Extracellular Binding Protein Research Articles

Growth factor control of bone mass.

Bone formation is determined by the number and function of osteoblasts. Cell number is governed by factors that regulate the replication and differentiation of pre-osteoblasts and factors that regulate osteoblastic cell death. Cell function is controlled by signals acting on the mature osteoblast. Platelet-derived and fibroblast growth factors are bone cell mitogens. Bone morphogenetic proteins (BMPs) and Wnt induce the differentiation of mesenchymal cells toward osteoblasts, and insulin-like growth factor (IGF)-I stimulates the function of mature osteoblasts and prevents their death. The activity of BMP, Wnt, and IGF-I is modulated by extracellular antagonists or binding proteins. Changes in growth factor synthesis and activity may play a role in the pathogenesis of selected forms of osteoporosis, and alterations in the expression or binding of the extracellular antagonists can be associated with changes in bone mass. Current approaches to bone anabolic therapies for osteoporosis include the administration of a growth factor, such as IGF-I, or the neutralization of an antagonist. Ideally, the targeting of an anabolic agent should be specific to bone to preclude non-skeletal unwanted side effects. Clinical trials are needed to determine the long-term effectiveness and safety of novel anabolic agents for the management of osteoporosis.

Quantitative Proteomics Analysis Integrated with Microarray Data Reveals That Extracellular Matrix Proteins, Catenins, and P53 Binding Protein 1 Are Important for Chemotherapy Response in Ovarian Cancers

Chemotherapy with carboplatin and paclitaxel is the standard treatment for ovarian cancer patients. Although most patients initially respond to this treatment, few are cured. Resistance to chemotherapy is the major cause of treatment failure. We applied a quantitative proteomic approach based on ICAT/MS/MS technology to analyze tissues harvested at primary debulking surgery before the initiation of combination chemotherapy in order to identify potential naive or intrinsic chemotherapy response proteins in ovarian cancers. We identified 44 proteins that are overexpressed, and 34 proteins that are underexpressed in the chemosensitive tissue compared to the chemoresistant tissue. The overexpressed proteins identified in the chemoresistant tissue include 10 proteins (25.6%) belonging to the extracellular matrix (ECM), including decorin, versican, basigin (CD147), fibulin-1, extracellular matrix protein 1, biglycan, fibronectin 1, dermatopontin, alpha-cardiac actin (smooth muscle actin), and an EGF-containing fibulin-like extracellular matrix protein 1. Interesting proteins identified as overexpressed in the chemosensitive tissue include gamma-catenin (junction plakoglobin) and delta-catenin, tumor suppressor p53-binding protein 1 (53BP1), insulin-like growth factor-binding protein 2 (IGFBP2), proliferating cell nuclear antigen (PCNA), annexin A11, and 53 kDa selenium binding protein 1. Integrative analysis with expression profiling data of eight chemoresistant tissues and 13 chemosensitive tissues revealed that 16 proteins showed consistent changes at both the protein and the RNA levels. These include P53 binding protein 1, catenin delta 1 and plakoglobin, EGF-containing fibulin-like extracellular matrix protein 1 and voltage-dependent anion-selective channel protein 1. Our results suggest that chemotherapy response may be determined by multiple and complex system properties involving extracellular-matrix, cell adhesion and junction proteins.

The genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiome

Following birth, the breast-fed infant gastrointestinal tract is rapidly colonized by a microbial consortium often dominated by bifidobacteria. Accordingly, the complete genome sequence of Bifidobacterium longum subsp. infantis ATCC15697 reflects a competitive nutrient-utilization strategy targeting milk-borne molecules which lack a nutritive value to the neonate. Several chromosomal loci reflect potential adaptation to the infant host including a 43 kbp cluster encoding catabolic genes, extracellular solute binding proteins and permeases predicted to be active on milk oligosaccharides. An examination of in vivo metabolism has detected the hallmarks of milk oligosaccharide utilization via the central fermentative pathway using metabolomic and proteomic approaches. Finally, conservation of gene clusters in multiple isolates corroborates the genomic mechanism underlying milk utilization for this infant-associated phylotype.

