Silencing of miR-101 Prevents Cartilage Degradation by Regulating Extracellular Matrix-related Genes in a Rat Model of Osteoarthritis.

Osteoarthritis (OA) is a common, degenerative joint disease characterized by articular cartilage degradation. Currently, clinical trials based on microRNA therapy have been performed to treat various diseases. However, no treatment has been found for arthritis. This study investigated the functions of miR-101 in cartilage degradation in vivo and evaluated the feasibility of using miR-101 as a therapeutic agent for OA. Mono-iodoacetate-induced arthritis (MIA) rats were used as an animal model of OA. miR-101 mimic or miR-101 inhibitor was injected into the rats' knees to evaluate its effects on cartilage degradation. Cartilage degradation aggravated at 14 days after the injection of miR-101 mimic. By contrast, miR-101 silencing reduced cartilage degradation. Moreover, the administration of miR-101 mimic is sufficient to cause cartilage degradation in the normal cartilage of rats. By contrast, miR-101 inhibitor could prevent this change. Microarray and qPCR were used to investigate the different expressed genes after injecting miR-101 mimic and miR-101 inhibitor in the rats' articular cartilage. Several cartilage degradation-related genes were selected and validated to function in cartilage degradation with miR-101. Our results demonstrated the therapeutic effect of miR-101 inhibition on cartilage degradation in MIA rats by regulating several cartilage degradation-related genes.

Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons.

Proteomic quantification and site-mapping of S-nitrosylated proteins using isobaric iodoTMT reagents.

S-Nitrosylation is a redox-based protein post-translational modification in response to nitric oxide signaling and is involved in a wide range of biological processes. Detection and quantification of protein S-nitrosylation have been challenging tasks due to instability and low abundance of the modification. Many studies have used mass spectrometry (MS)-based methods with different thiol-reactive reagents to label and identify proteins with S-nitrosylated cysteine (SNO-Cys). In this study, we developed a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive iodoTMTsixplex reagents to specifically detect and quantify protein S-nitrosylation. Irreversible labeling of SNO-Cys with the iodoTMTsixplex reagents enables immune-affinity detection of S-nitrosylated proteins, enrichment of iodoTMT-labeled peptides by anti-TMT resin, and importantly, unambiguous modification site-mapping and multiplex quantification by liquid chromatography-tandem MS. Additionally, we significantly improved anti-TMT peptide enrichment efficiency by competitive elution. Using ISA, we identified a set of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation in murine BV-2 microglial cells and revealed effects of S-allyl cysteine from garlic on LPS-induced protein S-nitrosylation in antioxidative signaling and mitochondrial metabolic pathways. ISA proved to be an effective proteomic approach for quantitative analysis of S-nitrosylation in complex samples and will facilitate the elucidation of molecular mechanisms of nitrosative stress in disease.

Three-component coupling reactions of arynes for the synthesis of benzofurans and coumarins.

The domino three-component coupling reaction of arynes with DMF and active methylenes or methines was studied as a highly efficient method for preparing heterocycles. Coumarin derivative 5 was formed when diethyl malonate (2) or α-bromomalonate (3) were used as a C2-unit. In contrast, dihydrobenzofurans 7a and 7b were obtained by using α-chloroenolates generated from α-chloromalonates 4a and 4b and Et2Zn. The benzofuran 15a could be obtained by using ethyl iodoacetate (14) as a C1-unit. The one-pot conversion of dihydrobenzofurans 7a, 7b and 8a into benzofurans 15a and 15b was also studied. The direct synthesis of benzofuran 15b was achieved by using the active methine 18 having ketone and ester groups.

Synthesis of a leucomitosane via a diastereoselective radical cascade.

The preparation of trans-2,3-disubstituted indolines from 1-azido-2-allylbenzene derivatives via a diastereoselective radical cascade using ethyl iodoacetate and triethylborane is described. Further lactamization afforded substituted benzopyrrolizidinones with excellent diastereomeric ratios. The radical cascade/lactamization sequence was efficiently applied to the synthesis of a 3-oxo-leucomitosane related to the mitomycin family of alkaloids.

