Topological side-chain classification of beta-turns: ideal motifs for peptidomimetic development.

PubMed

Tran, Tran Trung; McKie, Jim; Meutermans, Wim D F; Bourne, Gregory T; Andrews, Peter R; Smythe, Mark L

2005-08-01

Beta-turns are important topological motifs for biological recognition of proteins and peptides. Organic molecules that sample the side chain positions of beta-turns have shown broad binding capacity to multiple different receptors, for example benzodiazepines. