Transition of healthy to diseased synovial tissue in rheumatoid arthritis is associated with gain of mesenchymal/fibrotic characteristics

The healthy synovial lining layer consists of a single cell layer that regulates the transport between the joint cavity and the surrounding tissue. It has been suggested that abnormalities such as somatic mutations in the p53 tumor-suppressor gene contribute to synovial hyperplasia and invasion in rheumatoid arthritis (RA). In this study, expression of epithelial markers on healthy and diseased synovial lining tissue was examined. In addition, we investigated whether a regulated process, resembling epithelial to mesenchymal transition (EMT)/fibrosis, could be responsible for the altered phenotype of the synovial lining layer in RA. Synovial tissue from healthy subjects and RA patients was obtained during arthroscopy. To detect signs of EMT, expression of E-cadherin (epithelial marker), collagen type IV (indicator of the presence of a basement membrane) and alpha-smooth muscle actin (alpha-sma; a myofibroblast marker) was investigated on frozen tissue sections using immunohistochemistry. Fibroblast-like synoviocytes (FLSs) from healthy subjects were isolated and subjected to stimulation with synovial fluid (SF) from two RA patients and to transforming growth factor (TGF)-beta. To detect whether EMT/fibrotic markers were increased, expression of collagen type I, alpha-sma and telopeptide lysylhydroxylase (TLH) was measured by real time PCR. Expression of E-cadherin and collagen type IV was found in healthy and arthritic synovial tissue. Expression of alpha-sma was only found in the synovial lining layer of RA patients. Stimulation of healthy FLSs with SF resulted in an upregulation of alpha-sma and TLH mRNA. Collagen type I and TLH mRNA were upregulated after stimulation with TGF-beta. Addition of bone morphogenetic protein (BMP)-7 to healthy FLS stimulated with SF inhibited the expression of alpha-sma mRNA. The finding that E-cadherin and collagen type IV are expressed in the lining layer of healthy and arthritic synovium indicates that these lining cells display an epithelial-like phenotype. In addition, the presence of alpha-sma in the synovial lining layer of RA patients and induction of fibrotic markers in healthy FLSs by SF from RA patients indicate that a regulated process comparable to EMT might cause the alteration in phenotype of RA FLSs. Therefore, BMP-7 may represent a promising agent to counteract the transition imposed on synoviocytes in the RA joint.     

Characterization of a maltose transport system in Clostridium acetobutylicum ATCC 824

The utilization of maltose by Clostridium acetobutylicum ATCC 824 was investigated. Glucose was used preferentially to maltose, when both substrates were present in the medium. Maltose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity was detected in extracts prepared from cultures grown on maltose, but not glucose or sucrose, as the sole carbon source. Extract fractionation and PTS reconstitution experiments revealed that the specificity for maltose is contained entirely within the membrane in this organism. A putative gene system for the maltose PTS was identified (from the C. acetobutylicum ATCC 824 genome sequence), encoding an enzyme II^Mal and a maltose 6-phosphate hydrolase. Journal of Industrial Microbiology & Biotechnology (2001) 27, 298–306. Received 12 September 2000/ Accepted in revised form 30 November 2000     

Tumor Necrosis Factor-α +489G/A gene polymorphism is associated with chronic obstructive pulmonary disease

Pulmonary surfactant is a complex mixture of phospholipids and proteins, which is present in the alveolar lining fluid and is essential for normal lung function. Alterations in surfactant composition have been reported in several interstitial lung diseases (ILDs). Furthermore, a mutation in the surfactant protein C gene that results in complete absence of the protein has been shown to be associated with familial ILD. The role of surfactant in lung disease is therefore drawing increasing attention following the elucidation of the genetic basis underlying its surface expression and the proof of surfactant abnormalities in ILD.     

A structural role for Asp83 in the photoactivation of rhodopsin

Asp83 is a highly conserved residue in the second transmembrane domain of visual pigments and many members of other G protein-coupled receptor subfamilies. Upon illumination, the rod visual pigment rhodopsin proceeds through various intermediate states (Batho<-->BSI<-->Lumi<-->Meta I<-->Meta II). Meta II represents the active state of rhodopsin, which binds and activates the G protein transducin. Evidence has been presented that Asp83 participates in the formation of Meta II and undergoes a change in H-bonding. To investigate whether this role of Asp83 requires its proton-donating capacity and/or its H-bonding capability, we constructed the mutants D83C and D83N. Both mutants appear to effectively activate transducin, indicating that Asp83 is not essential for signal transduction. Differential effects of the mutations D83C and D83N are observed in the spectral properties and the pH sensitivity of the Meta I-->Meta II transition. In general, D83C behaves much more like wild-type than D83N. We conclude that the structural role of Asp83 also involves the acidic nature of its carboxyl group. In addition, the participation in Meta II formation of Cys83 in D83C manifests itself as a change in the vibrational properties of the sulfhydryl group, demonstrating that the -SH group can be used as a non-invasive probe for local structural changes.     

