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Introduction: Dexamethasone (Dex) caused impaired osteoblast differentiation and oxidative stress (OS) in bone marrow mesenchymal stem cells (BMSCs). This work sought to elucidate the precise molecular pathway through which Dex influences osteogenic differentiation (OD) and OS in BMSCs. Methods: The expression of Runt-related transcription factor 1 (RUNX1) and alpha-2 macroglobulin (A2M) was assessed in Dex-treated BMSCs using qRTPCR and Western Blot. Following the functional rescue experiments, cell proliferation was determined by MTT assay, reactive oxygen species (ROS) expression by DCFH-DA fluorescent probe, lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (Gpx) expression by kits, OD by alkaline phosphatase (ALP) staining and activity quantification, and the expression of OD-related proteins RUNX2, collagen type 1 alpha 1 (COL1A1), and osteocalcin (OCN) by qRT-PCR and Western Blot. The binding of RUNX1 to A2M was initially analyzed through Jaspar website and subsequently verified by dual-luciferase reporter and ChIP assays. Results: Dex-treated BMSCs had low RUNX1 and A2M expression. Dex treatment apparently elevated ROS and LDH levels, diminished cell proliferation rate and SOD, CAT, and Gpx expression, lightened intensity of ALP staining, and declined calcified nodules, ALP activity, and RUNX2, COL1A1, and OCN expression in BMSCs, which was counterweighed by RUNX1 or A2M overexpression. RUNX1 positively targeted A2M. A2M knockdown effectively nullified the ameliorative effects of RUNX1 overexpression on impaired OD and OS injury in Dex-induced BMSCs. Conclusions: Overexpression of RUNX1 attenuated Dex-induced impaired OD and OS injury in BMSCs by promoting A2M transcription. [ABSTRACT FROM AUTHOR]

While pregnancy-related proteins (PRP) are known to contribute to immunotolerance during pregnancy, their significance to development of invasive placenta is unclear. We compared PRP expression in humans and the common marmoset (Callithrix jacchus), a new-world monkey. Invasive placenta was observed at the maternal-foetal interface of marmoset placenta from green fluorescent protein (GFP)-expressing foetus and wild type mother. The pregnancy zone protein (PZP) and alpha-2 macroglobulin-like 1 (A2ML1) proteins exhibited the most prominent increase in expression during the second trimester in humans and marmoset, respectively. In humans, PZP accumulated at the maternal-foetal interface and A2ML1 accumulated in the amnion. Similarly, A2ML1 mRNA was detected in marmoset placenta. These proteins belong to the A2M family of protease inhibitors, and both PZP and A2ML1 share around 90% homology between human and marmoset and have highly conserved structures. However, the protease-reacting bait regions of the proteins had lower homology (56.8–60.7% in proteins) relative to the rest of the sequence. Notably, the cleavage site of a proinflammatory proline-endopeptidase was preserved in human PZP and marmoset A2ML1. These proteins contain multiple sites that are cleaved by proteases involving proline-endopeptidase. Systemic regulation of these A2M family proteins may be important in animals with invasive placenta. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to compare the influence of topical metronidazole gel application and scaling and root planing on gingival crevicular fluid variables. Method and Materials: In a split-mouth study, 39 volunteers with chronic periodontitis were treated by metronidazole gel or scaling and root planing. Clinical attachment level and probing depth were recorded, and aspartate aminotransferase (AST) and total/transformed alpha2-macroglobulin were determined in the gingival crevicular fluid at baseline, as well as after 3 and 6 months. Results: Both treatment procedures resulted in a gain of clinical attachment--0.67 mm for metronidazole and 0.50 mm for scaling and root planing (P < .001)--at the end of the study. The median probing depth was significantly reduced by 0.66 mm for metronidazole and 1.00 mm for scaling and root planing (P < .001) after 6 months. No change of AST was found. Alpha2-macroglobulin was significantly reduced for scaling and root planing and metronidazole after 3 and 6 months (P < .001). No significant difference was found between the 2 procedures at any variable. Conclusions: These data suggest that alpha2-macroglobulin reflects clinical changes better than AST and that metronidazole and scaling and root planing have the same influence on clinical outcome and biochemical variables in the gingival crevicular fluid. [ABSTRACT FROM AUTHOR]

Alpha-2-macroglobulin (α2M) is a molecule generally associated with inflammation, and chronic inflammation is associated with ageing and cancer. The degree of inflammation was recently proposed to be considered as a biomarker of biological ageing. In this study, glycans attached to α2M were analysed in a human population of different ages by lectin-based protein microarray. Higher reactivity of α2M with several lectins was detected in older individuals indicating an increased content of specific monosaccharides: α2,6 sialic acid, mannose and N-acetylglucosamine, and multiantennary complex type N-glycans. The increased glycosylation of α2M was accompanied by reduced binding of Zn ions and insulin-like growth factor-binding protein 2 (IGFBP-2). Glycosylation of α2M and its reactivity with IGFBP-2 is similarly affected by ageing and incidence of colon cancer, but the reactivity of α2M with Zn ions is differently affected, as the binding of Zn ions remains unaltered in patients with colon cancer compared to healthy middle-aged individuals. Thus, the binding of IGFBP-2 to α2M seems to be related to structural changes in the glycan moieties of α2M, whereas binding of Zn ions, most likely, is not. [ABSTRACT FROM AUTHOR]

Abstract Bicalutamide (BCT), a drug used in the treatment of prostate cancer, antagonises the actions of androgens, at the receptor level, thereby inhibiting the growth of prostate tumours. Alpha-2-macroglobulin (α 2 M), a pan-proteinase inhibitor, inhibits proteinase, regardless of specificity and catalytic mechanism. α 2 M is deficient in patients of advanced prostate cancer with bone metastases. Our studies explored the interaction of BCT with α 2 M and analysed the BCT induced structural alteration to the α 2 M. The result suggests that BCT decreases the antiproteolytic potential and causes structural and functional change in human α 2 M. UV–visible absorption spectroscopy confirms the formation of α 2 M-BCT complex. Fluorescence analysis shows significant quenching in fluorescence intensity of α 2 M upon binding with BCT. Synchronous fluorescence result suggests the interaction of BCT with α 2 M changed the microenvironment around tyrosine residues. Secondary structure of α 2 M also undergoes a slight change upon complexation with the drug as evident by shift in negative ellipticity in far UV CD spectroscopy. FTIR results confirm the alteration in secondary structure of α 2 M upon drug interaction. Molecular docking studies show that BCT bind to a monomer of α 2 M primarily through hydrophobic force. Thermodynamics parameters were determined by isothermal titration calorimetry found that the binding was exothermic in nature. [ABSTRACT FROM AUTHOR]

Cancer resistance is a major cause for longevity of the naked mole-rat. Recent liver transcriptome analysis in this animal compared to wild-derived mice revealed higher expression of alpha2-macroglobulin (A2M) and cell adhesion molecules, which contribute to the naked mole-rat’s cancer resistance. Notably, A2M is known to dramatically decrease with age in humans. We hypothesize that this might facilitate tumour development. Here we found that A2M modulates tumour cell adhesion, migration and growth by inhibition of tumour promoting signalling pathways, e.g. PI3K / AKT, SMAD and up-regulated PTEN via down-regulation of miR-21, in vitro and in tumour xenografts. A2M increases the expression of CD29 and CD44 but did not evoke EMT. Transcriptome analysis of A2M-treated tumour cells, xenografts and mouse liver demonstrated a multifaceted regulation of tumour promoting signalling pathways indicating a less tumorigenic environment mediated by A2M. By virtue of these multiple actions the naturally occurring A2M has strong potential as a novel therapeutic agent. [ABSTRACT FROM AUTHOR]

