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Human α2-macroglobulin (A2M) is the most characterized protease inhibitor in the alpha-macroglobulin (αM) superfamily, but the structure of its native conformation has not been determined. Here, we combined negative stain electron microscopy (EM), small-angle X-ray scattering (SAXS), and cross-linking–mass spectrometry (XL-MS) to investigate native A2M and its collapsed conformations that are obtained through aminolysis of its thiol ester by methylamine or cleavage of its bait region by trypsin. The combined interpretation of these data resulted in a model of the native A2M tetramer and its conformational changes. Native A2M consists of two crescent-shaped disulfide-bridged subunit dimers, which face toward each other and surround a central hollow space. In native A2M, interactions across the disulfide-bridged dimers are minimal, with a single major interface between the linker (LNK) regions of oppositely positioned subunits. Bait region cleavage induces both intrasubunit domain repositioning and an altered configuration of the disulfide-bridged dimer. These changes collapse the tetramer into a more compact conformation, which encloses an interior protease-trapping cavity. A recombinant A2M with a modified bait region was used to map the bait region’s position in native A2M by XL-MS. A second recombinant A2M introduced an intersubunit disulfide into the LNK region, demonstrating the predicted interactions between these regions in native A2M. Altogether, our native A2M model provides a structural foundation for understanding A2M’s protease-trapping mechanism, its conformation-dependent receptor interactions, and the dissociation of native A2M into dimers due to inflammatory oxidative stress., Graphical Abstract, Highlights • Native A2M is hollow and tube-like. • A2M’s bait regions are oriented inward and are accessed from inside A2M. • A2M tetramerizes through symmetrical interactions between its LNK regions. • The receptor-binding site is in an inaccessible position inside native A2M., In Brief The native conformation of the protease inhibitor A2M was investigated using negative stain electron microscopy, small-angle X-ray scattering, and cross-linking mass spectrometry. The low-resolution model built from these data describes a hollow tubular configuration that explains several aspects of A2M’s unique trapping mechanism. This model was further validated by two recombinantly expressed A2M mutants, which probed the location of the bait region and demonstrated the existence of a critical interface between A2M’s disulfide-bridged dimers.

Le placenta est un organe généré pendant la grossesse qui a pour objectif d'assurer le bon développement fœtal. L'un des principaux types cellulaires placentaires est le syncytiotrophoblaste formé par un processus appelé syncytialisation, consistant en la fusion et différentiation de cytotrophoblastes. Nous avons établi que la «glucose-regulated protein 78» (GRP78), une protéine connue pour réguler la «unfolded protein response» (UPR) et pour agir comme récepteur à la surface cellulaire, est impliquée dans la syncytialisation. Nous avons démontré que l'UPR est activée pendant la syncytialisation et qu'elle permet ainsi de réguler l'autophagie et la survie cellulaire nécessaires à la syncytialisation. Par ailleurs, nous avons démontré que la GRP78 à la surface cellulaire interagit avec l'α2-macroglobuline et permet ainsi l'activation de la voie de signalisation ERK1/2-CREB-UPR conduisant à la modulation de la fusion trophoblastique. En conclusion, nous avons démontré le rôle essentiel de la GRP78 dans la régulation de la syncytialisation trophoblastique.

Context: Type-2 diabetes mellitus (DM2) requires an adequate glycemic control to avoid diabetic complications. The best parameter available is glycosylated hemoglobin (HbA1c), as it gives us an overview of an individuals’ glycemic control of the previous 4 months. Salivary biomarkers used as a diagnostic tool can indicate the control or degree of progression of diseases. Studies indicate that salivary alpha-2-macroglobulin (A2MG) levels are elevated in diabetes patients. Aims: To study the relationship of salivary A2MG with glycosylated Hba1c among patients with DM2. Settings and Design: A total of 87 patients of DM2, age 35–65 years were recruited. Materials and Methods: The routine oral cavity examination and dental check-up was done to rule out any dental disease. The patients with hepatic diseases and inflammatory diseases of oral cavity and body were excluded. The values of HbA1c were collected from the records of patients. Salivary A2MG levels were estimated by enzyme-linked immunosorbent assay. Levels of fasting and postprandial blood sugar, serum creatinine, and A2MG were compared with the HbA1c groups (

