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.

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.

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.

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.

Proton donor in yeast pyruvate kinase: chemical and kinetic properties of the active site Thr 298 to Cys mutant.

The active site T298 residue of yeast pyruvate kinase (YPK), located in a position to serve potentially as the proton donor, was mutated to cysteine. T298C YPK was isolated and purified, and its enzymatic properties were characterized. Fluorescence and CD spectra indicate minor structural perturbations. A kinetic analysis of the Mg(2+)-activated enzyme demonstrates no catalytic activity in the absence of the heterotropic activator fructose 1,6-bisphosphate (FBP). In the presence of Mg(2+) and FBP, T298C has approximately 20% of the activity of wild-type (wt) YPK. The activator constant for FBP increases by 1 order of magnitude compared to this constant with the wt enzyme. T298C shows positive cooperativity by FBP with a Hill coefficient of 2.6 (wt, n(H,FBP) = 1). Mn(2+)-activated T298C behaves like Mn(2+)-activated wt YPK with a V(max) that is 20% of that for the wt enzyme with or without FBP. A pH-rate profile of T298C relative to that for wt YPK shows that pK(a,2) has shifted from 6.4 in wt to 5.5, indicating that the thiol group elicits an acidic pK shift. Inactivation of both wt and T298C by iodoacetate elicits a pseudo-first-order loss of activity with T298C being inactivated from 8 to 100 times faster than wt YPK. A pH dependence of the inactivation rate constant for T298C gives a value of 8.2, consistent with the pK for a thiol. Changes in fluorescence indicate that the T298C-Mg(2+) complex binds PEP, ADP, and both ligands together. This demonstrates that the lack of activity is not due to the loss of substrate binding but to the lack of ability to induce the proper conformational change. The mutation also induces changes in binding of FBP to all the relevant complexes. Binding of the metal and binding of PEP to the enzyme complexes are also differentially altered. Solvent isotope effects are observed for both wt and T298C. Proton inventory studies indicate that k(cat) is affected by a proton from water in the transition state and the effects are metal ion-dependent. The results are consistent with water being the active site proton donor. Active site residue T298 is not critical for activity but plays a role in the activation of the water and affects the pK that modulates catalytic activity.

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.

[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.

Dual role of cysteine 172 in redox regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase activity and degradation.

Alkylation and oxidation of cysteine residues significantly decrease the catalytic activity and stimulate the degradation of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). We analyzed the role of vicinal cysteine residues in redox regulation of RuBisCO from Synechocystis sp. strain PCC 6803. Cys172 and Cys192, which are adjacent to the catalytic site, and Cys247, which cross-links two large subunits, were replaced by alanine. Whereas all mutant cells (C172A, C192A, C172A-C192A, and C247A) and the wild type grew photoautotrophically at similar rates, the maximal photosynthesis rates of C172A mutants decreased 10 to 20% as a result of 40 to 60% declines in RuBisCO turnover number. Replacement of Cys172, but not replacement of Cys192, prominently decreased the effect of cysteine alkylation or oxidation on RuBisCO. Oxidants that react with vicinal thiols had a less inhibitory effect on the activity of either the C172A or C192A enzyme variants, suggesting that a disulfide bond was formed upon oxidation. Thiol oxidation induced RuBisCO dissociation into subunits. This effect was either reduced in the C172A and C192A mutant enzymes or eliminated by carboxypentitol bisphosphate (CPBP) binding to the activated enzyme form. The CPBP effect presumably resulted from a conformational change in the carbamylated CPBP-bound enzyme, as implied from an alteration in the electrophoretic mobility. Stress conditions, provoked by nitrate deprivation, decreased the RuBisCO contents and activities in the wild type and in the C192A and C247A mutants but not in the C172A and C172A-C192A mutants. These results suggest that although Cys172 does not participate in catalysis, it plays a role in redox regulation of RuBisCO activity and degradation.

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.

ATP nucleotidylation of creatine kinase.