Differential direct effects of cyclo-oxygenase-1/2 inhibition on proteoglycan turnover of human osteoarthritic cartilage: an <Emphasis Type="Italic">in vitro</Emphasis>study

Treatment of osteoarthritis (OA) with nonsteroidal anti-inflammatory drugs (NSAIDs) diminishes inflammation along with mediators of cartilage destruction. However, NSAIDs may exert adverse direct effects on cartilage, particularly if treatment is prolonged. We therefore compared the direct effects of indomethacin, naproxen, aceclofenac and celecoxib on matrix turnover in human OA cartilage tissue. Human clinically defined OA cartilage from five different donors was exposed for 7 days in culture to indomethacin, naproxen, aceclofenac and celecoxib – agents chosen based on their cyclo-oxygenase (COX)-2 selectivity. As a control, SC-560 (a selective COX-1 inhibitor) was used. Changes in cartilage proteoglycan turnover and prostaglandin E₂ production were determined. OA cartilage exhibited characteristic proteoglycan turnover. Indomethacin further inhibited proteoglycan synthesis; no significant effect of indomethacin on proteoglycan release was found, and proteoglycan content tended to decrease. Naproxen treatment was not associated with changes in any parameter. In contrast, aceclofenac and, prominently, celecoxib had beneficial effects on OA cartilage. Both were associated with increased proteoglycan synthesis and normalized release. Importantly, both NSAIDs improved proteoglycan content. Inhibition of prostaglandin E₂ production indirectly showed that all NSAIDs inhibited COX, with the more COX-2 specific agents having more pronounced effects. Selective COX-1 inhibition resulted in adverse effects on all parameters, and prostaglandin E₂ production was only mildly inhibited. NSAIDs with low COX-2/COX-1 selectivity exhibit adverse direct effects on OA cartilage, whereas high COX-2/COX-1 selective NSAIDs did not show such effects and might even have cartilage reparative properties.     

Large-scale overproduction, functional purification and ligand affinities of the His-tagged human histamine H1 receptor.

This report describes an efficient strategy for amplified functional purification of the human H1 receptor after heterologous expression in Sf9 cells. The cDNA encoding a C-terminally histidine-tagged (10xHis) human histamine H1 receptor was used to generate recombinant baculovirus in a Spodoptera frugiperda-derived cell line (IPLB-Sf9). As judged from its ligand affinity profile, functional receptor could be expressed at high levels (30-40 pmol per 10(6) cells). Rapid proteolysis in the cell culture led to limited fragmentation, without loss of ligand binding, but could be efficiently suppressed by including the protease inhibitor leupeptin during cell culture and all subsequent manipulations. Effective solubilization of functional receptor with optimal recovery and stability required the use of dodecylmaltoside as a detergent in the presence of a high concentration of NaCl and of a suitable inverse agonist. Efficient purification of solubilized receptor could be achieved by affinity chromatography over nickel(II) nitrilotriacetic acid resin. Functional membrane reconstitution of purified H1 receptor was accomplished in mixed soybean lipids (asolectin). The final proteoliposomic H1 receptor preparation has a purity greater than 90% on a protein basis and displays a ligand binding affinity profile very similar to the untagged receptor expressed in COS-7 cells. In conclusion, we are able to produce pharmacologically viable H1 receptor in a stable membrane environment allowing economic large-batch operation. This opens the way to detailed studies of structure-function relationships of this medically and biologically important receptor protein by 3D-crystallography, FT-IR spectroscopy and solid-state NMR spectroscopy.     

A molecular phylogeny for aplocheiloid fishes (Atherinomorpha, Cyprinodontiformes): the role of vicariance and the origins of annualism

Annual aplocheiloid killifish embryos possess a rare ability among vertebrates to enter stages of developmental arrest (diapause) when subjected to adverse environmental conditions. Previous morphological analyses have presented disparate hypotheses regarding the evolution of the intriguing life history associated with this phenomenon. We present a novel hypothesis of aplocheiloid relationships based on 1,009 bp of sequence data from three mitochondrial genes (cytochrome b, 12S rRNA, and 16S rRNA). Phylogenetic analysis using maximum parsimony, neighbor- joining, and maximum likelihood produce strongly congruent topologies. Our data confirm the monophyly of the Neotropical family Rivulidae, while demonstrating a paraphyletic Old World assemblage. The basal sister group position of Indo-Malaysian and Madagascaran taxa relative to a monophyletic South American/African dichotomy strongly indicates the role of vicariance in the diversification of these fishes in spite of their definition as secondary freshwater fish. The distribution of annualism onto this topology implies a single early origin for this suite of characters, prior to the divergence of South American and African taxa. If so, then annualism has since been lost several times during the evolution of genera now residing in permanent aquatic habitats. Paleoclimatic knowledge complements this scenario based on molecular characters.      