Structure–Function Aspects of PstS in Multi-Drug–Resistant Pseudomonas aeruginosa

The increasing prevalence of multi-drug–resistant (MDR) strains of Pseudomonas aeruginosa among critically ill humans is of significant concern. In the current study, we show that MDR clinical isolates of P. aeruginosa representing three distinct genotypes that display high virulence against intestinal epithelial cells, form novel appendage-like structures on their cell surfaces. These appendages contain PstS, an extracellular phosphate binding protein. Using anti-PstS antibodies, we determined that the PstS-rich appendages in MDR strains are involved in adherence to and disruption of the integrity of cultured intestinal epithelial cell monolayers. The outer surface–expressed PstS protein was also identified to be present in P. aeruginosa MPAO1, although to a lesser degree, and its role in conferring an adhesive and barrier disruptive phenotype against intestinal epithelial cells was confirmed using an isogenic ΔPstS mutant. Formation of the PstS rich appendages was induced during phosphate limitation and completely suppressed in phosphate-rich media. Injection of MDR strains directly into the intestinal tract of surgically injured mice, a known model of phosphate limitation, caused high mortality rates (60%–100%). Repletion of intestinal phosphate in this model completely prevented mortality. Finally, significantly less outer surface PstS was observed in the MPAO1 mutant ΔHxcR thus establishing a role for the alternative type II secretion system Hxc in outer surface PstS expression. Gene expression analysis performed by RT-PCR confirmed this finding and further demonstrated abundant expression of pstS analogous to pa5369, pstS analogous to pa0688/pa14–55410, and hxcX in MDR strains. Taken together, these studies provide evidence that outer surface PstS expression confers a highly virulent phenotype of MDR isolates against the intestinal epithelium that alters their adhesive and barrier disrupting properties against the intestinal epithelium.

Chordin knockdown enhances the osteogenic differentiation of human mesenchymal stem cells

IntroductionBone morphogenetic proteins (BMPs) are critical growth factors in the osteogenic differentiation of progenitor cells during development in embryos and fracture repair in adults. Although recombinant BMPs are in use clinically, their clinical efficiency needs to be improved. The biological activities of BMPs are naturally regulated by extracellular binding proteins. The specific hypotheses tested in this study were as follows: the BMP inhibitor chordin is produced endogenously during the osteogenic differentiation of human mesenchymal stem cells (MSCs); and blockade of the activity of the BMP inhibitor increases the rate of osteogenic differentiation of human MSCs in vitro.MethodsHuman MSCs were derived from bone marrow from an iliac crest aspirate and from patients undergoing hip hemiarthroplasty. The MSCs were induced down the osteogenic pathway using standard osteogenic differentiation media, and expressions of BMP-2 and chordin were determined by gene expression analysis. During osteogenic differentiation, chordin knockdown was induced using RNA interference. Osteogenic differentiation was assessed by measuring the expression of alkaline phosphatase and calcium deposition. The differences in expression of osteogenic makers between groups were compared by analysis of variance, followed by Gabriel post hoc test.ResultsWe demonstrate the expression of BMP-2 and chordin in human MSCs during osteogenic differentiation. Knockdown of chordin by RNA interference in vitro resulted in a significant increase in the expression of the osteogenic marker alkaline phosphatase and the deposition of extracellular mineral, in response to osteogenic stimulation.ConclusionWe conclude that endogenously produced chordin constrains the osteogenic differentiation of human MSCs. The targeting of BMP inhibitors, such as chordin, may provide a novel strategy for enhancing bone regeneration.