Objectives Investigation of the blood compatibility requires a number of sensitive assays to quantify the activation of the blood protein cascades and cells induced by biomaterials. A global assay measuring the blood compatibility of biomaterials could be a valuable tool in such regard. In this study, we investigated whether an enzyme-linked immunosorbent assay (ELISA), that specifically measures the electrophoretic “fast form” of α 2 -macroglobulin (F-α 2 M), could be a sensitive and global marker for activation of calcium dependent and in-dependent proteases in plasma exposed to biomaterials in vitro . Methods A F-α 2 M specific monoclonal antibody was generated and applied in an ELISA setup. Using the F-α 2 M ELISA, we investigated activation of calcium dependent and in-dependent proteases by polyvinylchloride (n = 10), polytetrafluoroethylene (n = 10) and silicone (n = 10) tubings as well as glass tubes (n = 10). Results We found that F-α 2 M is a sensitive marker for activation of both calcium dependent and in-dependent proteases. A significant difference between F-α 2 M concentrations in the control sample and plasma exposed to the artificial surfaces was found ( p > 0.001). This was observed both in the presence and absence of calcium. Furthermore, the highest F-α 2 M concentration was in both cases found in plasma incubated with glass. Conclusions Our findings demonstrate that F-α 2 M is a sensitive marker for detection of protease activation in plasma by artificial surfaces. Potentially, levels of F-α 2 M could be a global marker of the blood compatibility of biomaterials. [ABSTRACT FROM AUTHOR]

Background The intron 5 insertion/deletion polymorphism of Alpha-2-macroglobulin receptor-associated protein gene (Alpha-2-MRAP) has been implicated in numerous diseases. The current study was designed to analyze the association of intron 5 insertion/deletion polymorphism of Alpha-2-MRAP with nephrolithiasis patients. Methods PCR was conducted on genomic DNA of patients and control to look for Alpha-2-MRAP insertion/deletion polymorphism. Besides that, serum level of Alpha-2-MRAP, oxidative stress marker myeloperoxidase, Malondialdehyde (MDA), Advanced oxidation protein products (AOPP), and uric acid were determined. Results The D and I allele frequencies were 57.50% and 42.50% in patients, 77.50% and 22.50% in control, individually. The result showed that II genotype was associated with nephrolithiasis patients group. A significant decrease was observed in serum Alpha-2-MRAP,myeloperoxidase and TAS,while TOS,OSI,MDA,AOPP and uric acid were substantially increased in II and ID when compared to DD genotype in patients with nephrolithiasis. Conclusion Our results demonstrate for the first time that patients with II genotype had an increased risk of stones. Also, the results demonstrate that I allele of the 5 insertion/deletion polymorphism in the Alpha-2-MRAP gene is related with an increase of oxidative stress in nephrolithiasis patients and may possibly impose a risk for cardiovascular diseases in patients with II genotype of Alpha-2-MRAP. [ABSTRACT FROM AUTHOR]

Alpha-2-macroglobulin (α 2 M), a pan-proteinase inhibitor, inhibits a variety of endogenous and exogenous proteinases and constitutes an important part of body’s innate defense system. In the present study, we explored how trifluoroethanol (TFE) may modulate the structure, antiproteinase activity and aggregation of α 2 M. TFE was sequentially added over a range of 0–20% (v/v) and the effects induced were studied by activity assay, intrinsic fluorescence, ANS fluorescence, circular dichroism, turbidity assay, Rayleigh scattering measurement and ThT fluorescence measurement. Decrease in activity and increase in fluorescence intensity of α 2 M upon addition of TFE shows structural deviation from the native structure and suggests aggregation of protein upon solvent addition. Increase in turbidity and Rayleigh scattering of modified α 2 M confirms the formation of aggregates. Insignificant ThT fluorescence intensity of TFE treated α 2 M is indicative of amorphous or non-amyloid aggregation. Further, circular dichroism results indicate the changes in secondary structure of native α 2 M as negative ellipticity decreased on addition of the polar solvent to the inhibitor. The turbidometric analysis, Rayleigh scattering, ThT fluorescence intensity of modified α 2 M suggests that the protein might be driven towards non-amyloid or amorphous aggregation. Our studies provide important mechanistic insight how α 2 M undergoes conformational and functional changes when exposed to TFE. [ABSTRACT FROM AUTHOR]

Although quotient of alpha2 macroglobulin (Qα2MG) was previously reported to be useful for the evaluation of blood–brain barrier (BBB) function, it is not commonly used. We therefore evaluated BBB function among the various subsets of neuropsychiatric systemic lupus erythematosus (NPSLE) using quotient Q α2MG. Furthermore, we determined the correlation between Q α2MG and cerebrospinal (CSF) interleukin (IL)-6 level and quotient complement component 3 (Q C3). To determine intrathecal production of C3, the C3 index (Q C3/Q α2MG) was also calculated. Fifty-six patients with SLE were included in this study. Of these, 48 were diagnosed with NPSLE, consisting of 30 diffuse NPSLE patients (acute confusional state (ACS): n = 14, non-ACS: n = 16) and 18 patients with focal NPSLE. CSF IL-6 concentration, and paired serum and CSF levels of α2MG and C3, were measured by enzyme-linked immuno solvent assay (ELISA). The Q α2MG, Q C3, and C3 index were then calculated. Q α2MG, Q C3, and IL-6 concentrations in the CSF were significantly elevated in NPSLE compared with non-NPSLE. Among the subsets of NPSLE, significant increases in Q α2MG, CSF IL-6, and Q C3 were observed in ACS compared with non-ACS or focal NPSLE. There was a positive correlation between CSF IL-6 level and Q α2MG, as well as between Q C3 and Q α2MG, in diffuse NPSLE. There were no significant differences in C3 index between NPSLE and non-NPSLE, as well as among the subgroups of NPSLE. Our study suggests that BBB disruption is present in ACS, and elevated levels of IL-6 and C3 in CSF in diffuse NPSLE, especially in ACS, might result from their entry to the CSF from the systemic circulation through the damaged BBB, as well as increased intrathecal production. Furthermore, Q α2MG might be useful for the evaluation of BBB integrity. [ABSTRACT FROM AUTHOR]

Background There is a growing interest in the association between schizophrenia and the activation of inflammatory system with signs of acute phase (AP) response. Majority of such studies had focused on C-reactive protein (CRP). The aims of the present study were (i) to examine the gene expression profiles of other acute phase proteins (APP), namely haptoglobin (HP), alpha-1 antitrypsin (A1T), and alpha-2 macroglobulin (A2M) in patients with first episode psychosis (FEP) over a period of three months and (ii) to explore the association between APP levels and severity of symptoms. Methods In this study, HP, A1T and A2M gene expression levels from whole blood were measured at recruitment, 1- and 3-month follow-up visits using quantitative PCR (qPCR) in 43 patients with FEP and in 57 healthy controls. Diagnoses was ascertained on the Structured Clinical Interview for DSM-IV-TR. Severity of symptoms in patients was assessed on the Positive and Negative Syndrome Scale (PANSS) and a previously validated 5-factor PANSS structure was applied in the subsequent analyses. Results The FEP sample comprised of 28 (65.1%) individuals with schizophrenia, 12 (27.9%) with schizophreniform disorder and 3 (7%) with schizoaffective disorder. HP gene expression level was noted to be significantly higher in patients than controls at all three time points: recruitment (P = 0.049), 1-month follow up (P = 0.002) and 3-month follow up (P = 0.005). PANSS positive, depression, and excitement symptom factors showed significant associations with HP (P = 0.002), A1T (P = 0.016) and A2M (P = 0.034), respectively. These findings remained significant after controlling for age, gender, smoking status and accumulated chlorpromazine dosage. Conclusion The current study provides information on HP, A1T and A2M gene expression profiles in FEP patients and their associations with psychopathology. This provides support for the hypothesis that inflammation is related to schizophrenia and further encourages studies on immune-inflammatory markers to understand the relationship between inflammation and schizophrenia. [ABSTRACT FROM AUTHOR]

