The vitamin E isoforms α-tocopherol and γ-tocopherol have opposite associations with spirometric parameters: the CARDIA study

Background

Clinical studies of the associations of vitamin E with lung function have reported conflicting results. However, these reports primarily examine the α-tocopherol isoform of vitamin E and have not included the isoform γ-tocopherol which we recently demonstrated in vitro opposes the function of α-tocopherol. We previously demonstrated, in vitro and in animal studies, that the vitamin E isoform α-tocopherol protects, but the isoform γ-tocopherol promotes lung inflammation and airway hyperresponsiveness.

Methods

To translate these findings to humans, we conducted analysis of 4526 adults in the Coronary Artery Risk Development in Young Adults (CARDIA) multi-center cohort with available spirometry and tocopherol data in blacks and whites. Spirometry was obtained at years 0, 5, 10, and 20 and serum tocopherol was from years 0, 7 and 15 of CARDIA.

Results

In cross-sectional regression analysis at year 0, higher γ-tocopherol associated with lower FEV₁ (p = 0.03 in blacks and p = 0.01 in all participants) and FVC (p = 0.01 in blacks, p = 0.05 in whites, and p = 0.005 in all participants), whereas higher α-tocopherol associated with higher FVC (p = 0.04 in blacks and whites and p = 0.01 in all participants). In the lowest quartile of α-tocopherol, higher γ-tocopherol associated with a lower FEV₁ (p = 0.05 in blacks and p = 0.02 in all participants). In contrast, in the lowest quartile of γ-tocopherol, higher α-tocopherol associated with a higher FEV₁ (p = 0.03) in blacks. Serum γ-tocopherol >10 μM was associated with a 175–545 ml lower FEV₁ and FVC at ages 21–55 years.

Conclusion

Increasing serum concentrations of γ-tocopherol were associated with lower FEV1 or FVC, whereas increasing serum concentrations of α-tocopherol was associated with higher FEV1 or FVC. Based on the prevalence of serum γ-tocopherol >10 μM in adults in CARDIA and the adult U.S. population in the 2011 census, we expect that the lower FEV₁ and FVC at these concentrations of serum γ-tocopherol occur in up to 4.5 million adults in the population.     

The relationship between osteoclastogenic and anti-osteoclastogenic pro-inflammatory cytokines differs in human osteoporotic and osteoarthritic bone tissues

Background

Pro-inflammatory cytokines possess osteoclastogenic or anti-osteoclastogenic activities. They influence osteoclasts directly or via the receptor activator of nuclear factor κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG) system. Recent evidence suggests that inflammation may play a role in osteoporosis (OP) and osteoarthritis (OA). We aimed therefore to determine whether there is a difference between both groups: first, in the expression of the osteoclastogenic and anti-osteoclastogenic cytokines, second, in correlation of these cytokines with bone mineral density (BMD) and levels of bone turnover markers (BTM) and third, in correlation between the expression of these cytokines and osteoclast specific genes and RANK/RANKL/OPG genes.

Methods

Human bone samples from 54 age and sex matched patients with OP or OA were collected during hip arthroplasty surgery. The expression of 25 genes encoding pro-inflammatory cytokines, their receptors, osteoclast specific genes and RANK/RANKL/OPG genes was measured using quantitative real-time PCR. Total hip, femoral neck and lumbar spine BMD and BTM in blood samples were measured. The comparison between OP and OA was assessed using Student''s t-test or Mann-Whitney U test and correlations between gene expression, BMD and BTM were determined using nonparametric correlation.

Results

The results demonstrated a higher expression of interleukin (IL)-6 and IL-1α in OP, and interferon (IFN)-γ in OA (p < 0.0005). Negative correlations of total hip BMD with tumor necrosis factor-α (TNF-α) in OA and with RANKL/RANK in OP were found (p < 0.05). Significant correlations with BTM were shown for IL-1α and IFN-γ in OP (rho = 0.608 and -0.634) and for TNF-α, IL-6 and transforming growth factor-β1 (TGF-β1) in OA (rho = 0.591, -0.521 and 0.636). Results showed OP specific negative correlations (IFN-γ with ITGB3, IFN-β1 with CTSK, tartrate resistant acid phosphatase (TRAP), CALCR, RANK, RANKL, IL-1α with CTSK, OPG, IL-17A with CALCR) and positive (TGF-β1 with CTSK, TRAP, RANK), and OA specific negative (IL-1α with osteoclast associated immunoglobulin-like receptor (OSCAR), TNF-α with RANK, RANKL, OPG) and positive (IL-6 with RANK, RANKL, OPG) correlations.