Introduction: Low Carbohydrate High Protein diet represents a popular strategy to achieve weight loss. Objective: The aim of this study was to characterize effects of low carbohydrate, high protein diet (LCHP) on atherosclerotic plaque development in brachiocephalic artery (BCA) in $apoE/LDLR^{-/-}$ mice and to elucidate mechanisms of proatherogenic effects of LCHP diet. Materials and Methods: Atherosclerosis plaques in brachiocephalic artery (BCA) as well as in aortic roots, lipoprotein profile, inflammation biomarkers, expression of SREBP-1 in the liver as well as mortality were analyzed in Control diet (AIN-93G) or LCHP (Low Carbohydrate High Protein) diet fed mice. Results: Area of atherosclerotic plaques in aortic roots or BCA from LCHP diet fed mice was substantially increased as compared to mice fed control diet and was characterized by increased lipids and cholesterol contents (ORO staining, FT-IR analysis), increased macrophage infiltration (MOMA-2) and activity of MMPs (zymography). Pro-atherogenic phenotype of LCHP fed $apoE/LDLR^{-/-}$ mice was associated with increased plasma total cholesterol concentration, and in LDL and VLDL fractions, increased TG contents in VLDL, and a modest increase in plasma urea. LCHP diet increased SCD-1 index, activated SREBP-1 transcription factor in the liver and triggered acute phase response as evidence by an increased plasma concentration of haptoglobin, CRP or AGP. Finally, in long-term experiment survival of $apoE/LDLR^{-/-}$ mice fed LCHP diet was substantially reduced as compared to their counterparts fed control diet suggesting overall detrimental effects of LCHP diet on health. Conclusions: The pro-atherogenic effect of LCHP diet in $apoE/LDLR^{-/-}$ mice is associated with profound increase in LDL and VLDL cholesterol, VLDL triglicerides, liver SREBP-1 upregulation, and systemic inflammation.

Background: Although there are many available data about renal involvement in patients with beta thalassemia major (TM), the changes in renal functions of other types, such as thalassemia intermedia (TI) and thalassemia minor (TMin), were reported less. Therefore, we aimed to evaluate renal tubular and glomerular functions in patients with three types of beta thalassemia. Methods: This prospective case-control study was conducted on 118 beta-thalassemia patients (49 in TM, 18 in TI and 51 TMin) and 51 healthy controls. Glomerular functions [estimated glomerular filtration rate (GFR), serum cystatin C and urinary protein creatinine ratio] and tubular functions [fractioned sodium excretion (FENa), tubular reabsorption of phosphorus, urinary excretion of uric acid, levels of retinol-binding protein, alpha-1 macroglobulin (alpha-1M), and beta-2 microglobulin, calcium creatinine ratio] were assessed in all patients and controls. Results: The mean ages of the groups and controls at presentation were similar. Although GFR was similar in all patients and control groups, serum levels of cystatin C in patients with TM and TI were significantly higher compared to TMin and controls. Alpha-1M, FENa, urinary excretion of uric acid, and urine protein/creatinine ratio in TM and TI groups were significantly higher than the others. Mean cystatin C level was also higher in patients with TMin compared the controls. However, there were no significant differences according to all tubular and other glomerular functions between TMin and control groups. Conclusions: Although all types of beta thalassemia patients should be closely monitored to prevent further decrease in renal functions, the patients with TI should be considered to have a higher risk of glomerular and tubular deterioration as well as TM. © 2015 © 2015 Taylor & Francis.

Sporadic Alzheimer's disease (AD) is an emerging chronic illness characterized by a progressive pleiotropic pathophysiological mode of actions triggered during the senescence process and affecting the elderly worldwide. The complex molecular mechanisms of AD not only are supported by cholinergic, beta-amyloid, and tau theories but also have a genetic basis that accounts for the difference in symptomatology processes activation among human population which will evolve into divergent neuropathological features underlying cognitive and behaviour alterations. Distinct immune system tolerance could also influence divergent responses among AD patients treated by immunotherapy. The complexity in nature increases when taken together the genetic/immune tolerance with the patient's brain reserve and with neuropathological evolution from early till advance AD clinical stages. The most promising diagnostic strategies in today's world would consist in performing high diagnostic accuracy of combined modality imaging technologies using beta-amyloid 42 peptide-cerebrospinal fluid (CSF) positron emission tomography (PET), Pittsburgh compound B-PET, fluorodeoxyglucose-PET, total and phosphorylated tau-CSF, and volumetric magnetic resonance imaging hippocampus biomarkers for criteria evaluation and validation. Early diagnosis is the challenge task that needs to look first at plausible mechanisms of actions behind therapies, and combining them would allow for the development of efficient AD treatment in a near future. © 2013 Benoît Leclerc and Abedelnasser Abulrob.

Objective: To analyse matrix metalloproteinases (MMPs) and tissue inhibitor-1 of MMPs (TIMP-1) levels in the systemic circulation and synovial fluid (SF) of patients with RA and to compare these levels with inflammatory and collagen degradation markers. Methods: ProMMP-1, -2, -3, -8, -9, TIMP-1, levels of MMP/ α2-macroglobulin complexes, and collagen degradation products were measured by sandwich ELISA, activity assays, and HPLC in paired SF and serum samples from 15 patients with RA and 13 with OA. Results: MMPs were higher in SF of patients with RA than in OA or controls. MMP levels in SF of patients with OA were higher than in controls. In serum, levels of proMMP-3, -8 and -9 were higher in patients with RA than in OA or controls, whereas only proMMP-8 and -9 were higher in serum of patients with OA than in controls. A strong correlation was seen between serum and SF levels of MMP-8 and -9 in RA. Increased levels of MMP/α2-macroglobulin complexes indicated an MMP/TIMP imbalance in serum and SF in RA. SF hydroxyproline correlated significantly with SF levels of proMMP-9 in RA. Conclusions: Systemic MMP-8 and -9 levels represent the situation in the inflamed joint; MMP-9 is likely to be involved in degradation of joint collagen. The hypothesis of MMP/ TIMP imbalance in RA is strengthened. Chemicals / CAS: alpha 2 macroglobulin, 95568-41-5; gelatinase A, 146480-35-5; gelatinase B, 146480-36-6; hydroxyproline, 51-35-4, 6912-67-0; interstitial collagenase, 9001-12-1; stromelysin, 79955-99-0; tissue inhibitor of metalloproteinase 1, 140208-24-8; alpha-Macroglobulins; Collagenases, EC 3.4.24.-; Enzyme Precursors; Hydroxyproline, 51-35-4; Matrix Metalloproteinase 8, EC 3.4.24.34; Matrix Metalloproteinase 9, EC 3.4.24.35; Matrix Metalloproteinases, EC 3.4.24.-; Metalloendopeptidases, EC 3.4.24.-; MMP8 protein, human, EC 3.4.24.-; pro-matrix metalloproteinase 9, EC 3.4.24.-; prostromelysin, EC 3.4.24.-; Tissue Inhibitor of Metalloproteinase-1