Ferulic acid is a major phenolic acid found in numerous plant species in conjugated form. It binds to enzymes and oligomeric proteins and modifies their structure and function. This study was designed to examine the interaction of ferulic acid, an active ingredient of some important medicines, with α 2 M, a key serum proteinase, under physiological conditions. The mechanism of interaction was studied by spectroscopic techniques such as, UV–visible absorption, fluorescence spectroscopy, circular dichroism along with isothermal titration calorimetry. Fluorescence quenching of α 2 M by ferulic acid demonstrated the formation of α 2 M-ferulic acid complex by static quenching mechanism. Binding parameters calculated by Stern-Volmer method showed that ferulic acid binds to α 2 M with moderate affinity of the order of ∼10 4 M −1 . The thermodynamic signatures reveal that binding was enthalpy driven and hydrogen bonding played a major role in ferulic acid-α 2 M binding. CD spectra analysis suggests very little conformational changes in α 2 M on ferulic acid binding. [ABSTRACT FROM AUTHOR]

ABSTRACT Osteonecrosis of the femoral head (ONFH) is a potentially devastating complication that occurs in up to 40% of young adults receiving chronic glucocorticoid (GC) therapy. Through a validated GC therapy rat model, we have previously shown that Wistar Kyoto (WK ) rats exhibit a genetic susceptibility to GC-induced ONFH compared to Sasco Fischer (F344) rats. We have undertaken this study in order to investigate differences between these two strains for their bone parameters, alpha-2-macroglobulin (A2M) circulating levels and incidence of GC-induced osteonecrosis of the femoral head. WK and F344 rats were treated either with 1.5 mg/kg/day of prednisone or placebo for 6 months. Blood was taken every month. The femoral heads were harvested for histological examination to detect ONFH and analyzed with micro-computed tomography. After 3 months of GC-therapy, plasma A2M was elevated in treated rats only. GC-treated WK rats exhibited histological evidence of early ONFH through higher rates of cellular apoptosis and empty osteocyte lacunae in the subchondral bone compared to placebos and to F344 rats. Furthermore, micro-CT analysis exhibited femoral head collapse only in GC-treated WK rats. Interestingly, GC-treated F344 rats exhibited significant micro-CT changes, but such changes were less concentrated in the articular region and were accompanied histologically with increased marrow fat. These µCT and histological findings suggest that elevated A2M serum level is not predictive and suitable as an indicative biomarker for early GC-induced ONFH in rodents. Elevated A2M levels observed during GC treatment suggests that it plays role in the host reparative response to GC-associated effects. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1183-1194, 2017. [ABSTRACT FROM AUTHOR]

The broad-spectrum protease inhibitor alpha-2-macroglobulin (α2M) is widely found in vertebrates and invertebrates. However, no research regarding α2M has been described in sea cucumbers to date. In this study, the first sea cucumber α 2 M named Stmα 2 M was identified from Stichopus monotuberculatus, a tropical species with high edible and economic values in Southern China. The Stmα 2 M cDNA is 5276 bp in length, containing a 5′-untranslated region (UTR) of 279 bp, a 3′-UTR of 344 bp and an open reading frame of 4653 bp that encodes a protein of 1550 amino acids. The Stmα2M possesses all the three characteristics of known α2M proteins, including the bait region domain, thioester domain and receptor-binding domain. Higher levels of mRNA expression were noticed in intestine, respiratory tree, coelomocytes and tentacle of S. monotuberculatus. Transcriptional expression of Stmα 2 M showed the strongest response to lipopolysaccharides (LPS, 8.19-fold up-regulation) after 6 h post-challenge in coelomocytes. In addition, the α2M activity was detected in S. monotuberculatus and protein activity of α2M increased remarkably at 6 h after LPS stimulation. As a whole, this study indicated that Stmα2M is an important immune-related molecule, and the study on functions of Stmα2M may provide some new and valuable ideas for preventing and controlling diseases of cultured sea cucumbers. [ABSTRACT FROM AUTHOR]

Alpha-2-macroglobulin ( A2M) is essential for hosts defending against pathogen infections. In this study, two distinct A2Ms were identified from sea cucumbers Stichopus monotuberculatus and Holothuria atra, which belong to different genera in the Phylum Echinodermata, and named StmA2M and HaA2M, respectively. Structural analysis of StmA2M and HaA2M revealed that all contain the bait region domain, thioester domain, and receptor-binding domain. Phylogenetic analysis indicated that StmA2M and HaA2M all belong to the echinoderm A2Ms. The cleavage site RXXR and the catalytic histidine residue for complement component C3 were found in StmA2M and HaA2M. Compared with human A2M, the three-dimensional structures of StmA2M and HaA2M were more similar to that of common carp A2M. Both StmA2M and HaA2M showed the strongest response at transcriptional levels to lipopolysaccharide ( LPS) at 12 h and polyriboinosinic polyribocytidylic acid [Poly (I: C)] at 24 h, respectively. Activity of StmA2M showed the strongest response to Poly (I: C) at 6 h, while the strongest change of HaA2M activity was induced by LPS at 24 h. The differential mRNA expression and enzyme activity levels suggested that A2M in sea cucumbers might be involved in different immune responses to LPS or Poly (I: C) challenge. [ABSTRACT FROM AUTHOR]

The shrimp multifunctional protein alpha-2-macroglobulin (A2M) is abundantly expressed in plasma, highly up-regulated upon microbial infection and involved in several immune pathways such as blood clotting system, phagocytosis and melanization. Herein, the function of Lv A2M from Litopenaeus vannamei on the prophenoloxidase (proPO) system is reported. The recombinant (r) Lv A2M produced strongly and specifically inhibited trypsin and the PO activity in shrimp plasma in a dose-dependent manner. Silencing of Lv A2M led to an increase in the PO activity in shrimp plasma although the expression of proPO-associated genes, proPO-activating enzyme ( PPAE ) and prophenoloxidase ( proPO ) but not the proPO-activating factor ( PPAF ) was down-regulated. In Vibrio parahaemolyticus AHPND-infected shrimp, the Lv A2M activity was suppressed in an early phase of infection while the PO activity was increased. Thus, the proPO-activating system was regulated by the Lv A2M. [ABSTRACT FROM AUTHOR]

Rupture of carotid atherosclerotic plaque may cause stroke, while few biomarker in clinic can evaluate carotid plaque vulnerability. In this study, we divided the recruited participants into no plaque, stable plaque, and vulnerable plaque group according to carotid ultrasonography, and screened the differentially expressed proteins in plasma of these participants using isobaric tags for relative and absolute quantitation labeling coupled with liquid chromatography-tandem mass spectrometry. 28 proteins were identified differentially expressed, among which alpha-2-macroglobulin (α2M) and heparin cofactor II (HCII) were found to be at hub position in the interactions of these proteins by STRING analysis and were selected for enzyme-linked immunosorbent assay measurement to assess their relevance with carotid plaques vulnerability and diagnostic efficiency. The plasma level of α2M was found positively correlated, while HCII level was negatively correlated with higher vulnerability of carotid plaques. Both proteins were efficient in differentiating stable and vulnerable carotid plaques. These findings provide potential new targets for the research of carotid plaque vulnerability. Plasma α2M and HCII may be potential biomarkers for evaluation of the vulnerability of carotid plaques if further studied. [ABSTRACT FROM AUTHOR]

Anterior segment dysgeneses (ASDs) comprise a spectrum of developmental disorders affecting the anterior segment of the eye. Here, we describe three unrelated families affected by a previously unclassified form of ASD. Shared ocular manifestations include bilateral iris hypoplasia, ectopia lentis, corectopia, ectropion uveae, and cataracts. Whole-exome sequencing and targeted Sanger sequencing identified mutations in CPAMD8 (C3 and PZP-like alpha-2-macroglobulin domain-containing protein 8) as the cause of recessive ASD in all three families. A homozygous missense mutation in the evolutionarily conserved alpha-2-macroglobulin (A2M) domain of CPAMD8, c.4351T>C (p. Ser1451Pro), was identified in family 1. In family 2, compound heterozygous frameshift, c.2352_2353insC (p.Arg785Glnfs ∗ 23), and splice-site, c.4549-1G>A, mutations were identified. Two affected siblings in the third family were compound heterozygous for splice-site mutations c.700+1G>T and c.4002+1G>A. CPAMD8 splice-site mutations caused aberrant pre-mRNA splicing in vivo or in vitro. Intriguingly, our phylogenetic analysis revealed rodent lineage-specific CPAMD8 deletion, precluding a developmental expression study in mice. We therefore investigated the spatiotemporal expression of CPAMD8 in the developing human eye. RT-PCR and in situ hybridization revealed CPAMD8 expression in the lens, iris, cornea, and retina early in development, including strong expression in the distal tips of the retinal neuroepithelium that form the iris and ciliary body, thus correlating CPAMD8 expression with the affected tissues. Our study delineates a unique form of recessive ASD and defines a role for CPAMD8, a protein of unknown function, in anterior segment development, implying another pathway for the pathogenicity of ASD. [ABSTRACT FROM AUTHOR]