Rat model of lumbar facet joint osteoarthritis associated with facet-mediated mechanical hyperalgesia induced by intra-articular injection of monosodium iodoacetate.

BACKGROUND/PURPOSE: The relationship between lumbar facet joint (LFJ) osteoarthritis (OA) and back pain remains unclear. An OA model associated with joint pain was successfully induced by monosodium iodoacetate (MIA) in rat knees. We aimed to establish an experimental OA model with facet-mediated mechanical hyperalgesia in the LFJ in rats. METHODS: We established a rat experimental model of LFJ OA with facet-mediated mechanical hyperalgesia by injection of MIA into a single facet joint. After injection, changes in the LFJ structure were assessed histologically and mechanical hyperalgesia in the hind paw was determined using the von Frey test. In addition, interleukin-1ß and tumor necrosis factor-α levels in the synovium were measured by enzyme-linked immunosorbent assay, and the inhibitory effects of celecoxib and gabapentin on mechanical hyperalgesia were evaluated. RESULTS: Progressive cartilage degeneration and changes in subchondral bone were observed after injection. A biphasic pattern of mechanical hyperalgesia was noted in the hind paw. The concentrations of interleukin-1ß and tumor necrosis factor-α were significantly increased only on Days 1 and 3 when compared with controls. Celecoxib was effective only on Day 3 and ineffective on Days 21 and 35, whereas gabapentin kept its inhibitory effect on Days 3, 21 and 35. CONCLUSION: An experimental LFJ OA model associated with facet-mediated mechanical hyperalgesia can be established by intra-articular injection of MIA. This model might provide a useful tool for further study to ascertain the complex mechanism of facet joint pain.

Catalytic enantioselective Reformatsky reaction with ketones.

Chiral tertiary alcohols were obtained with good yields and enantioselectivities via a catalytic Reformatsky reaction with ketones, including the challenging diaryl ketones, using chiral BINOL derivatives.

Isolation and characterization of adipose tissue glycerol-3-phosphate dehydrogenase.

Recent studies on the differentiation of human preadipocytes have extensively used GPDH as a convenient marker enzyme to follow the development of the cells. Since the properties of human adipose tissue GPDH is largely unknown, it was considered of interest to characterize the purified enzyme. Glycerol-3-phosphate dehydrogenase was purified to homogeneity using blue Sepharose and hydroxylapatite chromatography. Monomeric molecular mass of GPDH was estimated using SDS-PAGE while the dimeric mass was estimated using non-denaturing PAGE. Fluorometric titrations were used to measure the binding of NADH to the enzyme. Inactivation experiments with proteolytic enzymes, urea and heat treatment were used to investigate a possible conformational change due to NADH binding. The purified enzyme displayed a monomeric molecular mass of 35,000 Da, a dimeric molecular mass of 74,000 Da and an isoelectric point (pI) of 5.85. The enzyme exhibited a pH optimum of 7.5 for the reduction of DHAP and 9.0 for G-3-P oxidation. Glycerol (50%) was found to stabilize the enzyme activity during storage, but altered the kinetic properties of the enzyme, acting as a competitive activator with respect to DHAP reduction. GPDH was inhibited by sulfhydryl modifying reagents and fatty acids. The effectiveness of inhibition by saturated fatty acids increased proportionately with chain length from decanoate to stearate. In addition preincubation of the enzyme, in the presence of oleate, resulted in a time dependent inactivation. Time dependent inactivation of GPDH by both iodoacetate and oleate was prevented by the presence of NADH but not NAD+.(ABSTRACT TRUNCATED AT 250 WORDS)

Conjugation to preactivated proteins using divinylsulfone and iodoacetic acid.

