A Scoping Review of the Use of Pioglitazone in the Treatment of Temporo-Mandibular Joint Arthritis.

Thiazolidinediones (TZDs) are a group of diabetes medications currently being investigated for anti-arthritis effectiveness, one of which is pioglitazone. The purpose of this scoping review is to evaluate the potential use of pioglitazone in the treatment of temporomandibular joint (TMJ) arthritis. The criteria of eligibility were studies with the diagnosis of arthritis and pioglitazone treatment with a change in any inflammation index as an outcome. Of the 1169 records initially identified following the selection process, two animal studies and four clinical studies were included in the review. Improvements from the baseline were observed in each treatment group for each inflammation indicator. The results of the animal studies on the temporomandibular joints and on patients with rheumatoid and psoriatic arthritis indicate that the drug in question may have potential to treat arthritis, including within the temporomandibular joint.

Spatiotemporal organisation of protein processing in the kidney.

The kidney regulates plasma protein levels by eliminating them from the circulation. Proteins filtered by glomeruli are endocytosed and degraded in the proximal tubule and defects in this process result in tubular proteinuria, an important clinical biomarker. However, the spatiotemporal organization of renal protein metabolism in vivo was previously unclear. Here, using functional probes and intravital microscopy, we track the fate of filtered proteins in real time in living mice, and map specialized processing to tubular structures with singular value decomposition analysis and three-dimensional electron microscopy. We reveal that degradation of proteins requires sequential, coordinated activity of distinct tubular sub-segments, each adapted to specific tasks. Moreover, we leverage this approach to pinpoint the nature of endo-lysosomal disorders in disease models, and show that compensatory uptake in later regions of the proximal tubule limits urinary protein loss. This means that measurement of proteinuria likely underestimates severity of endocytotic defects in patients.

Autologous Stem Cells Transplants in the Treatment of Temporomandibular Joints Disorders: A Systematic Review and Meta-Analysis of Clinical Trials.

This systematic review aims to analyze the outcomes of the treatment of temporomandibular joint (TMJ) articular pain (AP) and restricted maximum mouth opening (MMO) with intra-articular administration of mesenchymal stem cells (MSCs). The inclusion criteria allowed primary studies involving AP and/or MMO pre-treatment and post-intervention values. Medical databases that were covered by ACM Digital, BASE, EBSCOhost, Google Scholar, PubMed, Scopus, and Web of Science engines were searched. The risk of bias was assessed with RoB 2 and ROBINS-I tools. The results were tabulated, plotted, and analyzed for regression. A total of 5 studies involving 51 patients/69 TMJs were identified, and 4 studies on 50 patients/67 TMJs were synthesized. Interventions were each time effective in decreasing AP and increasing MMO in a 6-month follow-up period by an average of about 85% and over 40%, respectively. Regression analysis showed a good fit of the logarithmic model for AP relief (5.8 - 0.8 ln x; R2 = 0.90) and MMO increase (33.5 + 2.4 ln x; R2 = 0.89). The results for AP and MMO were based on 3 studies in 39 patients and 4 studies in 50 patients, respectively, all at high risk of bias. The intra-articular administration of MSCs to TMJs, based on weak evidence, may be highly effective in reducing AP and improving MMO. This study received no funding.

Metabolic Modifications in Terpenoid and Steroid Pathways Triggered by Methyl Jasmonate in Taxus × media Hairy Roots.

The in vitro cultures of Taxus spp. were one of the first plant in vitro systems proved to exert the positive effect of elicitation with methyl jasmonate (MeJA) on the biosynthesis of specialized metabolites. The main aim of the present study is to examine the effect of MeJA treatment on the steroid and triterpenoid content of two genetically different hairy root lines of Taxus × media, KT and ATMA. The results revealed that the two lines differed in the total content of steroids and triterpenoids (in the ATMA root line, their amounts were lower than those in the KT line by 43% and 30%, respectively), but not in the composition of these compounds. The metabolic response to elicitation with MeJA was different: in the KT root line, the content of steroids decreased by 18%, whereas it increased by 38% in the ATMA line. Several metabolic features were common, including the characteristic changes in the ratio of sitosterol to stigmasterol content, caused by the very sharp boost in stigmasterol levels, the increase in the amount of glycoside forms of sterols, as well as in triterpenoid and total phenolic content. It is the first report on modifications of the terpenoid biosynthetic pathway in Taxus hairy root cultures triggered by MeJA, concerning steroids and triterpenoids.

