Biochemical characterization of human osteoclast integrins. Osteoclasts express alpha v beta 3, alpha 2 beta 1, and alpha v beta 1 integrins

The study of osteoclast integrins has been previously hampered by the lack of a source of large numbers of purified osteoclasts. Osteoclastoma, a human giant cell tumor of bone, supplied a rich source of osteoclasts within a tissue containing many diverse cell types. Osteoclastoma integrin immunostaining confirmed the presence of the integrin alpha v beta 3 complex and the alpha 2 and beta 1 integrin subunits on osteoclasts. However, weak integrin expression, for example with alpha v beta 5, was difficult to interpret. Purification with magnetic beads coated with vitronectin receptor monoclonal antibody (13C2) enabled osteoclast membranes to be isolated with high purity and yield (57%) from osteoclastoma tissue. Positively (osteoclast-enriched) selected membranes were biochemically assessed for integrin expression by immunoprecipitation and visualization by non-radioactive enhanced chemiluminescence. alpha 1, alpha 4, alpha 6, alpha 8, alpha M, alpha X, gpIIb, beta 4, beta 6, and beta 8 integrin chains were undetectable at a sensitivity of 1 ng. alpha 3, alpha 5, alpha L, beta 2, and alpha v beta 5 were found in the negatively selected osteoclastoma tissue but not in the positively purified osteoclast membranes. The presence of alpha v beta 1 and alpha 2 beta 1 dimers was demonstrated biochemically on the immunoisolated osteoclast membranes. Osteoclast alpha v beta 3 isolation by Arg-Gly-Asp (RGD) affinity chromatography for NH2-terminal amino acid sequencing confirmed that the osteoclast vitronectin receptor was identical to that previously characterized on other cell types. In situ hybridization using human alpha v riboprobes in osteoclasts from human and rodent bone further demonstrated the high level and specificity of expression of alpha v vitronectin receptor in osteoclasts.     

Conserved structural features between HLA-DO beta and -DR beta

HLA-DO is a non-classical MHC class II molecule presumed to play a specialized role in the antigen processing pathway. We have modeled the HLA-DO beta-chain and found its overall structure compatible with the one of DR beta. Functional studies further highlighted the similarity between these beta-chains of the class II family of proteins. Indeed, a mixed heterodimer composed of the DR alpha and a chimeric DO beta-chains presented bacterial superantigens to T cells and was shown to interact with CD4. The implications of such structural conservation for the in vivo functions of HLA-DO are discussed.     

Demonstration and isolation of the hybrid hemoglobins alpha 2 A beta A beta C and alpha 2 A beta S beta C

The corss-linking reagent p,p'-difluoro-m,m'-dinitrodiphenylsulfone has been used to fix in the tetramer form the various species of hemoglobin present in mixtures of hemoglobin A and hemoglobin C and of hemoglobin S and hemoglobin C. Following reaction, the presence of the hybrid hemoglobins alpha 2 A beta A beta C and alpha 2 A beta S beta C in these hemoglobin mixtures was demonstrated electrophoretically and the hybrids were isolated by ion-exchange chromatography. The identity of the alpha 2 A beta A beta C hybrid was further verified by peptide analysis. The success in cross-linking alpha 2 A beta 2 C, alpha 2 A beta A beta C, and alpha 2 A beta S beta C with p,p'-difluoro-m,m'-dinitrodiphenylsulfone shows that the distance between the alpha chain amino terminals in solution for these hemoglobin species is the same as in normal hemoglobin.     

Site-directed mutagenesis of charged and potentially proton-carrying residues in the beta subunit of the proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli. Characterization of the beta H91, beta D392, and beta K424 mutants

