SEARCH · PubMed Health

Results for “Extracellular Traps”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 595 records · Page 33Linked to original sources

PubMed1997

CGP37157 modulates mitochondrial Ca2+ homeostasis in cultured rat dorsal root ganglion neurons.

The effects of 7-chloro-3,5-dihydro-5-phenyl-1H-4,1-benzothiazepine-2-on (CGP37157), an inhibitor of mitochondrial Na+/Ca2+ exchange, on depolarization-induced intracellular free Ca2+ concentration ([Ca2+]i) transients were studied in cultured rat dorsal root ganglion neurons with indo-1-based microfluorimetry. A characteristic plateau in the recovery phase of the [Ca2+]i transient resulted from mitochondrion-mediated [Ca2+]i buffering. It was blocked by metabolic poisons and was not dependent on extracellular Ca2+. CGP37157 produced a concentration-dependent decrease in the amplitude of the mitochondrion-mediated plateau phase (IC50 = 4 +/- 1 microM). This decrease in [Ca2+]i was followed by an increase in [Ca2+]i upon removal of the drug, suggesting that Ca2+ trapped in the matrix was released when the CGP37157 was removed from the bath. CGP37157 also inhibited depolarization-induced Ca2+ influx at the concentrations required to see effects on [Ca2+]i buffering. Thus, CGP37157 inhibits mitochondrial Na+/Ca2+ exchange and directly inhibits voltage-gated Ca2+ channels, suggesting caution in its use to study [Ca2+]i regulation in intact cells.

Animals↗

PubMed1999

Metabolites of diallyl disulfide and diallyl sulfide in primary rat hepatocytes.

The objectives of this study were to analyse and identify the metabolites of diallyl disulfide (DADS) and diallyl sulfide (DAS) in primary rat hepatocytes prepared by collagenase perfusion. According to the results, allyl mercaptan (AM) and allyl methyl sulfide (AMS) were the metabolites of DADS. The highest amount of AMS (0.93 +/- 0.08 microg/ml at 90 min) was much less than that of AM (46.2 +/- 6.6 microg/ml at 60 min). Combined with the Purge and Trap using a gas chromatography-mass spectrometry (GC-MS) system, it is very useful to detect the trace amounts of metabolites in primary rat hepatocytes. Results also showed that AMS was a metabolite of DAS. The highest amount of AMS in the extracellular fluid of hepatocytes was 0.63 +/- 0.16 microg/ml at 30 min of incubation.

Allyl Compounds↗

PubMed1979

Uptake and accumulation of putrescine and its lethality in Anacystis nidulans.

The rate of uptake of putrescine by Anacystis nidulans has been shown to depend on the external pH and the extracellular concentration of putrescine. Accumulation of exogenous putrescine was also proportional to the concentration of putrescine in the medium, suggesting that putrescine uptake was not subject to cellular regulation. An equation was derived to test the hypothesis that putrescine accumulation was due to ion trapping. Comparison of the predicted and observed intracellular concentrations of putrescine under various conditions showed a close correlation in support of the hypothesis of ion trapping. Under conditions leading to cell death (e.g., 150 microM putrescine, pH 9.8), the correlation did not hold as a result of leakage of accumulated putrescine.

Cyanobacteria↗

PubMed1993

Characterization of the nuclear translocation of acidic fibroblast growth factor.

The subcellular localization of human acidic FGF (aFGF; FGF-1) expressed to high levels by using a bacteriophage T7 RNA polymerase-driven vaccinia virus expression system was studied in BHK21 and HeLa cells. Acidic FGF was detected by immunoblotting or immunofluorescence using an affinity-purified rabbit polyclonal antibody. The nuclei of most transfected cells, but not nuclei of control cells, were strongly immunoreactive. The nuclear accumulation of aFGF was confirmed by subcellular fractionation and immunoblotting, indicating that about 50% of the expressed protein was located in the nuclei at 12 h after transfection. It has previously been reported that a putative N-terminal nuclear localization sequence (NLS) in aFGF is required for full mitogenic activity (Imamura et al., Science 249, 1567-1570, 1990). We found that deletion of the first 27 residues including the putative NLS did not prevent the nuclear translocation of aFGF in either cell type. This observation suggests that the putative NLS sequence is not essential for targeting aFGF to the cell nucleus. To analyze further the mechanism of nuclear import, purified aFGF was microinjected into the cytoplasm of growing BHK21 cells under various conditions. In chilled (4 degrees C) or ATP-depleted cells, the injected aFGF entered the nucleus with similar efficiency to that in control cells at 37 degrees C. This suggests that aFGF, which has a molecular mass of only 16,500, enters the cell nucleus by free diffusion, and possibly becomes trapped by binding to some nuclear structures. When added exogenously to growing BHK21 cells, aFGF was not localized to the nucleus. Instead, a punctate staining pattern in the cytosol was observed, reminiscent of that in the endosomal-lysosomal compartments. In addition, a diffuse extracellular surface-staining was evident. This result demonstrates that receptor-mediated endocytosis of aFGF does not result in its translocation to the nucleus, as has been reported for basic FGF.

