Translational regulation contributes to the secretory response of chondrocytic cells following exposure to interleukin-1ß.

Osteoarthritis is a chronic disease characterized by the loss of articular cartilage in synovial joints through a process of extracellular matrix destruction that is strongly associated with inflammatory stimuli. Chondrocytes undergo changes to their protein translational capacity during osteoarthritis, but a study of how disease-relevant signals affect chondrocyte protein translation at the transcriptomic level has not previously been performed. In this study, we describe how the inflammatory cytokine interleukin 1-ß (IL-1ß) rapidly affects protein translation in the chondrocytic cell line SW1353. Using ribosome profiling we demonstrate that IL-1ß induced altered translation of inflammatory-associated transcripts such as NFKB1, TNFAIP2, MMP13, CCL2, and CCL7, as well as a number of ribosome-associated transcripts, through differential translation and the use of multiple open reading frames. Proteomic analysis of the cellular layer and the conditioned media of these cells identified changes in a number of the proteins that were differentially translated. Translationally regulated secreted proteins included a number of chemokines and cytokines, underlining the rapid, translationally mediated inflammatory cascade that is initiated by IL-1ß. Although fewer cellular proteins were found to be regulated in both ribosome profiling and proteomic data sets, we did find increased levels of SOD2, indicative of redox changes within SW1353 cells being modulated at the translational level. In conclusion, we have produced combined ribosome profiling and proteomic data sets that provide a valuable resource in understanding the processes that occur during cytokine stimulation of chondrocytic cells.

Phaseolus vulgaris STP13.1 is an H+-coupled monosaccharide transporter, present in source leaves and seed coats, with higher substrate affinity at depolarized potentials.

Sugar transport proteins (STPs) are high-affinity H+-coupled hexose symporters. Recently, the contribution of STP13 to bacterial and fungal pathogen resistance across multiple plant species has garnered significant interest. Quantitative PCR analysis of source leaves, developing embryos, and seed coats of Phaseolus vulgaris L. (common bean) revealed that PvSTP13.1 was expressed in source leaves and seed coats throughout seed development. In contrast, PvSTP13.1 transcripts were detected at exceedingly low levels in developing embryos. To characterize the transport mechanism, PvSTP13.1 was expressed in Xenopus laevis oocytes, and inward-directed currents were analyzed using two-electrode voltage clamping. PvSTP13.1 was shown to function as an H+-coupled monosaccharide symporter exhibiting a unique high affinity for hexoses and aldopentoses at depolarized membrane potentials. Specifically, of the 31 assessed substrates, which included aldohexoses, deoxyhexoses, fructose, 3-O-methyl-D-glucose, aldopentoses, polyols, glycosides, disaccharides, trisaccharides, and glucuronic acid, PvSTP13.1 displayed the highest affinity (K 0.5) for glucose (43 µM), mannose (92 µM), galactose (145 µM), fructose (224 µM), xylose (1.0 mM), and fucose (3.7 mM) at pH 5.6 at a depolarized membrane potential of -40 mV. The results presented here suggest PvSTP13.1 contributes to retrieval of hexoses from the apoplasmic space in source leaves and coats of developing seeds.

The design and synthesis of novel, potent and orally bioavailable N-aryl piperazine-1-carboxamide CCR2 antagonists with very high hERG selectivity.

A novel N-aryl piperazine-1-carboxamide series of human CCR2 chemokine receptor antagonists was discovered. Early analogues were potent at CCR2 but also inhibited the hERG cardiac ion channel. Structural modifications which decreased lipophilicity and basicity resulted in the identification of a sub-series with an improved margin over hERG. The pharmacological and pharmacokinetic properties of the lead compound from this series, N-(3,4-dichlorophenyl)-4-[(2R)-4-isopropylpiperazine-2-carbonyl]piperazine-1-carboxamide, are described.

Gene expression analysis of subchondral bone, cartilage, and synovium in naturally occurring equine palmar/plantar osteochondral disease.

Osteoarthritis (OA) is a disease of the entire joint but the relationship between pathological events in various joint tissues is poorly understood. We examined concurrent changes in bone, cartilage, and synovium in a naturally occurring equine model of joint degeneration. Joints (n = 64) were grossly assessed for palmar/plantar osteochondral disease (POD) in racehorses that required euthanasia for unrelated reasons and assigned a grade of 0 (n = 34), 1 (n = 17), 2 or 3 (n = 13) using a recognized grading scheme. Synovium, cartilage, and subchondral bone were collected for histological and gene expression analysis. Relations between POD grade, cartilage histological score, and gene expression levels were examined using one-way analysis of variance or Kruskal-Wallis test and Spearman's correlation coefficient with corrections for multiple comparisons. Cartilage histological score increased in joints with POD grade 1 (p = 0.002) and 2 or 3 (p < 0.001) compared to 0. At grade 1, expression of COL1A1, COL2A1, and MMP1 increased and BGN decreased in subchondral bone while expression of BGN and ACAN decreased in cartilage. These changes further progressed at grades 2 and 3. POD grades 2 and 3 were associated with decreased expression of osteoclast inhibitor OPG and increased markers of cartilage degeneration (MMP13, COL1A1). Expression of the vascular endothelial growth factor decreased with POD grade and negatively correlated with cartilage histological score. Synovium showed no histological or transcriptomic changes related to pathology grade. Cartilage degeneration in POD is likely to be secondary to remodeling of the subchondral bone. Limited activation of proinflammatory and catabolic genes and moderate synovial pathology suggests distinct molecular phenotype of POD compared with OA.

