid, carrageenan, hyaluronic acid, and chondroitin sulfate. The amino acid sequence of Fct114 shared less than 25% identity and had no conserved motifs when compared with previously identified fucoidanases from other marine bacterial strains. These data suggest that Fct114 is a novel polysaccharide-degrading enzyme.

A physics-based approach to modeling real-fuel combustion chemistry - I. Evidence from experiments, and thermodynamic, chemical kinetic and statistical considerations

Real distillate fuels usually contain thousands of hydrocarbon components. Over a wide range of combustion conditions, large hydrocarbon molecules undergo thermal decomposition to form a small set of low molecular weight fragments. In the case of conventional petroleum-derived fuels, the composition variation of the decomposition products is washed out due to the principle of large component number in real, multicomponent fuels. From a joint consideration of elemental conservation, thermodynamics and chemical kinetics, it is shown that the composition of the thermal decomposition products is a weak function of the thermodynamic condition, the fuel-oxidizer ratio and the fuel composition within the range of temperatures of relevance to flames and high temperature ignition. Based on these findings, we explore a hybrid chemistry (HyChem) approach to modeling the high-temperature oxidation of real, distillate fuels. In this approach, the kinetics of thermal and oxidative pyrolysis of the fuel is modeled using lumped kinetic parameters derived from experiments, while the oxidation of the pyrolysis fragments is described by a detailed reaction model. Sample model results are provided to support the HyChem approach.

Radical Degradation Processes Initiated by Catalytic Nanoparticles of CoFe2O4 Towards Polymer Waste Application

Polymer waste presents a modern environmental challenge due to the long-term stability of these materials. Elastomer-based polymers (e.g., tires, pipelines and shoes) are unique in comprising numerous double bonds, which are typically sensitive to radical-initiated oxidative degradation leading to the scission of polymer chains. In this paper, we propose a new approach for radical-initiated oxidative degradation of polymers using cobalt ferrite (CoFe2O4) nanoparticles (NPs). We show that magnetic CoFe2O4 NPs can effectively catalyze the oxidative degradation of polybutadiene (PB), offering easy magnetic recycling and reuse without affecting their catalytic efficiency. The CoFe2O4 NPs were synthesized via a facile surfactant-free method based on a sonochemical reaction. We used methyl ethyl ketone peroxide (MEKP), a model system, to study the rate of radical decomposition catalyzed by the NPs. The radical decomposition rates were determined by following the discoloration of methylene blue (MB) using ultraviolet–visible (UV/Vis) spectroscopy; electron paramagnetic resonance (EPR) measurements were used to study radical formation. The radical-initiated oxidative degradation of PB was studied by thermogravimetric analysis with mass spectrometry detection (TGA–MS). Our results show that cobalt ferrite NPs lead to formation of polymers with low molecular weight fragments and crosslinking, indicating that these NPs are very effective catalysts for the radical degradation of PB. CoFe2O4 NPs are potentially suitable for other polymers, hence this approach may provide a novel route for the chemical (tertiary) recycling of polymers.

Exceptional visible-light photoelectrocatalytic activity of In2O3/In2S3/CdS ternary stereoscopic porous heterostructure film for the degradation of persistent 4-fluoro-3-methylphenol

Abstact In this work, In2O3/In2S3/CdS ternary stereoscopic porous heterostructure films were successfully fabricated via consecutive in situ ionic exchange reactions to degrade persistent 4-Fluoro-3-methylphenol through photoelectrocatalytic process. The optimized In2O3/In2S3/CdS ternary stereoscopic porous heterostructure film electrode showed much higher photoelectrocatalytic efficiency than the In2O3 and In2O3/In2S3 films. The resulting degradation reaction constant is about 3 and 2 times higher than that on In2O3 and binary In2O3/In2S3 films, respectively. The extraordinary higher degradation and mineralization efficiencies of the In2O3/In2S3/CdS composite film electrode come from the synergy of ternary components in the stereoscopic porous heterostructure. The existence of double type-II band alignment with nanosized interfaces in the ternary heterostructure can significantly improve charge transport and electron-hole separation. The stereoscopic porous architechture can greatly enhance visible light utilization and superhydrophilicity, and meanwhile facilitate the mass transport and accessibility of active sites. Ion chromatography, gas chromatography–mass spectrometry, total organic carbon analysis, active species trapping experiment as well as electron spin resonance techniques were employed to probe the degradation and the mineralization processes of 4-Fluoro-3-methylphenol. A unifying photoeletrocatalytic degradation route and mechanism is provided to explain the degradation and the mineralization processes. The generated more superoxide ions and holes promoted the defluorination process and favored the subsequent benzene ring cleavage and mineralization of low molecular weight fragments (e.g. carboxylic acids). This work provides a route to degrade persistent organic pollutants and realize efficient water contamination purification via efficient visible light photoelectrocatalytic process.

