Demonstration of α2-macroglobulin in cultured fibroblasts, using crossed immunoelectrophoresis

Normal human fibroblasts, cultured in medium supplemented with newborn calf serum (NCS), were examined using crossed immunoelectrophoresis, with antibodies directed against NCS. About ten serum components were detected in cell sonicates. The procedure by which the cells were dissociated (EDTA or trypsin) did not affect the number of peaks observed. The most prominent serum component present in cell sonicates was identified as α₂-macroglobulin.     

α1-Antitrypsin and α2-macroglobulin do not inhibit the kinin-releasing activity of kallikreins from human urine and saliva

α₁-Antitrypsin, α₂-macroglobulin and low-molecular weight kininogen were isolated from human serum and kallikreins from human urine and saliva.α₁-Antitrypsin and α₂-macroglobulin inhibited the activity of trypsin in releasing kinin from low-molecular weight kininogen, due to their binding with the enzyme, but did non inhibit or bind with urinary and salivary kallikreins.     

Electron microscopy of α2-macroglobulin with a thiol ester bound ligand

In order to covalently bind the hydrolyzed thiol ester groups of the human α₂-macroglobulin (α2M) transformed by methylamine, the phospholipase A2 (PLA2), a small enzyme (M_r = 13 000) from Naja nigricollis snake venom was activated by succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Average images determined from electron micrographs of the methylamine-transformed α2M, with and without activated PLA2, were determined by image processing and compared. A localization of the PLA2 was achieved by subtracting the average image of α2M transformed by methylamine from that containing PLA2. The results are consistent with previous work showing the central localization of chymotrypsin trapped in α2M. They also suggest that the four thiol esters are located near the center of the α2M. molecule.     

Endothelin-1 stimulates phospholipid hydrolysis and prostaglandin F2α production in primary human decidua cell cultures

The regulation of prostaglandin (PG) production by the uterine decidua may be an important mechanism controlling the onset and maintenance of human parturition. The present in vitro study has evaluated the potential for endothelin-1 (ET-1) to activate cell signalling and PGE2 alpha production in human primary decidua cell cultures. ET-1 stimulated a dose-dependent increase in inositol phospholipid hydrolysis and PG precursor release as evidenced by respective increases in [3H] inositol monophosphate accumulation and [14C] arachidonate release from radiolabelled decidua cells. PGF2 alpha production was increased in some but not all cell preparations in response to ET-1 alone. Pretreatment of decidua cells with interleukin-1 beta (IL-1 beta) enhanced PGF2 alpha production but not arachidonate release in response to ET-1. These in vitro observations support a possible role for ET-1 in the regulation of decidual PG production during parturition.     

A simple method for preparation of valency hybrid hemoglobins, (αβ)2 and (αβ)2

The improved methods for the preparation of valency hybrid hemoglobins, (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ were presented. The (α³⁺β²⁺)₂ valency hybrid was separated from the solutions of partially reduced methemoglobin with ascorbic acid, by using CM 32 column chromatography. The (α²⁺β³⁺)₂ valency hybrid was also isolated from hemoglobin solutions, which were partially oxidized with ferricyanide, by chromatography on CM 32 column. These valency hybrid hemoglobins were found to be single on isoelectric focusing electrophoresis. Present procedures are very simple and are suitable for the bulk preparation of (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ valency hybrids.     

Triiodo-L-thyronine stimulates glycogen synthesis in rat hepatocyte cultures

The hyperthyroid state is associated with low hepatic glycogen levels, but paradoxically with a high activity of glycogen synthase and low activity of glycogen phosphorylase. We determined the effects of triiodo-L-thyronine (T₃) on glycogen synthesis and glycogen synthase activity in rat hepatocytesin vitro. Culture of rat hepatocytes with T₃ (100 nM–1 M) for 16 h–40 h increases glycogen synthesis from glucose and gluconeogenic precursors. The stimulation of glycogen synthesis by T₃ was associated with an increase in the activity of glycogen synthase and was additive with the long-term effects of insulin but not with the short-term stimulation of glycogen synthesis by insulin. Culture of hepatocytes with T₃ (at concentrations up to 1 M) did not affect the responsiveness of glycogen synthesis to short-term stimulation by insulin but culture with 10 M-T₃ decreased the responsiveness to insulin without affecting the basal rate. It is suggested that the high activity of glycogen synthase in the hyperthyroid state is due to a direct effect of T₃ on the hepatocyte, but the low hepatic glycogen content is probably due to either secondary metabolite and/or endocrine changes or to impaired responsiveness to insulin. T₃ may have an anabolic role in the control of hepatic glycogen storage in the euthyroid postprandial state. (Mol Cell Biol120: 151–158, 1993)Abbreviations T₃ triiodo-L-thyronine     

