Streptavidin-horseradish Peroxidase Research Articles

Systemic tetracycline delays degradation of three different collagen membranes in rat calvaria

The aim of this study was to quantitatively evaluate the effect of systemic tetracycline (TTC) on the degradation of three different collagen membranes. Collagen membranes were cut into 5 mm diameter membrane discs and labeled with aminohexanoyl-biotin-N-hydroxy-succinimide ester. One membrane disc each of a non-cross-linked [BioGide (BG)], glutaraldehyde cross-linked [BioMend Extend (BM)], and ribose cross-linked [Ossix (OS)] was implanted on the calvaria of 40 Wistar rats. Another 10 biotinylated collagen membrane discs from each membrane type were processed for histologic observation and served as baseline; half of them (five from each group) were also treated with formic acid to inspect possible interference with biotinilazation of collagen by formic acid used during the decalcification process. A 10 mg/kg dose of TTC (50% of the minimal recommended antibacterial dose) to the experimental (20 animals) and saline to the control (20 animals) group was administered intramuscularly every 3 days. From each group, block sections were retrieved in half of the animals after 14 days and in the remaining after 28 days. Decalcified tissue histology was stained with streptavidin horseradish peroxidase. A computer-assisted program measured the membranes' collagen contents. Statistical analysis consisted of analysis of variance (ANOVA) with repeated measures. No statistically significant differences in collagen contents were appreciated between biotinylated non-implanted membranes treated or not treated by formic acid. Systemic TTC had a different effect on the bio-degradation of the membranes: while it significantly decreased the resorption of two of the membranes (BG and BM), it had minimal influence on the ribose cross-linked membrane (OS). ANOVA with repeated measures, tests of within-subjects effects, showed a statistically significant difference between the membranes (P<0.001), within the membranes at the different time-points (P<0.001), a significant interaction between membranes and time and between the membranes and administered TTC (P<0.001). Test of between-subject effects revealed a statistically significant interaction with time and with TTC (P<0.001). Systemically administered TTC in sub-antibacterial doses may offer a possible treatment alternative to reduce bio-degradation and enhance bio-durability of certain collagen membranes. The findings of the present study could have clinical application in large non-self-contained bone defects, where prolonged membrane barrier functions are desirable.

The Integrity of the Glycine Co-agonist Binding Site of N-Methyl-d-aspartate Receptors Is a Functional Quality Control Checkpoint for Cell Surface Delivery

N-Methyl-D-aspartate receptors are a subclass of ligand-gated, heteromeric glutamatergic neurotransmitter receptors whose cell surface expression is regulated by quality control mechanisms. Functional quality control checkpoints are known to contribute to cell surface trafficking of non-N-methyl-D-aspartate glutamate receptors. Here we investigated if similar mechanisms operate for the surface delivery of NMDA receptors. Point mutations in the glycine binding domain of the NR1-1a subunit were generated: D732A, a mutation that results in an approximately 3 x 10(4) decrease in glycine binding affinity; D732E, a conservative change; and D723A, a residue in the same NR1-1a domain that has no effect on glycine binding affinity. Each NR1-1a subunit was co-expressed with NR2A in mammalian cells. Immunoblotting and immunoprecipitations showed that all mutants were expressed to similar levels as wild-type NR1-1a and associated with NR2A. Cell surface expression measured by an enzyme-linked immunosorbent assay found that whereas NR1-1a (D732E)/NR2A and NR1-1a (D723A)/NR2A trafficked as efficiently as NR1-1a/NR2A, there was a 90% decrease in surface expression for NR1-1a (D732A)/NR2A. This was confirmed by confocal microscopy imaging and cell surface biotinylation. Further imaging showed that NR1-1a (D732A) and co-transfected NR2A co-localized with an endoplasmic reticulum marker. Dichlorokynurenic acid, a competitive glycine site antagonist, partially rescued surface expression. Mutation of the NR1-1a ER retention motif showed that the ligand binding checkpoint is an early event preceding endoplasmic reticulum sorting mechanisms. These findings demonstrate that integrity of the glycine co-agonist binding site is a functional checkpoint requisite for efficient cell surface trafficking of assembled NMDA receptors.

