Integrins regulate opioid receptor signaling in trigeminal ganglion neurons.

The binding of integrins to the extracellular matrix results in focal organization of the cytoskeleton and the genesis of intracellular signals that regulate vital neuronal functions. Recent evidence suggests that integrins modulate G-protein-coupled receptor (GPCR) signaling in hippocampal neurons. In this study we evaluated the hypothesis that integrins regulate the mu opioid receptor in rat trigeminal ganglion neurons. For these studies, primary cultures of adult rat trigeminal ganglion neurons were used to demonstrate the colocalization of beta1 and beta3 integrins with mu opioid receptor in caveolin-1-rich membrane fractions, and at focal adhesions sites generated by integrin ligand binding. Furthermore, we show that the mu opioid receptor agonist, DAMGO ([D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin), inhibits cyclic AMP (cAMP) accumulation in response to prostaglandin E2 (PGE(2)) stimulation in bradykinin-primed, but not unprimed, cultured trigeminal ganglia neurons. Application of soluble GRGDS (Gly-Arg-Gly-Asp-Ser) peptides that bind specific integrins (i.e. RGD-binding integrins) completely abolished the DAMGO effect in bradykinin-primed trigeminal ganglia neurons, but did not alter bradykinin-mediated hydrolysis of phosphatidylinositol. Likewise, monospecific anti-beta1 and anti-beta3 integrin subunit antibodies blocked this DAMGO effect in bradykinin-primed trigeminal ganglia neurons. Indeed, application of anti-beta1 integrin subunit actually reversed DAMGO signaling, resulting in increased cAMP accumulation in these cells. This suggests that the relative amounts of specific activated integrins at focal adhesions may govern signaling by the mu opioid receptor, perhaps by altering interactions with G proteins (e.g. Galphai vs. Galphas). Collectively, these data provide the first evidence that specific integrins regulate opioid receptor signaling in sensory neurons.

alpha-Crystallin facilitates the reactivation of hydrogen peroxide-inactivated rhodanese.

It was previously shown that rhodanese, inactivated with hydrogen peroxide, could only be reactivated in the presence of a reductant or the substrate thiosulfate if these reagents were added soon after inactivation and if the oxidant was removed. Here, we report on the facilitated reactivation (75%) of hydrogen peroxide-inactivated rhodanese by the chaperone alpha-crystallin. Reactivation by the chaperone still required a reductant and thiosulfate. Without alpha-crystallin, but in the presence of the reductant and thiosulfate, the inactivated enzyme regained about 39% of its original activity. The alpha-crystallin-assisted reactivation of hydrogen peroxide-inactivated rhodanese was independent of ATP. Further, we found, that alpha-crystallin interacted transiently, but could not form a stable complex with hydrogen peroxide-inactivated rhodanese. Unlike in prior studies that involved denaturation of rhodanese through chemical or thermal means, we have clearly shown that alpha-crystallin can function as a molecular chaperone in the reactivation of an oxidatively inactivated protein.

Iron-dependent generation of free radicals: plausible mechanisms in the progressive deterioration of the temporomandibular joint.

PURPOSE: The purposes of this study were 1) to determine whether iron concentrations detected in temporomandibular joint (TMJ) lavage fluid samples obtained from symptomatic patients are sufficient to catalyze the degradation of specific extracellular matrix (ECM) molecules in vitro, and 2) to provide evidence of oxidative stress in symptomatic TMJs by the detection of protein carbonyls in lavage fluids. PATIENTS AND METHODS: Iron concentrations in TMJ lavage samples (19 joints in 14 patients) were determined colorimetrically, and the ability of the sample to produce free radicals in the presence of hydrogen peroxide was determined with the chromogen 2,2'-azinobis (3-ethylbenzothizoline-6-sulfonic acid), diammonium salt (ABTS). The presence of oxidized proteins was measured fluorimetrically using Bodipy FL hydrazide (Molecular Probes, Eugene, OR). Degradation of fibronectin was visualized by Western blot. Relative susceptibilities of fibronectin and collagen I to free radical cleavage were measured with the Fenton reaction. RESULTS: Redox-active iron concentration in lavage samples was found to be as high as 3.66 micromol/L. A 70-kd protein band, presumed to be albumin, was found to contain higher levels of carbonyls than peripheral serum albumin, which correlated with a greater degree of oxidative damage. Fibronectin was found to be more susceptible than collagen I to free radical degradation, and fragments of the former were found in the lavage. The TMJ lavage fluid was capable of producing free radicals in the presence of peroxide. CONCLUSION: Circumstantial evidence is provided that the presence of modified and cleaved proteins isolated from lavage of symptomatic TMJs may have been subjected to oxidative stress.