Adenosine Triphosphate↗

PubMed2005

Decorin binds myostatin and modulates its activity to muscle cells.

Myostatin, a member of TGF-beta superfamily of growth factors, acts as a negative regulator of skeletal muscle mass. The mechanism whereby myostatin controls the proliferation and differentiation of myogenic cells is mostly clarified. However, the regulation of myostatin activity to myogenic cells after its secretion in the extracellular matrix (ECM) is still unknown. Decorin, a small leucine-rich proteoglycan, binds TGF-beta and regulates its activity in the ECM. Thus, we hypothesized that decorin could also bind to myostatin and participate in modulation of its activity to myogenic cells. In order to test the hypothesis, we investigated the interaction between myostatin and decorin by surface plasmon assay. Decorin interacted with mature myostatin in the presence of concentrations of Zn(2+) greater than 10microM, but not in the absence of Zn(2+). Kinetic analysis with a 1:1 binding model resulted in dissociation constants (K(D)) of 2.02x10(-8)M and 9.36x10(-9)M for decorin and the core protein of decorin, respectively. Removal of the glycosaminoglycan chain by chondroitinase ABC digestion did not affect binding, suggesting that decorin could bind to myostatin with its core protein. Furthermore, we demonstrated that immobilized decorin could rescue the inhibitory effect of myostatin on myoblast proliferation in vitro. These results suggest that decorin could trap myostatin and modulate its activity to myogenic cells in the ECM.

Animals↗

PubMed1998

Involvement of hydrogen peroxide in the differentiation of clonal HD-11EM cells into osteoclast-like cells.

The present study uses the osteoclast precursor clonal line, HD-11EM, to study the potential of hydrogen peroxide (H2O2) in mediating the differentiation of HD-11EM into osteoclast-like cells. HD-11EM cells are a newly established clonal cell line that, in response to 1alpha,25-(OH)2D3, differentiate into osteoclast-like cells that are multinucleated (more than three nuclei), express tartrate-resistant acid phosphatase (TRAP), and excavate resorption pits when cultured on dentin slices in the presence of osteoblasts (Hsia et al., 1995, J. Bone Miner. Res., 10(Suppl 1):S424; Hsia, and Hauschka, 1997, unpublished data). Here we demonstrate that HD-11EM express the reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase specific cytochrome b558 subunits, and that stimulation of HD-11EM with 1 or 10 nM 1alpha,25-(OH)2D3 increases the extracellular release of H2O2 within 5-10 min. Ours is the first report that stimulation of a cell with 1alpha,25-(OH)2D3 enhances the activation of NADPH-oxidase and increases the basal release of superoxide and the formation of its dismutation product, H2O2. To determine the possible involvement of H2O2 in the differentiation of HD-11EM, these cells were exposed to glucose/glucose oxidase. This enzyme system was used to deliver a pure and continuous source of H2O2 in nanomole amounts consistent with quantities produced by HD-11EM in response to 1alpha,25-(OH)2D3. Both 1alpha,25-(OH)2D3 and the exogenously generated H2O2 stimulated a dose- and time-dependent increase in TRAP activity/cell and the number of multinucleated cells 24-48 hr after treatment. Northern analysis confirmed an increase in expression of TRAP mRNA in response to either 1alpha,25-(OH)2D3 or H2O2. Decreases in cell proliferation and v-myc mRNA were also observed in response to these agents. Taken together, our findings indicate that production of H2O2 by HD-11EM is an important local factor involved in differentiation of HD-11EM into osteoclast-like cells, and suggest that H2O2 may play a role in native osteoclast differentiation.