Conclusions

Our results demonstrate that the relationship between osteoclastogenic and anti-osteoclastogenic pro-inflammatory cytokines differs in human OP and OA bone and could present an important factor for characteristics of OP and OA bone phenotypes.     

Extension of a de novo TIM barrel with a rationally designed secondary structure element

The ability to construct novel enzymes is a major aim in de novo protein design. A popular enzyme fold for design attempts is the TIM barrel. This fold is a common topology for enzymes and can harbor many diverse reactions. The recent de novo design of a four‐fold symmetric TIM barrel provides a well understood minimal scaffold for potential enzyme designs. Here we explore opportunities to extend and diversify this scaffold by adding a short de novo helix on top of the barrel. Due to the size of the protein, we developed a design pipeline based on computational ab initio folding that solves a less complex sub‐problem focused around the helix and its vicinity and adapt it to the entire protein. We provide biochemical characterization and a high‐resolution X‐ray structure for one variant and compare it to our design model. The successful extension of this robust TIM‐barrel scaffold opens opportunities to diversify it towards more pocket like arrangements and as such can be considered a building block for future design of binding or catalytic sites.     

Expression of SERPINA3s in cattle: focus on bovSERPINA3-7 reveals specific involvement in skeletal muscle

α₁-Antichymotrypsin is encoded by the unique SERPINA3 gene in humans, while it is encoded by a cluster of eight closely related genes in cattle. BovSERPINA3 proteins present a high degree of similarity and significant divergences in the reactive centre loop (RCL) domains which are responsible for the antiprotease activity. In this study, we analysed their expression patterns in a range of cattle tissues. Even if their expression is ubiquitous, we showed that the expression levels of each serpin vary in different tissues of 15-month-old Charolais bulls. Our results led us to focus on bovSERPINA3-7, one of the two most divergent members of the bovSERPINA3 family. Expression analyses showed that bovSERPINA3-7 protein presents different tissue-specific patterns with diverse degrees of N-glycosylation. Using a specific antibody raised against bovSERPINA3-7, Western blot analysis revealed a specific 96 kDa band in skeletal muscle. BovSERPINA3-7 immunoprecipitation and mass spectrometry revealed that this 96 kDa band corresponds to a complex of bovSERPINA3-7 and creatine kinase M-type. Finally, we reported that the bovSERPINA3-7 protein is present in slow-twitch skeletal myofibres. Precisely, bovSERPINA3-7 specifically colocalized with myomesin at the M-band region of sarcomeres where it could interact with other components such as creatine kinase M-type. This study opens new prospects on the bovSERPINA3-7 function in skeletal muscle and promotes opportunities for further understanding of the physiological role(s) of serpins.     

Rings and ribbons in protein structures: Characterization using helical parameters and Ramachandran plots for repeating dipeptides

Helical parameters displayed on a Ramachandran plot allow peptide structures with successive residues having identical main chain conformations to be studied. We investigate repeating dipeptide main chain conformations and present Ramachandran plots encompassing the range of possible structures. Repeating dipeptides fall into the categories: rings, ribbons, and helices. Partial rings occur in the form of “nests” and “catgrips”; many nests are bridged by an oxygen atom hydrogen bonding to the main chain NH groups of alternate residues, an interaction optimized by the ring structure of the nest. A novel recurring feature is identified that we name unpleated β, often situated at the ends of a β‐sheet strand. Some are partial rings causing the polypeptide to curve gently away from the sheet; some are straight. They lack β‐pleat and almost all incorporate a glycine. An example is the first glycine in the GxxxxGK motif of P‐loop proteins. Ribbons in repeating dipeptides can be either flat, as seen in repeated type II and type II′ β‐turns, or twisted, as in multiple type I and type I′ β‐turns. Hexa‐ and octa‐peptides in such twisted ribbons occur frequently in proteins, predominantly with type I β‐turns, and are the same as the “β‐bend ribbons” hitherto identified only in short peptides. One is seen in the GTPase‐activating protein for Rho in the active, but not the inactive, form of the enzyme. It forms a β‐bend ribbon, which incorporates the catalytic arginine, allowing its side chain guanidino group to approach the active site and enhance enzyme activity. Proteins 2014; 82:230–239. © 2013 Wiley Periodicals, Inc.     