S-Palmitoylation of γ-Secretase Subunits Nicastrin and APH-1

Proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases generates beta-amyloid (Abeta) peptides, which accumulate in the brains of individuals affected by Alzheimer disease. Detergent-resistant membrane microdomains (DRM) rich in cholesterol and sphingolipid, termed lipid rafts, have been implicated in Abeta production. Previously, we and others reported that the four integral subunits of the gamma-secretase associate with DRM. In this study we investigated the mechanisms underlying DRM association of gamma-secretase subunits. We report that in cultured cells and in brain the gamma-secretase subunits nicastrin and APH-1 undergo S-palmitoylation, the post-translational covalent attachment of the long chain fatty acid palmitate common in lipid raft-associated proteins. By mutagenesis we show that nicastrin is S-palmitoylated at Cys(689), and APH-1 is S-palmitoylated at Cys(182) and Cys(245). S-Palmitoylation-defective nicastrin and APH-1 form stable gamma-secretase complexes when expressed in knock-out fibroblasts lacking wild type subunits, suggesting that S-palmitoylation is not essential for gamma-secretase assembly. Nevertheless, fractionation studies show that S-palmitoylation contributes to DRM association of nicastrin and APH-1. Moreover, pulse-chase analyses reveal that S-palmitoylation is important for nascent polypeptide stability of both proteins. Co-expression of S-palmitoylation-deficient nicastrin and APH-1 in cultured cells neither affects Abeta40, Abeta42, and AICD production, nor intramembrane processing of Notch and N-cadherin. Our findings suggest that S-palmitoylation plays a role in stability and raft localization of nicastrin and APH-1, but does not directly modulate gamma-secretase processing of APP and other substrates.

Ziram Causes Dopaminergic Cell Damage by Inhibiting E1 Ligase of the Proteasome

The etiology of Parkinson disease (PD) is unclear but may involve environmental toxins such as pesticides leading to dysfunction of the ubiquitin proteasome system (UPS). Here, we measured the relative toxicity of ziram (a UPS inhibitor) and analogs to dopaminergic neurons and examined the mechanism of cell death. UPS (26 S) activity was measured in cell lines after exposure to ziram and related compounds. Dimethyl- and diethyldithiocarbamates including ziram were potent UPS inhibitors. Primary ventral mesencephalic cultures were exposed to ziram, and cell toxicity was assessed by staining for tyrosine hydroxylase (TH) and NeuN antigen. Ziram caused a preferential damage to TH+ neurons and elevated alpha-synuclein levels but did not increase aggregate formation. Mechanistically, ziram altered UPS function through interfering with the targeting of substrates by inhibiting ubiquitin E1 ligase. Sodium dimethyldithiocarbamate administered to mice for 2 weeks resulted in persistent motor deficits and a mild reduction in striatal TH staining but no nigral cell loss. These results demonstrate that ziram causes selective dopaminergic cell damage in vitro by inhibiting an important degradative pathway implicated in the etiology of PD. Chronic exposure to widely used dithiocarbamate fungicides may contribute to the development of PD, and elucidation of its mechanism would identify a new potential therapeutic target.

Nadir CD4 T-Cell Counts Continue to Influence Interferon-γ Responses in HIV Patients Who Began Antiretroviral Treatment With Advanced Immunodeficiency

Nadir CD4 T-Cell Counts Continue to Influence Interferon-γ Responses in HIV Patients Who Began Antiretroviral Treatment With Advanced Immunodeficiency

Caspase-3 Is Enriched in Postsynaptic Densities and Increased in Alzheimer's Disease

Progressive synaptic degeneration and neuron loss are major structural correlates of cognitive impairment in Alzheimer's disease (AD). The mechanisms by which synaptic degeneration in AD occurs have not been established. The activation of proteins within the caspase family has been implicated in AD-associated neurodegeneration, and synaptically localized caspase activity could play a role in the synaptic degeneration and loss found in AD. We used synaptosomal fractionation with Western blotting and immunohistochemistry to examine the anatomical, subcellular, and subsynaptic expression patterns of caspase 3 in both the anterior cingulate cortex and hippocampus of control and AD patients. In both control and AD cases, there was a selective enrichment of caspase- 3 at synapses, particularly in the postsynaptic density (PSD) fractions. Compared with controls, AD patients exhibited significant increases in synaptic procaspase- 3 and active caspase-3 expression levels that were most evident in the PSD fractions. These data demonstrate for the first time the preferential localization and increase of caspase-3 in the PSD fractions in AD and suggest an important role for caspase 3 in synapse degeneration during disease progression.