Acid Phosphatase↗

PubMed2004

Modulation of Mg2+ efflux from rat ventricular myocytes studied with the fluorescent indicator furaptra.

The fluorescent Mg(2+) indicator furaptra (mag-fura-2) was introduced into single ventricular myocytes by incubation with its acetoxy-methyl ester form. The ratio of furaptra's fluorescence intensity at 382 and 350 nm was used to estimate the apparent cytoplasmic [Mg(2+)] ([Mg(2+)](i)). In Ca(2+)-free extracellular conditions (0.1 mM EGTA) at 25 degrees C, [Mg(2+)](i) averaged 0.842 +/- 0.019 mM. After the cells were loaded with Mg(2+) by exposure to high extracellular [Mg(2+)] ([Mg(2+)](o)), reduction of [Mg(2+)](o) to 1 mM (in the presence of extracellular Na(+)) induced a decrease in [Mg(2+)](i). The rate of decrease in [Mg(2+)](i) was higher at higher [Mg(2+)](i), whereas raising [Mg(2+)](o) slowed the decrease in [Mg(2+)](i) with 50% reduction of the rate at approximately 10 mM [Mg(2+)](o). Because a part of the furaptra molecules were likely trapped inside intracellular organelles, we assessed possible contribution of the indicator fluorescence emitted from the organelles. When the cell membranes of furaptra-loaded myocytes were permeabilized with saponin (25 microg/ml for 5 min), furaptra fluorescence intensity at 350-nm excitation decreased to 22%; thus approximately 78% of furaptra fluorescence appeared to represent cytoplasmic [Mg(2+)] ([Mg(2+)](c)), whereas the residual 22% likely represented [Mg(2+)] in organelles (primarily mitochondria as revealed by fluorescence imaging). [Mg(2+)] calibrated from the residual furaptra fluorescence ([Mg(2+)](r)) was 0.6-0.7 mM in bathing solution [Mg(2+)] (i.e., [Mg(2+)](c) of the skinned myocytes) of either 0.8 mM or 4.0 mM, suggesting that [Mg(2+)](r) was lower than and virtually insensitive to [Mg(2+)](c). We therefore corrected furaptra fluorescence signals measured in intact myocytes for this insensitive fraction of fluorescence to estimate [Mg(2+)](c). In addition, by utilizing concentration and dissociation constant values of known cytoplasmic Mg(2+) buffers, we calculated changes in total Mg concentration to obtain quantitative information on Mg(2+) flux across the cell membrane. The calculations indicate that, in the presence of extracellular Na(+), Mg(2+) efflux is markedly activated by [Mg(2+)](c) above the normal basal level (approximately 0.9 mM), with a half-maximal activation of approximately 1.9 mM [Mg(2+)](c). We conclude that [Mg(2+)](c) is tightly regulated by an Mg(2+) efflux that is dependent on extracellular [Na(+)].

Animals↗

PubMed1997

HIV is trapped and masked in the cytoplasm of lymph node follicular dendritic cells.

To gain further insight into the pathogenesis of human immunodeficiency virus (HIV) infection, lymph nodes from seven asymptomatic HIV+ subjects were analyzed during the latent phase of disease. Both ultrastructural and immunohistochemical analyses revealed that, in all of the cases, plasma cells producing IgM/gamma were present in germinal centers. Secreted immunoglobulins formed extracellular deposits mimicking the follicular dendritic cell network. Immunoglobulin produced by germinal center plasma cells are specific for HIV because they bind the HIV env protein gp 120. Plasma cells producing antibodies with the same specificity were also abundant in the extrafollicular regions of lymph nodes. During the latent phase of infection, the virus largely accumulates within the germinal centers. Therefore, extracellular immunoglobulin may form immune complexes, as shown by the presence of HIV-specific antibodies, HIV particles, and complement components C3c, C3d, and C1q in the interdendritic spaces. When the ultrastructural localization of HIV in germinal centers was analyzed, abundant virus particles were found in the interdendritic spaces. In addition to this extracellular localization of HIV, receptor-mediated endocytosis of viral particles by follicular dendritic cells was observed. Complete HIV particles were found within the endosomal compartment of the follicular dendritic cells and, as complete viral particles, free in the cytoplasm, indicating that the virus may escape from the endocytic compartment. As the virus is abundant in the cytoplasm, this event leads to formation of a hidden reservoir within follicular dendritic cells. In this location, HIV escapes recognition by cytotoxic T lymphocytes. In contrast, virus budding indicating a productive infection of follicular dendritic cells that would render them susceptible to T-cell-mediated lysis has been seldom observed.