Inhibition of Period1 Gene Attenuates the Morphine-Induced ERK-CREB Activation in Frontal Cortex, Hippocampus, and Striatum in Mice

Objective: Recent studies demonstrated that the Period1 gene (Per1) is involved in behavioral alterations induced by addictive drugs. We explored the effects of inhibiting expression in brain of Per1 on morphine conditioned place preference (CPP) and morphine-induced phosphorylation of extracellular signal-regulated kinase (ERK) and cAMP-response element binding protein (CREB) in mice. Methods: During the first three sessions of conditioning, the male mice were intracerebroventricularlly (i.c.v.) injected with vehicle or deoxyribozyme 164 (DRz164) which cleaves per1 mRNA before subcutaneous (s.c.) injection morphine. The control group was given i.c.v. injection vehicle and s.c. injection saline instead of morphine. After testing CPP, mice were sacrificed and phosphorylated ERK and CREB in the frontal cortex, hippocampus, and striatum were examined by immunohistochemistry. Results: Mice pretreated with DRz164 did not acquire morphine CPP. Pretreatment with DRz164 significant attenuated the morphine-induced activation of ERK and CREB in the frontal cortex, hippocampus, and striatum. Conclusions: Our results indicated that per1 plays an important role in morphine reward, and ERK–CREB pathway was involved in the effects of per1. We suggested that per1 gene may be a potential treatment target for drug addition.

Streptococcal pyrogenic exotoxin B antibodies in a mouse model of glomerulonephritis

Streptococcal pyrogenic exotoxin B is an extracellular cysteine protease. Only nephritis-associated strains of group A streptococci secrete this protease and this may be involved in the pathogenesis of post-streptococcal glomerulonephritis. Mice were actively immunized with a recombinant protease inactive exotoxin B mutant or passively immunized with exotoxin B antibody. Characteristics of glomerulonephritis were measured using histology, immunoglobulin deposition, complement activation, cell infiltration, and proteinuria. None of the mice given bovine serum albumin or exotoxin A as controls showed any marked changes. Immunoglobulin deposition, complement activation, and leukocyte infiltration occurred only in the glomeruli of exotoxin B-hyperimmunized mice. One particular anti-exotoxin B monoclonal antibody, 10G, was cross-reactive with kidney endothelial cells and it caused kidney injury and proteinuria when infused into mice. This cross-reactivity may be involved in the pathogenesis of glomerulonephritis following group A streptococcal infection.

The Heme Transfer from the Soluble HasA Hemophore to Its Membrane-bound Receptor HasR Is Driven by Protein-Protein Interaction from a High to a Lower Affinity Binding Site

HasA is an extracellular heme binding protein, and HasR is an outer membrane receptor protein from Serratia marcescens. They are the initial partners of a heme internalization system allowing S. marcescens to scavenge heme at very low concentrations due to the very high affinity of HasA for heme (Ka = 5,3 x 10(10) m(-1)). Heme is then transferred to HasR, which has a lower affinity for heme. The mechanism of the heme transfer between HasA and HasR is largely unknown. HasR has been overexpressed and purified in holo and apo forms. It binds one heme molecule with a Ka of 5 x 10(6) m(-1) and shows the characteristic absorbance spectrum of a low spin heme iron. Both holoHasA and apoHasA bind tightly to apoHasR in a 1:1 stoichiometry. In this study we show that heme transfer occurs in vitro in the purified HasA.HasR complex, demonstrating that heme transfer is energy- and TonB complex-independent and driven by a protein-protein interaction. We also show that heme binding to HasR involves two conserved histidine residues.

A Drosophila Protein Specific to Pheromone-Sensing Gustatory Hairs Delays Males' Copulation Attempts

In insects, increasing evidence suggests that small secreted pheromone binding proteins (PBPs) and odorant binding proteins (OBPs) are important for normal olfactory detection of airborne pheromones and odorants far from their source. In contrast, it is unknown whether extracellular ligand binding proteins participate in perception of less volatile chemicals, including many pheromones, that are detected by direct contact with chemosensory organs. CheB42a, a small Drosophila melanogaster protein unrelated to known PBPs or OBPs, is expressed and likely secreted in only a small subset of gustatory sensilla on males' front legs, the site of gustatory perception of contact pheromones. Here we show that CheB42a is expressed specifically in the sheath cells surrounding the taste neurons expressing Gr68a, a putative gustatory pheromone receptor for female cuticular hydrocarbons that stimulate male courtship. Surprisingly, however, CheB42a mutant males attempt to copulate with females earlier and more frequently than control males. Furthermore, CheB42a mutant males also attempt to copulate more frequently with other males that secrete female-specific cuticular hydrocarbon pheromones, but not with females lacking cuticular hydrocarbons. Together, these data indicate that CheB42a is required for a normal gustatory response to female cuticular hydrocarbon pheromones that modulate male courtship.