Two methods for the preactivation of proteins and conjugation of peptides to proteins under mild conditions are presented. Preactivation of proteins with divinylsulfone (DVS) permits peptide conjugation through either amino, hydroxyl or sulphydryl groups depending on the coupling pH used, while preactivation with iodoacetic acid (IAA) N-hydroxy-succinimide ester permits selective conjugation through sulphydryl groups. In addition, the latter method allows quantitation of the conjugation ratio through determination of carboxymethyl cysteine after acid hydrolysis. The divinylsulfone activated proteins can be stored for extended periods of time at -20 degrees C until required for conjugation, while the iodoacetic acid activated protein can be stored for a few days at -20 degrees C. These conjugation methods were investigated with respect to obtaining peptide/protein conjugates for immunization purposes and for use as reagents in immunoassays. The DVS activated proteins permitted direct conjugation of luteinizing releasing hormone (LHRH) through its tyrosine side chain and allowed synthesis of well defined conjugates. The DVS derivatives of bovine serum albumin (BSA), reduced and carboxymethylated BSA and purified protein derivative (PPD) were compared with respect to their potential value as carriers for obtaining antibodies to LHRH (M(r) 1000) and epidermal growth factor (EGF, M(r) 5000). IAA-PPD was evaluated as a carrier for the conjugation of glutathione specifically through its cysteine side chain and for obtaining antibodies to glutathione. The antisera obtained were specific and of high titer, and the methods described here will thus allow the convenient synthesis of carrier conjugates with well defined characteristics.

Detection of subtle differences in the surface structure of lysozymes by use of an immobilized Fab fragment.

A method was developed to evaluate the association constant at physiological pH (pH 7.5) between a lysozyme and the Fab fragment derived from anti-lysozyme monoclonal antibody 37-7, which was immobilized to the adsorbent for HPLC. Comparison of the association constants between lysozymes and the immobilized Fab fragment indicated that mAb 37-7 recognized the prominently exposed regions (hills and ridges) around His15 of hen lysozyme, but His15 itself was not directly involved in the binding with mAb 37-7. Moreover, the epitope was confirmed by the reactivity of His15 with monoiodoacetic acid in the presence of mAb 37-7. The association constant of 15-carboxymethylated histidine lysozyme (15CM lysozyme) with the immobilized Fab fragment was smaller by one-seventh than that of 15-carboxamidated histidine lysozyme, though the side chains introduced were almost identical in size. From the pH titration of 15CM lysozyme with 13C-enriched carboxyl group by use of 13C-NMR, the pKa of the introduced carboxyl group was evaluated to be 5.06. Since the carboxyl group was fully ionized under the conditions of measurement (pH 7.5), electrostatic repulsion was found to disturb severely the association between mAb 37-7 and hen lysozyme. Moreover, it was demonstrated that, because of the high reproducibility of measurement, the immobilized Fab fragment could detect subtle differences in the surface structure of lysozymes.

Stereodivergent and regioselective synthesis of 3,4-cis- and 3,4-trans-pyrrolidinediols from alpha-amino acids.

[reaction: see text] Highly stereodivergent Woodward-Prevost reaction applied to iodoacetates derived from homochiral alpha-amino acids afforded enantiopure 3,4-cis- and 3,4-trans-pyrrolidinediol derivatives, with control over the protecting group, allowing for differential protection.

Carboxymethylation of the human estrogen receptor ligand-binding domain-estradiol complex: HPLC/ESMS peptide mapping shows that cysteine 447 does not react with iodoacetic acid.

Experiments were carried out to determine the degree of solvent and reagent accessibility of the cysteines in the ligand-binding domain of the human estrogen receptor (hER LBD). The cysteine residues were alkylated when human ER LBD was present in its ligand (estradiol)-bound conformation. Direct electrospray ionization mass spectrometry (ESMS) as well as liquid chromatography coupled with ESMS, and matrix-assisted laser ionization desorption time-of-flight mass spectrometry were used to determine the location and the yield of the derivatized residues after proteolysis with trypsin. We observed that the cysteine 447 was protected against alkylation under these conditions, whereas cysteines 381, 417, and 530 were fully derivatized.