Ligand-Directed Modification of Active Matrix Metalloproteases: Activity-based Probes with no Photolabile Group.

Activity-based probes enable discrimination between the active enzyme and its inactive or inactivated counterparts. Since metalloproteases catalysis is non-covalent, activity-based probes targeting them have been systematically developed by decorating reversible inhibitors with photo-crosslinkers. By exploiting two types of ligand-guided chemistry, we identified novel activity-based probes capable of covalently modifying the active site of matrix metalloproteases (MMPs) without any external trigger. The ability of these probes to label recombinant MMPs was validated in vitro and the identity of the main labelling sites within their S3 ' region unambiguously assigned. We also demonstrated that our affinity probes can react with rhMMP12 at nanogram scale (that is, at 0.07 % (w/w)) in complex proteomes. Finally, this ligand-directed chemistry was successfully applied to label active MMP-12 secreted by eukaryote cells. We believe that this approach could be transferred more widely to many other metalloproteases, thus contributing to tackle their unresolved proteomic profiling in vivo.

[Applications of synthetic and semisynthetic polymers (Kollidon K25, Kollidon K90 and Hydroksyethylocellulose) as carriers of ketoprofen in solid oral prolonged release dosage forms. The impact of selected non-ionic surfactants (Tween 80 and Rofam 70) on the release kinetics].

BACKGROUND: Hydrophilic matrices used as oral forms of sustained release drugs are a suitable application medium for short-acting nonsteroidal anti-inflammatory drugs (NSAID) - ketoprofen. A properly selected hydrophilic matrix in oral preparations may significantly increase efficacy and application safety of ketoprofen. OBJECTIVES: The aim of the research was to analyze the usefulness of polymers (synthetic Kollidon K25 and K90, semi-synthetic hydroxyethylcellulose) and calcium hydrogen phosphate dihydrate (as an inorganic filler) in manufacturing solid oral matrix forms of ketoprofen and to study of the effect of non-ionic surfactants (Tween 80, Rofam 70) on release kinetics. MATERIAL AND METHODS: Ketoprofen, HEC, Kollidon K25, and K90, calcium hydrogen phosphate, magnesium stearate. Incorporation. Studies on the tablet mass. Direct tableting. Studies on the pharmacopoeial parameters and pharmaceutical availability. Approximation of the results. RESULTS: The results of the granulometric studies on tablet mass were in accordance with pharmacopoeial standards. The results of morphological and biopharmaceutical studies of the obtained matrices (tablets) were consistent with the pharmacopoeial standards for formulations with HEC, K25 and K90. The release results most closely related to row 0 kinetics were obtained for the matrix containing HEC and K25. Tween 80 added to 0.1N HCl accelerated the release of ketoprofen, while Rofam 70 decelerated it. Tween 80 and Rofam 70 added to the pH 7.4 buffer accelerated the release of ketoprofen. CONCLUSIONS: The presented model system of preformulation studies showed the usefulness of HEC and Kolidon K25 in the technology of hydrophilic matrices with ketoprofen. Surfactants added to the medium do not affect the release rate of ketoprofen.

Social health insurance without corporate actors: changes in self-regulation in Germany, Poland and Turkey.

Social health insurance in Western Europe has for many years been characterized by self-regulation in which specific conditions of healthcare financing and provision have been regulated by social-insurance institutions through mutual self-governance. However, the principle of self-regulation has recently been weakened by increased state regulation and market competition, which were introduced in response to economic and social changes. Even in Germany, which has been regarded as an "ideal-type" health insurance system and in which self-regulation remains at the core of healthcare governance, more direct state intervention has gained in importance. On the other hand, in countries such as Poland and Turkey, where this tradition of self-regulation is missing, social health insurance is deemed a financing instrument but not an instrument of governance and corporate actors are not accorded a significant role in regulation. This article investigates how social health insurance systems are regulated in contexts in which corporate actors' role is either diminishing or absent by focusing on three crucial areas of regulation: financing, the remuneration of medical doctors, and the definition of the healthcare benefit package. In Germany, state regulation has increased in healthcare financing and remuneration while the role of corporate actors has grown in the definition of the benefits package. In Poland and Turkey, on the other hand, reforms have maintained the status quo in terms of the strong regulatory, budgetary, and managerial powers of the state and very limited involvement of corporate actors.