Effluents from oil production activities contain chemicals that interfere with normal function of intra- and extra-cellular estrogen binding proteins

Some environmental pollutants have the ability to alter the endocrine function in fish through interaction with the estrogen receptor (ER). Many of these chemicals are also able to interfere with the endocrine system through other mechanisms of action, however. The plasma sex steroid-binding protein (SBP), which is involved in regulating circulating levels of endogenous sex steroids, has recently been proposed to contribute to pollutant induced disruption of endocrine homeostasis. The objective of the present work was to determine whether industrial effluents contain chemicals that are able to modulate the endocrine system through interference with the function of the ER and SBP using in vitro biological assays (bioassays) from rainbow trout. The results show that solid phase extracts of process water (produced water) from an oil production facility in the North Sea and a land-based oil refinery contain chemicals that are able to induce estrogenic effects as well as displace natural sex steroid 17beta-estradiol from the SBP. The bioactive chemicals were found to be partly resistant to biological degradation, but the identity of the chemicals was not determined. The alkylphenol 4-tert-butylphenol, which is known to occur in effluents from various oil production facilities, was found to be estrogenic and displace 17beta-estradiol from the SBP and may thus contribute to the observed endocrine disrupting activity.

Impact of Extracellular Protein Binding on Passive and Active Drug Transport Across Caco-2 Cells

The objective of the study is to evaluate the mechanism behind alterations in passive and active transport of drugs in the presence of basolaterally applied extracellular protein using the Caco-2 cell model. The permeation across Caco-2 monolayers of two groups of compounds, transported either solely by passive diffusion or partly also by active transport in the secretory direction, was studied at two donor concentrations in the absence or presence of bovine serum albumin (BSA, 0-4%). Each group contained compounds with high or low protein binding (PB) capabilities and high or low absorption in humans (fraction absorbed, f(a)). The unbound fraction (f(u)) of each compound was determined by ultrafiltration. The transport rate of the passively permeating compounds was the same in both apical-to-basolateral (absorptive) and basolateral-to-apical (secretory) directions in the absence of BSA. Basolaterally applied BSA increased the transport rate in the absorptive direction and decreased it in the secretory direction for all compounds, in direct proportion to the extent of PB. The efflux ratios for the actively transported compounds were reduced in the presence of BSA. Basolaterally applied BSA, which mimics in vivo PB, alters both passive and active drug transport in the Caco-2 cell model. Using C(u) in the calculations of transport rate allowed elucidation of the different mechanisms behind these alterations. Our data also suggest that active secretory transport for highly protein-bound compounds might have less effect in vivo than predicted from traditional Caco-2 cell models (without BSA).

Biosynthesis of the IFN-γ binding protein of ectromelia virus, the causative agent of mousepox

Ectromelia virus (ECTV), the causative agent of mousepox, expresses an extracellular interferon-γ binding protein (IFN-γBP) with homology to the ligand binding domains of the IFN-γ high affinity receptor (IFN-γR1). Unlike the cellular receptor, the IFN-γBP binds IFN-γ from several species. The IFN-γBP is synthesized early after infection, accumulating in the extracellular milieu as dimers composed of two protein species with M r of 34.6 or 33.0 kDa. Homodimers are covalently linked by an interchain disulphide bond at position 216. The IFN-γBP has complex N-linked oligosaccharides at positions 41 and 149 as determined by site-directed mutagenesis and glycosidase treatment. Glycosylation at position 41 is required for secretion from mammalian cells and may play a role in the activity of the IFN-γBP. Glycosylation at position 149 is not required for secretion, and the lack of glycosylation at this site does not diminish ligand binding as measured by surface plasmon resonance (SPR) and ELISA.