Mapping of the α4 subunit gene (GABRA4) to human chromosome 4 defines an α2—α4—β1—γ1 gene cluster: further evidence that modern GABAA receptor gene clusters are derived from an ancestral cluster

We demonstrated previously that an α₁—β₂—γ₂ gene cluster of the γ-aminobutyric acid (GABA_A) receptor is located on human chromosome 5q34–q35 and that an ancestral α—β—γ gene cluster probably spawned clusters on chromosomes 4, 5, and 15. Here, we report that the α₄ gene (GABRA4) maps to human chromosome 4p14–q12, defining a cluster comprising the α₂, α₄, β₁, and γ₁ genes. The existence of an α₂—α₄—β₁—γ₁ cluster on chromosome 4 and an α₁—α₆—β₂—γ₂ cluster on chromosome 5 provides further evidence that the number of ancestral GABA_A receptor subunit genes has been expanded by duplication within an ancestral gene cluster. Moreover, if duplication of the α gene occurred before duplication of the ancestral gene cluster, then a heretofore undiscovered subtype of α subunit should be located on human chromosome 15q11–q13 within an α₅—α_x—β₃—γ₃ gene cluster at the locus for Angelman and Prader—Willi syndromes.     

Immunoelectron microscopy studies with a monoclonal antibody directed against a receptor recognition site epitope in human α2-macroglobulin

α₂-Macroglobulin (α₂M) is a plasma proteinase inhibitor that binds up to 2 mole of proteinase per mole of inhibitor. Proteinase binding or reaction with small primary amines causes a major conformational change in α₂M. As a result of this conformational change, a new epitope recognized by monoclonal antibody 7H11D6 is exposed. The association of α₂M-proteinase or α₂M-methylamine with α₂M cellular receptors is prevented by 7H11D6. In this investigation, the binding of 7H11D6 to α₂M was studied by electron microscopy. 7H11D6 bound to α₂M-methylamine and α₂M-trypsin but not to native α₂M. The structure of α₂M after conformational change resembled the letter “H.” 7H11D6 epitopes were identified near the apices of the four arms in the α₂M “H” structure. 7H11D6 that was adducted to colloidal gold (7HAu) retained the specificity of the free antibody (binding to α₂M-trypsin but not to native α₂M). α₂M conformational change intermediates prepared by sequential reaction with a protein crosslinker and trypsin also bound 7HAu. These results suggest that a complete α₂M conformational change is not necessary for 7H11D6 epitope exposure and may not be required for receptor recognition. 7HAu was used to isolate a preparation consisting primarily of binary α₂M-trypsin (1 mole trypsin per mole α₂M instead of 2). Structures resembling the letter “H” were most common; however, each field showed some atypical molecules with arms that were compacted instead of thin and elongated. These incompletely transformed structures were similar to the α₂M conformational intermediates described previously (S. L. Gonias and N. L. Figler (1989) J. Biol. Chem. 264, 9565–9570). We propose that lateral arm extension is a critical step in α₂M conformational change. Failure of lateral arm extension is probably a common property of different α₂M conformational intermediates.     

α2-macroglobulin ‘fast’ forms inhibit superoxide production by activated macrophages

Mouse peritoneal macrophages activated by bacillus Calmette-Guerin (BCG) were incubated with human α₂-macroglobulin converted to its ‘fast’ form with either trypsin or methylamine before being stimulated with phorbol myrystate acetate. Both α₂-macroglobulin-trypsin and α₂-macroglobulin-methylamine inhibited macrophage production of superoxide anion (O₂⁻) while native α₂-macroglobulin had little effect except at high concentration. The α₂-macroglobulin ‘fast’ forms, which bind with a K_d of about 8 nM, inhibited 50% generation of O₂⁻(ID₅₀) at a concentration of 7 nM while α₂-macroglobulin inhibited O₂⁻ production with an ID₅₀ of 141 nM. The ‘fast’ forms of α₂-macroglobulin may play a role in the feedback regulation of inflammatory reactions.     

Acidic fibroblast growth factor (HBGF-1) stimulates DNA synthesis in primary rat hepatocyte cultures.