DARPin-assisted crystallography of the CC2-LZ domain of NEMO reveals a coupling between dimerization and ubiquitin binding.

NEMO is an integral part of the IkappaB kinase complex and serves as a molecular switch by which the NF-kappaB signaling pathway can be regulated. Oligomerization and polyubiquitin (poly-Ub) binding, mediated through the regulatory CC2-LZ domain, were shown to be key features governing NEMO function, but the relationship between these two activities remains unclear. In this study, we solved the structure of this domain in complex with a designed ankyrin repeat protein, which helps its crystallization. We generated several NEMO mutants in this domain, including those associated with human diseases incontinentia pigmenti and immunodeficiency with or without anhidrotic ectodermal dysplasia. Analytical ultracentrifugation and thermal denaturation experiments were used to evaluate the dimerization properties of these mutants. A fluorescence-based assay was developed, as well, to quantify the interaction to monoubiquitin and poly-Ub chains. Moreover, the effect of these mutations was investigated for the full-length protein. We show that a proper folding of the ubiquitin-binding domain, termed NOA/UBAN/NUB, into a stable coiled-coil dimer is required but not sufficient for efficient interaction with poly-Ub. In addition, we show that binding to poly-Ub and, to a lesser extent, to monoubiquitin increases the stability of the NOA coiled-coil dimer. Collectively, these data provide structural insights into how several pathological mutations within and outside of the CC2-LZ's NOA ubiquitin binding site affect IkappaB kinase activation in the NF-kappaB signaling pathway.

Translation initiation from two in-frame AUGs generates mitochondrial and cytoplasmic forms of the p43 component of the multisynthetase complex.

In humans, nine aminoacyl-tRNA synthetases form a stable multiprotein complex with the three auxiliary proteins p18, p38, and p43. The N-terminal moiety of p43 is involved in its anchoring to the complex, and its C-terminal moiety has a potent tRNA binding capacity. The p43 component of the complex is also the precursor of p43(ARF), an apoptosis-released factor, and of p43(EMAPII), the endothelial-monocyte activating polypeptide II. Here we identified a new translation product of the gene of p43, which contains nine additional N-terminal amino acid residues. This gene product is targeted to the mitochondria and accounts for 2% of p43 expressed in human cells. The cytoplasmic and mitochondrial species of p43 are produced from the same mRNA by a mechanism of leaky scanning of the AUG codon at position -27, which is in an unfavorable sequence context for translation initiation. The finding that a mitochondrial species of p43 exists in human cells further exemplifies the multifaceted implications of p43 and opens new perspectives for the understanding of the role of p43 in the apoptotic cell.

Dynamic Organization of Aminoacyl-tRNA Synthetase Complexes in the Cytoplasm of Human Cells.

The localization in space and in time of proteins within the cytoplasm of eukaryotic cells is a central question of the cellular compartmentalization of metabolic pathways. The assembly of proteins within stable or transient complexes plays an essential role in this process. Here, we examined the subcellular localization of the multi-aminoacyl-tRNA synthetase complex in human cells. The sequestration of its components within the cytoplasm rests on the presence of the eukaryotic-specific polypeptide extensions that characterize the human enzymes, as compared with their prokaryotic counterparts. The cellular mobility of several synthetases, assessed by measuring fluorescence recovery after photobleaching, suggested that they are not freely diffusible within the cytoplasm. Several of these enzymes, isolated by tandem affinity purification, were copurified with ribosomal proteins and actin. The capacity of aminoacyl-tRNA synthetases to interact with polyribosomes and with the actin cytoskeleton impacts their subcellular localization and mobility. Our observations have conceptual implications for understanding how translation machinery is organized in vivo.