Structural Basis for the Binding of Compatible Solutes by ProX from the Hyperthermophilic Archaeon Archaeoglobus fulgidus

Compatible solutes such as glycine betaine and proline betaine serve as protein stabilizers because of their preferential exclusion from protein surfaces. To use extracellular sources of this class of compounds as osmo-, cryo-, or thermoprotectants, Bacteria and Archaea have developed high affinity uptake systems of the ATP-binding cassette type. These transport systems require periplasmic- or extracellular-binding proteins that are able to bind the transported substance with high affinity. Therefore, binding proteins that bind compatible solutes have to avoid the exclusion of their ligands within the binding pocket. In the present study we addressed the question to how compatible solutes can be effectively bound by a protein at temperatures around 83 degrees C as this is done by the ligand-binding protein ProX from the hyperthermophilic archaeon Archaeoglobus fulgidus. We solved the structures of ProX without ligand and in complex with both of its natural ligands glycine betaine and proline betaine, as well as in complex with the artificial ligand trimethylammonium. Cation-pi interactions and non-classical hydrogen bonds between four tyrosine residues, a main chain carbonyl oxygen, and the ligand have been identified to be the key determinants in binding the quaternary amines of the three investigated ligands. The comparison of the ligand binding sites of ProX from A. fulgidus and the recently solved structure of ProX from Escherichia coli revealed a very similar solution for the problem of compatible solute binding, although both proteins share only a low degree of sequence identity. The residues involved in ligand binding are functionally equivalent but not conserved in the primary sequence.

Extracellular receptor kinase and cAMP response element binding protein activation in the neonatal rat heart after perinatal cocaine exposure.

Prenatal exposure to cocaine has been shown to induce an increase in the myocardial expression and activation of the cAMP response binding protein (CREB), a transcriptional factor that has been shown to regulate gene expression. Several different kinases, including protein kinase A, calcium calmodulin kinase II, and mitogen-activated protein kinase can induce phosphorylation of CREB at serine 133, a necessary step for CREB activation. We examined whether the mitogen-activated protein kinase-extracellular receptor kinase (ERK) pathway may be involved in mediating the serine 133 CREB phosphorylation in cardiac nuclei after perinatal cocaine exposure. Pregnant rats were treated daily with saline or cocaine at 60 mg/kg (C60) by intragastric administration during the entire gestational period, and treatment was continued in the nursing dams after delivery until the time of the study. Nuclear extracts were isolated from hearts of 1-d- and 7-d-old neonatal rats. We performed immunoblotting experiments using an antibody that recognized CREB with phosphorylation specifically at the serine 133 site and an antibody that recognized both the phosphorylated and the unphosphorylated forms of CREB, as well as antibodies for total ERK, phospho-ERK, total ribosomal S6 kinase 1 (RSK1), RSK2, and phospho-RSK. We assessed the interaction of RSK with CREB or CREB-binding protein by performing co-immunoprecipitation experiments. We found that perinatal cocaine exposure increased both phospho-ERK and phospho-RSK expression, indicative of an increased activity of these two enzymes. Furthermore, we demonstrated that phospho-RSK was immunoprecipitated with CREB in all neonatal cardiac nuclei and that the greatest interaction was found in day 7 hearts after perinatal cocaine exposure. Our results thus illustrate that the ERK-RSK pathway was active in the postnatal rat heart at 1 and 7 d of age and that this pathway may mediate the increase in myocardial CREB activation after perinatal cocaine exposure in the day 7 hearts.

