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PubMed1992

Simultaneous electroelution of proteins from denaturing or native gels into a well matrix.

An electroelution device that is based on a semidry blotter and that allows the simultaneous elution of proteins or other charged macromolecules from one-dimensional gels is described. It consists of a central plate that has a matrix of oblong wells arranged in eight columns of 32 wells each, such that when a gel is placed on the plate each lane of bands is underlaid by a column of wells. The plate is placed between the electrodes of a semidry blotter and the wells are sealed by a dialysis membrane resting on polyacrylamide gel block, prior to being filled with transfer buffer. Using radiolabeled molecular weight standards resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, elution is shown to require 30-60 min for 90% or better of proteins between 10 and 120 kDa to be removed from the gel. The recovery volume is 200 microliters per well and losses due to adsorption onto the dialysis membrane are minimal. beta-Galactosidase eluted from a nonreducing, nondenaturing gel was shown to be quantitatively active. The advantages of the device include its ease of assembly and operation, its speed, its reproducibility, and the fact that no gel slicing is required since all proteins are eluted simultaneously, allowing large-scale screening of multiple protein fractions.

Electrophoresis, Polyacrylamide Gel↗

PubMed1992

A scanning electron-microscopic study of the effects of methylmercury on the neuronal cytoskeleton.

Cultured mouse neuroblastoma cells, Neuro-2a (c1300), were exposed to 2.5 and 5.0 microM methylmercury (MeHg) with or without the concomitant administration of 10 mM glutathione for 24 h. Treated cells viewed by scanning electron microscopy (SEM) appeared sponge-like and were surrounded by fragments of cytoplasmic processes. SEM cytoskeletal preparations of treated cells showed a collapsed matrix containing globular bodies. Microtubules were not seen in treated cells, but intermediate and microfilaments were observed. SDS-PAGE analysis of cytoskeletal extracts revealed bands ranging in size from 90 to 27 kDa in all treatment groups except in the 5.0 microM-MeHg-treated group. This group showed a single band co-migrating with actin. Cells exposed to glutathione alone or concomitantly with MeHg appeared similar to control cells under all experimental conditions. These observations suggest that MeHg may predominantly affect microtubules to form a condensation product.

Animals↗

PubMed1995

Quantitative assessment of dentin bridge formation following pulp-capping in miniature swine.

A quantitative assessment of dentin bridges was conducted on sections prepared from teeth capped with four pulp-capping agents (Bioglass, Life, Demineralized Dentin Matrix, and Teflon) in a micro-swine model. The degree of mineralization of the dentin bridges relative to the adjacent primary dentin was measured using a computer-based image analysis of microradiographs prepared from the sections. The rate of formation of the dentin bridge was measured from fluorescent bands formed in the same sections by a Demeclocycline dentin marker. There were no statistically significant differences in the relative mineral densities of the dentin bridges and the rate of dentin bridge formation under the four pulp-capping agents. These findings support the suggestion that components of the extracellular matrix rather than pulp-capping agents may be important in the formation and mineralization of dentin bridges in repairing dental pulps.

Analysis of Variance↗

PubMed2001

Resonance Raman spectroscopy of mass selected Al2 in an argon matrix.

In an excitation range of 620-760 nm, resonance Raman spectra of aluminum dimers (Al2) in an argon matrix have been obtained for the first time. Temperature annealing experiments were performed to remove Raman lines attributed site effects caused by the Al2/Ar matrix. We observe a single fundamental at 293.3 (5) cm(-1) along with a progression up to 1149 (1) cm(-1). Taking successive differences of band centers we obtain spectroscopic constants for the ground state fundamental, w(e) = 297.5 (5) cm(-1), the anharmonicity, e(e)x(e) = 1.68 (8) cm(-1). Our results are in close agreement with previous experimental results for Al2 which designate the ground state as a 3piu state, and may be considered as confirmation of this assignment.

Aluminum↗

PubMed1990

Construction and expression of a novel recombinant anaphylatoxin, C5a-N19, as a probe for the human C5a receptor.