Previous experiments have shown that human neoplastic and embryonic glial cell lines synthesize and secrete in culture, alpha 2 macroglobulin (alpha 2M), a broad spectrum proteinase inhibitor present in serum and extracellular fluids. The present study was aimed to investigate the presence of alpha 2M receptors on glial cell membrane, since several non-neural cell types producing alpha 2M also express alpha 2M receptors. By flow cytometric analysis, immunofluorescence and immunoelectronmicroscopy techniques we demonstrate an alpha 2M receptor-related immunoreactivity on the plasma membrane of a human glioma cell line. Ultrastructural experiments reveal a close colocalization of immunoreactivities for alpha 2M and its receptor in clathrin-coated pits and vesicles, structures typically involved in receptor-mediated endocytic pathways.

In this study, we applied the multivariate statistical tool Partial Least Squares (PLS) to analyze the relative importance of 83 plasma proteins in relation to coronary heart disease (CHD) mortality and the intermediate end points body mass index, HDL-cholesterol and total cholesterol. From a Dutch monitoring project for cardiovascular disease risk factors, men who died of CHD between initial participation (1987-1991) and end of follow-up (January 1, 2000) (N = 44) and matched controls (N = 44) were selected. Baseline plasma concentrations of proteins were measured by a multiplex immunoassay. With the use of PLS, we identified 15 proteins with prognostic value for CHD mortality and sets of proteins associated with the intermediate end points. Subsequently, sets of proteins and intermediate end points were analyzed together by Principal Components Analysis, indicating that proteins involved in inflammation explained most of the variance, followed by proteins involved in metabolism and proteins associated with total-C. This study is one of the first in which the association of a large number of plasma proteins with CHD mortality and intermediate end points is investigated by applying multivariate statistics, providing insight in the relationships among proteins, intermediate end points and CHD mortality, and a set of proteins with prognostic value. © 2009 American Chemical Society.

In this study, we applied the multivariate statistical tool Partial Least Squares (PLS) to analyze the relative importance of 83 plasma proteins in relation to coronary heart disease (CHD) mortality and the intermediate end points body mass index, HDL-cholesterol and total cholesterol. From a Dutch monitoring project for cardiovascular disease risk factors, men who died of CHD between initial participation (1987-1991) and end of follow-up (January 1, 2000) (N = 44) and matched controls (N = 44) were selected. Baseline plasma concentrations of proteins were measured by a multiplex immunoassay. With the use of PLS, we identified 15 proteins with prognostic value for CHD mortality and sets of proteins associated with the intermediate end points. Subsequently, sets of proteins and intermediate end points were analyzed together by Principal Components Analysis, indicating that proteins involved in inflammation explained most of the variance, followed by proteins involved in metabolism and proteins associated with total-C. This study is one of the first in which the association of a large number of plasma proteins with CHD mortality and intermediate end points is investigated by applying multivariate statistics, providing insight in the relationships among proteins, intermediate end points and CHD mortality, and a set of proteins with prognostic value. © 2009 American Chemical Society.

Objective: To analyse the effects of leflunomide and methotrexate treatment on matrix metalloproteinase (MMP) activity levels in a2 macroglobulin/MMP (α2M/MMP) complexes in the systemic circulation of rheumatoid arthritis (RA) patients. Methods: A total of 102 RA patients from a prospective, double-blind, randomised clinical trial comparing leflunomide and methotrexate were selected; clinical data and blood samples were collected at baseline, at 4 months and at 1 year. Serum MMP activity levels in α2M were quantified using low molecular weight fluorogenic substrates, indicating the proportion of activated MMPs that were not inhibited by specific tissue inhibitors of MMP (TIMP). Results: Patients had active disease as shown by high disease activity score (DAS, mean of 6.9 and 7.0 for methotrexate and leflunomide patients respectively), which was reduced over the study period (4.2 and 5.2 respectively, p