Processing and Transport of Matrix γ-Carboxyglutamic Acid Protein and Bone Morphogenetic Protein-2 in Cultured Human Vascular Smooth Muscle Cells: EVIDENCE FOR AN UPTAKE MECHANISM FOR SERUM FETUIN

Matrix gamma-carboxyglutamic acid protein (MGP) is a member of the vitamin K-dependent protein family with unique structural and physical properties. MGP has been shown to be an inhibitor of arterial wall and cartilage calcification. One inhibitory mechanism is thought to be binding of bone morphogenetic protein-2. Binding has been shown to be dependent upon the vitamin K-dependent gamma-carboxylation modification of MGP. Since MGP is an insoluble matrix protein, this work has focused on intracellular processing and transport of MGP to become an extracellular binding protein for bone morphogenetic protein-2. Human vascular smooth muscle cells (VSMCs) were infected with an adenovirus carrying the MGP construct, which produced non-gamma-carboxylated MGP and fully gamma-carboxylated MGP. Both forms of MGP were found in the cytosolic and microsomal fractions obtained from the cells by differential centrifugation. The crude microsomal fraction was shown to contain an additional, more acidic Ser-phosphorylated form of MGP believed to be the product of Golgi casein kinase. The data suggest that phosphorylation of MGP dictates different transport routes for MGP in VSMCs. A proteomic approach failed to identify a larger soluble precursor of MGP or an intracellular carrier protein for MGP. Evidence is presented for a receptor-mediated uptake mechanism for fetuin by cultured human VSMCs. Fetuin, shown by mass spectrometry not to contain MGP, was found to be recognized by anti-MGP antibodies. Fetuin uptake and secretion by proliferating and differentiating cells at sites of calcification in the arterial wall may represent an additional protective mechanism against arterial calcification.

Regulation of PDGF and its receptors in fibrotic diseases

Platelet-derived growth factor (PDGF) isoforms play a major role in stimulating the replication, survival, and migration of myofibroblasts during the pathogenesis of fibrotic diseases. During fibrogenesis, PDGF is secreted by a variety of cell types as a response to injury, and many pro-inflammatory cytokines mediate their mitogenic effects via the autocrine release of PDGF. PDGF action is determined by the relative expression of PDGF α-receptors (PDGFRα) and β-receptors (PDGFRβ) on the surface of myofibroblasts. These receptors are induced during fibrogenesis, thereby amplifying biological responses to PDGF isoforms. PDGF action is also modulated by extracellular binding proteins and matrix molecules. This review summarizes the literature on the role of PDGF and its receptors in the development of fibrosis in a variety of organ systems, including lung, liver, kidney, and skin.

Activins, myostatin and related TGF-beta family members as novel therapeutic targets for endocrine, metabolic and immune disorders.

Activins and inhibins were first identified by virtue of their ability to regulate follicle-stimulating hormone (FSH) secretion from the anterior pituitary. Activins are also powerful regulators of gonadal functions. However, the physiological functions of activins are not restricted to reproductive tissues. Activins are involved in apoptosis of hepatocytes and B cells, fibrosis, inflammation and neurogenesis. Activins are regarded as novel drug targets since blocking activins would provide benefits by preventing apoptosis, fibrosis, inflammation and growth of several cancers. Activins are members of the transforming growth factor-beta (TGF-beta) family, which has numerous peptide growth and differentiation factors including activins, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs) and TGF-betas. Among them, GDF8 is also known as myostatin and is structurally related to activins. Myostatin is specifically expressed in the skeletal muscle lineage and is a candidate for muscle chalone negatively regulating the growth of myoblasts. Myostatin is regarded as a good drug target since therapeutics that modulate skeletal muscle growth would be useful for disease conditions such as muscular dystrophy, sarcopenia, cachexia and even diabetes. Recent studies have revealed that activins and myostatin signal through activin type II receptors (ActRIIA and ActRIIB) and their activities are regulated by extracellular binding proteins, follistatins and follistatin-related gene (FLRG). Furthermore, signaling of activins, myostatin and related ligands is also controlled by intracellular receptor-interacting proteins by novel mechanisms. In this review, I would like to show the current progress in the field emphasizing the importance of activins and myostatin as novel drug targets for immune, endocrine and metabolic disorders.

Inhibition of antigen-presenting cell function and stimulation of human peripheral blood mononuclear cells to express an antiinflammatory cytokine profile by the stress protein BiP: relevance to the treatment of inflammatory arthritis.