Conserved and semiconserved acidic and basic residues of the beta subunit of the proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli potentially involved in proton pumping were investigated. Out of 16 charged residues studied, 6 have not been previously investigated. The most dramatic effects of mutation were observed with beta H91, beta D392, and beta K424. beta H91E showed a pronounced shift of the pH optimum for both reduction of thio-NADP+ by NADH (forward reaction) and reduction of 3-acetylpyridine-NAD+ by NADPH (reverse reaction) to lower pH. This mutant catalyzed a cyclic reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADP(H) with a pH profile also shifted toward a lower pH. These results are consistent with a mechanism where the normal forward and reverse reactions are indeed limited by protonation/deprotonation of beta H91. The cyclic reaction was affected by mutations of beta H91, probably through conformational changes involving the active NADP(H) site. The beta D392A mutant was inactive with regard to forward and reverse reactions, but showed a wild-type-like pH dependence for the partly active cyclic reaction. However, Km,app for NADP(H) in this reaction was elevated 50-100-fold, suggesting that beta D392 is located in or near the NADP(H)-binding site. Transhydrogenases contain a conserved beta K424-beta R425-beta S426 sequence that has been proposed to be important for NADP(H) binding. beta K424R was strongly inhibited and showed an 18-fold increased Km,app for NADPH in the reverse reaction as compared to wild type. Consequently, this mutation affected all NADP(H)-linked activities and essentially abolished the unspecific interaction of NAD(H) with this site. The pH dependences of the forward and reverse reactions, as well as the cyclic reaction, were shifted to a lower pH as compared to the wild-type enzyme, and the salt dependence was also altered.     

Oestrogen receptor beta (ER beta)

Idiosyncratic adverse drug reactions are unpredictable, target multiple organ systems, and often become life-threatening events. Although the causes of idiosyncratic adverse drug reactions are not known in most cases, evidence suggests that they may be mediated through immunological mechanisms. It is generally thought that for a drug to lead to an immune response, it must first become covalently bound to a carrier protein. Since most drugs are unreactive, it is usually a reactive metabolite that is expected to form covalent adducts. However, it is not clear why more people do not develop immune reactions against drug-protein adducts. One possible explanation is that orally administered drugs may lead to oral tolerance in most individuals through mechanisms similar to that found with orally administered antigens. However, very little is known regarding the interaction of drugs with gut-associated lymphoid tissue of the small intestine, where oral tolerance can develop. As an initial step to test this hypothesis, we have investigated whether diclofenac, a commonly used nonsteroidal antiinflammatory drug, can lead to protein adducts in rat small intestine. Diclofenac was administered to rats by gastric gavage. Immunoblot analysis of small intestine homogenates and isolated enterocyte subcellular fractions with drug-specific antiserum revealed 142-, 130-, 110-, and 55-kDa protein adducts of diclofenac. The 142- and 130-kDa adducts of diclofenac were identified as aminopeptidase N (CD13) and sucrase-isomaltase, respectively, by amino acid sequence analyses and by their reactions with protein-specific antibodies. The adducts were localized by immunohistochemistry and found primarily in the mid-villus and villus-tip enterocytes and also in the dome overlying Peyer's patches. Similar adducts were detected immunochemically in villus-tip enterocytes of animals treated with halothane or acetaminophen. These results show that intestinal protein adducts of drugs can be formed in gut-associated lymphoid tissue where they may lead to the down-regulation of drug-induced allergic reactions in many individuals.     

Immunohistochemical localization of G protein beta1, beta2, beta3, beta4, beta5, and gamma3 subunits in the adult rat brain

The regional distributions of the G protein beta subunits (Gbeta1-beta5) and of the Ggamma3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gbeta and Ggamma3 subunits were widely distributed throughout the brain, with most regions containing several Gbeta subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gbeta immunostaining. Negative immunostaining was observed in cortical layer I for Gbeta1 and layer IV for Gbeta4. The hippocampal dentate granular and CA1-CA3 pyramidal cells displayed little or no positive immunostaining for Gbeta2 or Gbeta4. No anti-Gbeta4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gbeta1 was absent from the cerebellar molecular layer, and Gbeta2 was not detected in the Purkinje cells. No positive Ggama3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Ggamma3 antibody and individual anti-Gbeta1-beta5 antibodies displayed regional selectivity with Gbeta1 (cortical layers V-VI) and Gbeta2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gbeta1-beta5 with Ggamma3, specific dimeric associations in situ were observed within discrete brain regions.     

Antibodies against beta1 and beta2 adrenergic receptors in myasthenia gravis

With ageing the membrane/soluble form ratio of membrane-boundenzymes increases especially in the ileum and that of peptidases changes rather insignificantly in both portions of the rat small intestine. The soluble forms are supposed to take part in assimilation of food substrates penetrating enterocytes in an unsplit form due to an enhanced permeability of the membranes, particularly those of the ileum.     