Fragment-based design, synthesis, biological evaluation, and SAR of 1H-benzo[d]imidazol-2-yl)-1H-indazol derivatives as potent PDK1 inhibitors

In this work, we describe the use of the rule of 3 fragment-based strategies from biochemical screening data of 1100 in-house, small, low molecular weight fragments. The sequential combination of in silico fragment hopping and fragment linking based on S160/Y161/A162 hinge residues hydrogen bonding interactions leads to the identification of novel 1H-benzo[d]imidazol-2-yl)-1H-indazol class of Phosphoinositide-Dependent Kinase-1 (PDK1) inhibitors. Consequent SAR and follow-up screening data led to the discovery of two potent PDK1 inhibitors: compound 32 and 35, with an IC50 of 80 nM and 94 nM, respectively. Further biological evaluation showed that, at the low nanomolar concentration, the drug had potent ability to inhibit phosphorylation of AKT and p70S6, and selectively kill the cancer cells with mutations in both PTEN and PI3K. The microarray data showed that DUSP6, DUSP4, and FOSL1 were down-regulated in the sensitive cell lines with the compound treatment. The in vivo test showed that 35 can significantly inhibit tumor growth without influencing body weight growth. Our results suggest that these compounds, especially 35, merit further pre-clinical evaluation.

Bio-degradable highly fluorescent conjugated polymer nanoparticles for bio-medical imaging applications

Conjugated polymer nanoparticles exhibit strong fluorescence and have been applied for biological fluorescence imaging in cell culture and in small animals. However, conjugated polymer particles are hydrophobic and often chemically inert materials with diameters ranging from below 50 nm to several microns. As such, conjugated polymer nanoparticles cannot be excreted through the renal system. This drawback has prevented their application for clinical bio-medical imaging. Here, we present fully conjugated polymer nanoparticles based on imidazole units. These nanoparticles can be bio-degraded by activated macrophages. Reactive oxygen species induce scission of the conjugated polymer backbone at the imidazole unit, leading to complete decomposition of the particles into soluble low molecular weight fragments. Furthermore, the nanoparticles can be surface functionalized for directed targeting. The approach opens a wide range of opportunities for conjugated polymer particles in the fields of medical imaging, drug-delivery, and theranostics.

HM015k, a Novel Silybin Derivative, Multi-Targets Metastatic Ovarian Cancer Cells and Is Safe in Zebrafish Toxicity Studies.

This study was designed to determine the in vitro mechanisms by which the novel silybin derivative, (E)-3-(3-(benzyloxy) phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (HM015k or 15k), produces its anticancer efficacy in ovarian cancer cells. Compound 15k induced apoptosis in ovarian cancer cells in a time-dependent manner by significantly upregulating the expression of Bax and Bak and downregulating the expression of Bcl-2. Interestingly, 15k induced the cleavage of Bax p21 into its more efficacious cleaved form, Bax p18. In addition, caspase 3 and caspase 9 were cleaved to their active forms, inducing the cleavage of poly ADP ribose polymerase (PARP) and β-catenin. Furthermore, in OV2008 cells, 15k induced significant cleavage in nuclear β-catenin to primarily inactive fragments of lower molecular weight. Furthermore, 15k reversed the metastatic potential of OV2008 cells by inhibiting their migration and invasiveness. The mesenchymal phenotype in OV2008 was reversed by 15k, causing cells to be rounder with epithelial—like phenotypes. The 15k-induced reversal was further confirmed by significant upregulation of the E-cadherin expression, an epithelial marker, while N-cadherin, a mesenchymal marker, was downregulated in OV2008 cells. Compound 15k inhibited the expression of the oncogenic c-Myc protein, downregulated proteins DVL3 and DVL2 and significantly upregulated cyclin B1. Also, 15k significantly downregulated the expression levels of ABCG2 and ABCB1 transporters in resistant ABCG2 overexpressing H460/MX20 and resistant ABCB1 overexpressing MDCK/MDR1 cells, respectively. Finally, 15k was safe in zebrafish in vivo model at concentrations up to 10 μM and induced no major toxicities in cardiac, morphology and swimming position parameters. Overall, 15k is a multi-targeted inhibitor with efficacy against metastatic and resistant ovarian cancer. Future in vivo studies will be conducted to determine the efficacy of 15k in tumor-bearing animals.

Delaying implant-based mammary reconstruction after radiotherapy does not decrease capsular contracture: An in vitro study