Sensitive detection of lip genes by electrochemical DNA sensor and its application in polymerase chain reaction amplicons from Phanerochaete chrysosporium

An electrochemical DNA sensor based on the sandwich hybridization recognition of target sequence of lignin peroxidase ( lip) genes on a gold electrode was developed. A monolayer of thiolated capture probe was formed on a gold electrode through self-assembling. Following hybridizations with target nucleic acid and biotinylated detection probe, streptavidin–horseradish peroxidase (HRP) conjugate was applied to the electrode. The DNA conformation and surface coverage on electrode were characterized by impedance spectroscopy and square wave voltammetry. The experimental variables were optimized to maximize the hybridization efficiency, detection sensitivity and speed up the assay time. The amperometric current response to HRP-catalyzed reaction was linearly related to the natural logarithm of the target nucleic acid concentration in the range from 0.6 to 30 nM, with the correlation coefficient of 0.9722. The detection limit was 0.03 nM. Synthesized oligonucleotide as well as Phanerochaete chrysosporium lip gene fragments amplified using polymerase chain reaction and digested by restriction endonucleases were tested. The DNA sensor exhibited good precision, stability, sensitivity, and selectivity, and discriminated satisfactorily against mismatched nucleic acid samples of similar lengths.

Rho Family GTPase Modification and Dependence on CAAX Motif-signaled Posttranslational Modification

Rho GTPases (20 human members) comprise a major branch of the Ras superfamily of small GTPases, and aberrant Rho GTPase function has been implicated in oncogenesis and other human diseases. Although many of our current concepts of Rho GTPases are based on the three classical members (RhoA, Rac1, and Cdc42), recent studies have revealed the diversity of biological functions mediated by other family members. A key basis for the functional diversity of Rho GTPases is their association with distinct subcellular compartments, which is dictated in part by three posttranslational modifications signaled by their carboxyl-terminal CAAX (where C represents cysteine, A is an aliphatic amino acid, and X is a terminal amino acid) tetrapeptide motifs. CAAX motifs are substrates for the prenyltransferase-catalyzed addition of either farnesyl or geranylgeranyl isoprenoid lipids, Rce1-catalyzed endoproteolytic cleavage of the AAX amino acids, and Icmt-catalyzed carboxyl methylation of the isoprenylcysteine. We utilized pharmacologic, biochemical, and genetic approaches to determine the sequence requirements and roles of CAAX signal modifications in dictating the subcellular locations and functions of the Rho GTPase family. Although the classical Rho GTPases are modified by geranylgeranylation, we found that a majority of the other Rho GTPases are substrates for farnesyltransferase. We found that the membrane association and/or function of Rho GTPases are differentially dependent on Rce1- and Icmt-mediated modifications. Our results further delineate the sequence requirements for prenyltransferase specificity and functional roles for protein prenylation in Rho GTPase function. We conclude that a majority of Rho GTPases are targets for pharmacologic inhibitors of farnesyltransferase, Rce1, and Icmt.

P1-366: Glycogen synthase kinase activity in platelets of patients with Alzheimer's disease, mild cognitive impairment and controls

