Effects of barbiturate anesthetics on calcium-induced marine sponge cell aggregation

1. Four barbiturates (sodium salts), thiopental, methitural, methohexital and pentobarbital, were tested for their ability to inhibit calcium-induced aggregation of single-cell suspensions of the marine sponge Microciona prolifera.2. Pentobarbital significantly inhibited aggregation at all concentrations tested whereas there was no significant inhibition by the others and significant potentiation was observed at some concentrations.3. These responses were similar to those of human platelets, previously reported. The possible role of calcium is discussed.     

Component analysis of the urushiol content of poison ivy and poison oak

The partial separation and quantitation of the components of the urushiol fraction of poison ivy and poison oak are discussed. The urushiol fraction of poison ivy is primarily composed of C₁₅ side-chain catechol, while the urushiol extract of poison oak is principally the C₁₇ homolog. The presence of a C₁₇ homolog in ivy and a C₁₅ homolog in poison oak urushiol is also detected. Each of these catechol derivatives contain a mixture of congeners which are partially separated by the GLC system used. In each case the tri-olefinic component occurs in greatest abundance and the mono-olefinic congener is least abundant; no saturated material was detected. The compounds were analyzed as trimethylsilyl derivatives and a qualitative analysis was accomplished by GC-MS.     

Stimulus-response coupling in marine sponge cell aggregation: lipid metabolism and the function of exogenously added arachidonic and docosahexaenoic acids

Cells of the marine sponge, Microciona prolifera, the most ancient of the animal cells which clump on recognition, resemble neutrophils and platelets in undergoing stimulus-response coupling when exposed to Ca2+ ionophores and phorbol esters. We have studied lipid content and remodelling in sponge cells by thin-layer, gas-liquid, and high-performance liquid chromatography (HPLC) analyses supplemented by ultraviolet and mass spectroscopy. Phosphatidylcholine (PC) (35.6%), phosphatidylethanolamine (PE) (27.4%) and phosphatidylserine (PS) (21.4%) constituted the bulk of phospholipids detected. The major fatty acids were all polyenoic; 22:6 (22%), 26:2 (17%) and 26:3 (15%). Arachidonic acid (20:4), present as 2.7% of total phospholipid, and docosahexanoic acid (22:6) were found to elicit aggregation of sponge cells when added (10 microM) in synergy with ionomycin (1 microM), resembling in their effects those of phorbol esters (but not phorbol) and 1-oleyl-2-acetylglycerol (OAG). Moreover, 20:4 and 22:6, as well as phorbol ester and OAG, overcame the block to aggregation imposed by colchicine and vinblastine. Kinetic studies of lipid remodelling showed that aggregating cells diverted [14C]22:6 or [14C]20:4 from triacylglycerol into diacylglycerol and phospholipids; appearance of label in phosphatidic acid and phosphatidylinositol (PI) anteceded labeling of phosphatidylcholine. In unstimulated cells, [14C]22:6 was rapidly incorporated into phosphatidylcholine with little accumulation in phosphatidate. Although 22:6 and 20:4 resembled OAG and phorbol esters in overcoming the effects of colchicine and vinblastine (which had no effects on overall lipid metabolism), they did not reverse the block to aggregation of nordihydroguaiaretic acid (NDGA) (which inhibited lipid metabolism). Under none of these circumstances was 22:6 or 20:4 converted to cyclooxygenase or lipoxygenase products in the course of aggregation: all labeled acyl groups remained present as unmodified fatty acids on alkaline hydrolysis. These data not only extend the observations of Muller et al. (J. Biol. Chem. 262 (1987) 9850-9858) on the role of phosphoinositides and C kinase in marine sponge cell aggregation, but also demonstrate that sponges form diacylglycerols in the process. We suggest that exogenous 22:6 and 20:4 (like phorbol esters or OAG) can substitute for endogenous diacylglycerol in the activation of protein kinase C.     