Particulate retrieval of hydrolytically degraded poly(lactide-co-glycolide) polymers.

This article describes a technique for the retrieval of polymeric particulate debris following advanced hydrolytic in vitro degradation of a biodegradable polymer and presents the results of the subsequent particle analysis. Granular 80/20 poly(L-lactide-co-glycolide) (PLG) was degraded in distilled, deionized water in Pyrextrade mark test tubes at 80 degrees C for 6 weeks. Subsequently, a density gradient was created by layering isopropanol over the water, followed by a 48-h incubation. Two opaque layers formed in the PLG tubes, which were removed and filtered through 0.2-micrometer polycarbonate membrane filters. In addition, Fourier transform IR spectroscopy (FTIR) was performed to confirm the presence of polymer in the removed layers. The filters were gold sputter coated, and scanning electron microscopy (SEM) images were made. FTIR analysis confirmed that the removed material was PLG. SEM images of the extracts from the upper (lowest density) opaque layer showed a fine, powderlike substance and globular structures of 500-750 nm. The SEM images of the lower (highest density) opaque layer showed particles with a crystalline-like morphology ranging in size from 4 to 30 micrometer. Particulate PLG debris generated with the described technique can be useful for further studies of its biological role in complications associated with poly(alpha-hydroxy)ester implants. This study shows the presence of very persistent nano- and microparticles in the degradation pathway of PLG.

Detection and preliminary characterization of matrix metalloproteinase activity in temporomandibular joint lavage fluid.

In this study, lavage fluid was fractionated from the superior joint space in patients with temporomandibular joint (TMJ) dysfunction. A hide powder azure protease assay was used to assess protease activity in lavage fluid. No correlation between a patient's pain and the level of protease activity was demonstrated. Latent as well as active proteases were detected in the sample lavage fluid. Latent matrix metalloproteinases (MMPs) were activated using trypsin. Stromelysin-1 was detected in an active form in lavage fluid by immunozymography. The presence of high molecular weight species with protease activity was also demonstrated. This study validates the presence of stromelysin-1 as well as other MMPs in TMJ lavage fluid and proposes a mechanism for their physiologic activation.

Oxidative stress and degenerative temporomandibular joint disease: a proposed hypothesis.

The molecular events that underlie degenerative temporomandibular joint diseases are poorly understood. Recent studies have provided evidence that a variety of molecular species, including cytokines, matrix degrading enzymes, neuropeptides, and arachidonic acid catabolites may be involved. This paper advances the theory that mechanical stresses lead to the accumulation of damaging free radicals in affected articular tissues of susceptible individuals. This condition is called oxidative stress. The authors postulate mechanisms that may be involved in the production of free radicals in the temporomandibular joint and in the subsequent induction of molecular events that may amplify damage of articular tissues initiated by free radicals. If the proposed model is correct, then future therapeutic strategies directed at the control of oxidative stress could be effective in the management of degenerative temporomandibular joint diseases.

Presence of denatured hemoglobin deposits in diseased temporomandibular joints.