Glycogen synthase kinase-3β (GSK3) is a highly expressed kinase in the central nervous system, and its activity is regulated by phosphorylation and desphosphorylation of serine-threonine epitopes. GSK3 activity is increased in the AD brain, being implicated in the hyperphosphorylation of TAU and abnormal APP processing. This abnormality has been recently detected in human leukocytes, and for this reason GSK3 has been suggested as a candidate biomarker of AD, which can be measured in peripheral tissues and may help indicate the risk of conversion from MCI to AD. Recent, unpublished determinations from our group showed that GSK3 is highly expressed in platelets, bearing both phosphorylated and non-phosphorylated forms. The objective of the present study is to validate a semi-quantitative method to determine GSK3 activity in human platelets, with the aid of Western Blot. Platelet samples from patients with AD, MCI and age-matched controls were obtained from peripheral blood. Aliquots holding 25μg of total protein in sample buffer were submitted to electrophoresis in 10% poliacrylamide gels and transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies raised against total- and phospho-GSK, following incubations with species-specific biotinilated secondary antibodies, and a streptavidin horseradish peroxidase conjugate. Development was performed with enhanced chemiluminescence (ECL-Plus), prior to densitometry of the correspondent bands. GSK3 activity was estimated by ratio between its inactive (phosphorylated) form and the total GSK amount in platelet samples (phospho-GSK3/ total-GSK3). In this preliminary analysis (n=27), the method proved adequate for the semi-quantitative determination of GSK3 expression and activity in human platelets. There was a clear tendency toward an increased GSK3 activity in patients with AD and MCI as compared to controls, although not within significant limits: AD (n=9), 0.64±0.09; MCI (n=9), 0.75±0.10; Controls (n=9), 0.85±0.08. Recruitment in under way (n=51 at present), and a larger sample of patients and controls will presented at the ICAD-Chicago. If confirmed in a larger sample, the current findings will support the notion that GSK3 expression and activity can be determined in human platelets, with findings largely similar to the ones presented in leukocytes.

Detection of Escherichia coli O157:H7, Salmonella typhimurium, and Legionella pneumophila in Water Using a Flow-Through Chemiluminescence Microarray Readout System

Fast, sensitive, and especially, multianalyte test systems are currently of high interest for the monitoring and quality control of drinking water, since traditional microbiological methods are labor intensive and can take days until a response is achieved. In this study, the first flow-through chemiluminescence microarray was developed and characterized for the rapid and simultaneous detection of Escherichia coli O157:H7, Salmonella typhimurium, and Legionella pneumophila in water samples using a semiautomated readout system. Therefore, antibody microarrays were produced on poly(ethylene glycol)-modified glass substrates by means of a contact arrayer. For capturing bacteria, species-specific polyclonal antibodies were used. Cell recognition was carried out by binding of species-specific biotinylated antibodies. Chemiluminescence detection was accomplished by a streptavidin-horseradish peroxidase (HRP) catalyzed reaction of luminol and hydrogen peroxide. The chemiluminescence reaction that occurred was recorded by a sensitive charge-coupled device (CCD) camera. The overall assay time was 13 min, enabling a fast sample analysis. In multianalyte experiments, the detection limits were 3 x 10(6), 1 x 10(5), and 3 x 10(3) cells/mL for S. typhimurium, L. pneumophila, and E. coli O157:H7, respectively. Quantification of samples was possible in a wide concentration range with good recoveries. The presented system is well suited for quick and automatic water analysis.

Genetically Programmed Biases in Th1 and Th2 Immune Responses Modulate Atherogenesis

Atherosclerotic lesions contain T lymphocytes, which orchestrate adaptive immunity and regulate many innate immune pathways. This study examined the influence of Th1 and Th2 helper cell subsets on atherogenesis in two ApoE(-/-) mouse strains, C57BL/6 and BALB/c, which display opposite T-cell subset polarizations. ApoE(-/-) BL/6 mice showed predominant Th1-like immune responses on polyclonal stimulation of splenic CD4(+) T cells and had IgG2a antibodies to oxidized low-density lipoprotein (a disease-related antigen) whereas ApoE(-/-) BALB/c mice displayed predominant Th2 responses by CD4(+) T cells and an IgG1 isotype response toward oxidized low-density lipoprotein. ApoE(-/-) BL/6 and BALB/c mice consumed a high-cholesterol diet for 10, 16, and 24 weeks with equivalent cholesterolemic responses. The Th1-slanted BL/6 mice developed significantly more atherosclerosis in the aortic root and abdominal aorta at all time points compared with BALB/c mice, supporting a proatherogenic role for Th1 response. Progression of atherosclerosis was associated with increased levels of interleukin (IL)-6 in mouse serum and CD4(+) T-cell culture supernatants and increased levels of the acute-phase protein, serum amyloid A (SAA). Both IL-6 and SAA levels rose significantly in ApoE(-/-) BL/6 mice compared with BALB/c mice. The circulating cytokine milieu (IL-6) and acute phase reactants such as SAA may reflect alterations in the Th1/Th2 balance. The results presented here illustrate how genetically determined modifiers of both immune and inflammatory responses can modulate atherogenesis independently of lipid levels.