Dissection of the structural organization of the aminoacyl-tRNA synthetase complex.

The spatio-temporal organization of proteins within the cytoplasm of eukaryotic cells rests in part on the assembly of stable and transient multiprotein complexes. Here we examined the assembly of the multiaminoacyl-tRNA synthetase complex (MARS) in human cells. This complex contains nine aminoacyl-tRNA synthetases and three auxiliary proteins and is a hallmark of metazoan species. Isolation of the complexes has been performed by tandem affinity purification from human cells in culture. To understand the rules of assembly of this particle, expression of the three nonsynthetase components of MARS, p18, p38, and p43, was blocked by stable small interfering RNA silencing. The lack of these components was not lethal for the cells, but cell growth was slightly reduced. The residual complexes that could form in vivo in the absence of the auxiliary proteins were isolated by tandem affinity purification. From the repertoire of the subcomplexes that could be isolated, a comprehensive map of protein-protein interactions mediating complex assembly is deduced. The data are consistent with a structural role of the three nonsynthetase components of MARS, with p38 connecting two subcomplexes that may form in the absence of p38.

Inhibition of NF-kappaB activation with designed ankyrin-repeat proteins targeting the ubiquitin-binding/oligomerization domain of NEMO.

The link between the NF-kappaB signal transduction pathway and cancer is now well established. Inhibiting this pathway is therefore a promising approach in the treatment of certain cancers through a pro-apoptotic effect in malignant cells. Owing to its central role in the pathway, the IkappaB kinase (IKK) complex is a privileged target for designing inhibitors. Previously, we showed that oligomerization of NEMO is necessary for IKK activation and defined a minimal oligomerization domain (CC2-LZ) for NEMO, and we developed NEMO peptides inhibiting NF-kappaB activation at the level of the IKK complex. To improve the low-affinity inhibitors, we used ribosome display to select small and stable proteins with high affinity against the individual CC2-LZ because the entire NEMO protein is poorly soluble. Several binders with affinities in the low nanomolar range were obtained. When expressed in human cells, some of the selected molecules, despite their partial degradation, inhibited TNF-alpha-mediated NF-kappaB activation while having no effect on the basal activity. Controls with a naive library member or null plasmid had no effect. Furthermore, we could show that this NF-kappaB inhibition occurs through a specific interaction between the binders and the endogenous NEMO, resulting in decreased IKK activation. These results indicate that in vitro selections with the NEMO subdomain alone as a target may be sufficient to lead to interesting compounds that are able to inhibit NF-kappaB activation.

Role of HIV-1 Vpr-induced apoptosis on the release of mitochondrial lysyl-tRNA synthetase.

Mitochondrial lysyl-tRNA synthetase (LysRS) is thought to be involved in the specific packaging of tRNA(3)(Lys) into HIV-1 viral particles. The HIV-1 auxiliary viral protein Vpr is an apoptogenic protein that affects the integrity of the mitochondrial membrane and has also been reported to interact with LysRS. In the present study, we show that HIV-1 Vpr expressed in E. coli and purified to homogeneity does not interact specifically with LysRS and does not impact its aminoacylation activity. However, we also show that the mitochondrial localization of LysRS in HeLa cells is altered after addition of Vpr in the culture medium. These results suggest that HIV-1 Vpr fulfills an essential role in the process of packaging of mitochondrial LysRS.

Characterization of p43(ARF), a derivative of the p43 component of multiaminoacyl-tRNA synthetase complex released during apoptosis.