Alpha macroglobulins (AM) are plasma proteins whose main function is to inactivate proteinases, protecting the tissues from the action of these enzymes. AM have influence on plasma viscosity (PV) and binds monofluorophosphate (MFP), which disturbs its homeostasis. The aim of this work was to evaluate whether the administration with MFP could modify blood viscosity. AM levels (?mol/l), PV (mPa·s), viscosity of red blood cells suspensions in NaCl 9 g/l (VES) and in autologue plasma (VEP) were measured in fifty-day old rats after a single dose of 80 ?mol MFP or after 30 days of treatment with 80 ?mol of MFP. Relative viscosity (RV) was calculated as the ratio VEP/PV. AM and PV increased significantly after 30 min of an oral dose of MFP. Controls (n=6), AM: 19.65±0.85, PV: 1.39±0.01, treated (n=6), AM: 22.88±0.75 (p less than 0.05), PV: 1.76±0.14 (p less than 0.05). After 30 days of treatment with MFP, AM and PV increased significantly. Controls (n=6), AM: 10.76±1.33, PV: 1.19±0.04, treated (n=6), AM: 17.66±1.27 (p less than 0.05), PV: 1.38±0.03 (p less than 0.05). The treatment with MFP modifies neither the VEP nor the RV. These results would indicate that AM and/or MFP did not interact with erythrocyte membrane and did not modify erythrocyte deformability. © 2008 - IOS Press and the authors. All rights reserved.

Objective: To analyse the effects of leflunomide and methotrexate treatment on matrix metalloproteinase (MMP) activity levels in a2 macroglobulin/MMP (α2M/MMP) complexes in the systemic circulation of rheumatoid arthritis (RA) patients. Methods: A total of 102 RA patients from a prospective, double-blind, randomised clinical trial comparing leflunomide and methotrexate were selected; clinical data and blood samples were collected at baseline, at 4 months and at 1 year. Serum MMP activity levels in α2M were quantified using low molecular weight fluorogenic substrates, indicating the proportion of activated MMPs that were not inhibited by specific tissue inhibitors of MMP (TIMP). Results: Patients had active disease as shown by high disease activity score (DAS, mean of 6.9 and 7.0 for methotrexate and leflunomide patients respectively), which was reduced over the study period (4.2 and 5.2 respectively, p

A new, easier and efficient purification method, using Sephacryl and DEAE-Sephacel, of the C-terminal fragment of two alpha-macroglobulins, alpha(2)-M and PZP, is presented. Two larger peptides were identified for each protein as the C-terminal fragment, with molecular weights of approximately 30 kDa and the N-terminal sequences were determined to be SSTQDTV for alpha(2)-M and VALHLS for PZP. The smaller peptides with molecular weights of 18 kDa correspond to a shorter C-terminal sequence of these proteins, and they were determined to be EEFPFA for alpha(2)-M and ALKVQTV for PZP, with no interfering sequences detected. The results confirmed the discriminatory capacity of the purification procedure and the purity of the fragments. This new methodology facilitates biological studies of alpha-macroglobulins, and will enable elucidation of the role the C-terminal region may exert to eliminate alpha-macroglobulin-proteinases complexes from the circulation by the LRP/receptor.

This study focuses on inflammatory aspect of Alzheimer's disease, and the role of AD-related proteins in mechanisms that underlie local chronic inflammation in the vicinity of amyloid plaques. Microglia, as the main immunoresponsive element of the brain, is central to the proposed model. Release of cyto/ chemokines (TNFa, IL-6, MIP-1a/ß, MCP-1, GROa/KC) and nitric oxide is followed as the main parameter of its activity. In order to test potency of several AD relevant proteins (IL-1ß, IL-18, IL-6, a2M) to amplify otherwise weak release- inducing activity of amyloid ß peptide, primary murine microglial culture is used. As expected, when used alone, Aß branches consisting of a fibrillar Aß 1-40/ Aß 1-42 mixture induce a minimal effect on primary microglia in vitro. A stronger response is elicited only when additional proinflammatory stimuli are combined with Aß preparations. Some of the physiologically critical soluble factors, present in chronic inflammatory media in AD, such as cytokine interleukin-1ß and even at the higher extent acute phase protein a2 macroglobulin, appear to be important synergistic partners of Aß. Regulation of microglial release activity by a2M, whose implication in the ongoing pathology was already considered but not really studied in context of occurring inflammatory process, is also reported., Der Fokus der vorliegenden Studie liegt auf dem Entzündungsaspekt im Verlauf der Alzheimererkrankung. Insbesondere wurde hierbei die Rolle der an der Erkrankung beteiligten Proteine, bezogen auf die Mechanismen die der lokalen chronischen Entzündung im Zusammenhang mit Amyloid Plaque zuzuordnen sind, untersucht. Im Zentrum des vorgesteltten Modells stehen die Mikrogliazellen, als die haupt- immunreaktiven Zellen des Gehirns. Die Ausschüttung von Zyto- und Chemokinen (TNFa, IL-6, MIP-1a/ß, MCP-1, GROa/KC) sowie Stickstoff-Monoxid (NO)wurde als Hauptparameter der Aktivität der Mikrogliazellen gemessen. Das Potenzial von verschiedenen Alzheimer relevanten Proteinen (IL-1ß, IL-18, IL-6, a2M) die gegebenenfalls schwache Sekretionssaktivität von Amyloid-ß-Peptid zu verstärken, wurde in primären murinen Mikrogliazellkulturen getestet. Wie zu erwarten, rufen Aß Proben (bestehend aus einer Mischung fibrilliären Aß 1-40/ Aß 1-42) - wenn alleine verwendet - einen minimalen Effekt in primären Mikrogliazellen in vitro hervor. Eine stärkere Reaktion wird nur dann ausgelöst, wenn zusätzliche pro-entzündliche Stimuli mit den Aß Präparaten kombiniert werden. Einige der ausschlaggebenden löslichen physiologischen Faktoren die in dem chronisch-entzündlichen Millieu im betroffenen Gewebe in der AD präsent sind, wie z.B. das Zytokin Interleukin-1ß und das akut-Phasen-Protein a2-Makroglobulin, erscheinen als wichtiger Synergiepartner von Aß. Über die Regulation der Sekrektionsaktivität von Mikrogliazellen durch a2M, dessen Beteiligung am Verlauf der Alzheimer- Erkrankung schon in Betracht gezogen wurde, welches aber nie im Kontext mit den im betroffenen Gewebe ablaufenden Entzündungsprozessen untersucht wurde, wird ebenfalls berichtet.