We have constructed a novel recombinant C5a anaphylatoxin (C5a-N19) containing a 19-residue amino-terminal extension peptide, using a plasmid vector which secretes the nascent polypeptide to the Escherichia coli periplasmic space. C5a-N19 was purified from cell lysates by immunoaffinity chromatography using a monoclonal antibody which recognizes a portion of the amino-terminal extension peptide. C5a-N19 was characterized as biologically indistinguishable from the unmodified recombinant anaphylatoxin for release of lysosomal enzymes from dibutyryl-cAMP-differentiated U937 cells. In contrast to unmodified C5a, which is not recognized by anti-C5a antibodies following binding to its cellular receptor, receptor-bound C5a-N19 is recognized by the monoclonal antibody directed against the amino-terminal extension sequence. Because the monoclonal antibody recognizes the C5a-receptor complex on cells, this methodology is useful in fluorescence sorting of C5a receptor-positive cells. A C5a receptor affinity column was constructed by saturating monoclonal antibody bound to agarose with C5a-N19. Digitonin-solubilized C5a receptor from dibutyryl-cAMP-induced U937 cells was adsorbed to the matrix and eluted by dissociation of the ligand-receptor complex from the antibody. Analysis by SDS-polyacrylamide gel electrophoresis revealed a unique protein band at 41K, consistent with the molecular weight predicted from cross-linking experiments when the contribution of C5a is subtracted. Development of this recombinant C5a derivative provides a useful probe previously unavailable for the C5a receptor molecule.

Anaphylatoxins↗

PubMed2005

Growth and photoluminescence characterization of highly oriented CuI/beta-cyclodextrin hybrid composite film.

A highly (111) oriented CuI/beta-cyclodextrin inorganic/organic composite film on glass substrates was deposited from a dispersion of CuI and beta-cyclodextrin in a mixture of acetonitrile and dimethylformamide (DMF) solvents, in which DMF plays an important role in the orientation of CuI film. The composite film exhibits obviously improved band gap photoluminescence compared with that of the pure CuI film due to the passivation of iodine-related defect sites by beta-cyclodextrin matrix. This result indicates that the optical properties of CuI film can be easily adjusted by the formation of composite film with proper ligand agents.

Journal Article↗

PubMed2003

Dominance of the final state in photoemission mapping of the fermi surface of Co/Cu(001).

The Fermi surface of tetragonally distorted fcc Co films grown on Cu(001) has been investigated with first-principles calculations and compared with an experimental determination using angle-resolved photoemission. The angular distributions for hnu=21-45 eV are dominated by the structure of the final states rather than by the shape of the Fermi surface. Theoretical estimates of the photoemission matrix elements support this observation. This suggests that photoemission can have limitations in mapping Fermi surfaces, especially for materials that exhibit flat, closely spaced valence bands.

Journal Article↗

PubMed1994

Molecular identification and phylogeny of parasitic wasp species (Hymenoptera: Trichogrammatidae) by mitochondrial DNA RFLP and RAPD markers.

Mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) were tested for their ability to discriminate between seven species of minute parasitic wasps belonging to the genus Trichogramma. They proved to be reliable species-diagnostic molecular markers. Pairwise comparisons of the mtDNA restriction maps revealed considerable differentiation among the seven species. Percentage of common restriction sites ranged from 30% to 83%. Phylogenetic analyses performed either on the mtDNA nucleotide distance matrix or on the matrix of the restriction site-state generated a tree congruent with those based on allozymes and morphology. RAPD procedures also revealed species-specific banding patterns and seem promising for a rapid and easy identification of Trichogramma species. Moreover, for some Trichogramma species, RAPD banding patterns might be informative of the phylogenetic relatedness.

Animals↗

PubMed1984

Extraction of enamel proteins.

Two multi-step extraction procedures were used to isolate matrix proteins from fetal bovine enamel. Using SDS-PAGE and gel isoelectric focusing it was concluded that extraction procedures influence the number of protein bands in the gel, their pI, apparent molecular weight and staining intensity.

Animals↗

PubMed1998

A new approach to utilize PCR-single-strand-conformation polymorphism for 16S rRNA gene-based microbial community analysis.

Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S rRNA gene (V-4 and V-5 regions) was amplified by PCR with universal primers, with one primer phosphorylated at the 5' end. The phosphorylated strands of the PCR products were selectively digested with lambda exonuclease, and the remaining strands were separated by electrophoresis with an MDE polyacrylamide gel, a matrix specifically optimized for SSCP purposes. By this means, reannealing and heteroduplex formation of DNA strands during electrophoresis could be excluded, and the number of bands per organism was reduced. PCR products from 10 of 11 different bacterial type strains tested could be differentiated from each other. With template mixtures consisting of pure-culture DNAs from 5 and 10 bacterial strains, most of the single strains could be detected from such model communities after PCR and SSCP analyses. Purified bands amplified from pure cultures and model communities extracted from gels could be reamplified by PCR, but by this process, additional products were also generated, as detected by further SSCP analysis. Profiles generated with DNAs of rhizosphere bacterial communities, directly extracted from two different plant species grown in the same field site, could be clearly distinguished. This study demonstrates the potential of the selected PCR-single-stranded DNA approach for microbial community analysis.

Bacteria↗

PubMed2006

Identification and characterization of p63 (CKAP4/ERGIC-63/CLIMP-63), a surfactant protein A binding protein, on type II pneumocytes.

Surfactant protein A (SP-A) binds to alveolar type II cells through a specific high-affinity cell membrane receptor, although the molecular nature of this receptor is unclear. In the present study, we have identified and characterized an SP-A cell surface binding protein by utilizing two chemical cross-linkers: profound sulfo-SBED protein-protein interaction reagent and dithiobis(succinimidylpropionate) (DSP). Sulfo-SBED-biotinylated SP-A was cross-linked to the plasma membranes isolated from rat type II cells, and the biotin label was transferred from SP-A to its receptor by reduction. The biotinylated SP-A-binding protein was identified on blots by using streptavidin-labeled horseradish peroxidase. By using DSP, we cross-linked SP-A to intact mouse type II cells and immunoprecipitated the SP-A-receptor complex using anti-SP-A antibody. Both of the cross-linking approaches showed a major band of 63 kDa under reduced conditions that was identified as the rat homolog of the human type II transmembrane protein p63 (CKAP4/ERGIC-63/CLIMP-63) by matrix-assisted laser desorption ionization and nanoelectrospray tandem mass spectrometry of tryptic fragments. Thereafter, we confirmed the presence of p63 protein in the cross-linked SP-A-receptor complex by immunoprobing with p63 antibody. Coimmunoprecipitation experiments and functional assays confirmed specific interaction between SP-A and p63. Antibody to p63 could block SP-A-mediated inhibition of ATP-stimulated phospholipid secretion. Both intracellular and membrane localized pools of p63 were detected on type II cells by immunofluorescence and immunobloting. p63 colocalized with SP-A in early endosomes. Thus p63 closely interacts with SP-A and may play a role in the trafficking or the biological function of the surfactant protein.

Amino Acid Sequence↗

PubMed1988

Kniest dysplasia is characterized by an apparent abnormal processing of the C-propeptide of type II cartilage collagen resulting in imperfect fibril assembly.

Epiphyseal and growth plate cartilages from four cases of Kniest dysplasia have been studied. In each case collagen fibril organization appeared abnormal by electron microscopy compared with age-matched normal cartilages: fibrils were much thinner, of irregular shape and did not exhibit the characteristic banding pattern. This was associated with the absence (compared with normal cartilage) of the C-propeptide of type II collagen (chondrocalcin) from the extracellular matrix of epiphyseal cartilages, although it was detected (as in normal cartilages) in the lower hypertrophic zone of the growth plate in association with calcifying cartilage. The C-propeptide was abnormally concentrated in intracellular vacuolar sites in Kniest cartilages and its total content was reduced in all cases but not in all cartilages. Moreover, it was not a part of the procollagen molecule. In contrast, type II collagen alpha-chain size was normal, indicating the formation of a triple helix. Also type II collagen content was normal and it was present in extracellular sites and only occasionally detected intracellularly. These observations suggest that the defect in Kniest dysplasia may result from the secretion of type II procollagen lacking the C-propeptide and abnormal fibril formation, and that the C-propeptide is normally required for fibril formation.

