Identification of 5S and 45S rDNA sites in Chrysanthemum species by using oligonucleotide fluorescence in situ hybridization (Oligo-FISH).

Fluorescence in situ hybridization (FISH) is a conventional method used to visualize the distribution of DNA elements within a genome. To examine the relationships within the Chrysanthemum genus, ribosomal DNA (rDNA), a popular cytogenetic marker, was utilized as a probe for FISH within this genus. Based on the genome data of Chrysanthemum nankingense, C. seticuspe and its allied genera in the Compositae(Asteraceae), we explored rDNA sequences to design oligonucleotide probes and perform oligonucleotide fluorescence in situ hybridization (Oligo-FISH) in eight Chrysanthemum accessions. The results showed that the majority of 5S rDNA signals were located in subterminal chromosome regions and that the number of 5S rDNA sites might be tightly associated with ploidy. For 45S rDNA sites, the number and intensity of signals differed from those of previously investigated Chrysanthemum resources. These findings may provide an optimally reliable method of examining the chromosome composition and structural variation of Chrysanthemum and its related species and allow researchers to understand the evolutionary history and phylogenetic relationships of Chrysanthemum.

Purification of α2-macroglobulin from Cohn Fraction IV by immobilized metal affinity chromatography: A promising method for the better utilization of plasma.

As an abundant plasma protein, α2-macroglobulin (α2-M) participates widely in physiological and pathological activities including coagulation regulation, antitumor activities, and regulation of cytokines. It also presents a therapeutic potential for radiation injury. A two-step isolation method for the purification of α2-M from Cohn Fraction IV is described. This process includes a salting-out method and immobilized metal affinity chromatography. The LC-ESI-MS/MS analysis and a comparison of the amino acid composition demonstrated that the final product was α2-M. The final protein, with a purity of approximately 95% and a yield of nearly 45%, was obtained from Cohn Fraction IV regardless of plasma haptoglobin type, although all but type 1-1 have previously been considered unfavorable for α2-M preparation. The effects of temperature, pH, and methylamine on α2-M activity were evaluated to avoid activity loss during preparation and preservation. The results suggested that α2-M activity could be readily inactivated at temperatures above 50°C, at pH levels above 9.0 or below 4.0, or in the presence of methylamine. Cohn Fraction IV is usually discarded as a biological waste product in the human serum albumin production process; because the simple process developed in this study is relatively inexpensive, the preparation of α2-M from Cohn Fraction IV may better utilize human plasma, a valuable resource.

Identification of a new alpha-2-macroglobulin: Multi-spectroscopic and isothermal titration calorimetry study.

A α2M homologue was isolated from sheep (Ovis aries) blood plasma, using a simple two-step procedure, ammonium sulphate fractionation and gel filtration chromatography. Sheep α2M was found to be a large tetrameric glycoprotein of 630 kDa with monomeric subunit of 133 kDa each. Each subunit of sheep α2M 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 79Ǻ, 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 (ΔH), entropy change (ΔS) and Gibb's free energy change (ΔG), along with number of binding sites (N) and affinity constant (K) are explored for α2M-trypsin binding for the first time for any known α2M molecule. The thermodynamics of proteinase-antiproteinase association suggests that trypsin-α2M interaction is enthalpy driven event.

Purification, characterization and molecular cloning of alpha-2-macroglobulin in cobia, Rachycentron canadum.

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.

Crystallization and preliminary X-ray diffraction analysis of eukaryotic α2 -macroglobulin family members modified by methylamine, proteases and glycosidases.

α2 -Macroglobulin (α2 M) has many functions in vertebrate physiology. To understand the basis of such functions, high-resolution structural models of its conformations and complexes with interacting partners are required. In an attempt to grow crystals that diffract to high or medium resolution, we isolated native human α2 M (hα2 M) and its counterpart from chicken egg white (ovostatin) from natural sources. We developed specific purification protocols, and modified the purified proteins either by deglycosylation or by conversion to their induced forms. Native proteins yielded macroscopically disordered crystals or crystals only diffracting to very low resolution (>20 Å), respectively. Optimization of native hα2 M crystals by varying chemical conditions was unsuccessful, while dehydration of native ovostatin crystals improved diffraction only slightly (10 Å). Moreover, treatment with several glycosidases hindered crystallization. Both proteins formed spherulites that were unsuitable for X-ray analysis, owing to a reduction of protein stability or an increase in sample heterogeneity. In contrast, transforming the native proteins to their induced forms by reaction either with methylamine or with peptidases (thermolysin and chymotrypsin) rendered well-shaped crystals routinely diffracting below 7 Å in a reproducible manner.