Objective: To determine protein and activity levels of matrix metalloproteinases 1 and 3 (MMP-1 and MMP-3) in synovial fluid of patients with knee joint injury, primary osteoarthritis, and acute pyrophosphate arthritis (pseudogout). Methods: Measurements were done on knee synovial fluid obtained in a cross sectional study of cases of injury (n=283), osteoarthritis (n=105), and pseudogout (n=65), and in healthy controls (n=35). Activity of MMP-1 and MMP-3 in α2 macroglobulin complexes was measured using specific low molecular weight fluorogenic substrates. ProMMP-1, proMMP-3, and TIMP-1 (tissue inhibitor of metalloproteinase 1) were quantified by immunoassay. Results: Mean levels of proMMP-1, proMMP-3, and TIMP-1 were increased in injury, osteoarthritis, and pseudogout compared with controls. MMP-1 activity was increased in pseudogout and injury groups over control levels, whereas MMP-3 activity was increased only in the pseudogout group. The increase in MMP-1 activity coincided with a decrease in TIMP-1 levels in the injury group. Conclusions: Patients with joint injury have a persistent increase in proMMP-1 and proMMP-3 in synovial fluid and an increase in activated MMPs, which are not inhibited by TIMP. The differences in activation and inhibition patterns between the study groups are consistent with disease specific patterns of MMP activation and/or inhibition in joint pathology. Chemicals / CAS: alpha 2 macroglobulin, 95568-41-5; interstitial collagenase, 9001-12-1; stromelysin, 79955-99-0; tissue inhibitor of metalloproteinase 3, 145809-21-8, 164781-40-2; alpha-Macroglobulins; Matrix Metalloproteinases, EC 3.4.24.-; Tissue Inhibitor of Metalloproteinase-1

Objective: To determine protein and activity levels of matrix metalloproteinases 1 and 3 (MMP-1 and MMP-3) in synovial fluid of patients with knee joint injury, primary osteoarthritis, and acute pyrophosphate arthritis (pseudogout). Methods: Measurements were done on knee synovial fluid obtained in a cross sectional study of cases of injury (n=283), osteoarthritis (n=105), and pseudogout (n=65), and in healthy controls (n=35). Activity of MMP-1 and MMP-3 in α2 macroglobulin complexes was measured using specific low molecular weight fluorogenic substrates. ProMMP-1, proMMP-3, and TIMP-1 (tissue inhibitor of metalloproteinase 1) were quantified by immunoassay. Results: Mean levels of proMMP-1, proMMP-3, and TIMP-1 were increased in injury, osteoarthritis, and pseudogout compared with controls. MMP-1 activity was increased in pseudogout and injury groups over control levels, whereas MMP-3 activity was increased only in the pseudogout group. The increase in MMP-1 activity coincided with a decrease in TIMP-1 levels in the injury group. Conclusions: Patients with joint injury have a persistent increase in proMMP-1 and proMMP-3 in synovial fluid and an increase in activated MMPs, which are not inhibited by TIMP. The differences in activation and inhibition patterns between the study groups are consistent with disease specific patterns of MMP activation and/or inhibition in joint pathology. Chemicals / CAS: alpha 2 macroglobulin, 95568-41-5; interstitial collagenase, 9001-12-1; stromelysin, 79955-99-0; tissue inhibitor of metalloproteinase 3, 145809-21-8, 164781-40-2; alpha-Macroglobulins; Matrix Metalloproteinases, EC 3.4.24.-; Tissue Inhibitor of Metalloproteinase-1

To investigate the effect of modification of biological environmental conditions, one of the factors influencing the healing of anterior cruciate ligament rupture, we performed experimental anterior cruciate ligament ruptures on New Zealand rabbits. After experimental rupture, intra-articular alpha-2 macroglobulin was injected into the knees of the rabbits in the experiment group to prevent structural changes resulting from the enzymatic reactions in the ruptured anterior cruciate ligament. At the end of 10th day of the experiment, we observed that the anterior cruciate ligaments in the experiment group had retained their prerupture brightness and volume when compared with the control group in which intraarticular alpha-2 macroglobulin had not been injected. We also noted that the anterior cruciate ligaments in the experiment group had not retracted or swollen, the incision sites were regular and clean, and they did not show any signs of degeneration. In the histological examination, the anterior cruciate ligaments in the control groups showed disruption of the collagen network and a significant diminution in number of fibroblasts and fibrocytes ( p