Antigen-Antibody Complex↗

PubMed2005

Online microdialysis-dynamic nanoelectrospray ionization-mass spectrometry for monitoring neuropeptide secretion.

Although mass spectrometric approaches offer a sensitive method for identifying cell-cell signaling peptides, the high salt-containing environment of extracellular solutions often complicates characterization of these microscale samples. Accordingly, we have developed a miniature hollow-fiber microdialysis device optimized for desalting small-volume neuronal samples online, with the device directly connected to a modified dynamic nanoelectrospray ionization assembly interfaced with an ion trap mass spectrometer. Improvements over existing designs include placement of a capillary insert within the microdialysis fiber to minimize volume, as well as the use of a microinjector that enables 1 microl sample injections. We present detailed evaluation of peptide recoveries within the microdialysis fiber by liquid chromatography-electrospray ionization-ion trap-mass spectrometry analysis of tissue homogenate in artificial seawater with and without microdialysis. Analyte recoveries after microdialysis ranged from 6 to 78% with higher recoveries of more hydrophilic peptides, while little correlation between mass and percentage recovery was observed in the range studied (2000 to 6000 Da). Recoveries of peptides were the lowest for the analytes with the highest initial mass spectrometry signal intensity. Finally, we illustrate the utility of this microdialysis device for desalting neuropeptides secreted from preparations of the peptidergic bag cell neurons of the marine mollusk, Aplysia californica. Without microdialysis, the high concentration of salts ( approximately 0.5 M) prevented detection of peptides, whereas following online microdialysis-dynamic nanoelectrospray mass spectrometry of stimulated releasate, three peptides (acidic peptide, acidic peptide 1-24 and delta-bag cell peptide) were detected.

Animals↗

PubMed1992

Ultrastructural evidence for neurogenically mediated changes in blood vessels of the rat dura mater and tongue following antidromic trigeminal stimulation.

We investigated the effects of unilateral electrical trigeminal ganglion stimulation (0.1 or 1.0 mA, 5 Hz, 5 ms, 5 min) on the morphology of blood vessels within the rat dura mater and tongue using light and transmission electron microscopy. Stimulation at both intensities caused changes which were confined to the ipsilateral post-capillary venules except in the tongue where arterioles were affected as well. Changes were more marked after 1.0 mA. Dramatic increases in the numbers of endothelial pinocytotic vesicles were found along the luminal and abluminal surfaces ipsilateral to the stimulation. Tight junctions remained largely intact, except that injected ferritin particles were occasionally trapped inside these junctions. Cytoplasmic microvilli and endothelial blebs were sometimes present as well. Approximately 80% of the examined dural post-capillary venules showed one or more of these endothelial changes. Horseradish peroxidase injected intravenously 5 min prior to stimulation was detected in the extracellular space surrounding dural blood vessels and within pinocytotic vesicles. Ferritin injected similarly, was also localized in post-capillary venule walls, interstitial spaces, intraendothelial vesicles and in vacuoles. Platelet accumulation and aggregation were present in approximately 10% of post-capillary venules in dura and tongue. These changes were associated with mast cell secretion, but neither vascular nor mast cell activation was observed in adult rats in whom C-fibers were destroyed during the neonatal period with capsaicin. The present observations provide morphological evidence which supports findings from previously reported albumin tracer studies suggesting enhanced transport and endothelial activation following electrical stimulation of small caliber afferent fibers.

Animals↗

PubMed1985

Potassium channels in isolated presynaptic nerve terminals from rat brain.