The most common complications of irradiated implant-based mammary reconstruction are fibrosis and capsular contracture. The indications for postmastectomy adjuvant radiotherapy have considerably broadened. Facing an increased number of patients who will require radiotherapy, most guidelines recommend delaying reconstruction after radiotherapy to prevent long-term fibrotic complications. Does radiotherapy permanently alter cellular properties which will adversely affect implant-based reconstruction? If so, is there a benefit in delaying reconstruction after radiotherapy? Our invitro model simulates two implant-based mammary reconstruction approaches: the irradiated implant and the delayed implant reconstructions by using an implant inset beneath healthy non-irradiated tissue post radiotherapy. We performed cell culture of fibroblasts and endothelial cells to simulate these two surgical conditions. Irradiated fibroblasts simulate the capsular tissue seen around the breast implant. The delayed reconstruction approach is simulated by non-irradiated fibroblasts conditioned with supernatant culture media obtained from irradiated endothelial cells. Irradiation induced fibrosis through fibroblast differentiation into myofibroblasts, as demonstrated by increased α-smooth-muscle actin levels in fibroblasts. This constitutes the basis for scar tissue contraction observed in irradiated implant-based breast reconstruction. Irradiation of endothelial cells induced irreversible cell cycle arrest known as senescence and secretion of the profibrotic connective tissue growth factor. Non-irradiated fibroblasts conditioned with culture media obtained from irradiated endothelial cells exhibited myofibroblast differentiation and the expression of fibrotic phenotype akin to capsular contracture. Our results demonstrate that radiotherapy causes irreversible cellular changes, which permanently alter the microenvironment in favor of fibrosis. Given that these changes are permanent, delaying reconstruction does not present an advantage in preventing capsular contracture.

Physiochemical and functional properties of chum salmon (Oncorhynchus keta) skin gelatin extracted at different temperatures.

Aquatic source gelatins are gaining more attention due to the advantages in safety and religion acceptability compared with mammalian sources. For understanding the effects of extracting temperature on gelatins from chum salmon (Oncorhynchus keta) skins (GCSS), gelatins were extracted at temperatures from 40 to 90°C and the physiochemical properties of GCSS were investigated. GCSS yield increased while imino acids content declined as the increase of temperature. GCSS40, 50 and 60 showed strong β-, α1- and α2-chains but the three faded in GCSS70, 80 and 90, with the presence of low molecular weight fragments. Amides A, I and III were shifted to higher wavenumber in GCSS70, 80 and 90 compared with that of GCSS40, 50 and 60. X-ray diffraction showed lower intensity of peak at 7° in GCSS80 and 90 than in the other GCSS. Gel strength declined while a*, b* and ΔE* value increased as temperature increased. Foam expansion and stability of GCSS40, 50 and 60 were lower than those of GCSS70, 80 and 90. Emulsion activity and stability decreased as temperature increased. Extracting temperature greatly affected yield, molecular composition and functionalities of GCSS. A temperature lower than 50°C is recommended for GCSS extraction. © 2017 Society of Chemical Industry.

Polysaccharides and their depolymerized fragments from Costaria costata: Molecular weight and sulfation-dependent anticoagulant and FGF/FGFR signal activating activities

Crude polysaccharides from Costaria costata were extracted by hot water and further fractionated by anion exchange chromatography into three polysaccharide fractions. Three low molecular weight fragments were then prepared by degradation of the polysaccharides with hydrogen peroxide and ascorbic acid. The structural features of the polysaccharides and their low molecular weight fragments were elucidated for the first time based on the HGPC, FT-IR, NMR, MS, monosaccharide composition, and other chemical analyses. Their anticoagulant and FGF-1, -2, -7, -8, -9, -10/FGFR1c signaling activation activities in BaF3 cells were also examined. Our studies showed that the polysaccharides were sulfated at different positions of galactose and fucose residues. The APTT-, PT- and TT-based anticoagulant assay results indicated that a high molecular weight and a higher degree of sulfation were essential for their anticoagulant activities. In contrast, not only the polysaccharides but also the depolymerized fragments showed significant FGF/FGFR signal activating activities in a FGF-, molecular weight-, and sulfation-dependent manner. The results presented in current study demonstrated the potential use of the polysaccharides and their fragments as anticoagulants and FGF signal regulators.

Osteopontin–metallothionein I/II interactions in experimental autoimmunune encephalomyelitis

Osteopontin (OPN), an extracellular matrix (ECM) glyco-phosphoprotein, plays an important role in autoimmune-mediated demyelinating diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). As an integrin and CD44 binding protein it participates in bidirectional communication between the ECM and target cells and affects transduction pathways that maintain neuronal and immune cell homeostasis. Its biological activity is also heavily influenced by microenvironment, which stimulates the cleavage of OPN and changes its functions.In this study we estimated the expression profile of OPN in neural tissues of DA rats during the first relapse of chronic relapsing EAE and investigated the relationship of OPN to metallothionein I+II (MTs), which play pivotal role in zinc-related cell homeostasis and in protection of CNS against cytokine-induced injury. The data showed that in EAE rats OPN mRNA and protein levels increased concurrently with the transcription of MTs and that within the spinal cord (SC) lysates EAE-afflicted rats had a higher content of OPN fragments of low molecular weight than untreated and CFA-treated rats. The expression of OPN and MTs was upregulated on ependymal, lymphoid and astroglial cells and on multiple αvβ3+ neurons in SC and in the brain (cortex, white matter, hippocampus, and cerebellum). Besides, multiple cells co-expressed OPN and MTs. Granular OPN signals were detected in secretory vesicles of Golgy (αvβ3 neurons) and in patches adjacent to the plasma membrane (subventricular zone).The findings imply that in demyelinating lesions are generated proteolytic OPN fragments and that OPN/MT interactions contribute to tissue remodeling during an autoimmune attack.