Creatine kinase (CK) will autoincorporate radiolabel from [gamma32P]ATP and has thus been reported to be autophosphorylated. Also, in contrast to normal brain enzyme, CK in Alzheimer-diseased brain homogenate shows greatly decreased activity, abolished photolabeling with [32P]8N3ATP, and no detectable autoincorporation of radiolabel by [gamma32P]ATP. Surprisingly, our studies with both human brain and purified CK showed that [alpha32P]ATP, [gamma32P]ATP, [alpha32P]ADP, [2,8H3]ATP, [gamma32P]2',3'-O-(2,4, 6-trinitrophenyl)-ATP, and [gamma32P]benzophenone-gammaATP all autoincorporate radiolabel into CK with good efficiency. This demonstrates that the gamma-phosphate and the 2' and 3' hydroxyls are not involved in the covalent linkage and that all three phosphates, the ribose and base of the ATP molecule are retained upon autoincorporation (nucleotidylation). Treatment with NaIO3 to break the 2'-3' linkage effected total loss of radiolabel indicating that nucleotidylation resulted in opening of the ribose ring at the C1' position. Nucleotidylation with increasing [alpha32P]ATP at 37 degrees C gives an approximate k0.5 of 125 microM and saturates at 340 microM nucleotide. Modification of 8-10% of the copy numbers occurs at saturation, and CK activity is inhibited to approximately the same degree. Low micromolar levels of native substrates such as ADP, ATP, and phosphocreatine substantially reduce [alpha32P]ATP nucleotidylation. In contrast, AMP, GTP, GMP, NADH, and creatine did not effectively reduce nucleotidylation. When [alpha32P]ATP-nucleotidylated or [alpha32P]8N3ATP-photolabeled CK is treated with trypsin a single, identical radiolabeled peptide (V279-R291) is generated that comigrates on reverse phase HPLC and Tris-tricine electrophoresis. Nucleotidylation into this peptide was prevented 86% by the presence of ATP. We conclude that CK is nucleotidylated within the active site by modification at the C1'position and that autophosphorylation of this enzyme does not occur.

Purification and characterization of cysteine protease from Pleurotus ostreatus.

Cysteine protease activity in mycelial culture increased 7.7-fold after fruit body formation in Pleurotus ostreatus, using the Leu pNA (LPNA) cleavage assay. The enzyme was purified from fruit bodies and its M(r) was 97,000 by gel filtration and 48,500 by SDS-PAGE, indicating that it is a dimer. The enzyme was sensitive to iodoacetic acid, p-chloromercuribenzoate, N-ethylmaleimide, and HgCl2. The sequence of the first 9 N-terminal amino acids of cysteine protease was ASGLXXAIL.

Monitoring protein refolding induced by disulfide formation using capillary isoelectric focusing-electrospray ionization mass spectrometry.

Rapid growth in the biotechnology industry has led to a dramatic increase in attention to the protein folding problem. Understanding protein-folding pathways is essential to the production of biopharmaceuticals since commercial production of recombinant proteins often requires a protein-refolding process for recovery of high yields. Protein folding coupled to the formation of disulfide bonds presents one of the simplest approaches to studying folding intermediates. On-line capillary isoelectric focusing-electrospray ionization mass spectrometry (CIEF-ESIMS) is demonstrated for kinetic studies of disulfide bond-induced protein refolding. Refolding intermediates of bovine pancreatic ribonuclease A, a model system for this study, are blocked at different stages by alkylating free thiols with iodoacetate. The alkylation reaction results in the introduction of charge (-1) and mass (59) differences for each alkylation site, providing the means for predictable separation and direct identification of refolding intermediates using CIEF-ESIMS. Besides the observation of refolding intermediates containing different numbers of disulfide bonds and even mixed disulfides, the two-dimensional resolving power of CIEF-ESIMS allows the determination of conformational heterogeneity among groups of refolding intermediates.

The three-dimensional structure of Cys-47-modified mouse liver glutathione S-transferase P1-1. Carboxymethylation dramatically decreases the affinity for glutathione and is associated with a loss of electron density in the alphaB-310B region.

The three-dimensional structure of mouse liver glutathione S-transferase P1-1 carboxymethylated at Cys-47 and its complex with S-(p-nitrobenzyl)glutathione have been determined by x-ray diffraction analysis. The structure of the modified enzyme described here is the first structural report for a Pi class glutathione S-transferase with no glutathione, glutathione S-conjugate, or inhibitor bound. It shows that part of the active site area, which includes helix alphaB and helix 310B, is disordered. However, the environment of Tyr-7, an essential residue for the catalytic reaction, remains unchanged. The position of the sulfur atom of glutathione is occupied in the ligand-free enzyme by a water molecule that is at H-bond distance from Tyr-7. We do not find any structural evidence for a tyrosinate form, and therefore our results suggest that Tyr-7 is not acting as a general base abstracting the proton from the thiol group of glutathione. The binding of the inhibitor S-(p-nitrobenzyl)-glutathione to the carboxymethylated enzyme results in a partial restructuring of the disordered area. The modification of Cys-47 sterically hinders structural organization of this region, and although it does not prevent glutathione binding, it significantly reduces the affinity. A detailed kinetic study of the modified enzyme indicates that the carboxymethylation increases the Km for glutathione by 3 orders of magnitude, although the enzyme can function efficiently under saturating conditions.