86Rb efflux from pinched-off rat brain presynaptic nerve terminals (synaptosomes) was used to measure the K permeability of the terminals. Synaptosomes were pre-loaded with 86Rb and the suspensions were then filtered on glass fibre filters. The terminals trapped on the filters were superfused with 'efflux solutions', and the effluent and filters were then counted. 86Rb efflux into physiological saline (PSS) containing 5 mM-K and 145 mM-Na was about 0.4% of the 86Rb load per second (component 'R'). Increasing extracellular K concentration [( K]o), or adding veratridine and sea anemone toxin, stimulated efflux; presumably by depolarizing the nerve terminals. The K-stimulated 86Rb efflux was a graded function of [K]o. High [K]o evoked at least three components of efflux: a 'fast phase' (T) that apparently inactivated in less than 1 s, a 'slower phase' (S) that was linear for 3-5 s, and a Ca-dependent phase (C). Some, but not all, of the slow phase 86Rb efflux (component S) may be attributable to increased efflux mediated by the 'resting' K permeability mechanism when the driving force is increased by depolarization. K efflux was also studied and was found to be qualitatively similar to 86Rb efflux. 86Rb: 42K permeability ratios were 0.6-0.8 for most components of the efflux. Raising the Mg concentration in the efflux solution shifted the 86Rb efflux versus [K]o curve in the direction of increased [K]o. This shift may be the result of screening of surface charges by Mg. Several agents that block various K channels in other preparations inhibited K-stimulated 86Rb efflux in synaptosomes: tetraethylammonium (TEA), tetrabutylammonium (TBA), and 4-aminopyridine (4-AP). The fast component (T) of high [K]o-stimulated 86Rb efflux was selectively blocked by low concentrations of 4-AP (apparent half-maximal inhibition, KI = 0.1-0.2 mM); it was also blocked by TEA (KI = 0.6 mM) and TBA (KI = 0.8-1.0 mM). Dose-response curves for inhibition of component T by all three agents were monophasic. the slow component (S) of the K-stimulated 86Rb efflux was much less sensitive to all three agents, than was component T; the broad dose-response curves were consistent with the view that two (or more) different K conductances may contribute to component S.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed2001

Reelin is a serine protease of the extracellular matrix.

Reelin is an extracellular matrix protein that plays a pivotal role in development of the central nervous system. Reelin is also expressed in the adult brain, notably in the cerebral cortex, where it might play a role in synaptic plasticity. The mechanism of action of reelin at the molecular level has been the subject of several hypotheses. Here we show that reelin is a serine protease and that proteolytic activity is relevant to its function, since (i) Reelin expression in HEK 293T cells impairs their ability to adhere to fibronectin-coated surfaces, and adhesion to fibronectin is restored by micromolar concentrations of diisopropyl phosphorofluoridate, a serine hydrolase inhibitor; (ii) purified Reelin binds FP-Peg-biotin, a trap probe which irreversibly binds to serine residues located in active catalytic sites of serine hydrolases; (iii) purified Reelin rapidly degrades fibronectin and laminin, while collagen IV is degraded at a much slower rate; fibronectin degradation is inhibited by inhibitors of serine proteases, and by monoclonal antibody CR-50, an antibody known to block the function of Reelin both in vitro and in vivo. The proteolytic activity of Reelin on adhesion molecules of the extracellular matrix and/or receptors on neurons may explain how Reelin regulates neuronal migration and synaptic plasticity.

Cell Adhesion↗

PubMed1997

Intracellular and extracellular forms of alkaline pullulanase from an alkaliphilic Bacillus sp. S-1.

Bacillus sp.S-1 alkaline pullulanase (AP) exists in two forms: a precursor form (PUL-Ia, M(r) 180,000) and a processed form (PUL-Ib, M(r) 140,000). PUL-Ia was accumulated intracellularly in large amounts, and PUL-Ib was detected in both the membrane fraction and the fraction trapped between the cytoplasmic membrane and the cell wall. Two forms of AP were purified to homogeneity and their properties were compared with previously purified PUL-E (140 kDa). PUL-Ib showed similar properties, such as the M(r) value, the pI value (5.7), specific activity, substrate specificity, the NH2-terminal amino acid sequence (Phe-Leu-Asn-Met-Ser), and biophysical characters. However, in the case of PUL-Ia, even though the patterns of optimum pH and temperature, substrate specificity, and enzyme inhibition and activation were similar to those for PUL-Ib and PUL-E, the M(r) value and the pI value (5.97) were different. Furthermore, the NH2-terminal amino acid sequence of PUL-Ia was completely blocked, and the stabilities over pH and temperature ranges were decreased. The catalytic activities of PUL-Ia were distinguishable in the Km and Vmax values for various substrates and in the specific activity (71.4 U/mg) for pullulan hydrolysis. PUL-Ib and PUL-E showed 10-fold higher specific activities (744.6 for PUL-E and 736 for PUL-Ib) than PUL-Ia. However, PUL-Ia was immunologically identical to PUL-E and PUL-Ib. Therefore, it was concluded that PUL-Ib and PUL-E are the same form of the enzyme, suggesting that PUL-Ia is initially synthesized and proteolytically processed to the mature form of PUL-E. On the other hand, the translocation of AP required processing of the AP protein and the processing facilitated enzymatic activation and stabilization through a complete conformational change, resulting in an increase in affinity for substrates of PUL-E.