PURPOSE: The purpose of this study was to test the hypotheses that hemoglobin recovered by arthrocentesis of the superior joint space of symptomatic human temporomandibular joints (TMJs) is "old" hemoglobin that was not iatrogenically introduced by the arthrocentesis procedure and that it exists primarily in a non-native or denatured conformation state that may be sufficient to catalyze a reaction leading to the formation of damaging free radicals. PATIENTS AND METHODS: Twelve patients diagnosed with a unilateral articular disk displacement with TMJ arthralgia were included in this study. A superior joint space arthrocentesis was performed in the affected TMJ, and outflow lavage volumes were collected in serial 2-mL fractions. alpha-Hemoglobin/albumin ratios were determined for each collected fraction by densitometric analysis of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, 3,3',5,5'-tetramethylbenzidine (TMB) assays were used to determine the conformation state of the recovered hemoglobin. RESULTS: High alpha-hemoglobin/albumin ratios relative to that of serum (at least 10 times greater) were observed in several collected fractions of TMJ lavage fluid in all subjects studied. Because the tissue half-life of hemoglobin is significantly longer than that of albumin, these findings indicate that much of the hemoglobin recovered by arthrocentesis of symptomatic TMJs represents "old" hemoglobin that was present in the joint before the procedure. Furthermore, based on reactivity in the TMB assay, we estimate that up to 89% of the alpha-hemoglobin present in TMJ lavage fluid samples exists in a denatured state. CONCLUSIONS: These results indicate that a significant amount of hemoglobin recovered by arthrocentesis of symptomatic TMJs exists in a denatured state and was present in the joint before arthrocentesis. Recent studies suggest that denatured hemoglobin may contribute redox active iron that can catalyze a reaction, leading to the formation of damaging free radicals. Such a process may represent one of the earliest molecular events involved in the pathogenesis of degenerative TMJ disease.

Elution of proteins by continuous temporomandibular joint arthrocentesis.

PURPOSE: The purpose of this study was to determine whether specific proteins recovered from human temporomandibular joints (TMJs) by superior space arthrocentesis are eluted at different outflow volumes. MATERIALS AND METHODS: Twenty subjects with unilateral TMJ pain and restricted mandibular range of motion underwent superior space arthrocentesis of the affected TMJ. Sixteen serial fractions of the arthrocentesis outflow volume were collected for analysis. The protein content of each fraction was determined by a BCA protein assay and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, samples from each collected fraction were assayed for protease activity. RESULTS: The average amount of protein recovered in the total 32 mL of collected arthrocentesis fluid was 1.5 mg (0.72 to 2.1 mg). Significant differences (P = .03) in total protein recovered from arthrocentesis fluid were observed between males (0.824 +/- 0.43 mg/20 mL) and females (1.389 +/- 0.54 mg/20 mL). In general, protein concentration declined serially in collected TMJ lavage fluid fractions. Specific proteins and proteases detected in the lavage fluid were eluted at different outflow volumes. CONCLUSIONS: Although specific proteins are eluted from the TMJ at different outflow volumes during arthrocentesis, the procedure effectively reduces the protein concentration of the lavage fluid in a volume-dependent manner. Based on empirical assumptions, it is estimated that approximately 100 mL of total arthrocentesis volume is sufficient for a therapeutic lavage of the superior joint space of the human TMJ.

Protein interactions with particulate Teflon: implications for the foreign body response.