Tumor Suppressor Protein p53 Regulates Megakaryocytic Polyploidization and Apoptosis

The molecular mechanisms underlying differentiation of hematopoietic stem cells into megakaryocytes are poorly understood. Tumor suppressor protein p53 can act as a transcription factor affecting both cell cycle control and apoptosis, and we have previously shown that p53 is activated during terminal megakaryocytic (Mk) differentiation of the CHRF-288-11 (CHRF) cell line. Here, we use RNA interference to reduce p53 expression in CHRF cells and show that reduced p53 activity leads to a greater fraction of polyploid cells, higher mean and maximum ploidy, accelerated DNA synthesis, and delayed apoptosis and cell death upon phorbol 12-myristate 13-acetate-induced Mk differentiation. In contrast, reduced p53 expression did not affect the ploidy or DNA synthesis of CHRF cells in the absence of phorbol 12-myristate 13-acetate stimulation. Furthermore, primary Mk cells from cultures initiated with p53-null mouse bone marrow mononuclear cells displayed higher ploidy compared with wild-type controls. Quantitative reverse transcription-PCR analysis of p53-knockdown CHRF cells, compared with the "scrambled" control CHRF cells, revealed that six known transcriptional targets of p53 (BBC3, BAX, TP53I3, TP53INP1, MDM2, and P21) were down-regulated, whereas BCL2 expression, which is known to be negatively affected by p53, was up-regulated. These studies show that the functional role of the intrinsic activation of p53 during Mk differentiation is to control polyploidization and the transition to endomitosis by impeding cell cycling and promoting apoptosis.

Controllable biotinylated poly(ethylene-co-glycidyl methacrylate) (PE-co-GMA) nanofibers to bind streptavidin–horseradish peroxidase (HRP) for potential biosensor applications

Poly(ethylene-co-glycidyl methacrylate) (PE-co-GMA) nanofibers with abundant active epoxy groups on surfaces were fabricated through a novel manufacturing process. The prepared PE-co-GMA nanofibers with different average diameters ranging from 100 to 400 nm were aminated by reacting the epoxy groups with 1,3-diaminopropane. The resulting aminated PE-co-GMA nanofibers were subsequently biotinylated and then successfully applied to immobilize streptavidin–horseradish peroxidase (HRP) conjugate via specific, strong and rapid binding of biotin and streptavidin. The streptavidin–HRP immobilized PE-co-GMA nanofibers showed high activity, efficiency, sensitivity as well as good reusability. The results demonstrated that PE-co-GMA nanofibers prepared could be a promising candidate as solid support materials for potential biosensor applications.

Biodegradation of Three Different Collagen Membranes in the Rat Calvarium: A Comparative Study

Collagen barrier membranes are commonly applied in periodontal and bone-regenerative procedures. Membranes differ in their resorption pattern following implantation, thus influencing clinical outcome. The purpose of this study was to quantitatively evaluate the biodegradation of three different commercially available collagen membranes. Collagen membranes were cut into 5-mm-diameter disks and labeled with aminohexanoyl-biotin-N-hydroxy-succinimide ester. One membrane disk of each type (non-cross-linked [NCL], glutaraldehyde cross-linked [GCL], and ribose cross-linked [RCL]) was implanted on the calvaria of 20 Wistar rats. Block sections were retrieved after 2 days (baseline, two animals), 14 days (10 animals), or 28 days (eight animals). Decalcified histologic sections were stained with streptavidin horseradish peroxidase. Residual membrane thickness and area were measured. Statistical analysis consisted of analysis of variance (ANOVA) with repeated measures. Statistically significant differences in the amount of residual membrane material were recorded within each membrane (among different time points) and among different membranes at the same time points (P <0.001). At 28 days, the least amount of residual collagen area, expressed as the percentage of baseline, was observed in the NCL group (13.9% +/- 10.25%), followed by the GCL (24.7% +/- 35.11%) and RCL (91.3% +/- 10.35%) groups. Residual membrane thickness, expressed as the percentage of baseline thickness, presented a similar pattern (31% +/- 16.55%, 37% +/- 41.90%, and 94.1% +/- 12.22%, respectively). ANOVA with repeated measures showed a significant interaction between membranes and time (P <0.001). The tested membranes differed in their degradation patterns and collagen contents. Membranes should be chosen for each clinical case according to the desired biodegradation characteristics.