Intestinal secretory cell ER stress and inflammation

Data from animal models and human inflammatory bowel diseases have implicated the ER (endoplasmic reticulum) stress pathway in intestinal inflammation. We have characterized the development of inflammation in Winnie mice in which ER stress arises due to a single missense mutation in the MUC2 mucin produced by intestinal goblet cells. This model has allowed us to explore the genesis of inflammation ensuing from a single gene polymorphism affecting secretory cells. In these mice, a proportion of MUC2 misfolds during biosynthesis, leading to ER stress and activation of the unfolded protein response. Winnie mice develop spontaneous complex progressive inflammation that is most severe in the distal colon. Inflammation involves TH1, TH2 and TH17 T-cells, with a progressive development of a TH17-dominated response, but also involves innate immunity, in a pattern not dissimilar to human colitis. Experimental inhibition of tolerance in this model severely exacerbates colitis, demonstrating active effective suppression of inflammation. Even though the misfolding of MUC2 is a consequence of an inherited mutation, as inflammation develops, the molecular markers of ER stress increase further and goblet cell pathology becomes worse, suggesting that inflammation itself exacerbates ER stress.     

Role of aggregation factor and cell type in sponge cell adhesion.

A pair of sponge species, Microciona prolifera and Halichondria bowerbanki, which lack mutual species specificity in their aggregation "factor", are useful in establishing the mechanisms of action of these factors. These sponges were dissociated both mechanically, which leaves the factor on the cell surface, and by Humphrey's (1963) method, which isolates the factor from the cells. The adhesive specificities which arose, in the various combinations tested, point to an intercellular factor bridge consisting of a single symmetrical unit. An analysis of most other workers' results is consistent with this interpretation. However, MacLennan and Dodd's (1967) results using other species would require a bridge consisting of two or more asymmetrical units. Differences were found in the specificity of adhesion of various types of cells within a single species. This presents a heretofore unconsidered problem in assesing the adhesive factor's mechanism of action. Three structurally distinct cell types were separated from a suspension of dissociated Microciona cells by velocity sedimentation. These cells differ greatly in adhesiveness. The differences in adhesion are correlated with numbers and positions of cells incorporated into aggregates. Such differences are considered in explaining the mechanism of action of the factors.     

Two cell surface proteins bind the sponge Microciona prolifera aggregation factor

Two extracellular matrix cell surface proteins which bind the proteoglycan-like aggregation factor from the marine sponge Microciona prolifera (MAF) and which may function as physiological receptors for MAF were identified and characterized for the first time. By probing nitrocellulose blots of nonreducing sodium dodecyl sulfate gels containing whole sponge cell protein with iodinated MAF, a 210- and a 68-kDa protein, which have native molecular masses of approximately 200-400 and 70 kDa, were identified. MAF binding to blots is species-specific. It is also sensitive to reduction and is completely abolished by pretreatment of live cells with proteases, as was cellular aggregation, indicating that the 210- and 68-kDa proteins may be located on the cell surface. The additional observations that the 68 kDa is an endoglycosidase F-sensitive glycoprotein and that antisera against whole sponge cells or membranes can immunoprecipitate the 210 kDa when prebound to intact cells are consistent with a cell surface location. Both proteins can be isolated from sponge cell membranes and from the sponge skeleton (insoluble extracellular matrix), but the 210-kDa MAF-binding protein can also be found in the soluble extracellular matrix (buffer washes of cells and skeleton) as well. A third MAF-binding protein of molecular mass 95 kDa was also found in the sponge extracellular matrix but rarely on cells. Both of the cell-associated 210- and 68-kDa proteins are nonintegral membrane proteins, based on Triton X-114 phase separation, flotation of liposomes containing sponge membrane lysates, and their extraction from membranes by buffer washes. Both proteins bind MAF affinity resins, indicating that they each exhibit a moderate affinity for MAF under native conditions. They can also be separated from each other and from the bulk of the protein in an octylpolyoxyethylene extract of membranes by fast protein liquid chromatography Mono Q anion exchange chromatography, as assessed by native dot blot and denaturing Western blot assays. Although neither protein bound to heparin, gelatin, hexosamine, or uronic acid-Sepharose resins, their affinity for an invertebrate proteoglycan, their roles in sponge cell adhesion, and their peripheral membrane protein natures suggest that they may represent early invertebrate analogs of cell-associated vertebrate extracellular matrix adhesion proteins, such as fibronectin or vitronectin, or else an entirely novel set of cell adhesion molecules.     