Background: Previously rare A2ML1 variants were identified to confer otitis media susceptibility in an indigenous Filipino community and in otitis-prone US children. The goal of this study is to describe differences in the middle ear microbiome between carriers and non-carriers of an A2ML1 duplication variant that increases risk for chronic otitis media among indigenous Filipinos with poor health care access. Methods: Ear swabs were obtained from 16 indigenous Filipino individuals with chronic otitis media, of whom 11 carry the A2ML1 duplication variant. Ear swabs were submitted for 16S rRNA gene sequencing. Results: Genotype-based differences in microbial richness, structure, and composition were identified, but were not statistically significant. Taxonomic analysis revealed that the relative abundance of the phyla Fusobacteria and Bacteroidetes, and genus Fusobacterium were nominally increased in carriers compared to non-carriers, but were non-significant after correction for multiple testing. We also detected rare bacteria including Oligella that was reported only once in the middle ear. Conclusions: These findings suggest that A2ML1-related otitis media susceptibility may be mediated by changes in the middle ear microbiome. Knowledge of middle ear microbial profiles according to genetic background can be potentially useful for therapeutic and prophylactic interventions for otitis media and can guide public health interventions towards decreasing otitis media prevalence within the indigenous Filipino community. [ABSTRACT FROM AUTHOR]

Alpha-2 macroglobulin (α2M) is a ubiquitous protease inhibitor and considered to be an evolutionarily conserved constituent of innate host defence system. Here, an α2M gene (designated as Pfα2M ) was obtained from the pearl oyster Pinctada fucata by RT-PCR, PCR walking and rapid amplification of cDNA ends (RACE). The Pfα2M cDNA consists of 6394 bp with an open reading frame (ORF) of 5745 bp encoding a protein of 1914 amino acids with a 19 residues signal peptide. Pfα2M sequence contains three putative functional domains, including a bait region, a thiol ester domain and a receptor-binding domain. Phylogenetic analysis revealed that Pfα2M is closely related to the α2Ms from other molluscs. Pfα2M was expressed in all tested tissues including digestive gland, gill, adductor muscle, mantle and foot, while the highest expression was found in hemocytes. Following challenge with Vibrio alginolyticus , Pfα2M expression in hemocytes was significantly up-regulated at 2 h and then returned to the original level at 48 h. Knockdown of Pfα2M by RNA interference significantly reduced the phagocytosis of V. alginolyticus by hemocytes in vivo , and similar results were obtained upon chemical inactivation of the reactive thioester bond in Pfα2M by methylamine treatment. Taken together, it is suggested that Pfα2M is an immune-relevant molecule and involved in phagocytosis of V. alginolyticus by P. fucata hemocytes, and the function of Pfα2M in phagocytosis is dependent on the active thioester bond. [ABSTRACT FROM AUTHOR]

The non-specific protease inhibitor alpha-2-macroglobulin (A 2 M) is a key macromolecular glycoprotein that involved in host immune defense against pathogens in vertebrates and invertebrates. However, no research regarding A 2 M has been developed in echinoderms to date. In this study, the full-length cDNA of A 2 M was cloned from the sea cucumber ( Holothuria atra ), which is a tropical species widely distributed along the coasts of the South China Sea and designated HaA 2 M . HaA 2 M possesses all three conserved functional domains of known A 2 M proteins, including the bait region domain, thioester domain and receptor-binding domain. Compared to fish and shrimp A 2 Ms, the histidine residue from the catalytical regions is well conserved in HaA 2 M. HaA 2 M mRNA was predominantly expressed in coelomocytes and, to a lesser extent, in the body wall, intestine and respiratory tree. A 2 M activity was detected in the coelomic fluids of H. atra . The mRNA expression and activity levels were investigated in the major immune tissues and coelomic fluids of H. atra after challenge with inactivated Vibrio alginolyticus or polyriboinosinic polyribocytidylic acid [Poly (I: C)]. RNA interference (RNAi)-mediated knockdown of HaA 2 M resulted in a significant reduction of HaA 2 M gene transcript level (86%). RNAi-mediated silencing of HaA 2 M gene significantly decreased the A 2 M activity (38%) and increased the number of viable bacteria (2.8-fold) in the coelomic fluids of H. atra infected by V. alginolyticus . Our study, as a whole, supplied the evidences for HaA 2 M as an immune-relevant molecule and it might have multiple functions in the innate immune system of H. atra . [ABSTRACT FROM AUTHOR]

Background: Gastric contents aspiration in humans is a risk factor for severe respiratory failure with elevated mortality. Although aspiration-induced local lung inflammation has been studied in animal models, little is known about extrapulmonary effects of aspiration. We investigated whether a single orotracheal instillation of whole gastric fluid elicits a liver acute phase response and if this response contributes to enrich the alveolar spaces with proteins having antiprotease activity.Methods: In anesthetized Sprague-Dawley rats receiving whole gastric fluid, we studied at different times after instillation (4 h -7 days): changes in blood cytokines and acute phase proteins (fibrinogen and the antiproteases alpha1-antitrypsin and alpha2-macroglobulin) as well as liver mRNA expression of the two antiproteases. The impact of the systemic changes on lung antiprotease defense was evaluated by measuring levels and bioactivity of antiproteases in broncho-alveolar lavage fluid (BALF). Markers of alveolar-capillary barrier derangement were also studied. Non-parametric ANOVA (Kruskall-Wallis) and linear regression analysis were used.Results: Severe peribronchiolar injury involving edema, intra-alveolar proteinaceous debris, hemorrhage and PMNn cell infiltration was seen in the first 24 h and later resolved. Despite a large increase in several lung cytokines, only IL-6 was found elevated in blood, preceding increased liver expression and blood concentration of both antiproteases. These changes, with an acute phase response profile, were significantly larger for alpha2-macroglobulin (40-fold increment in expression with 12-fold elevation in blood protein concentration) than for alpha1-antitrypsin (2-3 fold increment in expression with 0.5-fold elevation in blood protein concentration). Both the increment in capillary-alveolar antiprotease concentration gradient due to increased antiprotease liver synthesis and a timely-associated derangement of the alveolar-capillary barrier induced by aspiration, contributed a 58-fold and a 190-fold increase in BALF alpha1-antitrypsin and alpha2-macroglobulin levels respectively (p < 0.001).Conclusions: Gastric contents-induced acute lung injury elicits a liver acute phase response characterized by increased mRNA expression of antiproteases and elevation of blood antiprotease concentrations. Hepatic changes act in concert with derangement of the alveolar capillary barrier to enrich alveolar spaces with antiproteases. These findings may have significant implications decreasing protease burden, limiting injury in this and other models of acute lung injury and likely, in recurrent aspiration. [ABSTRACT FROM AUTHOR]

In fish, a series of maternal derived immune components have been identified in their eggs or embryos at very early stages, which are proposed to provide protections to themselves against pathogenic attacks from hostile environment. The phenomenon of maternal immunity has been also recorded in several invertebrate species, however, so far, very limited information about the maternal immune molecules are available. In this study, it was demonstrated maternal alpha2 macroglobulin (A2m) protein, an important innate immune factor, exists in the fertilized eggs of amphioxus Branchiostoma japonicum , an invertebrate chordate. Maternal mRNA of A2m was also detected in amphioxus embryos at very early developing stages. In addition, it was recorded that the egg lysate prepared from the newly fertilized eggs can inhibit the growth of both Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus in a concentration dependent manner. The bacteriostatic activity can be reduced notably after precipitated A2m with anti-A2m antibody. Thus maternal A2m is partly attributed to the bacteriostatic activity. It was further demonstrated that recombinant A2m can bind to E. coli cells directly. All these points come to a result that A2m is a maternal immune factor existing in eggs of invertebrate chordate, which may be involved in defense their embryos against harmful microbes' attacks. [ABSTRACT FROM AUTHOR]

Purpose: Due to the controversial results in assessment of the association between α-2-Macroglobulin gene (A2M) polymorphisms and Parkinson's disease (PD), we performed a meta-analysis to evaluate the relationship between two A2M variants (rs669 and rs3832852) and PD.Methods: All eligible studies were retrieved in PubMed, Web of Science, Embase and PDGene databases. Data were extracted by two investigators independently. All the four genetic models were used for rs669 and rs3832852.Results: A total of 877 PD patients and 1296 controls from six studies were included for rs669 polymorphisms. The combined odds ratio (OR) indicated that rs669 polymorphisms were likely associated increased risk of PD only in dominant genetic models (OR = 1.41, CI = 1.03–1.92), especially in Asian subgroup (OR = 1.97, CI = 1.03–3.75). Five studies containing 772 PD patients and 1178 controls were identified for rs3832852 polymorphisms. The result suggested that rs3832852 polymorphisms were not associated with PD in all genetic models.Conclusions: Our data indicate that the rs669 (A/G) polymorphisms inA2Mgene are associated with increased risk in PD. To further validate the association of A2M polymorphisms with PD, more studies with larger samples are required. [ABSTRACT FROM PUBLISHER]

A α 2 M homologue was isolated from sheep ( Ovis aries ) blood plasma, using a simple two-step procedure, ammonium sulphate fractionation and gel filtration chromatography. Sheep α 2 M was found to be a large tetrameric glycoprotein of 630 kDa with monomeric subunit of 133 kDa each. Each subunit of sheep α 2 M was found to be made up of two fragments of 102 and 31 kDa respectively. The proteinase inhibitor from sheep was found to have Stokes radius of 79 Ǻ, which makes it much more compact than its human homologue. It entraps only 1 mol of trypsin per mole of inhibitor, like its caprine counterpart. The use of isothermal titration calorimetry has become gold standard for exploring thermodynamics of binding interactions. In this study, binding interaction of trypsin with alpha-2-macroglobulin is studied using ITC. The thermodynamic signatures – enthalpy change (Δ H ), entropy change (Δ S ) and Gibb's free energy change (Δ G ), along with number of binding sites ( N ) and affinity constant ( K ) are explored for α 2 M-trypsin binding for the first time for any known α 2 M molecule. The thermodynamics of proteinase-antiproteinase association suggests that trypsin–α 2 M interaction is enthalpy driven event. [ABSTRACT FROM AUTHOR]

Synthetic microstructures can be engineered to deliver bioactive compounds impacting on their pharmacokinetics and pharmacodynamics. Herein, we applied dextran-based layer-by-layer (LbL) microcapsules to deliver alpha-2-macroglobulin (α2MG), a protein with modulatory properties in inflammation. Extending recent observations made with dextran-microcapsules loaded with α2MG in experimental sepsis, we focused on the physical and chemical characteristics of these microstructures and determined their biology on rodent and human cells. We report an efficient encapsulation of α2MG into microcapsules, which enhanced i) human leukocyte recruitment to inflamed endothelium and ii) human macrophage phagocytosis: in both settings microcapsules were more effective than soluble α2MG or empty microcapsules (devoid of active protein). Translation of these findings revealed that intravenous administration of α2MG-microcapsules (but not empty microcapsules) promoted neutrophil migration into peritoneal exudates and augmented macrophage phagocytic functions, the latter response being associated with alteration of bioactive lipid mediators as assessed by mass spectrometry. The present study indicates that microencapsulation can be an effective strategy to harness the complex biology of α2MG with enhancing outcomes on fundamental processes of the innate immune response paving the way to potential future development in the control of sepsis. [ABSTRACT FROM AUTHOR]

Background Controlling prekallikrein activation by C1 inhibitor ( C1 Inh) represents the most essential mechanism for angioedema patient protection. C1 Inh function in the plasma is usually measured based on the residual activity of the C1s protease not involved in the pathological process. We have hereby proposed an alternative enzymatic measurement of C1 Inh function based on contact-phase activation and correlation with angioedema diagnostic requirements. Methods The contact phase was reconstituted using the purified components, with C1 Inh standard or plasma sample. The kinetics of the amidase activity were monitored using Pro- Phe- Arg- p NA, independently of alpha2-macroglobulin. We prevented any interference from a possible high plasma kininogenase activity by preincubating the samples with protease inhibitor. Receiver operating characteristics ( ROC) were used to calculate the assay's diagnostic performance. Results The calibration curve was built using C1 Inh standard (threshold limit 0.10 × 10−3 U, i.e., 0.2 pmol), and C1 Inh function was quantified in the sample, with a reference interval established based on healthy individuals ( n = 281; men: 0.61-1.10 U/ml, median: 0.85 U/ml; women: 0.42-1.08 U/ml, median: 0.74 U/ml). The median values of female donors were lower than those of the others due to estrogen, yet C1Inh function remained within the reference interval. The ROC curve calculation provided the following optimum diagnostic cutoff values: women 0.36 U/ml (area under curve [ AUC]: 0.99; sensitivity: 93.48%; specificity: 99.37%); and men 0.61 U/ml ( AUC: 1; sensitivity: 100.0%; specificity: 100.0%). Conclusion The performance outcome provided features suitable for angioedema diagnostic or follow-up. Established by means of the kinin formation process, this assay should be preferred over the method based on a C1s protease target. [ABSTRACT FROM AUTHOR]

Lack of diagnostic capacity has been a crucial barrier preventing an effective response to the challenges of malnutrition and tuberculosis (TB). Point-of-care diagnostic tests for TB in immuno-incompetent, malnourished population are thus needed to ensure rapid and accurate detection. The aim of the study was to identify potential biomarkers specific for TB infection and progression to overt disease in the malnourished population of Melghat. A prospective cohort study was conducted in the year 2009 through 2011 in six villages of the Melghat region. 275 participants consisting of malnourished cases with a) active TB (n = 32), b) latent TB infection (n = 90), c) with no clinical or bacteriological signs of active or latent TB (n = 130) and healthy control subjects (n = 23) were recruited for the study. The proteome changes of the host serum in response to Mycobacterium tuberculosis (M.tb) infection were investigated using one dimensional electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Three most differentially expressed proteins; alpha-2-macroglobulin (A-2-M), sero-transferrin and haptoglobin were identified by MALDI-TOF MS analysis, which were up-regulated in the malnourished patients with active TB and down-regulated in the malnourished patients compared with the healthy controls. Additionally, follow-up studies indicated that the expression of these proteins increased to nearly two folds in patients who developed active disease from latent state. Our preliminary results suggest that A-2-M, sero-transferrin and haptoglobin may be clinically relevant host biomarkers for TB diagnosis and disease progression in the malnourished population. This study provides preliminary framework for an in-depth analysis of the biomarkers in larger well-characterized cohorts. Evaluation of these biomarkers in follow-up cases may further aid in improving TB diagnosis. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (EOAD, LOAD), mild cognitive impairment (MCI), Parkinson's disease (PD) and healthy controls were included to determine the serum interleukin-1s (IL-1α, IL-1β), IL-6 and alpha-2-macroglobulin (α 2 M) levels using ELISA. IL-6 might be a significant contributor to the inflammatory response in LOAD. The MCI data indicate that IL-1s, α 2 M and BDNF are somehow related, and this relationship might allow MCI patients to be more similar to the healthy controls. A correlation analysis of multiple biomarkers in different neurodegenerative disorders might be more useful than determining the levels of a single cytokine in a single disorder. [ABSTRACT FROM AUTHOR]