Protein Detection Enhanced by 3DNA Dendrimer Signal Amplification

DNA dendrimers, conjugated with both anti-biotin antibodies and up to 350 labeling entities, were designed and adapted to protein microarray and enzyme-linked immunosorbent assay (ELISA) to improve the limits of protein detection with no additional steps or equipment. Application of conjugated dendrimers to standard ELISA cytokine detection resulted in up to threefold improvement of the limits of detection with no significant increase in the inter- and intra-assay coefficient of variation (CV) compared to streptavidin horseradish peroxidase (SA-HRP) detection. The adaptation of conjugated dendrimers to protein microarray cytokine detection resulted in up to 10-fold improvement of the limits of detection, but assay conditions would have to be optimized to decrease the intra- and inter-assay %CVs.

Dematin and Adducin Provide a Novel Link between the Spectrin Cytoskeleton and Human Erythrocyte Membrane by Directly Interacting with Glucose Transporter-1

Dematin and adducin are actin-binding proteins located at the spectrin-actin junctions, also called the junctional complex, in the erythrocyte membrane. Here we propose a new model whereby dematin and adducin link the junctional complex to human erythrocyte plasma membrane. Using a combination of surface labeling, immunoprecipitation, and vesicle proteomics approaches, we have identified glucose transporter-1 as the receptor for dematin and adducin in the human erythrocyte membrane. This finding is the first description of a transmembrane protein that binds to dematin and adducin, thus providing a rationale for the attachment of the junctional complex to the lipid bilayer. Because homologues of dematin, adducin, and glucose transporter-1 exist in many non-erythroid cells, we propose that a conserved mechanism may exist that couples sugar and other related transporters to the actin cytoskeleton.

Circulating Alloreactive T Cells Correlate with Graft Function in Longstanding Renal Transplant Recipients

Monitoring for alloreactive memory T cells after organ transplantation may allow individualization of immunosuppression. Two pathways of T cell allorecognition have been implicated in chronic graft dysfunction: Direct (recipient T cells respond to donor peptides presented by donor antigen-presenting cells) and indirect (donor peptides are processed and presented by recipient antigen-presenting cells). Previous studies have assessed these alloresponses only during the first 2 yr after kidney transplantation,so this study correlated the presence of circulating donor-reactive memory/effector T cells, primed by both pathways, in 34 longstanding living-donor renal transplant recipients using the highly sensitive IFN-gamma Elispot assay. Remarkably, 59% of patients had directly primed donor-reactive T cells, and their presence correlated directly with serum creatinine (P = 0.001) and inversely with estimated GFR (P = 0.042). Multivariate analysis revealed that hyporesponsiveness of direct, donor-specific T cells was the only variable that significantly correlated with graft function and that antidonor indirect alloreactivity was the only variable that significantly correlated with proteinuria. Interestingly, when both allorecognition pathways were considered together, patients with undetectable direct alloreactivity had better longterm graft function, independent of allosensitization by the indirect pathway. In conclusion, circulating donor-specific alloreactive T cells primed by both pathways are detectable long after transplantation and are associated with graft injury. Assessment of alloreactive memory/effector T cells might be helpful to tailor individual immunosuppression regimens for transplant recipients in the future.

Distinct Effects of N-Acetylgalactosamine-4-sulfatase and Galactose-6-sulfatase Expression on Chondroitin Sulfates