The relationship of raw broiler breast meat color and pH to cooked meat color and pH.

Three replicate trials were conducted to determine the influence of raw breast meat color and pH on subsequent cooked meat color and pH. In each trial, approximately 50 breast fillets were obtained from a commercial processing plant based on being either normal, lighter than normal, or darker than normal. Color (L* = lightness, a* = redness, and b* = yellowness) of each fillet was determined in triplicate on the underside surface of the fillet (to avoid scalding effects), and the pH was determined on a tissue sample removed from the posterior portion of each fillet. Fillets were then cooked in steam at 98 C for 20 min and cooled to room temperature, and a second sample was removed from the posterior section for cooked meat pH. Cooked meat color was measured on an exposed surface, to avoid cooking-related discoloration. The data were subjected to linear regression analysis to determine the relationship between raw and cooked values. Results indicated a significant linear relationship between raw and cooked values for each color parameter as well as pH. Model R2 values were 0.43, 0.40, 0.64, and 0.78 for L*, a*, b*, and pH, respectively. There were also significant linear relationships between raw meat L* and raw muscle pH (R2 = 0.59) as well as cooked meat L* and raw meat pH (R2 = 0.36). These results indicate that raw breast meat color and pH affect cooked breast meat color and pH but that cooking reduces the degree of color variation. Moreover, cooked meat lightness is more closely associated with raw breast meat pH than with cooked meat pH.

[DNA duplexes containing 2'-deoxy-2'-iodoacetamidouridine as reagents for affinity modification of proteins]. / DNK-dupleksy, soderzhashchie 2'-dezoksi-2'-iodatsetamidouridin, kak reagenty dlia affinnoi modifikatsii belkov.

DNA duplexes containing the iodoacetamido group at position 2' of the ribose moiety were proposed for affinity modification of Cys in DNA-binding proteins. Reactive DNA derivatives were obtained with iodoacetic anhydride and synthetic oligodeoxyribonucleotides containing 2'-amino-2'-deoxyuridine in place of thymine at various positions. The derivatives were tested for reaction with amino acids and peptides and shown to specifically interact with Cys-containing proteins. The possibility of using the modified DNA duplexes to probe the protein SH group close to the DNA sugar-phosphate backbone in DNA-protein complexes was demonstrated with the example of subunit p50 of human transcription factor NF-kappa B.

Improving the stability of the EC1 domain of E-cadherin by thiol alkylation of the cysteine residue.

The objective of this work was to improve chemical and physical stability of the EC1 protein derived from the extracellular domain of E-cadherin. In solution, the EC1 protein has been shown to form a covalent dimer via a disulfide bond formation followed by physical aggregation and precipitation. To improve solution stability of the EC1 protein, the thiol group of the Cys13 residue in EC1 was alkylated with iodoacetate, iodoacetamide, and maleimide-PEG-5000 to produce thioether derivatives called EC1-IA, EC1-IN, and EC1-PEG. The physical and chemical stabilities of the EC1 derivatives and the parent EC1 were evaluated at various pHs (3.0, 7.0, and 9.0) and temperatures (0, 3, 70 °C). The structural characteristics of each molecule were analyzed by circular dichroism (CD) and fluorescence spectroscopy and the derivatives have similar secondary structure as the parent EC1 protein at pH 7.0. Both EC1-IN and EC1-PEG derivatives showed better chemical and physical stability profiles than did the parent EC1 at pH 7.0. EC1-PEG had the best stability profile compared to EC1-IN and EC1 in solution under various conditions.

Rapid DNA chemical ligation for amplification of RNA and DNA signal.