PURPOSE: This study examined the nature of protein interactions with particulate polytetrafluoroethylene (PTFE, Teflon) to elucidate possible mechanisms involved in the foreign body response directed against failed Proplast/Teflon implants. MATERIALS AND METHODS: Fifty milligrams PTFE prepared to particle sizes ranging from < 32 microns to > 300 microns was incubated with newborn bovine serum. The total amount of protein adsorbed to the PTFE particles was determined using a standard colorimetric assay. The structural and functional integrity of the proteins adsorbed to PTFE was also examined. For these studies, xanthine oxidase was substituted for serum, and the enzymatic activity of xanthine oxidase adsorbed to PTFE was determined. Finally, primary interactions between protein and PTFE particles were assessed in experiments using water, 2 or 8 mol/L urea, 1 mol/L Nacl, or 1% sodium dodecyl sulfate in an attempt to dissociate bound protein from the surfaces of PTFE particles. RESULTS: Serum proteins bind almost instantly to the surface of PTFE particles. The effective surface area of PTFE increases dramatically with reduction of the material to small particles, as does the total amount of protein adsorbed by the particulate PTFE. Proteins bind to PTFE principally by hydrophobic interactions, and their three-dimensional structure is significantly perturbed by this interaction. In the case of xanthine oxidase, adsorption to PTFE distorts protein structure to the extent that biologic activity is eliminated. CONCLUSIONS: The amount of serum protein adsorbed to PTFE particles varies inversely with particle size for a constant mass of material. It is believed that the foreign body response directed against this material is related to the amount and relative distortion of proteins adsorbed to its surface. If so, it appears that reduction of an implant to small particles (typically 50 micron or less) will dramatically increase the biologic signal to local cell populations. Thus, the severity of the biologic response to PTFE debris may be dependent largely on the size of the debris particles.

Protein refolding at high concentrations using detergent/phospholipid mixtures.

Refolding of the sulfurtransferase enzyme rhodanese (EC 2.8.1.1) at high concentrations (0.2 mg/ml) was obtained at significant yields (> or = 45%) by using mixtures composed of detergents (either Triton X-100 or lauryl maltoside) and phospholipids. It is presumed that the ratio of detergent to phospholipid used in these mixtures results in the formation of very large micellar structures. Protein folding at lower concentrations (0.02 mg/ml) gave high yields (94%) when using the Triton X-100/phosphatidylglycerol micelle and these yields were higher than those obtained with either detergents or chaperonins. Differences between mixed micelle and liposome-mediated protein folding were clarified. We postulate that the method described here is successful at preventing misfolding and/or aggregation, and promoting correct folding, because the mixed micelle has both polar and nonpolar moieties which can bind to various exposed interactive sites in either unfolded proteins or protein folding intermediates. In this report, we detail the factors that allow the recovery of large amounts of active protein. This methodology should facilitate the large-scale production of biologically active, medically important proteins.

Physical characterization of a reactivatable liposome-bound rhodanese folding intermediate.

Recently, we described the formation of a complex between liposomes and the unfolded protein rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1), which could be liberated and efficiently reactivated after treatment of the complex with detergents [Zardeneta, G., & Horowitz, P. M. (1992) Eur. J. Biochem. 210, 831-837]. Previous data suggested that liposome-bound rhodanese was in the form of a folding intermediate. We have characterized in greater detail the nature of the conformation of the bound rhodanese. Physical characterization of the bound rhodanese intermediate was carried out using proteolysis, fluorescence studies with 1,8-anilinonapthalene-8-sulfonic acid, a probe for hydrophobic site exposure, intrinsic fluorescence to determine tryptophan accessibility using the quenchers acrylamide and iodide, and circular dichroism to detect extent of secondary structure. These studies show that the rhodanese intermediates bound to either cardiolipin or phosphatidylserine liposomes are not identical, the former being in a less compact conformation yet having more secondary structure than the latter, an observation which may explain why the reactivation of the former intermediate is more effective. Finally, turbidimetric and proteolytic studies raise the possibility that each rhodanese intermediate binds to several liposomes. This finding suggests that a possible reason for the differential reactivation yields obtained may be due to the fact that unfolded rhodanese has more binding sites for cardiolipin than for phosphatidylserine liposomes. A greater number of binding sites would result in better anchoring of rhodanese's interactive surfaces and thus reduce the likelihood of misfolding.

GroEL and GroES increase the specific enzymatic activity of newly-synthesized rhodanese if present during in vitro transcription/translation.