Background: The naked mole-rat (NMR) is a long-lived and cancer resistant species. Identification of potential anti-cancer and age related mechanisms is of great interest and makes this species eminent to investigate anti-cancer strategies and understand aging mechanisms. Since it is known that the NMR expresses higher liver mRNA-levels of alpha 2-macroglobulin than mice, nothing is known about its structure, functionality or expression level in the NMR compared to the human A2M. Results: Here we show a comprehensive analysis of NMR- and human plasma-A2M, showing a different prediction in glycosylation of NMR-A2M, which results in a higher molecular weight compared to human A2M. Additionally, we found a higher concentration of A2M (8.3±0.44 mg/mL vs. and 4.4±0.20 mg/mL) and a lower total plasma protein content (38.7±1.79 mg/mL vs. 61.7±3.20 mg/mL) in NMR compared to human. NMR-A2M can be transformed by methylamine and trypsin resulting in a conformational change similar to human A2M. NMR-A2M is detectable by a polyclonal antibody against human A2M. Determination of tryptic and anti-tryptic activity of NMR and human plasma revealed a higher anti-tryptic activity of the NMR plasma. On the other hand, less proteolytic activity was found in NMR plasma compared to human plasma. Conclusion: We found transformed NMR-A2M binding to its specific receptor LRP1. We could demonstrate lower protein expression of LRP1 in the NMR liver tissue compared to human but higher expression of A2M. This was accompanied by a higher EpCAM protein expression as central adhesion molecule in cancer progression. NMR-plasma was capable to increase the adhesion in human fibroblast in vitro most probably by increasing CD29 protein expression. This is the first report, demonstrating similarities as well as distinct differences between A2M in NMR and human plasma. This might be directly linked to the intriguing phenotype of the NMR and suggests that A2M might probably play an important role in anti-cancer and the anti-aging mechanisms in the NMR. [ABSTRACT FROM AUTHOR]

Alpha-2-macroglobulin is an abundant secreted protein that is of particular interest because of its diverse ligand binding profile and multifunctional nature, which includes roles as a protease inhibitor and as a molecular chaperone. The activities of alpha-2-macroglobulin are typically dependent on whether its conformation is native or transformed (i.e. adopts a more compact conformation after interactions with proteases or small nucleophiles), and are also influenced by dissociation of the native alpha-2-macroglobulin tetramer into stable dimers. Alpha-2-macroglobulin is predominately present as the native tetramer in vivo; once purified from human blood plasma, however, alpha-2-macroglobulin can undergo a number of conformational changes during storage, including transformation, aggregation or dissociation. We demonstrate that, particularly in the presence of sodium chloride or amine containing compounds, freezing and/or lyophilization of alpha-2-macroglobulin induces conformational changes with functional consequences. These conformational changes in alpha-2-macroglobulin are not always detected by standard native polyacrylamide gel electrophoresis, but can be measured using bisANS fluorescence assays. Increased surface hydrophobicity of alpha-2-macroglobulin, as assessed by bisANS fluorescence measurements, is accompanied by (i) reduced trypsin binding activity, (ii) increased chaperone activity, and (iii) increased binding to the surfaces of SH-SY5Y neurons, in part, via lipoprotein receptors. We show that sucrose (but not glycine) effectively protects native alpha-2-macroglobulin from denaturation during freezing and/or lyophilization, thereby providing a reproducible method for the handling and long-term storage of this protein. [ABSTRACT FROM AUTHOR]

Two detailed reviews of the management of neonatal thrombosis were published in 2012; one was an up-dated version of guidance first issued in 2004 and the other was a comprehensive review. Both of these publications gave very similar advice regarding the practical aspects of the indications, dosage and management of antithrombotic therapy. The authors stated that the evidence supporting most of their recommendations for anti-thrombotic therapy in neonates remained weak and so the therapy for a neonate with a thrombosis has to be based on an individualized assessment of estimated risk versus potential benefit. The aim of this present review is to give the treating physician an outline of the unique physiology of neonatal coagulation and how this affects the monitoring, dosing and even the choice of therapeutic strategy for the management of thrombosis in the neonate. [ABSTRACT FROM AUTHOR]

Eukaryotic cells have evolved robust mechanisms to counter excess cholesterol including redistribution of lipids into different compartments and compensatory up-regulation of phospholipid biosynthesis. We demonstrate here that excess cellular cholesterol increased the activity of the endoplasmic reticulum (ER) enzyme serine palmitoyl-CoA transferase (SPT), the rate-limiting enzyme in sphingomyelin synthesis. This increased SPT activity was not due to altered levels of SPTLC1 or SPTLC2, the major subunits of SPT. Instead, cholesterol loading decreased the levels of ORMDL1, a negative regulator of SPT activity, due to its increased turnover. Several lines of evidence demonstrated that free-cholesterol- induced autophagy, which led to increased turnover of ORMDL1. Cholesterol loading induced ORMDL1 redistribution from the ER to cytoplasmic p62 positive autophagosomes. Coimmunoprecipitation analysis of cholesterol-loaded cells showed increased association between ORMDL1 and p62. The lysosomal inhibitor chloroquine or siRNA knockdown of Atg7 inhibited ORMDL1 degradation by cholesterol, whereas proteasome inhibitors showed no effect. ORMDL1 degradation was specific to free-cholesterol loading as autophagy induced by serum starvation or general ER stress did not lead to ORMDL1 degradation. ORMDL proteins are thus previously unidentified responders to excess cholesterol, exiting the ER to activate SPT and increase sphingomyelin biosynthesis, which may buffer excess cellular cholesterol. [ABSTRACT FROM AUTHOR]

Alpha-2-macroglobulin (α-2-M) is a broad spectrum protease inhibitor which is abundant in the plasma of vertebrates and several invertebrates. The α-2-M was purified from cobia ( Rachycentron canadum ) plasma by a four-step procedure: poly ethylene glycol fractionation, affinity chromatography, hydrophobic interaction chromatography and ion exchange chromatography on Fast Protein liquid chromatography system in the present study. It migrated as one protein band with a molecular mass of about 360 kDa in the native state, whereas in SDS-PAGE it was about 180 kDa under non-reducing condition. This result revealed that the native protein was a dimer. In addition, it was cleaved into two different fragments of molecular mass about 93 and 87 kDa when reduced by dithiothreitol (DTT). The anti-protease activity of the purified α-2-M was apparently decreased as temperature elevated above 50 °C. The α-2-M exhibited highest protease inhibitory activity at pH 9. The results indicate that the α-2-M is a heat-labile and alkaline protease inhibitor. The purified α-2-M exhibited more than 50％ protease inhibitory activity against extracellular products (ECP) of Vibrio alginolytius isolated from diseased cobia. It seems that the protease activities in ECP may be affected by the plasma α-2-M. The protease inhibitory activities of cobia plasma or purified α-2-M were decreased when incubated with 10 mM methylamine for 30 min. The α-2-M cDNA consisted of 4611 bp with an open reading frame of 4374 bp had been cloned from cobia liver. This sequence contained thioester domain (GCGEQ) and thirteen predicted N-linked glycosylation sites. In addition, the amino acid sequence of thioester domain and genes of adjacent regions of cobia α-2-M were further compared with sequences of known fish species in GenBank. The unweighted pair group method using arithmetic average (UPGMA) was employed to construct the phylogenetic trees of α-2-M among different fish species (freshwater fish, sea water fish and primitive fish), and all these fish species were then clustered into three groups. The cobia α-2-M was closer to that of sea water fish than that of freshwater fish compared basing on its similarity of amino acid sequence and phylogenetic analysis of the partial gene. [ABSTRACT FROM AUTHOR]