Calcium-Binding Proteins↗

PubMed2003

Altered pH regulation during enamel development in the cystic fibrosis mouse incisor.

Regulation of pH is necessary to the production of an environment conducive to enamel growth and mineralization. We hypothesize that abnormal extracellular pH in the enamel matrix of mice with the cystic fibrosis gene knocked out (CF mice) results in altered enamel mineralization. The enamel matrix pH during amelogenesis was studied in 10 normal and 10 CF mice. Freshly dissected incisors were immersed in pH indicator or glyoxal bis (2-hydro-xyanil) (GBHA). The normal mouse enamel matrix pH was generally higher and modulated differently than did the CF mouse enamel. GBHA staining showed that normal mice had 2 well-demarcated bands in the maturation zone that correlated to the neutral pH zones, while CF mice showed no staining. These results indicate that CFTR plays a role in pH regulation during enamel development and that a reduced pH results in a lack of calcium influx during enamel maturation and hypomineralization of the CF incisor enamel.

Amelogenesis↗

PubMed1981

The occurrence of phase separation structures in biological membranes as derived from freeze-etch observations.

Some structural specializations revealed in biological membranes by freeze-fracturing are probably attributed to phase separation in the lipid matrix. The visibility of rigid domains within the fluid bilayer has been demonstrated in model membranes. Characteristics of rigid domains are the ripple- or band-pattern or a change of the surface curvature. Additionally in biological membranes an exclusion of particles or a regular incorporation of particles are the most important properties of rigid domains. By the combination of different features 8 basic domain types can be postulated. Examples for 6 of these types have been found as normally occurring structures or by induction. These are in the group of smooth particle-free domains the flat circular type (SFC), the bulged circular type (SBC) and the bulged elongated type (SBE). In the group with regularly arranged particles are known the flat circular type (RFC), the flat elongated type (RFE) and the bulged elongated type (RBE).

Animals↗

PubMed1994

Palmitoylation of luteinizing hormone/human choriogonadotropin receptors in transfected cells. Abolition of palmitoylation by mutation of Cys-621 and Cys-622 residues in the cytoplasmic tail increases ligand-induced internalization of the receptor.

We have examined whether the two cysteine residues (621 and 622) of the carboxyl-terminal cytoplasmic domain of the rat luteinizing hormone (LH/hCG) receptor are potential sites for palmitoylation. The full-length LH/hCG receptor cDNA was cloned into an expression vector (hCGR-pCMV4). A human embryonic kidney cell line expressing large T antigen (293T cells) was transiently transfected with hCGR-pCMV4 by the calcium phosphate precipitation technique. The functional expression of the receptor was confirmed by 35S-labeled cysteine incorporation into the receptor as well as by 125I-human chorionic gonadotropin (hCG) binding. The transfected cells were then labeled with [3H]palmitic acid, and the labeled receptors purified on hCG-Affi-Gel matrix and subjected to SDS-polyacrylamide gel electrophoresis and autoradiography. The hCGR-pCMV4-transfected cells incorporated [3H]palmitic acid into a 92-kDa band corresponding to the mature form of the LH/hCG receptor; this band was absent in cells transfected with vector alone. Site-directed mutagenesis of either cysteine 621 or 622 to serine residue was partially inhibitory, whereas mutation of both cysteine residues (621 and 622) completely abolished palmitoylation. Scatchard analyses revealed that the mutant and wild type receptors have similar affinities for 125I-hCG. The biological function of palmitoylation was then examined in the transfected cells. The results showed that although the intracellular trafficking of the receptor and the ability to stimulate cyclic AMP production were unaffected, the rate of ligand-induced internalization of the receptor was higher in palmitoylation-deficient mutants compared to the wild type receptors. The first order rate constants of internalization of the mutant receptors were over 2-fold higher than the wild type. Intracellular degradation of the receptor-bound ligand was also higher in the mutants. These studies suggest that the native LH/hCG receptor is palmitoylated at cysteine residues 621 and 622, creating a membrane-anchoring site at the putative cytoplasmic domain. This palmitic acid-mediated anchoring decreases the ligand-induced receptor internalization thereby prolonging the retention of the ligand-bound receptor on the cell surface.