From the test tube to the cell: exploring the folding and aggregation of a beta-clam protein

A crucial challenge in present biomedical research is the elucidation of how fundamental processes like protein folding and aggregation occur in the complex environment of the cell. Many new physico-chemical factors like crowding and confinement must be considered, and immense technical hurdles must be overcome in order to explore these processes in vivo. Understanding protein misfolding and aggregation diseases and developing therapeutic strategies to these diseases demand that we gain mechanistic insight into behaviors and misbehaviors of proteins as they fold in vivo. We have developed a fluorescence approach using FlAsH labeling to study the thermodynamics of folding of a model beta-rich protein, cellular retinoic acid binding protein (CRABP) in Escherichia coli cells. The labeling approach has also enabled us to follow aggregation of a modified version of CRABP and chimeras between CRABP and huntingtin exon 1 with its glutamine repeat tract. In this article, we review our recent results using FlAsH labeling to study in-vivo folding and present new observations that hint at fundamental differences between the thermodynamics and kinetics of protein folding in vivo and in vitro.     

Intestinal epithelial cell signalling and chronic inflammation: From the proteome to specific molecular mechanisms

Advancing knowledge regarding the cellular mechanisms of intestinal inflammation has led to a better understanding of the disease pathology in patients with inflammatory bowel disease (IBD) including Crohn's disease and ulcerative colitis. It has become clear from numerous studies that enteric bacteria are a critical component in the development and prevention/treatment of chronic intestinal inflammation. An emerging new paradigm suggests that changes in the homeostasis of bacteria- and host-derived signal transduction at the intestinal epithelial cell (IEC) level may lead to a break in barrier function and the development of adaptive immune disturbances. The functional loss of anti-inflammatory host-derived signals in the gut including the immunosuppressive cytokines Interleukin 10 (IL-10) and transforming growth factor (TGF)-beta are of high relevance to the pathogenesis of IBD. The development of analytical tools including two-dimensional (2D) high-resolution protein separation techniques and peptide mass fingerprinting via high-sensitivity mass-spectrometers (MS) allows the quantitative assessment of protein expression changes in disease-relevant cell types. By using these advanced methods, the characterization of the epithelial cell proteome from murine models of experimental colitis and human IBD patients identified novel disease-related mechanisms with respect to the regulation of the glucose-regulated endoplasmic reticulum stress response protein 78 (grp-78). In conclusion, the identification and functional analysis of differentially expressed proteins in purified intestinal target cell types will help to add important insights to the understanding of the molecular pathogenesis of these immune-mediated chronic intestinal disorders.     

Identification and further characterization of the specific cell binding fragment from sponge aggregation factor

Monoclonal antibodies (McAbs) were raised against the aggregation factor (AF) from the marine sponge Geodia cydonium. Two clones were identified that secrete McAbs against the cell binding protein of the AF complex. Fab fragments of McAbs: 5D2-D11 completely abolished the activity of the AF to form secondary aggregates from single cells. The McAbs were determined to react with the AF in vitro; this interaction was prevented by addition of the aggregation receptor, isolated and purified from the same species. After dissociation of the AF by sodium dodecyl sulfate and 2-mercaptoethanol, followed by electrophoretical fractionation, a 47-kD protein was identified by immunoblotting which interacted with the McAbs: 5D2-D11. During this dissociation procedure, the sunburst structure of the AF was destroyed. In a second approach, the 47-kD protein was isolated by immunoprecipitation; 12 molecules of this protein species were calculated to be associated with the intact AF particle. The 47-kD AF fragment bound to dissociated Geodia cells with a high affinity (Ka of 7 X 10(8) M-1) even in the absence of Ca++ ions; the number of binding sites was approximately 4 X 10(6)/cell. This interaction was prevented by addition of the aggregation receptor to the 47-kD protein in the homologous cell system. Moreover, it was established that this binding occurs species-specifically. The 47-kD fragment of the AF was localized only extracellularly by indirect immunofluorescence staining in cryostat slices. These data suggest that the 47-kD protein is the cell binding molecule of the AF from Geodia.     