In human, nine aminoacyl tRNA synthetases are associated with the three auxiliary proteins, p18, p38, and p43, to form a stable multiprotein complex. The p43 component, which has a potent tRNA binding capacity, is associated to the complex via its N-terminal moiety. This protein is also the precursor of the endothelial monocyte-activating polypeptide II (p43(EMAPII), corresponding to the C-terminal moiety of p43), a cytokine generated during apoptosis. Here we examined the cellular pathway that, starting from the p43 subunit of the complex, leads to this extracellular cytokine. We identified a new intermediate in this pathway, named p43(ARF) for Apoptosis-released Factor. This intermediate is produced in cellulo by proteolytic cleavage of endogenous p43 and is rapidly recovered in the culture medium. This p43 derivative was purified from the medium of human U937 cells subjected to serum starvation. It contains 40 additional N-terminal amino acid residues as compared with the cytokine p43(EMAPII) and may be generated by a member of the matrix metalloproteinase family. Recombinant p43(ARF) is a monomer in solution and binds tRNA with a Kd of approximately 6 nM, 30-fold lower than that of p43. Highly purified p43(ARF) or p43(EMAPII) do not stimulate the expression of E-selectin by human umbilical vein endothelial cells. Our results suggest that the cleavage of p43 and its cellular delocalization, and thus the release of this tRNA binding subunit from the complex, is one of the molecular mechanisms leading to the shut down of protein synthesis in apoptosis.

Viral hijacking of mitochondrial lysyl-tRNA synthetase.

The primer for reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome is tRNA3(Lys). During assembly of HIV-1 particles, tRNA3(Lys) is taken up from the host cell along with lysyl-tRNA synthetase (LysRS), the tRNA binding protein that specifically aminoacylates the different tRNA(Lys) isoacceptors. In humans, the cytoplasmic and mitochondrial species of LysRS are encoded by a single gene by means of alternative splicing. Here, we show that polyclonal antibodies directed to the full-length cytoplasmic enzyme equally recognized the two enzyme species. We raised antibodies against synthetic peptides that allowed discrimination between the two enzymes and found that mitochondrial LysRS is the only cellular source of LysRS detected in the virions. These results open new routes for understanding the molecular mechanisms involved in the specific packaging of tRNA3(Lys) into viral particles.

Transition metal ions significantly decrease phospholipase C activity degrading phosphatidylinositol-4,5-bisphosphate in the brain cortex.

Highly reactive transition metals, such as copper and iron play an obligatory role in generating of reactive oxygen species (ROS). Many neurodegenerative diseases including Alzheimer's disease (AD) and Parkinson's disease (PD) show increased accumulation of these metals. Phosphoinositide metabolism is altered in neurodegenerative diseases. In the present study, we examined the effect of CuSO(4) and FeCl(2) on phospholipase C (PLC) activity degrading phosphatidylinositol-4,5-bisphosphate (PIP(2)) and phosphatidylinositol (PI) in synaptic plasma membranes (SPM) from the rat brain cortex. We report that 25 microM CuSO(4) and FeCl(2) decreased PIP(2)-PLC activity by 60% and 75%, respectively. However, both compounds had no effect on PI-PLC activity. These data indicated that exclusively PIP(2)-PLC is sensitive to transition metal ions. We suggest that chelators of these metals may protect brain against alteration of phosphoinositide metabolism and might be beneficial in the treatment of neurodegenerative diseases.

The N-terminal domain of mammalian Lysyl-tRNA synthetase is a functional tRNA-binding domain.

Lysyl-tRNA synthetase from higher eukaryotes possesses a lysine-rich N-terminal polypeptide extension appended to a classical prokaryotic-like LysRS domain. Band shift analysis showed that this extra domain provides LysRS with nonspecific tRNA binding properties. A N-terminally truncated derivative of LysRS, LysRS-DeltaN, displayed a 100-fold lower apparent affinity for tRNA(3)Lys and a 3-fold increase in K(m) for tRNA(3)Lys in the aminoacylation reaction, as compared with the native enzyme. The isolated N-domain of LysRS also displayed weak affinity for tRNA, suggesting that the catalytic and N-domains of LysRS act synergistically to provide a high affinity binding site for tRNA. A more detailed analysis revealed that LysRS binds and specifically aminoacylates an RNA minihelix mimicking the amino acid acceptor stem-loop structure of tRNA(3)Lys, whereas LysRS-DeltaN did not. As a consequence, merging an additional RNA-binding domain into a bacterial-like LysRS increases the catalytic efficiency of the enzyme, especially at the low concentration of deacylated tRNA prevailing in vivo. Our results provide new insights into tRNA(Lys) channeling in eukaryotic cells and shed new light on the possible requirement of native LysRS for triggering tRNA(3)Lys packaging into human immunodeficiency virus, type 1 viral particles.