Purification of hen egg white ovomacroglobulin using one-step chromatography.

Hen egg white (EW) are one of the most ideal sources of active proteins, and ovomacroglobulin, as a protease inhibitor, has been demonstrated to possess numerous biological properties including antibacterial and anti-inflammatory properties as well as activity for the treatment of keratitis. The objective of this study was to develop a simple and rapid method for the purification of ovomacroglobulin from hen EW on a laboratory scale. Hen EW was diluted with an equal volume of distilled water followed by a two-step PEG precipitation to remove ovomucin and to obtain ovomacroglobulin-rich precipitate. The precipitate was dissolved and further purified by gel filtration chromatography. Ovomacroglobulin was collected with a purity of 97.0 ± 0.3% by HPLC and a yield of 62.5%. The atomic force microscopy images showed that ovomacroglobulin molecules on a mica surface emerged as an "oval-shaped plate" with a molecular volume of 1536.9 ± 330.0 nm(3) , indicating that purified ovomacroglobulin has an integrated molecular structure. With the improvement of PEG precipitation and the simplification of the chromatography, the whole purification process could be finished well within one working day. This protocol has an advantage of rapidity, and would facilitate studies of ovomacroglobulin.

Analysis of trypsin inhibition activity in human plasma proteins after separation by non-denaturing two-dimensional electrophoresis.

BACKGROUND: The functions of proteins can be retained following separation by non-denaturing two-dimensional electrophoresis (2-DE). The trypsin inhibition activities can then be examined following the separation and immobilization of the proteins under non-denaturing conditions. METHODS: Human plasma proteins were separated using 2-DE and transferred onto a polyvinylidene difluoride membrane and stained using Ponceau S. The trypsin inhibition activity of the membrane-bound proteins was qualitatively examined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The activities were also quantitatively examined by analyzing the release of the azo-chromophore when azocasein was the substrate. RESULTS: Trypsin activity was inhibited by the haptoglobin and α2-macroglobulin spots located on the membrane, whereas the protease activity was retained for the spots containing albumin and transferrin. The inhibition activities of the α2-macroglobulin and haptoglobin spots were 4.81- and 4.83-fold higher, respectively, when compared with the inhibition activity of the albumin spot. An axis of the relative activities of trypsin inhibition was added to the 2-DE pattern of human plasma proteins to construct a non-denaturing 3-D map of human plasma proteins. CONCLUSION: This 3-D map should represent a suitable diagnostic tool for the qualitative and quantitative analyses of the trypsin inhibition activities of proteins.

[The prognostic value of biochemical indicators under neonatal hepatitis of different etiology].

Recently, the share of children with verified neonatal hepatitis induced by genetic predisposition, malformations of biliary tracts, inborn infections with affection of hepatobiliary system increased. The comprehensive biochemical examination of 62 children aged from 1.5 months to 2 years old with diagnosis of neonatal hepatitis. The changes of standard indicators of cytolysis, cholestasis and protein metabolism were on average moderate in group with reliable increase of protein concentration of acute phase of inflammation. The peak changes of biochemical indicators during primary examination are revealed in group of children with malformations of hepatobiliary system conditioned by viruses of herpetic group and in the process of development of expressed fibrosis of liver up to first year of life. The detection of proteins of acute phase makes it possible to objectively evaluate the presence of prolonged inflammatory process in liver and to promote prognosis of course of neonatal hepatitis in children of early age and timely correction of therapy and improvement of outcomes of disease. The detection of C-reactive protein, alpha2-macroglobulin and alpha1-antitripsin is recommended to be included into algorithm of examination of children with neonatal hepatitis.

Application of champedak mannose-binding lectin in the glycoproteomic profiling of serum samples unmasks reduced expression of alpha-2 macroglobulin and complement factor B in patients with nasopharyngeal carcinoma.