Kinetic studies of a soluble alpha beta complex of nitrate reductase A from Escherichia coli. Use of various alpha beta mutants with altered beta subunits

A soluble alpha beta complex of nitrate reductase can be obtained from a strain of Escherichia coli that lacks the narI gene and expresses only the alpha and beta subunits. The beta subunit contains four Fe-S centres and the alpha subunit contains the molybdenum cofactor, which is the site at which nitrate is reduced. Despite the lack of the gamma subunit of the complete enzyme, this complex can still catalyse the reduction of nitrate with artificial electron donors such as benzyl viologen, so that it is suitable for studying the transfer of electrons between these two types of redox centre. To examine whether the electrons from reduced benzyl viologen are initially delivered to the Fe-S centres, or directly to the molybdenum cofactor, or both, we have studied the steady-state kinetics and the binding of benzyl viologen to the alpha beta complex and mutants alpha beta* with altered beta subunits. Reduction of the enzyme by reduced benzyl viologen in the absence of nitrate showed that all four Fe-S centres and the molybdenum cofactor could be reduced. Two classes of site with different equilibrium constants could be distinguished. The kinetic results suggest that benzyl viologen supplies its electrons directly to the molybdenum cofactor, at a rate showing a hyperbolic dependence on the square of the concentration of the electron donor. A reaction mechanism is proposed for the reduction of nitrate catalysed by the alpha beta complex of nitrate reductase with artificial electron donors.     

The profile of soluble amyloid beta protein in cultured cell media. Detection and quantification of amyloid beta protein and variants by immunoprecipitation-mass spectrometry

To study the metabolism of amyloid beta protein (Abeta) in Alzheimer's disease, we have developed a new approach for analyzing the profile of soluble Abeta and its variants. In the present method, Abeta and its variants are immuno-isolated with Abeta-specific monoclonal antibodies. The identities of the Abeta variants are determined by measuring their molecular masses using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The levels of Abeta variants are determined by their relative peak intensities in mass spectrometric measurements by comparison with internal standards of known identities and concentrations. We used this method to examine the Abeta species in conditioned media of mouse neuroblastoma cells transfected with cDNAs encoding wild type or mutant human amyloid precursor protein. In addition to human Abeta-(1-40) and Abeta-(1-42), more than 40 different human Abeta variants were identified. Endogenous murine Abeta and its variants were also identified by this approach. The present approach is a new and sensitive method to characterize the profile of soluble Abeta in conditioned media and biological fluids. Furthermore, it allows direct measurement of each individual peptide in a peptide mixture and provides comprehensive information on the identity and concentration of Abeta and Abeta variants.     

Sequence and expression of chicken beta A2- and beta B3-crystallins

Two novel point mutations have been identified in the low density lipoprotein receptor (LDLR) gene of a South African Indian patient with a clinical diagnosis of homozygous familial hypercholesterolemia (FH). The patient is a compound heterozygote, whose paternally-inherited allele has a single base substitution of A to T at position + 1. This conversion of the initiation codon ATG (methionine) to TTG (leucine) would abolish initiation of translation at the normal site, and consequently the synthesis of any normal LDLR molecules. The second mutation identified is a C to A base change at nucleotide position 1176 in exon 8, which creates a stop codon at cysteine-371. Except for previously-described polymorphisms in specific regions of the LDLR gene, the mutations identified in exons 1 and 8 were the only variants observed by screening enzymatically amplified genomic DNA comprising the entire coding and promoter region of the LDLR gene by combined heteroduplex-single-strand conformation polymorphism analysis and by direct sequencing. Cultured cells from the proband expressed no functional LDLR activity and contained no receptor protein that could be detected by antibody binding. These findings are consistent with the nature of the two base changes identified and provide evidence that the mutations cause FH in the proband and his affected family members. The mutations, designated M-21L and C371X, were absent in 17 apparently unrelated Indian hypercholesterolemics and 200 normal chromosomes screened.     