Enzymatically active mammalian rhodanese, a mitochondrial matrix enzyme, which has been found to require assistants for efficient refolding in vitro, has been synthesized from a plasmid in a cell-free, fractionated, coupled transcription/translation system derived from Escherichia coli. The bacterial chaperonins, GroEL and GroES, along with the rhodanese substrate thiosulfate greatly enhance the specific enzymatic activity of the rhodanese polypeptide that is formed. Indirect evidence suggests that the effect of the GroEL/ES chaperonins is on ribosome-bound nascent peptides. The in vitro transcription/translation system produces sufficient amounts of rhodanese to provide a system for studying factors that control the initial steps in folding of nascent proteins.

Analysis of the perturbation of phospholipid model membranes by rhodanese and its presequence.

The ability of the cytoplasmically synthesized mitochondrial enzyme rhodanese and its putative import signal sequence to interact with model phospholipid membranes was characterized. Membrane perturbation assays were used to test a current hypothesis that the initial step in protein translocation may involve binding of signal sequences with membrane lipids. Here we show comparative studies on the effect of native and various forms of denatured rhodanese, as well as two peptides, rho(1-23) and rho(11-23), derived from its NH2-terminal sequence, on the perturbation of 6-carboxyfluorescein-containing large unilamellar vesicles composed of either cardiolipin, phosphatidylcholine, or phosphatidylserine. We monitored the degree of perturbation by measuring dye leakage and found differential perturbation by either peptide or protein. Unfolded rhodanese perturbed vesicles in the order phosphatidylserine > cardiolipin >> phosphatidylcholine. Denatured rhodanese was approximately 25 times more effective (on a molar basis) than rho(1-23) in the disruption of anionic liposomes. Rho(11-23) was unable to perturb liposomes. We found an inverse correlation between degree of activity of rhodanese folding intermediates and their ability to perturb liposomes. On urea denaturation, enzymatic activity was completely lost before membrane perturbation ability reached significant levels. Analysis of the peptides by circular dichroism showed that anionic liposomes can induce alpha-helical structure only in rho(1-23) and denatured rhodanese. Intrinsic peptide fluorescence studies showed that only rho(1-23) and denatured rhodanese partitioned into these model membranes. Results obtained here imply that peptides from naturally occurring alpha-helical structures may need adjacent motifs for helical structure induction in lipid environments, and the subsequent secondary structure may, in turn, promote partitioning of these segments into the lipid phase and ultimately lead to membrane perturbation.

Cardiolipin liposomes sequester a reactivatable partially folded rhodanese intermediate.

The interaction was studied between the mitochondrial enzyme thiosulfate sulfurtransferase and liposomes, in the form of large unilamellar vesicles (LUV), prepared from either cardiolipin (CL), PtdCho or PtdSer. At equivalent concentrations of lipid, more partially folded thiosulfate sulfurtransferase bound to CL/LUV than to PtdSer/LUV, and only traces were bound to PtdCho/LUV. Native thiosulfate sulfurtransferase did not bind to any of these LUV. We show that CL/LUV-sequestered thiosulfate sulfurtransferase is inactive but may be reactivated (approximately 56%) with the aid of detergents, thiosulfate, beta-mercaptoethanol and phosphate buffer. Reactivations in the presence of PtdSer/LUV or PtdCho/LUV was only 9% or 1%, respectively. Analysis of the complex by protease digestion and fluorescence spectroscopy indicated that thiosulfate sulfurtransferase was held by CL/LUV and PtdSer/LUV as a folding intermediate. Data presented here suggest that detergents may not interact directly with the protein, but, rather, their primary role in reactivation is to disrupt the LUV, allowing flexibility to the anchored thiosulfate sulfurtransferase molecule, thereby promoting folding. These studies complement other reports which imply a possible role for CL in protein translocation across the mitochondria, since we find that CL binds to thiosulfate sulfurtransferase and sequesters it in a translocation-competent prefolded conformation, which may readily lead to a correctly folded enzyme.

Immunohistological localization of cell adhesion proteins and integrins in the periodontium.

