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.     

Interaction of gangliosides with B cells in splenic fragment cultures

The effect of gangliosides upon murine adult B cells at the single precursor cell level was examined using the splenic focus assay. Adult B cells were stimulated in in vitro organ fragment culture by a hapten-modified carrier protein in the presence of an excess of carrier-primed T cells. The addition of a potential tolerogen in the form of antigen coupled to a carrier not previously presented to the T cells resulted in a temporary unresponsiveness of the onset of antibody production in adult B cells, but not a permanent state of tolerance. This delay could be eliminated by the addition of purified murine gangliosides during the presentation of the hapten coupled to the unrecognized carrier. The ganglioside preparation was fractionated by ion-exchange chromatography and the active fraction was found to be a disialoganglioside. These results suggest that the ganglioside may interfere with membrane receptor-related events occurring during or after antigen binding or cross-linking to responding B cells.     

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.     

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.     

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.      

Phosphorylation of CD45 by casein kinase 2. Modulation of activity and mutational analysis

CD45 is a receptor-type protein-tyrosine phosphatase (PTP) that is required for antigen-specific stimulation and proliferation in lymphocytes. This study was designed to determine the nature of specific kinases in lymphocytes that phosphorylate CD45 and to determine the effect of phosphorylation on CD45 PTP activity. A major cytoplasmic lymphocyte kinase that phosphorylated CD45 was identified as casein kinase 2 (CK2) by use of an in-gel kinase assay in combination with immunoprecipitation, immunodepletion, and specific inhibition. Mutational analysis of CK2 consensus sites showed that the target for CK2 was in an acidic insert of 19 amino acids in the D2 domain, and Ser to Ala mutations at amino acids 965, 968, 969, and 973 abrogated CK2 phosphorylation of CD45. CK2 phosphorylation increased CD45 activity 3-fold toward phosphorylated myelin basic protein, and this increase was reversible by PP2A treatment. Mutation of Ser to Glu at the CK2 sites had the same effect as phosphorylation and also tripled the Vmax of CD45. CD45 isolated in vivo was highly phosphorylated and could not be phosphorylated by CK2 without prior dephosphorylation with phosphatase PP2A. We conclude that CK2 is a major lymphocyte kinase that is responsible for in vivo phosphorylation of CD45, and phosphorylation at specific CK2 sites regulates CD45 PTP activity.     

GENETIC CONSEQUENCES OF RARITY IN ASTER FURCATUS (ASTERACEAE), A THREATENED,SELF-INCOMPATIBLE PLANT

Aster furcatus is a rare, self-incompatible plant with fewer than 50 known populations throughout its range. We verified self-incompatibility in A. furcatus by conducting experimental self- and cross-pollinations and by examining seed set in a small population comprised of a single clonal genet. We examined variation at 22 electrophoretic loci in 23 populations of A. furcatus from across its range in Wisconsin, Illinois, Indiana, and Missouri. Except for two rare alleles found in single individuals in three populations, all loci but one of those examined were fixed for single alleles. The only variable locus (triosephosphate isomerase, TPI-1) tended to exhibit genotype frequencies in Hardy-Weinberg equilibrium or with a slight excess of heterozygotes. Although overall gene diversity was extremely low, TPI genotype frequencies were indicative of an outcrossing plant. We examined the subpopulation genetic structure among clonal plants within one Wisconsin population in greater detail. F statistics indicated that much of the genetic variation at the polymorphic TPI locus was due to differentiation among populations. We discuss the implications of self-incompatibility and low levels of genetic variation for the evolution and conservation of Aster furcatus and other rare plants with similar breeding systems.     

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     

Differential Orientation of 10T1/2 Mesenchymal Cells on Non-Uniform Stretch Environments

Non-uniform stress and strain fields are prevalent in many tissues in vivo, and often exacerbated by disease or injury. These mechanical gradients potentially play a role in contributing to pathological conditions, presenting a need for experimental tools to allow investigation of cell behavior within non-uniformly stimulated environments. Herein, we employ two in vitro cell-stretching devices (one previously published; one newly presented) capable of subjecting cells to cyclic, non-uniform stretches upon the surface of either a circular elastomeric membrane or a cylindrical PDMS tube. After 24 hours of cyclic stretch, 10T1/2 cells on both devices showed marked changes in long-axis orientation, with tendencies to align parallel to the direction of minimal deformation. The degree of this response varied depending on location within the stretch gradients. These results demonstrated the feasibility of conducting cell mechanobiology investigations with the two novel devices, while also highlighting the experimental capabilities of non-uniform mechanical environments for these types of studies. Such capabilities include robust data collection for developing mechanobiological dose-response curves, signal threshold identification, and potential spatial targeting for drug delivery.