G protein beta gamma subunits. Simplified purification and properties of novel isoforms

The beta and gamma subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) form tightly associated complexes. To examine functional differences among the large number of possible combinations of unique beta and gamma subunits, we have synthesized and characterized beta gamma complexes containing gamma 5 and gamma 7, two widely distributed gamma subunits. When either gamma 5 or gamma 7 is expressed concurrently with beta 1 or beta 2 subunits in a baculovirus/Sf9 cell system, all four subunit complexes support pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1 (where "r" indicates recombinant), indicating formation of functional complexes. Each of the complexes was purified by subunit exchange chromatography, using the G203A mutant of rGi alpha 1 as the immobilized ligand. The purified preparations were compared with other recombinant beta gamma subunits, including beta 1 gamma 1 and beta 1 gamma 2, for their ability to modulate type I and II adenylyl cyclase activities; stimulate phosphoinositide-specific phospholipase C beta; support pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1 and Go alpha; and inhibit steady-state GTP hydrolysis catalyzed by Gs alpha, Go alpha, and myristoylated rGi alpha 2. The results emphasize the unique properties of beta 1 gamma 1. The properties of the complexes containing gamma 5 or gamma 7 were similar to each other and to those of beta 1 gamma 2.     

Effects of beta blockade with metoprolol on simple and complex task performance.

[Correction Notice: An erratum for this article was reported in Vol 8(5) of Health Psychology (see record 2008-09113-001). The reference and citations for Merrill, Robbins, Schultz, Streeten, and Elias (1987) were incorrect. The correct reference is: Elias, M. F., Robbins, M. A., Schultz, N. R., Streeten, D. H. P., & Elias, P. K. (1987). Clinical significance of cognitive performance by hypertensive patients. Hypertension, 9, 192-197.] Measured the effect of treatment with the beta-blocker metoprolol on several indicants of task performance in 25 mildly to moderately hypertensive men (aged 24–63 yrs) treated for 2 wks each with drug and placebo, compared with 25 untreated normotensive men (aged 23–64 yrs). Ss responded twice to the Mood Adjective Check List (MACL) and the Stroop Color and Word Test. In addition, Ss participated in 2 parallel decision-making simulations. Results indicate that normotensives and hypertensives on drug or placebo did not differ on MACL and Stroop scores. Comparisons on 12 validated simulation measures showed that treatment with metoprolol aided complex task performance. Treatment improved strategic capacity and the ability to deal effectively with emergencies. Preliminary evidence that treatment with metoprolol might help restore performance capacity that may have been lost due to hypertensive disease was also obtained. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Differences in interferon alpha and beta signaling. Interferon beta selectively induces the interaction of the alpha and betaL subunits of the type I interferon receptor

All Type I interferons (IFNalpha, IFNbeta, IFNomega) bind to the Type I IFN receptor (IFNR) and elicit a common set of signaling events, including activation of the Jak/Stat and IRS pathways. However, IFNbeta selectively induces the association of the alpha subunit of the Type I IFNR with p100, a tyrosyl phosphoprotein, to transduce IFNbeta-specific signals. Using antibodies raised against the different components of the Type I IFNR, we identified p100 as the long form of the beta subunit (betaL subunit) of the Type I IFNR. This was also confirmed in experiments with mouse L-929 cells transfected with truncated forms of betaL. Thus, IFNbeta stimulation of human cells or mouse L-929 transfectants expressing the human alpha and betaL subunits, selectively induces the formation of a signaling complex containing the alpha and betaL subunits of the receptor. The IFNbeta-regulated interaction of the alpha and betaL chains is rapid and transient and follows a similar time course with the tyrosine phosphorylation of these receptor components. These data demonstrate that the signaling specificity for different Type I IFNs is established early in the signaling cascade, at the receptor level, and results from distinct interactions between components of the Type I IFNR.     

V beta repertoire of murine hepatic T cells. Implication for selection of double negative alpha beta + T cells