From cell senescence to age-related diseases: differential mechanisms of action of senescence-associated secretory phenotypes

Cellular senescence is a process by which cells enter a state of permanent cell cycle arrest. It is commonly believed to underlie organismal aging and age-associated diseases. However, the mechanism by which cellular senescence contributes to aging and age-associated pathologies remains unclear. Recent studies showed that senescent cells exert detrimental effects on the tissue microenvironment, generating pathological facilitators or aggravators. The most significant environmental effector resulting from senescent cells is the senescence-associated secretory phenotype (SASP), which is constituted by a strikingly increased expression and secretion of diverse pro-inflammatory cytokines. Careful investigation into the components of SASPs and their mechanism of action, may improve our understanding of the pathological backgrounds of age-associated diseases. In this review, we focus on the differential expression of SASP-related genes, in addition to SASP components, during the progress of senescence. We also provide a perspective on the possible action mechanisms of SASP components, and potential contributions of SASP-expressing senescent cells, to age-associated pathologies. [BMB Reports 2015; 48(10): 549-558]     

Involvement of a highly polyvalent glycan in the cell-binding of the aggregation factor from the marine sponge Microciona prolifera

A proteoglycan-like aggregation factor from the marine sponge Microciona prolifera (MAF) mediates cell-cell recognition via a cell-binding and a self-association domain. After repetitive and prolonged treatment of MAF with glycopeptide-N-glycosidase (PNGase) the specific binding of MAF to homotypic cells was decreased by 72%. Polyacrylamide gel electrophoresis and gel filtration analysis of such PNGase digests showed that: 1) the enzyme released a single glycan type of Mr = 6 X 10(3) (G-6) from MAF, 2) 1 mole of MAF contains at least 830 moles of N-linked chains of G-6 glycan. The correlation between the loss of the binding activity of MAF and the extent of the release of the repetitive G-6 polysaccharide strongly suggests its involvement in MAF-cell association via highly polyvalent interactions.     

Identification and isolation of the primary aggregation factor from the cell membrane of the sponge Geodia cydonium

Summary The primary aggregation factor (pAF) of sponge cells is a glycoprotein that is firmly associated with the cell membrane. Polyspecific antibodies (anti-GM) prepared from sera raised against membranes of cells from the siliceous sponge Geodia cydonium were found to inhibit initial aggregation of homologous cells. The inhibition of aggregation, caused by anti-GM was neutralized by pAF. The pAF had been successfully solubilized and enriched by affinity chromatography, gel filtration and density gradient centrifugation, if checked by polyacrylamide gel electrophoresis in the presence of urea. The M_r of the native pAF was approximately 40 000 as estimated by gel filtration; under denaturing conditions three protein species (M_r: 16 500, 15 500 and 13 500) were identified in the pAF preparation. The pAF was precipitable by Ca⁺⁺ and did not cross-react with antisera against homologous purified secondary aggregation factor and lectin. It is mainly composed of protein (48.0%) and carbohydrate (50.2%). The isolated pAF restored the aggregation potency not only of factor-depleted Geodia cells but also of cells from other Demospongiae. However, the pAF displayed no aggregation enhancing effect on urea-treated cells from species belonging to the Calcispongiae or Hexactinellida. We hypothesize that in contrast to the secondary aggregation, the initial aggregation of Geodia cells is mediated by the one-component system, the bivalent and bifunctional pAF.