Our previous data revealed that viral particles of yellow head virus (YHV) specifically interacted with granule-containing hemocytes. After isolation of targeted hemocytes, biotinylation was performed using Biotin–NSH–LC. Biotinylated protein was extracted and separated by 2-D PAGE. Electro-transferred proteins on a nitrocellulose membrane were probed with streptavidin-HRP complex to detect biotinylated proteins. The data from 2-D PAGE combined with affinity pull down purification revealed 8 and 6 biotinylated proteins specific to hyaline and granule containing hemocytes, respectively. Four proteins were found in common for both two hemocytes. The majority of proteins detected in granular hemocytes are membrane-associated proteins and immune-related proteins such as alpha-2-macroglobulin (A2M), kazal-type serine protease inhibitor (SPI) and crustin. Crustin Pm 1 was found to bind to YHV as shown with biotinylation pull-down assay and confirmed with two-dimensional virus overlay protein binding assay (2-D VOPBA). The expression of crustin Pm 1 was observed in semigranular and granular hemocytes whereas very low or no expression occurred in hyaline hemocytes. Crustin Pm 1 appears to either be directly involved in cellular binding or mediating virus internalization into permissive hemocytes. [ABSTRACT FROM AUTHOR]

Plasma from hibernating (HIB) woodchucks (Marmota monax) or 13-lined ground squirrels (Ictidomys tridecemlineatus) suppressed ³H-thymidine uptake in mouse spleen cell cultures stimulated with Concanavalin A (ConA); plasma from non-hibernating animals were only slightly inhibitory. Maximum inhibition occurred when HIB plasma was added to the cultures prior to ConA. After HPLC size exclusion chromatography of the HIB ground squirrel plasma, a single fraction (fraction-14) demonstrated inhibitory activity. Assay of fraction-14 from 8 HIB squirrels showed inhibition ranging from 13 to 95%; inhibition was correlated to the time the squirrels were exposed to cold prior to hibernation. Western blot analysis showed the factor to be a large molecular weight protein (>300 kDa), and mass spectrometry identified sequences that were 100% homologous with alpha-2-macroglobulin from humans and other species. These findings indicate a hibernation-related protein that may be responsible for immune system down regulation. [ABSTRACT FROM AUTHOR]

Highlights: [•] The oxidation of the α2M adsorbed on BDDE was found the pH dependent. [•] Native and denatured α2M could be discriminated using voltammetric methods. [•] LODs were found as 1.69μgmL−1 based on amino acids residues for denatured α2M. [ABSTRACT FROM AUTHOR]

The complement terminal pathway clears pathogens by generating cytotoxic membrane attack complex (MAC) pores on target cells. For more than 40 years, biochemical and cellular assays have been used to characterize the lytic nature of the MAC and to define its protein composition. Although models for pore formation have been inferred from structures of bacterial cytolysins, it was only recently that we were able to visualize how complement components come together during MAC assembly. This review highlights structural analyses of terminal pathway complexes to explore molecular mechanisms underlying MAC formation. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this study was to independently validate proteomic biomarkers previously reported to be differentially expressed in women with Polycystic Ovary Syndrome (PCOS) compared with controls. This study focused on plasma proteomic biomarkers. Methods: This was a cross-sectional study at the University of Nottingham, in which samples from 30 PCOS and 30 control women were analysed by Western blotting. Results: Mean abundance ratios from Western blots of plasma total haptoglobin and haptoglobin beta proteins were 1.25 (CI 1.11-1.4) and 1.24 (CI 1.04-1.44). The mean abundance ratio from the blots of alpha-2 macroglobulin was however 1.05 (CI, 1-1.1). The mean PCOS/control BMI ratio was 1.18 (CI 1.17-1.20). There was no correlation between PCOS/control BMI ratio and alpha-2 macroglobulin, total haptoglobin and haptoglobin beta abundance ratios. There was also no correlation between PCOS/control insulin ratio and alpha-2 macroglobulin, total haptoglobin and haptoglobin beta abundance ratios. Conclusions: Total haptoglobin and haptoglobin beta chain protein abundance was found to be elevated in women with PCOS compared with controls. We were unable to confirm decreased alpha-2 macroglobulin levels as reported in a previous study. Independent validation studies are required to validate early promising proteomic biomarkers in PCOS. [ABSTRACT FROM AUTHOR]

Abstract: Alpha 2 macroglobulin (α2M) is a non-specific protease inhibitor involved in host defence mechanism in invertebrates. By c-DNA cloning and sequencing, α2M gene was identified from the haemocytes of Indian white shrimp Fenneropenaeus indicus with an open reading frame of 4599bp encoding 1532 amino acids with a signal sequence of 26 amino acids. The calculated molecular mass of the mature protein is 170.6kDa with an estimated pI of 6.13. The F. indicus α2M (Fi-α2M) sequence contains putative conserved functional domains including a GCGEQNM thioester region and a receptor-binding domain which are present in other invertebrate and vertebrate α2Ms. Sequence comparison and phylogenetic analysis revealed that Fi-α2M displays the highest similarity with Litopenaeus vannamei (96%). QRT-PCR analysis of challenge experiments by Vibrio harveyi, GFP tagged Vibrio parahaemolyticus (GFP-Vp) and WSSV infection of F. indicus significantly increased the α2M mRNA transcript up to 24h in the haemocytes. In addition, confocal laser scanning microscopy (CLSM) confirms that the GFP-Vp treated F. indicus have high proliferation of GFP-Vp in haemolymph and intestine at 24h. These results suggest that α2M is an important molecule in shrimp immune system. [Copyright &y& Elsevier]

Intrapleural processing of prourokinase (scuPA) in tetracycline (TCN)-induced pleural injury in rabbits was evaluated to better understand the mechanisms governing successful scuPA-based intrapleural fibrinolytic therapy (IPFT), capable of clearing pleural adhesions in this model. Pleural fluid (PF) was withdrawn 0-80 min and 24 h after IPFT with scuPA (0-0.5 mg/kg), and activities of free urokinase (uPA), plasminogen activator inhibitor-1 (PAI-1), and uPA complexed with α-macroglobulin (αM) were assessed. Similar analyses were performed using PFs from patients with empyema, parapneumonic, and malignant pleural effusions. The peak of uPA activity (5-40 min) reciprocally correlated with the dose of intrapleural scuPA. Endogenous active PAI-1 (10-20 nM) decreased the rate of intrapleural scuPA activation. The slow step of intrapleural inactivation of free uPA (t1/2 β= 40 ± 10 min) was dose independent and 6.7-fold slower than in blood. Up to 260 ± 70 nM of M/uPA formed in vivo [second order association rate (kass) 580 ± 60 M1·s1]. αM/uPA and products of its degradation contributed to durable intrapleural plasminogen activation up to 24 h after IPFT. Active PAI-1, active α2M, and α2M/uPA found in empyema, pneumonia, and malignant PFs demonstrate the capacity to support similar mechanisms in humans. Intrapleural scuPA processing differs from that in the bloodstream and includes 1) dose-dependent control of scuPA activation by endogenous active PAI-1; 2) two-step inactivation of free uPA with simultaneous formation of αM/uPA; and 3) slow intrapleural degradation of M/uPA releasing active free uPA. This mechanism offers potential clinically relevant advantages that may enhance the bioavailability of intrapleural scuPA and may mitigate the risk of bleeding complications. [ABSTRACT FROM AUTHOR]

Highlights: [•] Chronic restraint stress decreased high anxiety rats (HR) activity in Porsolt test. [•] HR restraint rats had decreased alpha-2 GABA-A receptors density in hippocampus. [•] HR restraint rats had decreased alpha-2 GABA-A receptors density in prefrontal cortex. [•] HR restraint rats had increased alpha-2 GABA-A receptors density in basolateral amygdala. [•] HR restraint rats had a lower corticosterone concentration in prefrontal cortex. [Copyright &y& Elsevier]