Objective: To analyse matrix metalloproteinases (MMPs) and tissue inhibitor-1 of MMPs (TIMP-1) levels in the systemic circulation and synovial fluid (SF) of patients with RA and to compare these levels with inflammatory and collagen degradation markers. Methods: ProMMP-1, -2, -3, -8, -9, TIMP-1, levels of MMP/ α2-macroglobulin complexes, and collagen degradation products were measured by sandwich ELISA, activity assays, and HPLC in paired SF and serum samples from 15 patients with RA and 13 with OA. Results: MMPs were higher in SF of patients with RA than in OA or controls. MMP levels in SF of patients with OA were higher than in controls. In serum, levels of proMMP-3, -8 and -9 were higher in patients with RA than in OA or controls, whereas only proMMP-8 and -9 were higher in serum of patients with OA than in controls. A strong correlation was seen between serum and SF levels of MMP-8 and -9 in RA. Increased levels of MMP/α2-macroglobulin complexes indicated an MMP/TIMP imbalance in serum and SF in RA. SF hydroxyproline correlated significantly with SF levels of proMMP-9 in RA. Conclusions: Systemic MMP-8 and -9 levels represent the situation in the inflamed joint; MMP-9 is likely to be involved in degradation of joint collagen. The hypothesis of MMP/ TIMP imbalance in RA is strengthened. Chemicals / CAS: alpha 2 macroglobulin, 95568-41-5; gelatinase A, 146480-35-5; gelatinase B, 146480-36-6; hydroxyproline, 51-35-4, 6912-67-0; interstitial collagenase, 9001-12-1; stromelysin, 79955-99-0; tissue inhibitor of metalloproteinase 1, 140208-24-8; alpha-Macroglobulins; Collagenases, EC 3.4.24.-; Enzyme Precursors; Hydroxyproline, 51-35-4; Matrix Metalloproteinase 8, EC 3.4.24.34; Matrix Metalloproteinase 9, EC 3.4.24.35; Matrix Metalloproteinases, EC 3.4.24.-; Metalloendopeptidases, EC 3.4.24.-; MMP8 protein, human, EC 3.4.24.-; pro-matrix metalloproteinase 9, EC 3.4.24.-; prostromelysin, EC 3.4.24.-; Tissue Inhibitor of Metalloproteinase-1

The peroxisome proliferator-activated receptor α (PPARα), which is highly expressed in liver, plays key roles in lipid metabolism and inflammation. Interleukin-6 (IL-6) is the principal inducer of acute phase response (APR) gene expression. In the present study, we demonstrate that chronic treatment with the PPARα agonist fenofibrate fully prevents the IL-6-induced APR gene expression in wild-type but not in PPARα-deficient mice. PPARα prevents the IL-6-induced expression of the positive APR genes fibrinogen-α, -β, γ, haptoglobulin, and serum amyloid A and the IL-6-induced suppression of the negative APR gene, major urinary protein. Furthermore, the effect of PPARα on the APR gene expression does not simply consist in a delayed systemic response to IL-6 but occurs directly at the transcriptional level. This global suppression of acute phase gene transcription may be explained by two PPARα-dependent in vivo effects: 1) PPARα activation results in the down-regulation of the IL-6 receptor components gp80 and gp130 in the liver, thereby reducing the phosphorylation and activation of the downstream transcription factors STAT3 and c-Jun that transduce the IL-6 signal; and 2) PPARα reduces the basal expression of the transcription factors CCAAT enhancer-binding protein-α, -β, -δ, which are responsible for immediate and maintained transcription of APR genes. A similar global effect of fenofibrate on acute phase protein expression is observed in hyperlipidemic patients chronically treated with fenofibrate, which displayed decreased plasma concentrations of the positive APR proteins fibrinogen, C-reactive protein, serum amyloid A, plasminogen, and α2-macroglobulin and increased plasma concentrations of the negative APR albumin, underlining the clinical significance of our findings. Chemicals / CAS: alpha 2 macroglobulin, 95568-41-5; fenofibrate, 49562-28-9; fibrinogen, 9001-32-5; peroxisome proliferator activated receptor alpha, 147258-70-6; stress activated protein kinase, 155215-87-5; Antilipemic Agents; Apolipoproteins; Fibrinogen, 9001-32-5; Haptoglobins; Interleukin-6; Procetofen, 49562-28-9; Receptors, Cytoplasmic and Nuclear; Serum Amyloid A Protein; Transcription Factors