Enzymatic ligation methods are useful in the diagnostic detection of DNA sequences. Here, we describe the investigation of nonenzymatic phosphorothioate--iodoacetyl DNA chemical ligation as a method for the detection and identification of RNA and DNA. The specificity of ligation on the DNA target is shown to allow the discrimination of a single point mutation with a drop in the ligation yield of up to 16.1-fold. Although enzymatic ligation has very low activity for RNA targets, this reaction is very efficient for RNA targets. The speed of the chemical ligation with an RNA target achieves a 70% yield in 5 s, which is equal to or better than that of ligase-enzyme-mediated ligation with a DNA target. The reaction also exhibits a significant level of signal amplification under thermal cycling in periods as short as 100-120 min, with the RNA or DNA target acting in a catalytic way to ligate multiple pairs of probes.

Multiple chemical ligation under thermal cycle.

Enzymatic ligation methods are useful in diagnostic detection of DNA sequence. Here we describe the investigation of nonezymatic phosphorothioate-iodoacetyl DNA chemical ligation as a method for detection and identification of RNA and DNA. Specificity of ligation on DNA target is shown to yield discrimination of single point mutation as a drop in two magnitude. Although enzymatic ligation shows very low activity for RNA target, this reaction is found to be very efficient on RNA target. This chemical ligation with RNA target completes 70% within a few seconds, which equal or overcome ligase enzyme-mediated ligation with DNA target. The reaction is also shown to exhibit a significant level of signal amplification under thermal cycle for short time. Further, we found recently that ligation fidelity changed in function of chemical reactivity of probe. This trend was systematically investigated.

Synthesis of hyaluronan haloacetates and biology of novel cross-linker-free synthetic extracellular matrix hydrogels.

Hyaluronan (HA) derivatives containing thiol-reactive electrophilic esters were prepared to react with thiol-modified macromolecules to give cross-linker-free hydrogels. Specifically, HA was converted to two haloacetate derivatives, HA bromoacetate (HABA) and HA iodoacetate (HAIA). In cytotoxicity assays, these reactive macromolecules predictably induced cell death in a dose-dependent manner. Cross-linker-free synthetic extracellular matrix (sECM) hydrogels were prepared by thiol alkylation using HAIA and HABA as polyvalent electrophiles and thiol-modified HA (CMHA-S) with or without thiol-modified gelatin (Gtn-DTPH) as polyvalent nucleophiles. When primary human fibroblasts were seeded on the surface of the sECMs containing only the electrophilic HA haloacetate and nucleophilic CMHA-S components, no significant cytoadherence was observed. Cell attachment and viability was 17% (HABA) to 30% (HAIA) lower on HA haloacetate cross-linked hydrogels than on CMHA-S that had been oxidatively cross-linked via disulfide-bonds. In contrast, sECMs that included Gtn-DTPH allowed fibroblasts to attach, spread, and proliferate. Taken together, the HA haloacetates are attractive candidates for producing cross-linker-free sECM biomaterials that can function either as anti-adhesive barriers or as cytoadhesive sECMs for cell culture in pseudo-3-D.

Reversed-phase high-pressure liquid chromatographic tryptic peptide mapping for the comparison and study of monoclonal antibodies.

We have examined and optimized several parameters to generate efficient, high-resolution, high-information tryptic peptide maps of monoclonal antibodies and their Fab fragments, without separating the H and L chains, using reversed-phase high-pressure liquid chromatography. Use of a high protease-to-substrate ratio with optimized digestion time and HPLC gradient conditions led to a reproducible mapping of the reduced, carboxymethylated Fab fragments of two antibodies. The technique was then used to screen Fab lots for batch-to-batch consistency, and for examining the effect of 10 mM cysteine on papain cleavage of whole antibody. The technique was modified by labeling cysteine with chromophoric analogues of iodoacetamide instead of radiolabeled iodoacetamide, resulting in a three-dimensional peptide map. With multiwavelength detection, this consisted of simultaneous observation of all chromophores at 214 nm, those with aromatic residues at 280 nm, and those with cysteine at 422 nm, without collecting and counting each peak to identify cysteine-containing peptides.