The distribution of the cell adhesion proteins vitronectin, fibronectin, tenascin, and laminin as well as several integrin subunits, alpha 2, alpha 5, and alpha v, was studied in primate periodontal tissues. Full baboon mandibular sections were analyzed by immunohistochemical methods in order to localize the molecules studied in both soft and hard tissues. Vitronectin was associated with the connective tissue of the marginal gingiva, the periodontal ligament, as well as the endosteum and periosteum. A notable finding was the particularly high staining intensity of vitronectin in the periodontal ligament. Fibronectin was widely distributed in the periodontal connective tissue and was also localized to the pericellular matrix of osteocytes and blood vascular elements. Epithelial basement membranes stained positively for both fibronectin and tenascin. These proteins were also expressed in the periosteal and endosteal connective tissues and the periodontal ligament. The staining intensity for tenascin was higher in zones along the cementum and bone surfaces. Laminin was, characteristically, limited to basement membranes of epithelium and endothelium. The distribution of fibronectin, tenascin, and laminin is related to previous findings in other species. The localization of the several integrin alpha-subunits is also described in full baboon mandibular sections. The vitronectin receptor (alpha v) had a uniquely strong expression in osteoclasts of the alveolar bone and was found, at lesser intensity, on periodontal ligament fibroblasts. The fibronectin receptor alpha subunit, alpha 5, was also observed on osteoclasts, and, in addition, was widely distributed on fibroblasts, cementoblasts, and osteoblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Micelle-assisted protein folding. Denatured rhodanese binding to cardiolipin-containing lauryl maltoside micelles results in slower refolding kinetics but greater enzyme reactivation.

Unfolded (inactive) rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) can be reactivated in the presence of detergents, e.g. lauryl maltoside (LM). Here, we report the reactivation of urea-unfolded rhodanese in the presence of mixed micelles containing LM and the anionic mitochondrial phospholipid, cardiolipin (CL). Reactivation times increased as the number of CL molecules/micelle was increased. A maximum of 94% of the activity was recovered at 2.2 CL/micelle. Only 71% of the activity was recovered in the absence of CL. The major zwitterionic mitochondrial phospholipid, phosphatidylcholine (PC), had no effect on the LM-assisted reactivation of rhodanese. Size exclusion chromatography showed that denatured, but not native, rhodanese apparently binds to micellar amounts of LM and CL/LM, but not to PC/LM micelles. The lifetime of the enzyme-micelle complex increased with the number of CL molecules/micelle. Furthermore, chromatographic fractions containing micelle-bound enzyme had no activity, while renatured rhodanese-containing fractions were active. These results suggest that transient complexes form between enzyme and both LM and CL/LM micelles, and that this complex formation may be necessary for reactivation. For CL/LM micelles, interactions may occur between the positively charged amino-terminal sequence of rhodanese and the negatively charged CL phosphate. Finally, this work shows that there are similarities between "micelle-assisted" and chaperonin-assisted rhodanese refolding.

Quantitative assay for morphogenesis indicates the role of extracellular matrix components and G proteins.

A quantitative assay for morphogenesis is described that involves counting the organizing centers (swirling patterns) formed by many cultured fibroblasts. Organizing centers, which are found in vivo, represent one of the smallest units of morphogenesis. We show that macroscopically visible organizing centers form by the merger of smaller organizing centers. Parallel orientation of cells on plastic substrata requires cell-cell contact, but organizing centers can develop without cell-cell contact on collagen gels. On collagen gels, the orientation of collagen fibers determines the orientation of cells with respect to one another. Although organizing centers resemble fingerprints, we have shown that a stochastic process determines the spatial orientation of organizing centers. Treatment of transformed cell lines with agents that increase cAMP levels or alter the activity of guanine nucleotide binding proteins resulted in the generation of organizing centers. Cholesterol precursors involved in protein isoprenylation were found to be potent reverse-transformation agents that could alter the two-dimensional morphogenesis of cells. The simple assay described should permit the analysis of morphogenesis at the molecular and cellular levels.