The type B-specific lectin from the mushroom Marasmius oreades was immobilized onto Sepharose 4B. The immobilized lectin bound murine laminin and bovine thyroglobulin, glycoproteins that contain the Galalpha1,3Galbeta1,4GlcNAc epitope. This epitope is responsible for hyperacute rejection of xenotransplants from lower mammals to humans, Old World monkeys, or apes. The immobilized lectin also bound a fraction of serum proteins from type B human serum but little or none from type A or O(H) serum. The major protein bound from human B serum was a portion of the alpha2-macroglobulin. Treatment of this fraction with N-glycosidase F resulted in decreased molecular weight of bands associated with alpha2-macroglobulin and loss of their M. oreades lectin reactivity, whereas on treatment with coffee bean alpha-galactosidase, this bound fraction also lost reactivity with M. oreades lectin but became reactive with Ulex europaeus I lectin, suggesting the presence of L-fucosyl-alpha1,2-terminated structures. The presence of blood group epitopes on alpha2-macroglobulin has been detected previously by immunological methods, but this is the first isolation and characterization of the specifically glycosylated fraction of this serum protein. The immobilized lectin also bound a number of proteins from pig, rabbit, and rat serum that were distinct in electrophoretic mobility from the human B-serum components and presumably contain the xenotransplantation epitope among their glycan structures. This report further demonstrates the utility of immobilized lectins in isolating and characterizing glycan structures of naturally occurring glycoproteins.

Objective. The aim of the present study was to analyze α2Macroglobulin/MMP (α2M/MMP) complex formation and to investigate whether MMP activity in α2M/MMP complexes in serum can be used as a disease marker in rheumatoid arthritis (RA). Methods. High and low molecular weight (H/LMW) substrates and inhibitors and size exclusion were used to analyze α2M/MMP complex formation. LMW fluorogenic substrates were used to quantify the level of MMPs in α2M/MMP complexes in the serum of RA patients and healthy controls. Results. Active MMPs were fully inhibited by LMW inhibitor BB94 in the presence of α2M, whereas no inhibition was achieved by HMW inhibitor TIMP-1. Size exclusion analysis showed α2M/MMP complex formation in buffer and in normal plasma spiked with activated MMPs, whichFrom Subject Received Size Categories Beheer-TNO-SharePoint NDIA Weekly Insider 8:00 18 KB indicated α2M/MMP complex formation in the systemic circulation. MMP activity in α2M/MMP complexes in the serum of RA patients was significantly higher than in the serum of healthy controls (P

Elevated MMP activities are implicated in tissue degradation in, e.g., arthritis and cancer. The present study was designed to measure MMP enzyme activity in plasma. Free active MMP is unlikely to be present in plasma: upon entering the circulation, active MMP is expected to be captured by the proteinase inhibitor α2-macroglobulin (α2M). Reconstituted MMP-13/ α2M complex was unable to degrade collagen (MW 300,000) in contrast to the low-molecular-weight fluorogenic substrate (MW < 1500). Limited access of high-MW substrates to the active site of MMPs captured by α2M presents the most likely explanation. Consistently, the high-MW inhibitor TIMP (MW ~ 28,000) was unable to inhibit MMP/α2M enzyme activity, whereas the low-MW inhibitor BB94 (MW ~ 500) effectively suppressed enzyme activity. By using fluorogenic substrates with Dabcyl/Fluorescein as quencher/fluorophore combin-ation, sensitive MMP-activity assays in plasma were achieved. Spiking of active MMP-13 and MMP-13/α2M complex, and inhibitor studies with TIMP-1 and BB94, indicated that active MMPs are efficiently captured by (α2M in plasma. MMP activity was even detected in control plasma, and was significantly increased in plasma from rheumatoid arthritis patients.

Elevated MMP activities are implicated in tissue degradation in, e.g., arthritis and cancer. The present study was designed to measure MMP enzyme activity in plasma. Free active MMP is unlikely to be present in plasma: upon entering the circulation, active MMP is expected to be captured by the proteinase inhibitor α2-macroglobulin (α2M). Reconstituted MMP-13/ α2M complex was unable to degrade collagen (MW 300,000) in contrast to the low-molecular-weight fluorogenic substrate (MW < 1500). Limited access of high-MW substrates to the active site of MMPs captured by α2M presents the most likely explanation. Consistently, the high-MW inhibitor TIMP (MW ~ 28,000) was unable to inhibit MMP/α2M enzyme activity, whereas the low-MW inhibitor BB94 (MW ~ 500) effectively suppressed enzyme activity. By using fluorogenic substrates with Dabcyl/Fluorescein as quencher/fluorophore combin-ation, sensitive MMP-activity assays in plasma were achieved. Spiking of active MMP-13 and MMP-13/α2M complex, and inhibitor studies with TIMP-1 and BB94, indicated that active MMPs are efficiently captured by (α2M in plasma. MMP activity was even detected in control plasma, and was significantly increased in plasma from rheumatoid arthritis patients.