Azides↗

PubMed1975

The secretion of enzymes into the pericellular environment.

Connective tissue cells are capable of both synthesizing and degrading the macromolecular components of the extracellular matrix. The degradation of proteoglycan and collagen has been shown to be associated with the extracellular release of proteolytic enzymes, some of which are of lysosomal origin. The identity in carilage of two previously unrecognized proteases, capable of proteoglycan breakdown (CPGases), has recently been achieved by the use of a new assay for proteoglycan degradation. These enzymes have been shown to be synthesized and released in response to vitamin A. The third proteoglycan degrading enzyme of connective tissue cells, cathepsin D, has been located in the pericellular environment by trapping with specific antibody and the pattern of release studied in organ culture, experimental arthritis and in human rheumatoid tissues. The secretion of this enzyme and possibly also of the other CPGases is thought to be of importance in the local (pericellular) turnover of matrix macromolecules and, in association with collagenase, to be the cause of the excessive degradation in the pannus erosion of articular cartilage in rheumatoid arthritis.

Animals↗

PubMed2004

Adherence of Staphylococcus aureus fibronectin binding protein A mutants: an investigation using optical tweezers.

Bacterial adhesion to extracellular matrix proteins plays a major role in infections of host tissue and medical devices. In some species of gram-positive cocci, this adhesion is mediated by specific molecules present on the bacterial cell surface. We have used optical tweezers to dynamically measure the adhesive force between an individual Staphylococcus aureus bacterium and a fibronectin-coated surface. A bacterium was optically trapped and brought in contact with a 10-microm diameter polystyrene microsphere coated with fibronectin. The force required to detach the cell from the microsphere was measured by tracking the displacement signals of the trapped cell on a quadrant photodiode throughout the detachment process for a series of S. aureus strains expressing fibronectin-binding proteins with various degrees of mutation. The single-bond rupture forces ranged between 15 and 26 pN depending on the extent of mutation. No binding was observed in the strain with the highest degree of mutation. These results confirm that multiple regions of the S. aureus fibronectin adhesin participate in the binding process and provide further insight into the role of these regions in the adhesive process.

Adhesins, Bacterial↗

PubMed1993

Significance of the extracellular domain and the carbohydrates of the human neutrophil N-formyl peptide chemotactic receptor for the signal transduction by the receptor.

The N-formyl peptide chemotactic receptor of human neutrophils possesses a 2-kDa papain-removable extracellular domain that contains two N-linked oligosaccharide side chains and is not required for the high-affinity ligand binding. In the present study, the significance of the extracellular domain and the carbohydrates for signal transduction was elucidated by measuring the N-formyl hexapeptide-induced intracellular free calcium ([Ca2+]i) and the change in myeloperoxidase secretion in the control and papain-treated human neutrophils. [Ca2+]i was monitored both in cell suspension and individual cells with intracellularly trapped Fura 2 acetoxymethyl ester, using spectrofluorometric analysis and fluorescence ratio image analysis, respectively. The exposure of the cells in suspension to N-formyl hexapeptide resulted in an immediate, dose-dependent burst of elevated [Ca2+]i, which was virtually identical in both control and papain-treated cells with respect to the extent and kinetics. The maximum burst was 1.6-fold and was obtained at 10(-6) M hexapeptide. The individual control and papain-treated cells responded to 10(-6) M hexapeptide in a similar manner with several successive transients of [Ca2+]i, the maximum level being 3.0-3.5 microM. In both groups the [Ca2+]i transient began initially in the cell periphery, expanding rapidly throughout the cells. Concomitantly, the cells became polarized, and their chemokinesis increased. The secretion of myeloperoxidase was monitored as a physiological end response to N-formyl chemotactic peptides. The exposure of the control and papain-treated cells in suspension to hexapeptide resulted in a dose-dependent secretion of myeloperoxidase. The maximum secretion after exposure to 10(-8)-10(-6) M hexapeptide was equal in control and papain-treated neutrophils. These results indicate that the functional properties of the membrane-inserted N-formyl peptide chemotactic receptor are inherent to the receptor's transmembrane and intracellular domains, as far as binding of the ligand and subsequent receptor activation are concerned.