The sulfatase enzymes, N-acetylgalactosamine-4-sulfatase (arylsulfatase B (ASB)) and galactose-6-sulfatase (GALNS) hydrolyze sulfate groups of CS. Deficiencies of ASB and GALNS are associated with the mucopolysaccharidoses. To determine if expression of ASB and GALNS impacts on glycosaminoglycans (GAGs) and proteoglycans beyond their association with the mucopolysaccharidoses, we modified the expression of ASB and GALNS by overexpression and by silencing with small interference RNA in MCF-7 cells. Content of total sulfated GAG (sGAG), chondroitin 4-sulfate (C4S), and total chondroitin sulfates (CSs) was measured following immunoprecipitation with C4S and CS antibodies and treatment with chondroitinase ABC. Following silencing of ASB or GALNS, total sGAG, C4S, and CS increased significantly. Following overexpression of ASB or GALNS, total sGAG, C4S, and CS declined significantly. Measurements following chondroitinase ABC treatment of the cell lysates demonstrated no change in the content of the other sGAG, including heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. Following overexpression of ASB and immunoprecipitation with C4S antibody, virtually no sGAG was detectable. Total sGAG content increased to 23.39 (+/-1.06) microg/mg of protein from baseline of 12.47 (+/-0.68) microg/mg of protein following ASB silencing. mRNA expression of core proteins of the CS-containing proteoglycans, syndecan-1 and decorin, was significantly up-regulated following overexpression of ASB and GALNS. Soluble syndecan-1 protein increased following increases in ASB and GALNS and reduced following silencing, inversely to changes in CS. These findings demonstrate that modification of expression of the lysosomal sulfatases ASB and GALNS regulates the content of CSs.

VE-cadherin is a critical endothelial regulator of TGF-β signalling

VE-cadherin is an endothelial-specific transmembrane protein concentrated at cell-to-cell adherens junctions. Besides promoting cell adhesion and controlling vascular permeability, VE-cadherin transfers intracellular signals that contribute to vascular stabilization. However, the molecular mechanism by which VE-cadherin regulates vascular homoeostasis is still poorly understood. Here, we report that VE-cadherin expression and junctional clustering are required for optimal transforming growth factor-beta (TGF-beta) signalling in endothelial cells (ECs). TGF-beta antiproliferative and antimigratory responses are increased in the presence of VE-cadherin. ECs lacking VE-cadherin are less responsive to TGF-beta/ALK1- and TGF-beta/ALK5-induced Smad phosphorylation and target gene transcription. VE-cadherin coimmunoprecipitates with all the components of the TGF-beta receptor complex, TbetaRII, ALK1, ALK5 and endoglin. Clustered VE-cadherin recruits TbetaRII and may promote TGF-beta signalling by enhancing TbetaRII/TbetaRI assembly into an active receptor complex. Taken together, our data indicate that VE-cadherin is a positive and EC-specific regulator of TGF-beta signalling. This suggests that reduction or inactivation of VE-cadherin may contribute to progression of diseases where TGF-beta signalling is impaired.

Hyaluronan Facilitates Transforming Growth Factor-β1-mediated Fibroblast Proliferation

This study aims to understand the role of the matrix polysaccharide hyaluronan (HA) in influencing fibroblast proliferation and thereby affecting wound healing outcomes. To determine mechanisms that underlie scarred versus scar-free healing, patient-matched dermal and oral mucosal fibroblasts were used as models of scarring and non-scarring fibroblast phenotypes. Specifically, differences in HA generation between these distinct fibroblast populations have been examined and related to differences in transforming growth factor-beta(1) (TGF-beta(1))-dependent proliferative responses and Smad signaling. There was a differential growth response to TGF-beta(1), with it inducing proliferation in dermal fibroblasts but an anti-proliferative response in oral fibroblasts. Both responses were Smad3-dependent. Furthermore, the two fibroblast populations also demonstrated differences in their HA regulation, with dermal fibroblasts generating increased levels of HA, compared with oral fibroblasts. Inhibition of HA synthesis in dermal fibroblasts was shown to abrogate the TGF-beta(1)-mediated induction of proliferation. Inhibition of HA synthesis also led to an attenuation of Smad3 signaling in dermal fibroblasts. Microarray analysis demonstrated no difference in the genes involved in TGF-beta(1) signaling between dermal and oral fibroblasts, whereas there was a distinct difference in the pattern of genes involved in HA regulation. In conclusion, these two distinct fibroblast populations demonstrate a differential proliferative response to TGF-beta(1), which is associated with differences in HA generation. TGF-beta(1) regulates proliferation through Smad3 signaling in both fibroblast populations; however, it is the levels of HA generated by the cells that influence the outcome of this response.