Synthesis of pyroglutamic acid derivatives via double michael reactions of alkynones.

In the presence of substoichiometric quantities of potassium tert-butoxide and an additional metal salt, amide-tethered diacids undergo double Michael reactions with alkynones to provide highly functionalized pyroglutamic acid derivatives. The metal salt was found to play an important role in improving the diastereoselectivities of the reactions.

Transcriptome-wide analysis of messenger RNA decay in normal and osteoarthritic human articular chondrocytes.

OBJECTIVE: Messenger RNA (mRNA) decay rates control not only gene expression levels, but also responsiveness to altered transcriptional input. We undertook this study to examine transcriptome-wide posttranscriptional regulation in both normal and osteoarthritic (OA) human articular chondrocytes. METHODS: Human articular chondrocytes were isolated from normal or OA tissue. Equine articular chondrocytes were isolated from young or old horses at a commercial abattoir. RNA decay was measured across the transcriptome in human cells by microarray analysis following an actinomycin D chase. Messenger RNA levels in samples were confirmed using quantitative reverse transcription-polymerase chain reaction. RESULTS: Examination of total mRNA expression levels demonstrated significant differences in the expression of transcripts between normal and OA chondrocytes. Interestingly, almost no difference was observed in total mRNA expression between chondrocytes from intact OA cartilage and those from fibrillated OA cartilage. Decay analysis revealed a set of rapidly turned over transcripts associated with transcriptional control and programmed cell death that were common to all chondrocytes and contained binding sites for abundant cartilage microRNAs. Many transcripts exhibited altered mRNA half-lives in human OA chondrocytes compared to normal cells. Specific transcripts whose decay rates were altered were generally less stable in these pathologic cells. Examination of selected genes in chondrocytes from young and old healthy horses did not identify any change in mRNA turnover. CONCLUSION: This is the first investigation into the "posttranscriptome" of the chondrocyte. It identifies a set of short-lived chondrocyte mRNAs likely to be highly responsive to altered transcriptional input as well as mRNAs whose decay rates are affected in OA chondrocytes.

Pharmacokinetic benefits of 3,4-dimethoxy substitution of a phenyl ring and design of isosteres yielding orally available cathepsin K inhibitors.

Rational structure-based design has yielded highly potent inhibitors of cathepsin K (Cat K) with excellent physical properties, selectivity profiles, and pharmacokinetics. Compounds with a 3,4-(CH3O)2Ph motif, such as 31, were found to have excellent metabolic stability and absorption profiles. Through metabolite identification studies, a reactive metabolite risk was identified with this motif. Subsequent structure-based design of isoteres culminated in the discovery of an optimized and balanced inhibitor (indazole, 38).

Cap-dependent and hepatitis C virus internal ribosome entry site-mediated translation are modulated by phosphorylation of eIF2alpha under oxidative stress.

Chronic hepatitis C is often associated with oxidative stress. Hepatitis C virus (HCV) utilizes an internal ribosome entry site (IRES) element for translation, in contrast to cap-dependent translation of the majority of cellular proteins. To understand how virus translation is modulated under oxidative stress, HCV IRES-mediated translation was compared with cap-dependent translation using a bicistronic reporter construct and hydrogen peroxide (H2O2) as a stress inducer. In H2O2-sensitive HeLa cells, H2O2 repressed translation in a time- and dose-dependent manner, concomitant with the kinetics of eIF2alpha phosphorylation. A phosphomimetic of eIF2alpha, which mimics the structure of the phosphorylated eIF2alpha, was sufficient to repress translation in the absence of H2O2. In H2O2-resistant HepG2 cells, H2O2 activated both HCV IRES-mediated and cap-dependent translation, associated with an increased level of phospho-eIF2alpha. It was postulated that H2O2 might stimulate translation in HepG2 cells via an eIF2alpha-independent mechanism, whereas the simultaneous phosphorylation of eIF2alpha repressed part of the translational activities. Indeed, the translational repression was released in the presence of a non-phosphorylatable mutant, eIF2alpha-SA, resulting in further enhancement of both translational activities after exposure to H2O2. In HuH7 cells, which exhibited an intermediate level of sensitivity towards H2O2, both HCV IRES-mediated and cap-dependent translational activities were upregulated after treatment with various doses of H2O2, but the highest level of induction was achieved with a low level of H2O2, which may represent the physiological level of H2O2. At this level, the HCV IRES-mediated translation was preferentially upregulated compared with cap-dependent translation.