Calcium↗

PubMed2003

Delayed puberty and primary amenorrhea associated with a novel mutation of the human follicle-stimulating hormone receptor: clinical, histological, and molecular studies.

Inactivating mutations of the FSH receptor have been described in rare cases of premature ovarian failure. Only one mutation was associated with a complete phenotype, including delayed puberty, primary amenorrhea, and small ovaries. We describe here a new patient presenting a similar complete phenotype of premature ovarian failure, with high plasma FSH levels associated with very low estrogen and inhibin B levels. No biological response to high doses of recombinant FSH was detected. A novel homozygous Pro(519)Thr mutation was found in this patient. This mutation is located in the second extracellular loop of the FSH receptor, within a motif highly conserved in gonadotropin and TSH receptors. The mutation totally impairs adenylate cyclase stimulation in vitro. FSH binding experiments and confocal microscopy showed that this mutation alters the cell surface targeting of the mutated receptor, which remains trapped intracellularly. Histological studies of the ovaries of the patient showed an increase in the density of small follicles compared with age-matched normal women. A complete block in follicular maturation after the primary stage was also observed. Immunocytochemical studies allowed detection of the expression of c-Kit and proliferation cellular nuclear antigen, whereas no apoptosis was shown by the 3'-end-labeling method. This observation supports the concept that in humans FSH seems mandatory for the initiation of follicular growth only after the primary stage. In our patient complete FSH resistance yields infertility, which is remarkably associated with the persistence of a high number of small follicles.

Adult↗

PubMed2004

Protease degradomics: mass spectrometry discovery of protease substrates and the CLIP-CHIP, a dedicated DNA microarray of all human proteases and inhibitors.

The biological role of most proteases in vivo is largely unknown. Therefore, to develop robust techniques to analyze the protease degradome in cells and tissues and to elucidate their substrate degradomes we have developed a dedicated and complete human protease and inhibitor microarray that we have called the CLIP-CHIP Oligonucleotides (70-mers) for identifying all 715 human proteases, inactive homologs and inhibitors were spotted in triplicate onto glass slides with a dedicated subarray containing oligonucleotides for specific human breast carcinoma genes. Initial analyses revealed the elevated expression of a number of proteases in invasive ductal cell carcinoma including ADAMTS17, carboxypeptidases A5 and M, tryptase-gamma and matriptase-2. Matrix metalloproteinases (MMPs) showed a restricted expression pattern in both normal and cancerous breast tissues with most expressed at low levels. However, of the several MMPs expressed in significant quantities, the carcinoma samples showed only slightly elevated amounts other than for MMP-28 which was strongly elevated. To discover new protease substrates we developed a novel yeast two-hybrid approach we term 'inactive catalytic domain capture' (ICDC). Here, an inactive mutant protease catalytic domain lacking the propeptide was used as a yeast two hybrid bait to screen a human fibroblast cDNA library for interactor proteins as a substrate trap. Wnt-induced signaling protein-2 (WISP-2) was identified by ICDC and was biochemically confirmed as a new MMP substrate. In another approach we used isotope-coded affinity tag (ICAT) labeling with tandem mass spectrometry to quantitate the levels of secreted or shed extracellular proteins in MDA-MB-231 breast carcinoma cell cultures in the presence or absence of membrane type 1-MMP (MT1-MMP) overexpression. By this proteomic approach we identified and biochemically confirmed that IL-8, the serine protease inhibitor SLPI, the death receptor-6, pro-TNF-alpha and CTGF are novel substrates of MT1-MMP. The utility and quantitative nature of ICAT with MS/MS analysis as a new screen for protease substrate discovery based on detection of cleaved or shed substrate products should be readily adaptable to other classes of protease for assessing proteolytic function in a cellular context.

Breast Neoplasms↗