Theoretical investigations on circular and chain-like hydrogen bonded structures found in two crystal forms of α-cyclodextrin hexahydrate: Models for hydration and water clusters

Hydration of macromolecules and the structure of water of crystallization are not understood in detail because in these complex systems. H-atoms cannot be located and the hydrogen bonding schemes are not known. X-ray and neutron diffraction studies on a hydrated oligosaccharide, α-cyclodextrin 6H₂O, ((C₆H₁₀O₅)₆·6H₂O), crystals forms A and B, gave insight into the chain-like and circular arrangement of hydrogen bonds. In the circles, homodromic (unidirectional) and antidromic (counter-running) orientation of five to six hydrogen bounds is observed. PCILO calculations showed that homodromic circles and chains are approx. 8% per hydrogen bond more stable than antidromic circles, that the changes in electronic charges on H and O atoms are greater in homo than in antidromic systems and that the dipole moments are only approx. 3 D in the homodromic circles but 6–8 D in chain-like and antidromic arrangement. These results have been interpreted in terms of cooperative effect. Circular systems are considered as structural elements in hydration shells of macromolecules and in the assembly of ‘flickering’ water clusters.     

Modularity in Protein Evolution: Modular Organization and De Novo Domain Evolution in Mollusk Metallothioneins

Metallothioneins (MTs) are proteins devoted to the control of metal homeostasis and detoxification, and therefore, MTs have been crucial for the adaptation of the living beings to variable situations of metal bioavailability. The evolution of MTs is, however, not yet fully understood, and to provide new insights into it, we have investigated the MTs in the diverse classes of Mollusks. We have shown that most molluskan MTs are bimodular proteins that combine six domains—α, β1, β2, β3, γ, and δ—in a lineage-specific manner. We have functionally characterized the Neritimorpha β₃β₁ and the Patellogastropoda γβ₁ MTs, demonstrating the metal-binding capacity of the new γ domain. Our results have revealed a modular organization of mollusk MT, whose evolution has been impacted by duplication, loss, and de novo emergence of domains. MTs represent a paradigmatic example of modular evolution probably driven by the structural and functional requirements of metal binding.     

Convergence of alpha(v)beta(3) integrin- and macrophage colony stimulating factor-mediated signals on phospholipase Cgamma in prefusion osteoclasts

The macrophage colony stimulating factor (M-CSF) and alpha(v)beta(3) integrins play critical roles in osteoclast function. This study examines M-CSF- and adhesion-induced signaling in prefusion osteoclasts (pOCs) derived from Src-deficient and wild-type mice. Src-deficient cells attach to but do not spread on vitronectin (Vn)-coated surfaces and, contrary to wild-type cells, their adhesion does not lead to tyrosine phosphorylation of molecules activated by adhesion, including PYK2, p130(Cas), paxillin, and PLC-gamma. However, in response to M-CSF, Src(-/-) pOCs spread and migrate on Vn in an alpha(v)beta(3)-dependent manner. Involvement of PLC-gamma activation is suggested by using a PLC inhibitor, U73122, which blocks both adhesion- and M-CSF-mediated cell spreading. Furthermore, in Src(-/-) pOCs M-CSF, together with filamentous actin, causes recruitment of beta(3) integrin and PLC-gamma to adhesion contacts and induces stable association of beta(3) integrin with PLC-gamma, phosphatidylinositol 3-kinase, and PYK2. Moreover, direct interaction of PYK2 and PLC-gamma can be induced by either adhesion or M-CSF, suggesting that this interaction may enable the formation of integrin-associated complexes. Furthermore, this study suggests that in pOCs PLC-gamma is a common downstream mediator for adhesion and growth factor signals. M-CSF-initiated signaling modulates the alpha(v)beta(3) integrin-mediated cytoskeletal reorganization in prefusion osteoclasts in the absence of c-Src, possibly via PLC-gamma.     