Flagellar adhesion in chlamydomonas induces synthesis of two high molecular weight cell surface proteins

Because our previous studies (Snell, W.J., and W.S. Moore, 1980, J. Cell Biol. 84:203- 210) on the mating reaction of chlamydomonas reinhardtii showed that there was an adhesion-induced turnover of proteins whose synthesis is induced during aggregation. Analysis by SDS PAGE and autoradiography showed that proteins of 220,000 M(r) and 165, 000 M(r) (designated A(1) and A(2) respectively) consistently showed a high rate of synthesis only in flagella or flagellar membrane-enriched fractions prepared from aggregating gametes. Since the two proteins were soluble in the non-ionic detergent NP-40 and were removed from intact cells by a brief pronase treatment, it is likely that A(1) and A(2) are membrane proteins expose on the cell surface. A(1) and A(2) were each synthesized by gametes of both mating types (mt(-) and mt(+)) and synthesis of these two proteins could be detected in the normal mating reaction (wild type mt(-) and mt(+)), in mixtures of mt(-) and impotent mt(+) gametes (which could aggregate but not fuse), and in mixtures of gametes of a single mating type with isolated flagella of the opposite mating type. Cells aggregating in tunicamycin, an inhibitor of protein glycosylation, lost their adhesiveness during aggregation and did not synthesize the 220,000 M(r) protein but instead produced a protein (possibly an underglycosylated form of A(1)) of slightly lower mol wt. The 220,000 and 165,000 M(R) proteins appeared to be flagellar proteins and not cell wall proteins because A(1) and A(2) did not co-migrate with previously identified cell wall proteins, and synthesis of the two proteins could not be detected in flagella-less (bald-2) mutant cells. Analysis of the adhesive activity of sucrose gradient fraction of detergent (octyl glucoside)-solubilized flagellar membranes revealed that fractions containing A(1) and A(2) did not have detectable adhesive activity. The possibility remains that A(1) and A(2) are adhesion molecules whose activity could not be measured in the assay we used. Alternatively, the 220,000 and 165,000 M(r) proteins may be inactivated adhesion molecules or else they may be flagellar surface proteins involved only indirectly in the adhesion process.     

Participation of plasma membrane proteins in the formation of tight junction by cultured epithelial cells

Measurements of the transepithelial electrical resistance correlated with freeze-fracture observations have been used to study the process of tight junction formation under various experimental conditions in monolayers of the canine kidney epithelial cell line MDCK. Cells derived from previously confluent cultures and plated immediately after trypsin- EDTA dissociation develop a resistance that reaches its maximum value of several hundred ohms-cm(2) after approximately 24 h and falls to a steady-state value of 80-150 ohms- cm(2) by 48 h. The rise in resistance and the development of tight junctions can be completely and reversibly prevented by the addition of 10 μg/ml cycloheximide at the time of plating, but not when this inhibitor is added more than 10 h after planting. Thus tight junction formation consists of separable synthetic and assembly phases. These two phases can also be dissociated and the requirement for protein synthesis after plating eliminated if, following trypsinization, the cells are maintained in spinner culture for 24 h before plating. The requirement for protein synthesis is restored, however, if cells maintained in spinner culture are treated with trypsin before plating. Actinomycin D prevents development of resistance only in monolayers formed from cells derived from sparse rather than confluent cultures, but new mRNA synthesis is not required if cells obtained from sparse cultures are maintained for 24 h in spinner culture before plating. Once a steady-state resistance has been reached, its maintenance does not require either mRNA or protein synthesis; in fact, inhibition of protein synthesis causes a rise in the resistance over a 30-h period. Following treatments that disrupt the junctions in steady- state monolayers recovery of resistance also does not require protein synthesis. These observations suggest that proteins are involved in tight junction formation. Such proteins, which do not turn over rapidly under steady-state conditions, are destroyed by trypsinization and can be resynthesized in the absence of stable cell-cell or cell-substratum contact. Messenger RNA coding for proteins involved in tight junction formation is stable except when cells are sparsely plated, and can also be synthesized without intercellular contacts or cell-substratum attachment.