Acidic fibroblast growth factor (aFGF) stimulated DNA synthesis in primary rat hepatocyte cultures in a dose-dependent manner with maximal effect at 10-50 ng ml-1. This activity was dependent on the presence of heparin at a concentration of 10-50 micrograms.ml-1. Insulin interacted synergistically with aFGF, as it did with epidermal growth factor (EGF). The response to aFGF was only 50% that found with EGF. The disparity was not due to different kinetics of DNA synthesis, since the peak response for both growth factors occurred at 36-72 hr after plating of the hepatocytes. The potential relevance of this novel hepatocyte mitogen to normal and pathological liver growth is discussed.     

A novel serine protease complexed with α2-macroglobulin from skeletal muscle of lizard fish (Saurida undosquamis)

A novel fish muscle serine protease named muscle soluble serine protease (MSSP) was purified from the soluble fraction of lizard fish (Saurida undosquamis: Synodontidae) muscle by ammonium sulfate fractionation followed by four steps of column chromatographies. In native-PAGE, the purified enzyme appeared as a single band with an estimated mol. mass of approximately 380 kDa by gel filtration. In SDS-PAGE under reducing conditions, the purified enzyme migrated as two protein bands at 110 and 100 kDa, named subunits A and B, respectively. The 20 residues of N-terminal amino acid sequence of subunit B showed 70% of homology to β-chain of carp α₂-macroglobulin-1. Moreover, both subunits A and B showed immunoreactivity with anti carp α₂-macroglobulin antibody. Purified MSSP was inactivated by Pefabloc SC, aprotinin, benzamidine and TLCK, but not by α₁-antitrypsin. After acid treatment (pH 2, 24 h), however, the enzyme activity eluted at 14 kDa from Sephacryl S-200 carried out under acidic conditions was inhibited by α₁-antitrypsin. Lizard fish MSSP most rapidly hydrolyzed Boc-Val-Pro-Arg-MCA and Boc-Gln-Arg-Arg-MCA, but did not hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA and Suc-Ala-Ala-Pro-Phe-MCA, and was not suppressed either by E-64, pepstatin A and ethylenediaminetetraacetic acid (EDTA). These results indicate that the purified MSSP is a serine protease complexed with α₂-macroglobulin, and the entrapped protease was dissociated by the acid treatment. Purified and free MSSPs were most active at pH 10.0 and 9.0, respectively. Purified MSSP degraded myofibrillar proteins and casein but time courses of degradation of these substrates by the enzyme differed.     

Immunochemical study of β-glucuronidase inhibitor from porcine sublingual gland. Interaction of β-glucuronidase inhibitor with α2n-macroglobulin

Antiserum to the inhibitor of β-glucuronidase isolated from porcine sublingual gland was prepared in rabbits. Double immunodiffusion with the inhibitor produced a single precipitin line. However, neutralization of the inhibitor was produced by the antiserum and also by normal serum.Anti-β-glucuronidase inhibitor isolated from human serum, by fractionation with (NH₄)₂SO₄ followed by DEAE-cellulose, Sephadex G-200 and Sepharose 4B chromatography, was identified as α₂-macroglobulin by using ultracentrifuge analysis and immunoelectrophoresis. The mechanism of interaction of β-glucuronidase inhibitor with α₂-macroglobulin was also studied.     

The β4Integrin Subunit Is Expressed in Mouse Fibroblasts and Modulated by Transforming Growth Factor-β1

Integrin β₄subunit is present in association with α₆chain on both normal and transformed epithelial cells. Recently α₆β₄heterodimer was found on the endothelium of medium-sized blood vessels and on immature thymocytes. In this report we show, by Northern blotting, indirect immunofluorescence, immunoprecipitation, and Western blotting, that β₄subunit is expressed also on cells of mesenchymal origin such as fibroblasts, myoblasts, and myotubes. Increased expression of α₆β₄has been related to the aggressive metastatic phenotype of human and murine carcinomas. The transforming growth factor β₁(TGF-β₁) has been found to modulate the expression of several integrins and intracellular matrix proteins, as well as to stimulate cell invasion and metastatic potential. To evaluate whether α₆β₄expression is modulated by TGF-β₁, we transfected 3T3 fibroblasts with an expression vector carrying the human TGF-β₁cDNA driven by the SV40 early promoter. We observed by indirect immunofluorescence a modification in the subcellular distribution of β₄subunit, which acquires a perinuclear localization. This finding suggests this integrin subunit correlates with the cytoskeletal reorganization induced by TGF-β₁.     