Hepcidin regulates iron metabolism by down-regulating ferroportin-1 (Fpn1). We demonstrated that hepcidin is complexed to the blood transport protein, α2-macroglobulin (α2M) (Peslova,G., Petrak, J.,Kuzelova, K., Hrdy, I., Halada, P., Kuchel, P.W., Soe-Lin, S., Ponka, P., Sutak, R., Becker, E., Huang, M. L., Suryo Rahmanto, Y., Richardson, D. R., and Vyoral, D. (2009) Blood 113, 6225-6236). However, nothing is known about the mechanism of hepcidin binding to α2M or the effects of the α2M ·hepcidin complex in vivo. We show that decreased Fpn1 expression can be mediated by hepcidin bound to native α2M and also, for the first time, hepcidin bound to methylamine-activated α2M(α2M-MA). Passage of high molecular weight α2M hepcidin or α2M-MA · hepcidin complexes (≈725 kDa) through a Sephadex G-25 size exclusion column retained their ability to decrease Fpn1 expression. Further studies using ultrafiltration indicated that hepcidin binding to α2M and α2M-MA was labile, resulting in some release from the protein, and this may explain its urinary excretion. To determine whether α2M- MA·hepcidin is delivered to cells via the α2M receptor (Lrp1), we assessed α2M uptake and Fpn1 expression in Lrp1-/- and Lrp1+/+ cells. Interestingly, α2M hepcidin or α2M-MAhepcidin demonstrated similar activities at decreasing Fpn1 expression in Lrp1-/- and Lrp1+/+ cells, indicating that Lrp1 is not essential for Fpn1 regulation. In vivo, hepcidin bound to α2Mo r α2M-MA did not affect plasma clearance of α2M/2M-MA. However, serum iron levels were reduced to a significantly greater extent inmice treated with α2M hepcidin or α2M-MA hepcidin relative to unbound hepcidin. This effect could be mediated by the ability of α2Mo r α2M-MA to retard kidney filtration of bound hepcidin, increasing its half-life. A model is proposed that suggests that unlike proteases, which are irreversibly bound to activated α2M, hepcidin remains labile and available to down-regulate Fpn1. [ABSTRACT FROM AUTHOR]

Abstract: Alpha-2-macroglobulin (α-2-M) is a protease inhibitor broadly present in the plasma of vertebrates and invertebrates, and is an important non-specific humoral factor in defence system of the animals. This study conducted the immuno-analysis and mass spectrometric analysis methods to investigate the characteristics of the protease inhibitor, α-2-M, among groupers and related species. Rabbit antiserum to the purified α-2-M of Epinephelus coioides was used in different immunological methods to determine the immune cross-reactions of the α-2-M in samples. Plasma of Epinephelus bruneus, Epinephelus fuscoguttatus, Epinephelus lanceolatus, and Epinephelus quoyanus exhibited high protease inhibitory activities by BAPNA-trypsin assay. To purify the α-2-M protein, plasma protein of grouper E. coioides was first precipitated by using PEG 6000, then Blue Sepharose 6 Fast Flow, DEAE Sephacel, Con A Separose 4B and Phenyl Sepharose High Performance columns were used on FPLC system for purification. The molecular mass of grouper plasma α-2-M was determined as a 180 kDa protein on non-reduced SDS-PAGE. In addition, it was determined as 97 and 80 kDa protein on reduced SDS-PAGE. Enzymatic and chemical deglycosylation of glycogen revealed that the contents of glycogen in 97 and 80 kDa subunits were 12.4% and 15%, respectively, and were all belonging to N-linked type. Only one precipitation arc was visualized in all plasma of Epinephelus spp. using the rabbit antiserum to the purified α-2-M of E. coioides, on crossed immunoelectrophoresis (CIE) gels. The plasma of Epinephelus spp. and seawater fish species showed stronger responses than freshwater fish species while that of other animal species showed no response by dot-blot assay. One single band was detected on Native PAGE-Western blotting assay, one single 180 kDa band was detected on non-reduced SDS-PAGE-Western blotting, and four bands (80, 97, 160, 250 kDa) were detected on reduced SDS-PAGE when various grouper plasma was performed respectivity. However, no band was detected using plasma from the freshwater fish species and other animal species. Thus, further indicates that the protein structure of α-2-M of Epinephelus spp. was closely related among seawater fish species. In addition the identity of the two subunits was identified using LC/MS/MS which was similar to α-2-M of grass carp (Ctenopharyngodon idella) and bluegill sunfish (Lepomis macrochirus) on the protein hit. [Copyright &y& Elsevier]

Alpha macroglobulins are large glycoproteins which are present in the body fluids of both invertebrates and vertebrates. Alpha-2-macroglobulin (α2M), a key member of alpha macroglobulin superfamily, is a high-molecular weight homotetrameric glycoprotein. α2M has many diversified and complex functions, but it is primarily known by its ability to inhibit a broad spectrum of proteases without the direct blockage of the protease active site. α2M is also known to be involved in the regulation, transport, and a host of other functions. For example, apart from inhibiting proteinases, it regulates binding of transferrin to its surface receptor, binds defensin and myelin basic protein, etc., binds several important cytokines, including basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), interleukin-1β (IL-1β), and interleukin-6 (IL-6), and modify their biological activity. α2M also binds a number of hormones and regulates their activity. α2M is said to protect the body against various infections, and hence, can be used as a biomarker for the diagnosis and prognosis of a number of diseases. However, this multipurpose antiproteinse is not 'fail safe' and could be damaged by reactive species generated endogenously or exogenously, leading to various pathophysiological conditions. J. Cell. Physiol. 228: 1665-1675, 2013. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background: Autoimmune hepatitis (AIH) is a chronic liver disease caused by inflammation of the liver. The etiology of AIH remains elusive, and there are no reliable serum biomarkers. Methods: In order to identify candidate biomarkers, 2-DE analysis of serum proteins was performed using a mouse model of AIH induced by treatment with concanavalin A (ConA). To enrich samples for low abundance molecules a commercial albumin removal reagent was used. In an independent analysis, candidate biomarkers were identified in AIH patient's serum by a targeted iTRAQ (isobaric tags for relative and absolute quantification) identification. Candidates were validated in independent cohorts of ConA treated mice and AIH patients by ELISA (enzyme-linked immuno sorbent assay). Results: Nine proteins were differentially expressed in AIH mice treated with con-A. Two of these, the third component of complement (C3) and alpha-2-macroglobulin (A2M) were also up-regulated in AIH patient's sera by a targeted iTRAQ identification. In separate validation studies, serum C3 and A2M levels were increased in mice with ConA treatment after 20-40 h and in 34 AIH patients in a subgroup analysis, females with AIH aged 20-50 years old displayed the largest increases in serum A2M level. Biological network analysis implements the complement cascade and protease inhibitors in the pathogenesis of AIH. Conclusion: The serum proteins C3 and A2M are increased both in a mouse model and in patients with AIH by both 2-DE and iTRAQ methods. This integrated serum proteomics investigation should be applicable for translational researchers to study other medical conditions. [ABSTRACT FROM AUTHOR]

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Background: The gene ORMDL3 was shown to be associated with early-onset asthma susceptibility in multiple independent genome-wide and candidate-gene association studies. Asthmatic patients have elevated expression levels of this gene. ORMDL3 encodes a transmembrane protein localized in the endoplasmic reticulum (ER) that may be involved in ER stress and inflammation. It is essential to validate the genetic associations linking ORMDL3 with asthma through functional studies that confirm the biological relevance of this gene in disease. We investigated the effects of manipulating ORMDL3 expression levels in vitro in airway cells on innate immune inflammatory responses, ER stress and activation of the unfolded protein response (UPR). Methods: ORMDL3 expression levels were manipulated in airway cells using an overexpression plasmid and siRNA technologies. Successful modulation of ORMDL3 was confirmed at both the gene and protein level. The functional impact of modulation of ORMDL3 expression levels on inflammatory responses and activation of the UPR were quantified using complementary cellular and molecular immunology techniques. Results: Cells with altered ORMDL3 levels responded equally well to innate immune stimuli and produced similar levels of pro-inflammatory cytokines compared to wild-type cells. Treatment with ER stress inducers, thapsigargin and tunicamycin, resulted in activation of the unfolded protein response (UPR). However, we observed no difference in UPR activation in cells with ORMDL3 knockdown compared to cells with normal ORMDL3 levels. Conclusions: Our results suggest that ORMDL3 variation in the airway epithelium is unlikely to play a significant role in modulating innate immune responses and the UPR in the lung. [ABSTRACT FROM AUTHOR]