To further define the origin, selection, and diversity of hepatic T cells, we have determined V beta gene expression profiles in double negative (DN, CD4-8-) and single positive (SP, CD4+8- or CD8+4-) alpha beta + liver T cells of DBA/2 mice. These I-E+ mice express mouse mammary tumor (Mtv) provirus-encoded endogenous superantigens of the Mlsa,c type, and thus display deletions/depletions of several V beta-bearing SP cells. Total liver alpha beta + T cells of these mice exhibited an overall V beta expression profile similar to splenic T cells, with the notable exception of high V beta 7 and V beta 8.1 expression. As previously reported, DN alpha beta + T cells were enriched highly in the liver. This subset exhibited a V beta expression profile similar to thymic DN alpha beta + cells with deletions/depletions in several V beta s, but high V beta 7 expression in both populations. Surprisingly, hepatic CD4+ cells also displayed high V beta 7 expression compared with splenic T cells, suggesting that hepatic DN alpha beta + and CD4+ T cells are selected via a common pathway. The V beta 7-expressing DN alpha beta + and CD4+ liver T cell populations were polyclonal, as evidenced by cloning and sequencing. High V beta 7 expression in these cells was undiminished with age. On the basis of V beta repertoire and surface phenotype, DN alpha beta + and/or certain CD4+ T cells seem to constitute a distinct population primarily found in the liver, thymus, and bone marrow. These cells may originate from SP T cells that have down-regulated their accessory molecules under certain activation conditions and, because of the accompanying expression of particular adhesion molecules, they accumulate in tissues such as the liver and thymus.     

Association of beta-agonists with corresponding beta 2- and beta 1-adrenergic pentapeptide sequences

Synthesized beta 1- and beta 2-pentapeptide sequences corresponding to published adrenoceptor transmembrane activation site subtypes were investigated in vitro for selectivity in association for drug ligands of known selectivity. Both nuclear magnetic resonance spectroscopy and molecular mechanics demonstrated that structural differences among the corresponding pentapeptide activation-site sequences can explain agonist selectivity. Results suggest the agonists bind across the activation site loop on the second transmembrane alpha-helix by dipole/dipole interactions between a ligand and the peptide. Since electrostatic interactions within the membrane may determine the rate of intercellular ion flux, agonist association across the activation site sequence could thereby decrease electrostatic resistance to positive ion flux into the cell. Interactions between the peptides and the ligands may provide insight into the structures and mechanisms involved in association of ligands for the identical sequences on the beta-adrenoreceptors.     

The selective inhibition of beta 1 and beta 7 integrin-mediated lymphocyte adhesion by bacitracin

Integrins play an important role in lymphocyte adhesion to cellular and extracellular components of their microenvironment. The regulation of such adhesion often involves changes in the functional state of the integrins rather than alterations in their expression levels. Although the functional basis for such transitions is unknown, a possible role for disulfide exchange might be postulated based on the observations that integrin function can be activated by bifunctional reducing agents or by Abs that react with areas adjacent to predicted long-range disulfide bonds in integrins. Recently, it has been reported that enzymes that catalyze disulfide exchanges such as protein disulfide isomerase (PDI) are present on the surface of lymphoid cells, raising the possibility that such enzymes might be involved in the control of lymphocyte adhesion. A number of inhibitors of PDI function were examined for their effects on integrin-mediated adherence of T cells. The results did not support role for PDI in the regulation of integrin function, as the inhibitors somatostatin A, tocinoic acid, dithiobisnitrobenzoic acid, and anti-PDI mAb did not interfere with adherence. However, one of the PDI inhibitors, bacitracin, selectively interfered with the beta1 integrin-mediated adherence of lymphoid cells to collagen, fibronectin, laminin, and VCAM-1, and with alpha4beta7-dependent adherence to fibronectin and to VCAM-1. In contrast, alpha(v)beta3- and alpha(L)beta2-mediated adherence were not inhibited. Thus, it appears that bacitracin may be a selective inhibitor of beta1 and beta7 integrin functions by an as yet unknown mechanism.     

Lipolytic effects of beta1, beta2 and beta3-adrenergic agonists in isolated human fat cells from omental and retroperitoneal adipose tissues

The presence of beta1- and beta2-adrenoceptors has been clearly established in human fat cells. There is some controversy about the presence and function of beta3-adrenoceptors. It is well established that there are marked regional variations in catecholamine-induced lipolysis. In this work the possibility that a beta3-adrenoceptor plays a significant role in the control of lipid mobilization is studied and also its importance in comparison to beta1- and beta2-adrenoceptors in isolated human fat cells, is evaluated, by measuring the in vitro lipolysis induced by dobutamine, salbutamol, metaproterenol, BRL 37344 and CGP 12177A. Human adipocytes from omental and retroperitoneal fat deposits exhibited an "atypical" beta-adrenergic response but, given the small lipolytic effect initiated by BRL 37344 and CGP 12177A, they are probably poorly equipped in functional beta3-adrenoceptors.