Amino Acid Sequence↗

PubMed1983

Nuclear matrix preparations from liver tissue and from cultured vertebrate cells: differences in major polypeptides.

The major polypeptides of nuclear matrix preparations from vertebrate tissues are rather similar. However, nuclear matrix fractions isolated from cultured vertebrate cells of different origin show variations in their major polypeptides. We demonstrate that cytoplasmic intermediate filaments copurify with nuclear matrices from these cells. Because of their abundance, their subunit proteins form major bands on SDS-polyacrylamide gels. The tissue specificity of the intermediate filament proteins then gives rise to the variations observed. To explain the differences in major proteins of nuclear matrices isolated from vertebrate cells grown in tissue and in culture we have analyzed the distribution of intermediate filaments during isolation of nuclei from liver tissue. We show that during homogenization of liver intermediate filaments are torn off and can be separated from the nuclei. Nuclear matrix preparations from these nuclei, therefore, do not contain intermediate filaments and true nuclear matrix proteins (e.g. lamins) are the major protein species. Our results suggest that the major nuclear matrix polypeptides (lamins) are similar in all vertebrate cells, since lamin like proteins were identified in cultured cells, too. Using antisera we demonstrate an immunological difference between lamins A/C and lamin B.

Animals↗

PubMed1986

Distribution of interstitial retinol-binding protein (IRBP) in the vertebrates.

Immunoblots of interphotoreceptor matrix preparations from 20 species belonging to six vertebrate classes were probed with antibodies against bovine interstitial retinol-binding protein (b-IRBP). Each preparation displayed an immunoreactive protein band. In the Osteichthyes, the apparent Mr of this band was 67,600 +/- 2,700 (mean +/- SD, n = 8). In two of the Osteichthyes, the band was resolved into a closely spaced doublet. Including previously published data for five mammals and one amphibian, species from the other classes (Chondrichthyes, one species; Amphibia, four species; Reptilia, one species; Aves, one species; Mammalia, nine species) had IRBPs with Mr that averaged 2.0 times that of the Osteichthyes, namely 134,200 +/- 8,600 (mean +/- SD, n = 17). Frog IRBP was very similar to mammalian IRBP in terms of its immunohistochemical distribution (determined with rabbit anti-frog IRBP antibodies), its molecular weight (sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel-filtration chromatography), retinol- and concanavalin A-binding ability, and because it was synthesized and secreted in vitro by the isolated retina but not by the pigmented layers of eye. Goldfish IRBP apparently binds exogenous (3H)-retinol but does not bind concanavalin A and has about half the Mr of frog IRBP. The occurrence of IRBP-like proteins cross-reacting with anti b-IRBP antibodies in the interphotoreceptor matrix of all six major vertebrate classes is consistent with the hypothesis that IRBP is an important element in the vertebrate visual cycle.

Animals↗

PubMed1990

Bovine osteogenic protein is composed of dimers of OP-1 and BMP-2A, two members of the transforming growth factor-beta superfamily.

A bone-inductive protein has been purified from bovine bone and designated as osteogenic protein (OP). The purified OP induces new bone at less than 5 ng with half-maximal bone differentiation activity at about 20 ng/25 mg of matrix implant in a subcutaneous bone induction assay. The purified osteogenic protein is composed of disulfide-linked dimers that migrate on sodium dodecyl sulfate gels as a diffuse band with an apparent molecular weight of 30,000. Upon reduction, the dimers yield two subunits that migrate with molecular weights of 18,000 and 16,000. Both subunits are glycosylated. After chemical or enzymatic deglycosylation, the dimers migrate as a diffuse 27-kDa band that upon reduction yields two polypeptides that migrate at 16 kDa and 14 kDa, respectively. The carbohydrate moiety does not appear to be essential for biological activity since the deglycosylated proteins are capable of inducing bone formation in vivo. Amino acid sequences of peptides generated by proteolytic digestion show that the subunits are distinct but related members of the transforming growth factor-beta super-family. The 18-kDa subunit is the protein product of the bovine equivalent of the human OP-1 gene and the 16-kDa subunit is the protein product of the bovine equivalent of the human BMP-2A gene.

Alkaline Phosphatase↗