The use of lectin affinity chromatography prior to 2-DE separation forms an alternative method to unmask the expression of targeted glycoproteins of lower abundance in serum samples. Reduced expression of alpha-2 macroglobulin (AMG) and complement factor B (CFB) was detected in sera of patients with nasopharyngeal carcinoma (NPC) when pooled serum samples of the patients and those of healthy individuals were subjected to affinity isolation using immobilized champedak mannose-binding lectin and analyzed by 2-DE and densitometry. The AMG and CFB spots were not detected in the 2-DE protein profiles when the same pooled serum samples were subjected to albumin and IgG depletion and neither were they detected when the depleted samples were analyzed by western blotting and lectin detection. Together with other acute-phase response proteins that were previously reported to be altered in expression in NPC patients, AMG and CFB may serve as useful complementary biomarkers for NPC.

Antigen delivery by alpha(2)-macroglobulin enhances the cytotoxic T lymphocyte response.

alpha(2)M* targets antigens to APCs for rapid internalization, processing, and presentation. When used as an antigen-delivery vehicle, alpha(2)M* amplifies MHC class II presentation, as demonstrated by increased antibody titers. Recent evidence, however, suggests that alpha(2)M* encapsulation may also enhance antigen-specific CTL immunity. In this study, we demonstrate that alpha(2)M*-delivered antigen (OVA) enhances the production of specific in vitro and in vivo CTL responses. Murine splenocytes expressing a transgenic TCR specific for CTL peptide OVA(257-264) (SIINFEKL) demonstrated up to 25-fold greater IFN-gamma and IL-2 secretion when treated in vitro with alpha(2)M*-OVA compared with soluble OVA. The frequency of IFN-gamma-producing cells was increased approximately 15-fold, as measured by ELISPOT. Expansion of the OVA-specific CD8+ T cell population, as assayed by tetramer binding and [3H]thymidine incorporation, and OVA-specific cell-mediated cytotoxicity, as determined by a flow cytometric assay, were also enhanced significantly by alpha(2)M*-OVA. Furthermore, significant CTL responses were observed at antigen doses tenfold lower than those required with OVA alone. Finally, we also observed enhanced humoral and CTL responses by naïve mice following intradermal immunization with alpha(2)M*-OVA. These alpha(2)M*-OVA-immunized mice demonstrated increased protection against a s.c.-implanted, OVA-expressing tumor, as demonstrated by delayed tumor growth and prolonged animal survival. The observation that alpha(2)M*-mediated antigen delivery elicits specific CTL responses suggests the cross-presentation of antigen onto MHC class I. These results support alpha(2)M* as an effective antigen-delivery system that may be particularly useful for vaccines based on weakly immunogenic subunits or requiring dose sparing.

Alpha 2-macroglobulin capture allows detection of mast cell chymase in serum and creates a reservoir of angiotensin II-generating activity.

Human chymase is a highly efficient angiotensin II-generating serine peptidase expressed by mast cells. When secreted from degranulating cells, it can interact with a variety of circulating antipeptidases, but is mostly captured by alpha(2)-macroglobulin, which sequesters peptidases in a cage-like structure that precludes interactions with large protein substrates and inhibitors, like serpins. The present work shows that alpha(2)-macroglobulin-bound chymase remains accessible to small substrates, including angiotensin I, with activity in serum that is stable with prolonged incubation. We used alpha(2)-macroglobulin capture to develop a sensitive, microtiter plate-based assay for serum chymase, assisted by a novel substrate synthesized based on results of combinatorial screening of peptide substrates. The substrate has low background hydrolysis in serum and is chymase-selective, with minimal cleavage by the chymotryptic peptidases cathepsin G and chymotrypsin. The assay detects activity in chymase-spiked serum with a threshold of approximately 1 pM (30 pg/ml), and reveals native chymase activity in serum of most subjects with systemic mastocytosis. alpha(2)-Macroglobulin-bound chymase generates angiotensin II in chymase-spiked serum, and it appears in native serum as chymostatin-inhibited activity, which can exceed activity of captopril-sensitive angiotensin-converting enzyme. These findings suggest that chymase bound to alpha(2)-macroglobulin is active, that the complex is an angiotensin-converting enzyme inhibitor-resistant reservoir of angiotensin II-generating activity, and that alpha(2)-macroglobulin capture may be exploited in assessing systemic release of secreted peptidases.

Involvement of glycans in the immunological cross-reaction between alpha-macroglobulin and hemocyanin of the gastropod Helix pomatia.