Knowledge-based protein secondary structure assignment

We have developed an automatic algorithm STRIDE for protein secondary structure assignment from atomic coordinates based on the combined use of hydrogen bond energy and statistically derived backbone torsional angle information. Parameters of the pattern recognition procedure were optimized using designations provided by the crystallographers as a standard-of-truth. Comparison to the currently most widely used technique DSSP by Kabsch and Sander (Biopolymers 22:2577-2637, 1983) shows that STRIDE and DSSP assign secondary structural states in 58 and 31% of 226 protein chains in our data sample, respectively, in greater agreement with the specific residue-by-residue definitions provided by the discoverers of the structures while in 11% of the chains, the assignments are the same. STRIDE delineates every 11th helix and every 32nd strand more in accord with published assignments. © 1995 Wiley-Liss, Inc.     

The Structure of a Full-length Membrane-embedded Integrin Bound to a Physiological Ligand

Increased ligand binding to integrin (“activation”) underpins many biological processes, such as leukocyte trafficking, cell migration, host-pathogen interaction, and hemostasis. Integrins exist in several conformations, ranging from compact and bent to extended and open. However, the exact conformation of membrane-embedded, full-length integrin bound to its physiological macromolecular ligand is still unclear. Integrin α_IIbβ₃, the most abundant integrin in platelets, has been a prototype for integrin activation studies. Using negative stain electron microscopy and nanodisc-embedding to provide a membrane-like environment, we visualized the conformation of full-length α_IIbβ₃ in both a Mn²⁺-activated, ligand-free state and a Mn²⁺-activated, fibrin-bound state. Activated but ligand-free integrins exist mainly in the compact conformation, whereas fibrin-bound α_IIbβ₃ predominantly exists in a fully extended, headpiece open conformation. Our results show that membrane-embedded, full-length integrin adopts an extended and open conformation when bound to its physiological macromolecular ligand.     

Plasmin-sensitive dibasic sequences in the third fibronectin-like domain of L1-cell adhesion molecule (CAM) facilitate homomultimerization and concomitant integrin recruitment

L1 is a multidomain transmembrane neural recognition molecule essential for neurohistogenesis. While moieties in the immunoglobulin-like domains of L1 have been implicated in both heterophilic and homophilic binding, the function of the fibronectin (FN)-like repeats remains largely unresolved. Here, we demonstrate that the third FN-like repeat of L1 (FN3) spontaneously homomultimerizes to form trimeric and higher order complexes. Remarkably, these complexes support direct RGD-independent interactions with several integrins, including alpha(v)beta(3) and alpha(5)beta(1). A pep- tide derived from the putative C-C' loop of FN3 (GSQRKHSKRHIHKDHV(852)) also forms trimeric complexes and supports alpha(v)beta(3) and alpha(5)beta(1) binding. Substitution of the dibasic RK(841) and KR(845) sequences within this peptide or the FN3 domain limited multimerization and abrogated integrin binding. Evidence is presented that the multimerization of, and integrin binding to, the FN3 domain is regulated both by conformational constraints imposed by other domains and by plasmin- mediated cleavage within the sequence RK( downward arrow)HSK( downward arrow)RH(846). The integrin alpha(9)beta(1), which also recognizes the FN3 domain, colocalizes with L1 in a manner restricted to sites of cell-cell contact. We propose that distal receptor ligation events at the cell-cell interface may induce a conformational change within the L1 ectodomain that culminates in receptor multimerization and integrin recruitment via interaction with the FN3 domain.     

Spectroscopic and electronic structure studies of the role of active site interactions in the decarboxylation reaction of α-keto acid-dependent dioxygenases

The α-ketoglutate (α-KG)-dependent dioxygenases are a large class of mononuclear non-heme iron enzymes that require Fe^II, α-KG and dioxygen for catalysis, with the α-KG cosubstrate supplying the two additional electrons required for dioxygen activation. A sub-class of these enzymes exists in which the α-keto acid is covalently attached to the substrate, including (4-hydroxy)mandelate synthase (HmaS) and (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) which utilize the same substrate but exhibit two different general reactivities (H-atom abstraction and electrophilic attack). Previous kinetic studies of Streptomyces avermitilis HPPD have shown that the substrate analog phenylpyruvate (PPA), which only differs from the normal substrate (4-hydroxyphenyl)pyruvate (HPP) by the absence of a para-hydroxyl group on the aromatic ring, does not induce a reaction with dioxygen. While an Fe^IVO intermediate is proposed to be the reactive species in converting substrate to product, the key step utilizing O₂ to generate this species is the decarboxylation of the α-keto acid. It has been generally proposed that the two requirements for decarboxylation are bidentate coordination of the α-keto acid to Fe^II and the presence of a 5C Fe^II site for the O₂ reaction. Circular dichroism and magnetic circular dichroism studies have been performed and indicate that both enzyme complexes with PPA are similar with bidentate α-KG coordination and a 5C Fe^II site. However, kinetic studies indicate that while HmaS reacts with PPA in a coupled reaction similar to the reaction with HPP, HPPD reacts with PPA in an uncoupled reaction at an 10⁵-fold decreased rate compared to the reaction with HPP. A key difference is spectroscopically observed in the n → π^* transition of the HPPD/Fe^II/PPA complex which, based upon correlation to density functional theory calculations, is suggested to result from H-bonding between a nearby residue and the carboxylate group of the α-keto acid. Such an interaction would disfavor the decarboxylation reaction by stabilizing electron density on the carboxylate group such that the oxidative cleavage to yield CO₂ is disfavored.     