Background: Lipopolysaccharide (endotoxin) has been shown to increase the expression of plasminogen activator inhibitor type-1 (PAI-1) in the vessel wall. Endotoxin is known to increase PAI-1 production in endothelial cells, but its action on smooth muscle cells (SMCs) is presently not clear. In this study we determined the effect of endotoxin on PAI-1 and tissue plasminogen activator (t-PA) production by aortic SMCs in vivo in two animal species, and in culture. Methods: The aortas of Sprague Dawley rats and of New Zealand White rabbits were rapidly excised after parenteral administration of endotoxin. Total RNA was extracted from the aortic media, and PAI-1 and t-PA mRNA levels were quantified after Northern blotting. In addition, cultured rat aortic SMCs were treated with endotoxin. PAI activity in the conditioned medium was determined with a spectrophotometric assay, and total RNA was extracted from the cells and analyzed. Results: A rapid and strong induction in the aortic media of PAI-1 mRNA was observed by endotoxin in both rat (50 mg/kg) and rabbit (1 mg/kg). t-PA mRNA was barely detectable and was not increased by endotoxin. Studies in cultured SMCs showed low expression of PAI-1 mRNA under serum-free conditions and little PAI activity in the cell-conditioned medium. Endotoxin did not increase the levels of PAI-1 mRNA nor PAI activity under serum-free conditions. The effect of endotoxin (10 mg/ml) in the presence of 10% (v/v) newborn calf serum on PAI-1 mRNA was negligible; PAI activity, however, increased by 50.3 ± 7.3% compared with controls. mRNA levels of t-PA and low-density lipoprotein/receptor-related protein/α2-macroglobulin receptor also increased after endotoxin administration. PAI activity was identified as PAI-1 by immunoblotting. Fibrin zymography showed that t-PA was present only in complex with PAI-1. Conclusions: A strong increase in PAI-1 gene expression by endotoxin was observed in aortic SMCs in vivo but not in culture. This suggests that the effect of endotoxin on SMCs is indirect. The fibrinolytic/proteolytic potential of the SMCs in the vessel wall is likely to have important implications for the migration of cells during vessel wall remodeling, such as neointima formation, during tumor cell metastasis, and for the fate of intramural thrombi. © Kluwer Academic Publishers. Chemicals/CAS: alpha 2 macroglobulin, 95568-41-5; fibrin, 9001-31-4; plasminogen activator inhibitor 1, 140208-23-7; RNA, 63231-63-0; tissue plasminogen activator, 105913-11-9

Background: Lipopolysaccharide (endotoxin) has been shown to increase the expression of plasminogen activator inhibitor type-1 (PAI-1) in the vessel wall. Endotoxin is known to increase PAI-1 production in endothelial cells, but its action on smooth muscle cells (SMCs) is presently not clear. In this study we determined the effect of endotoxin on PAI-1 and tissue plasminogen activator (t-PA) production by aortic SMCs in vivo in two animal species, and in culture. Methods: The aortas of Sprague Dawley rats and of New Zealand White rabbits were rapidly excised after parenteral administration of endotoxin. Total RNA was extracted from the aortic media, and PAI-1 and t-PA mRNA levels were quantified after Northern blotting. In addition, cultured rat aortic SMCs were treated with endotoxin. PAI activity in the conditioned medium was determined with a spectrophotometric assay, and total RNA was extracted from the cells and analyzed. Results: A rapid and strong induction in the aortic media of PAI-1 mRNA was observed by endotoxin in both rat (50 mg/kg) and rabbit (1 mg/kg). t-PA mRNA was barely detectable and was not increased by endotoxin. Studies in cultured SMCs showed low expression of PAI-1 mRNA under serum-free conditions and little PAI activity in the cell-conditioned medium. Endotoxin did not increase the levels of PAI-1 mRNA nor PAI activity under serum-free conditions. The effect of endotoxin (10 mg/ml) in the presence of 10% (v/v) newborn calf serum on PAI-1 mRNA was negligible; PAI activity, however, increased by 50.3 ± 7.3% compared with controls. mRNA levels of t-PA and low-density lipoprotein/receptor-related protein/α2-macroglobulin receptor also increased after endotoxin administration. PAI activity was identified as PAI-1 by immunoblotting. Fibrin zymography showed that t-PA was present only in complex with PAI-1. Conclusions: A strong increase in PAI-1 gene expression by endotoxin was observed in aortic SMCs in vivo but not in culture. This suggests that the effect of endotoxin on SMCs is indirect. The fibrinolytic/proteolytic potential of the SMCs in the vessel wall is likely to have important implications for the migration of cells during vessel wall remodeling, such as neointima formation, during tumor cell metastasis, and for the fate of intramural thrombi. © Kluwer Academic Publishers. Chemicals/CAS: alpha 2 macroglobulin, 95568-41-5; fibrin, 9001-31-4; plasminogen activator inhibitor 1, 140208-23-7; RNA, 63231-63-0; tissue plasminogen activator, 105913-11-9

It was reported that C-reactive protein (CRP) levels increase in parallel with the progression of chronic liver diseases, such as chronic hepatitis and liver cirrhosis. Inflammatory markers, such as high sensitive C-reactive protein (hsCRP), ferritin, transferrin, albumin, alpha-1 acid glycoprotein (AAG), alpha-2 macroglobulin (AMG), alpha-1 anti-trypsin (AAT) and lipoprotein a [Lp(a)] were measured in coronary artery disease patients (CAD) and CAD patients with non-alcoholic steatohepatitis (NASH). In the present preliminary study an attempt was made to study whether there is an increase in the levels of CRP in CAD patients associated with NASH. CAD patients showed an increase in CRP and serum ferritin levels. In CAD patients with NASH along with an increase in the levels of serum ferittin (p