By tandem-crossed immunoelectrophoresis and ELISA experiments an immunological relationship was observed between alpha-macroglobulin (alphaM) and hemocyanin (Hc) of the terrestrial snail Helix pomatia. Both glycoproteins occur in the hemolymph: alphaM (minor component) as a specific proteinase inhibitor, Hc (consisting of three components: alpha(D)-HpH, alpha(N)-HpH and beta-HpH) as oxygen transport protein. The cross-reaction was found to be correlated with glycosylation. (i) With beta-HpH, which is richer in carbohydrates than alpha(D)-HpH and alpha(N)-HpH, mainly due to a higher 3-O-methyl-d-galactose content, the cross-reaction with HpalphaM was highest. (ii) From the 8 functional units, designated a-h, isolated from beta-HpH, two that lack carbohydrates (c and f) were not recognized by antibodies against HpalphaM, while the six glycosylated ones were strongly cross-reacting. The nearly complete loss of the cross-reactivity upon deglycosylation of functional units d and g and the inhibition in competitive ELISA experiments by glycopeptides isolated from both beta-HpH and HpalphaM are further evidence that glycans are involved in the immunological relationship between HpH and HpalphaM. Carbohydrate analyses indicated that the glycan structures present on HpalphaM are very similar (or identical) to those found on HpH, suggesting that glycans are common epitopes on both proteins. Especially d-xylose and 3-O-methyl-d-galactose seem to be responsible for the cross-reactivity since the alpha-macroglobulin and hemocyanin of the cephalopod Sepia officinalis, which lack these two monosaccharides in their glycan structures, do not immunologically cross-react.

Effect of high fiber density ratio polysulfone dialyzer on protein removal.

BACKGROUND/AIMS: A dialyzer (APS-EX) with a higher hollow fiber density ratio was manufactured using the highest performance polysulfone hollow fiber from Asahi-Kasei Medical. METHODS: We compared the performance of this device in comparison with hemodialysis (HD; APS-S) and hemodiafiltration (HDF) conditions (APS-S, 10 l post-HDF) to evaluate its merit as an internal filtration-enhanced dialyzer. RESULTS: With low molecular weight proteins, APS-EX had a reduction ratio of 74.3% for beta(2)-microglobulin (beta(2)-MG), and 31.0% for alpha(1)-MG. APS-EX had a significant higher removal amount of alpha(1)-MG compared to APS-S (HDF). Significant differences were seen in albumin loss, 4.0 g for APS-EX, 3.0 g for APS-S (HDF), and 0.9 g for APS-S (HD). Using HD mode, APS-EX demonstrated a performance which was more than equivalent to approximately 10 l post-HDF. CONCLUSIONS: The results suggested the possibility that HD equivalent to HDF can be performed safely with the ultrapure dialysate when using APS-EX with internal filtration.

High-resolution 2-DE for resolving proteins, protein adducts and complexes in plasma.

A 2-DE system has been devised in which proteins are first separated in their native state followed by separation according to mass under denaturing conditions (Nat/SDS-PAGE). Hydrophilic properties of the gel and the presence of dihydroxybisacrylamide in the first dimension allowed a good resolution for high-molecular-weight proteins and maintained interactions. With this method 252 plasma spots have been resolved and 140 have been characterized by MS as isoforms of 60 proteins, a relevant part of which (12) were not detected by traditional 2-D gels or by other nondenaturing 2-D techniques. The list includes complement factors (C4d, C7), coagulation factors (coagulation factor II, fibrin beta), apolipoproteins (apolipoprotein B) and cell debris (vinculin, gelsolin, tropomyosin, dystrobrevin beta, fibrinectin I). Nat/SDS PAGE also allowed separation of nicked forms of albumin, Apo B100 and alpha2-macroglobulin and showed the presence of atypical albumin adducts corresponding to post-translational and oxidation products. Our system provides therefore new tools for resolving proteins, protein aggregates and complexes and amplifies the potentiality of traditional electrophoretic analysis.

Alpha2-macroglobulin is a potential facilitator of prion protein transformation.

Cellular prion protein changes conformation during transformation to an infectious scrapie isoform. One measure of transformation is the development of partial resistance to protease treatment. A fraction of human and bovine plasma was identified containing activity that facilitates transformation of cellular prion protein to a protease resistant isoform in the presence of RNA in the absence of seeded scrapie prion protein. Purification of proteins from this fraction led to the identification of alpha2-macroglobulin as an active component suggesting that it may facilitate conformational changes in prion protein in spontaneous forms of prion disease.