Solvent-induced differentiation of protein backbone hydrogen bonds in calmodulin

In apo and holoCaM, almost half of the hydrogen bonds (H-bonds) at the protein backbone expected from the corresponding NMR or X-ray structures were not detected by h3JNC' couplings. The paucity of the h3JNC' couplings was considered in terms of dynamic features of these structures. We examined a set of seven proteins and found that protein-backbone H-bonds form two groups according to the h3JNC' couplings measured in solution. H-bonds that have h3JNC' couplings above the threshold of 0.2 Hz show distance/angle correlation among the H-bond geometrical parameters, and appear to be supported by the backbone dynamics in solution. The other H-bonds have no such correlation; they populate the water-exposed and flexible regions of proteins, including many of the CaM helices. The observed differentiation in a dynamical behavior of backbone H-bonds in apo and holoCaM appears to be related to protein functions.     

Key Residues in the Nicotinic Acetylcholine Receptor β2 Subunit Contribute to α-Conotoxin LvIA Binding

α-Conotoxin LvIA (α-CTx LvIA) is a small peptide from the venom of the carnivorous marine gastropod Conus lividus and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. It can distinguish the α3β2 nAChR subtype from the α6β2* (* indicates the other subunit) and α3β4 nAChR subtypes. In this study, we performed mutational studies to assess the influence of residues of the β2 subunit versus those of the β4 subunit on the binding of α-CTx LvIA. Although two β2 mutations, α3β2[F119Q] and α3β2[T59K], strongly enhanced the affinity of LvIA, the β2 mutation α3β2[V111I] substantially reduced the binding of LvIA. Increased activity of LvIA was also observed when the β2-T59L mutant was combined with the α3 subunit. There were no significant difference in inhibition of α3β2[T59I], α3β2[Q34A], and α3β2[K79A] nAChRs when compared with wild-type α3β2 nAChR. α-CTx LvIA displayed slower off-rate kinetics at α3β2[F119Q] and α3β2[T59K] than at the wild-type receptor, with the latter mutant having the most pronounced effect. Taken together, these data provide evidence that the β2 subunit contributes to α-CTx LvIA binding and selectivity. The results demonstrate that Val¹¹¹ is critical and facilitates LvIA binding; this position has not previously been identified as important to binding of other 4/7 framework α-conotoxins. Thr⁵⁹ and Phe¹¹⁹ of the β2 subunit appear to interfere with LvIA binding, and their replacement by the corresponding residues of the β4 subunit leads to increased affinity.     

In vitro and in silico studies of bis (indol-3-yl) methane derivatives as potential α-glucosidase and α-amylase inhibitors

In this paper, bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated for their inhibitory activity against α-glucosidase and α-amylase. All synthesised compounds showed potential α-glucosidase and α-amylase inhibitory activities. Compounds 5 g (IC₅₀: 7.54 ± 1.10 μM), 5e (IC₅₀: 9.00 ± 0.97 μM), and 5 h (IC₅₀: 9.57 ± 0.62 μM) presented strongest inhibitory activities against α-glucosidase, that were ∼ 30 times stronger than acarbose. Compounds 5 g (IC₅₀: 32.18 ± 1.66 µM), 5 h (IC₅₀: 31.47 ± 1.42 µM), and 5 s (IC₅₀: 30.91 ± 0.86 µM) showed strongest inhibitory activities towards α-amylase, ∼ 2.5 times stronger than acarbose. The mechanisms and docking simulation of the compounds were also studied. Compounds 5 g and 5 h exhibited bifunctional inhibitory activity against these two enzymes. Furthermore, compounds showed no toxicity against 3T3-L1 cells and HepG2 cells.