The stress protein and endoplasmic reticulum chaperone, immunoglobulin binding protein (BiP), is an autoantigen in rheumatoid arthritis (RA). Stress proteins, however, may have extracellular functions, mediated via cell surface receptors, that may include immunomodulatory functions. We sought to determine whether cell-free BiP is present in the synovial fluid (SF) of patients with RA and to further investigate the possible extracellular antiinflammatory and immunomodulatory properties of BiP in peripheral blood mononuclear cells (PBMCs) in vitro. The presence of BiP in SF was established by Western blotting. PBMCs were stimulated with exogenous recombinant human BiP, and cytokine production and cell proliferation were measured in the presence and absence of cell signaling inhibitors or neutralizing anti-interleukin-10 (anti-IL-10) monoclonal antibody. Cytokine levels were quantified by enzyme-linked immunosorbent assay, cell proliferation by tritiated thymidine uptake, and cell surface molecule expression by flow cytometry. PBMCs responded to BiP with secretion of an antiinflammatory profile of cytokines. Although BiP stimulated the early production of tumor necrosis factor alpha (TNF alpha), the major cytokine induced was IL-10. Soluble TNF receptor II and IL-1 receptor antagonist secretion was also increased. Addition of SB203580, the MAPK p38 pathway inhibitor, partially inhibited the production of IL-10 and TNF alpha, whereas they were unaffected by the MAPK ERK-1/2 inhibitor PD98059. BiP also inhibited the recall antigen response by PBMCs to tuberculin purified protein derivative. Further investigation showed that incubation of monocytes in the presence of either BiP or IL-10 down-regulated CD86 and HLA-DR expression. The effect observed with IL-10 was transient compared with the long-lasting reduction induced by BiP. Extracellular BiP may stimulate immunomodulatory and antiinflammatory pathways, which are only partly due to the production of IL-10. These properties may be of relevance for the treatment of diseases such as RA.

The diageotropica mutation of tomato disrupts a signalling chain using extracellular auxin binding protein 1 as a receptor.

The diageotropica ( dgt) mutant of tomato ( Lycopersicon esculentum Mill.) is known to lack a number of typical auxin responses. Here we show that rapid auxin-induced growth of seedling hypocotyls is completely abolished by the mutation over the full range of auxin concentrations tested, and also in early phases of the time course. Protoplasts isolated from wild-type hypocotyls respond to auxin by a rapid increase in cell volume, which we measured by image analysis at a high temporal resolution. A similar swelling could be triggered by antibodies directed against a part of the putative auxin-binding domain (box-a) of the auxin-binding protein 1 (ABP1). Induction of swelling both by auxin and by the antibody was not observed in the protoplasts isolated from the dgt mutant. However, dgt protoplasts are able to respond to the stimulator of the H(+)-ATPase, fusicoccin, with normal swelling. We propose that dgt is a signal-transduction mutation interfering with an auxin-signalling pathway that uses ABP1 as a receptor.

Gene expression changes in the human fibroblast induced by Centella asiatica triterpenoids.

The molecular pathways underlying the diverse biological activity of the triterpeniod compounds isolated from the tropical medicinal plant Centella asiatica were studied with gene microarrays and real-time reverse transcription polymerase chain reaction (real-time RT-PCR) to quantify the expression of 1053 human genes in human fibroblasts. Fibroblast cells grown in culture were used as a model system to evaluate the stimulation of wound healing by titrated extract from Centella asiatica (TECA) as well as by the four principal triterpenoid components of Centella. TECA treatment effects the expression of genes involved in angiogenesis and the remodeling of extracellular matrix, as well as diverse growth factor genes. The extent of expression change of TNFAIP6, an extracellular hyaluronan binding protein, was found to be largely dose-dependent, to respond most strongly to the free acids asiatic acid and madecassic acid, and to increase in expression over 48 hours of treatment. These results show that Centella triterpenes evoke a gene-expression response consistent with their prevailing medical uses in the treatment of connective tissue disorders such as wound healing and microangiopathy. The identification of genes modulated by these compounds provides the basis for a molecular understanding of Centella's bioactivity, and opportunities for the quantitative correlation of this activity with clinical effectiveness at a molecular level.