Three complement-like repeats compose the complete alpha2-macroglobulin binding site in the second ligand binding cluster of the low density lipoprotein receptor-related protein.

Given the importance of the low density lipoprotein receptor-related protein (LRP) as an essential endocytosis and signaling receptor for many protein ligands, and of alpha2-macroglobulin (alpha2M)-proteinase complexes as one such set of ligands, an understanding of the specificity of their interaction with LRP is an important goal. A starting point is the known role of the 138-residue receptor binding domain (RBD) in binding to LRP. Previous studies have localized high affinity alpha2M binding to the eight complement repeat (CR)-containing cluster 2 of LRP. In the present study we have identified the minimum CR domains that constitute the full binding site for RBD and, hence, for alpha2M on LRP. We report on the ability of the triple construct of CR3-4-5 to bind RBD with an affinity (Kd = 130 nM) the same as for isolated RBD to intact LRP. This Kd is 30-fold smaller than for RBD to CR5-6-7, demonstrating the specificity of the interaction with CR3-4-5. Binding requires previously identified critical lysine residues but is almost pH-independent within the range of pH values encountered between extracellular and internal compartments, consistent with an earlier proposed model of intracellular ligand displacement by intramolecular YWTD domains. The present findings suggest a model to explain the ability of LRP to bind a wide range of structurally unrelated ligands in which a nonspecific ligand interaction with the acidic region present in most CR domains is augmented by interactions with other CR surface residues that are unique to a particular CR cluster.

The properties of rabbit alpha1-macroglobulin upon activation are distinct from those of rabbit and human alpha2-macroglobulins.

We have characterized native and activated forms of rabbit alpha1M and compared them to rabbit and human alpha2M. Similar to human alpha2M, rabbit alpha1M is a tetramer associated via disulfide bonds and non-covalent interactions that exhibits autolysis into two fragments when heated. Like human alpha2M, rabbit alpha1M is cleaved by trypsin at one site; however, rabbit alpha1M shares characteristics with rabbit alpha2M that are different from the properties of human alpha2M. Amine or trypsin treatment of rabbit alpha-macroglobulins does not result in a significant conformational change or cleavage of four thiolester bonds. Full thiolester cleavage is only observed for rabbit alpha1M after exposure to both trypsin and a small amine. Additionally, amine-treated rabbit alpha-macroglobulins retain trypsin inhibitory potential and do not fully shield bound proteinases. Methylamine and trypsin treatment of rabbit alpha1M results in two dissimilar conformations that display differing exposure of the receptor-recognition site. While ammonia- and methylamine-modified rabbit alpha1M bind to macrophages with similar affinity to that of human alpha2M, trypsin-treated rabbit alpha1M exhibits dramatically lower affinity. This suggests that rabbit alpha1M may not play the same proteinase-inhibiting physiological role as human alpha2M.

Purification of alpha2-macroglobulin and the construction of immunogenic alpha2-macroglobulin-peptide complexes for use as cancer vaccines.

A simple method for purification of immunogenic alpha2-macroglobulin from murine serum is described. Also described is a method for creating alpha2-macroglobulin-antigen complexes using purified alpha2-macroglobulin and peptides derived from tumor cell lysates. The application of such alpha2-macroglobulin-antigen complexes for eliciting cancer immunity is discussed.

Elevated levels of serum alpha(2) macroglobulin in wild black bears during hibernation.

Bear serum alpha(2) macroglobulin (alpha(2)M) was purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and partially characterized by tryptic digestion of alpha(2)M and analysis of the peptides by peptide mass fingerprinting. The molecular weight of bear serum alpha(2)M was 181 kDa, same as for human serum alpha(2)M, on SDS-PAGE. However, the MALDI mass spectrum of the tryptic digested bear serum alpha(2)M showed that it is different from human alpha(2)M or other data bank proteins. Liquid chromatography (LC)/mass spectrometry (MS)/MS of the proteolytic products of bear serum alpha(2)M showed eight peptides that had similarities to human alpha(2)M suggesting that the protein of interest was indeed alpha(2)M of bear. The polyclonal antibody against bear serum alpha(2)M recognized only one protein from the western blot of bear serum proteins. It also recognized human alpha(2)M. The levels of serum alpha(2)M were significantly increased during hibernating state as compared to active state of bears indicating its protective role from the consequences of the metabolic depression during hibernation.