Synthesis of Benzodioxinopyrroles as selective α2-adrenoceptor antagonists

A novel series of tetrahydrobenzodioxinopyrroles has been identified as potent and selective α₂-adrenoceptor antagonists. Convergent syntheses have been developed that allowed the preparation of analogues and their enantiomers. A compound of particular interest is the 5-fluoro substituted analogue (fluparoxan).     

β1 And β3 integrins have different roles in the adhesion and migration of vascular smooth muscle cells on extracellular matrix

Extracellular matrix receptors on ductus arteriosus smooth muscle cells (SMC) must enable the cells to migrate through both interstitial and basement membrane matrices to form intimal mounds during postnatal ductus closure. We examined the role of β₁ and β₃ integrin receptors on SMC adhesion and migration. Using a new assay to measure cell migration, we found that lamb ductus arteriosus SMC attach to and migrate over surfaces coated with fibronectin (FN), laminin (LN), vitronectin (VN), and collagens I (I) and IV (IV). Blocking antibodies, specific to different integrin complexes, showed that SMC adhesion to FN, LN, I, and IV depended exclusively on functioning β₁ integrins with little, if any, contribution by the α_vβ₃ integrin; on the other hand, cell migration over these substrates depended to a large extent on the α_vβ₃ receptor. Immunofluorescent staining demonstrated that during the early phase of SMC migration, the β₁ integrins organized rapidly into focal plaques that, with time, gradually covered the cell's basal surface; on the other hand, the β₃ receptor remained concentrated at all times at the cell's margins. Ligand affinity chromatography and immunoprecipitation techniques identified a unique series of β₁ integrins binding to each matrix component: FN (α₅β₁, α₃β₁, α_vβ₁), LN (α₁β₁, α₇β₁), VN (α_vβ₁), I (α₁β₁, α₂β₁), and IV (α₁β₁). In contrast, the β₃ integrin, α_vβ₃, bound to all the substrates tested: FN, LN, VN, I, and IV. The results indicate that β₁ and β₃ integrins may play different roles in attachment and migration as SMC move through the vascular extracellular matrix to produce obliteration of the ductus arteriosus lumen.     

Expression, purification and characterization of recombinant Z α1-Antitrypsin—The most common cause of α1-Antitrypsin deficiency

α₁-Antitrypsin (α₁AT), the most abundant proteinase inhibitor circulating in the blood, protects extracellular matrix proteins of the lung against proteolytic destruction by neutrophil elastase. α₁AT deficiency predisposes patients to emphysema, juvenile cirrhosis and hepatocellular carcinoma. Over 90% of clinical cases of severe α₁AT deficiency are caused by the Z variant (E342K) of α₁AT. The presence of the Z mutation results in misfolding and polymerization of α₁AT. Due to its inherent propensity to polymerize there are no reported cases of recombinant Z α₁AT production. This has created a major impediment to studying the effect of the Z mutation on α₁AT. Here we report our attempts to produce recombinant Z α₁AT using both Escherichia coli and Pichia pastoris as host systems. Using a range of expression vectors in E. coli we were unable to produce soluble active Z α₁AT. Cytosolic expression of the Z α₁AT gene in P. pastoris was successful. Monomeric and active recombinant Z α₁AT was purified from the yeast cytosol using affinity chromatography and anion exchange chromatography. Biochemical analyses demonstrated that the recombinant Z α₁AT has identical properties to its native counterpart purified from plasma of patients homozygous for the Z allele. A recombinant source of pathological Z α₁AT will increase the chances of elucidating the mechanism of its polymerization and thus the development of therapeutic strategies.     

From the {Cu(μ2-S)N}4 butterfly architecture to the {Cu(μ3-S)N}12 double wheel

Copper(I) complexes with {Cu(μ₂-S)N}₄ and {Cu(μ₃-S)N}₁₂ core portions of butterfly-shaped or double wheel architectures have been isolated in the reaction of Cu(I) with the Schiff base ligand C₆H₄(CHNC₆H₄S)₂, “iso-abt”, under different conditions. containing the tetranuclear electroneutral complex is formed by the reaction of CuI in acetonitrilic solution and recrystallization from DMF, whereas containing dodecanuclear wheels is accessible starting from CuBF₄. Complexes 2 and 4 represent the first examples of cyclic complexes with the same overall stoichiometry but different ring sizes. The ligand induces two different coordination environments around copper(I) by switching between μ₂- and μ₃-sulfur bridging modes.