Highlights

1. A series of bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated inhibitory activities against α-glucosidase and α-amylase.
2. Compound 5g exhibited promising activity (IC₅₀ = 7.54 ± 1.10 μM) against α-glucosidase.
3. Compound 5s exhibited promising activity (IC₅₀ = 30.91 ± 0.86 μM) against α-amylase.
4. In silico studies were performed to confirm the binding interactions of synthetic compounds with the enzyme active site.

     

Crystallographic structure of a helical lipopeptaibol antibiotic analogue

Summary An X-ray diffraction analysis of the [Fmoc⁰, TOAC^4,8, Leu-OMe¹¹]analogue of the lipopeptaibol antibiotic trichogin A Iv shows that the undecapeptide is folded in a right-handed, mixed α/3₁₀-helix. The helical molecules are connected in a head-to-tail arrangement along the b-axis through C=O...H-N intermolecular H-bonding. This packing mode generates a hydrophobic cavity where the Fmoc N^α-protecting groups are accommodated. The distances and angles between the nitroxide groups of the two TOAC residues, separated by one turn of the α-helix, have been determined.     

Recent advances in selective alpha1-adrenoreceptor antagonists as antihypertensive agents

Hypertension is one of the most serious health problems of the modern world with a continuous rise in the number of patients. Selective α₁-adrenoreceptor antagonists though have many advantages and uses in the management of arterial hypertension, their lack of specificity at the level of α₁-adr subtypes leads to multiple side effects. Existence of multiple α₁-adr subtypes holds great promise for the discovery and development of more specific and selective drug molecules, targeting only one α₁-adr subtype at a time and thus relative freedom from side effects. Herein, the research done on the discovery and evaluation of a variety of chemically diverse structures as selective antagonists of α₁-adr and α₁-adr subtypes in recent years has been reviewed.     

Effect of β2-adrenergic receptor gene (ADRB2) 3′ untranslated region polymorphisms on inhaled corticosteroid/long-acting β2-adrenergic agonist response

Background

Evidence suggests that variation in the length of the poly-C repeat in the 3′ untranslated region (3′UTR) of the β₂-adrenergic receptor gene (ADRB2) may contribute to interindividual variation in β-agonist response. However, methodology in previous studies limited the assessment of the effect of sequence variation in the context of poly-C repeat length. The objectives of this study were to design a novel genotyping method to fully characterize sequence variation in the ADRB2 3′UTR poly-C repeat in asthma patients treated with inhaled corticosteroid and long-acting β₂-adrenergic agonist (ICS/LABA) combination therapy, and to analyze the effect of the poly-C repeat polymorphism on clinical response.

Methods

In 2,250 asthma patients randomized to treatment with budesonide/formoterol or fluticasone/salmeterol in a six-month study (AstraZeneca study code: SD-039-0735), sequence diversity in the ADRB2 poly-C repeat region was determined using a novel sequencing-based genotyping method. The relationship between the poly-C repeat polymorphism and the incidence of severe asthma exacerbations, and changes in pulmonary function and asthma symptoms from baseline to the average during the treatment period, were analyzed.

Results

Poly-C repeat genotypes were assigned in 97% (2,192/2,250) of patients. Of the 13 different poly-C repeat alleles identified, six alleles occurred at a frequency of >5% in one or more population in this study. The repeat length of these six common alleles ranged from 10 to 14 nucleotides. Twelve poly-C repeat genotypes were observed at a frequency of >1%. No evidence of an association between poly-C repeat genotype and the incidence of severe asthma exacerbations was observed. Patients’ pulmonary function measurements improved and asthma symptoms declined when treated with ICS/LABA combination therapy regardless of poly-C repeat genotype.

Conclusions

The extensive sequence diversity present in the poly-C repeat region of the ADRB2 3′UTR did not predict therapeutic response to ICS/LABA therapy.     

Characterization, size estimation and solubilization of α-macroglobulin complex receptors in liver membranes

Receptors for α₂-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of ¹²⁵I-labelled α₂-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α₂-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of ¹²⁵I-labelled rat α₁-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α₂-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α₂-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound ¹²⁵I-α₁-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α₂-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.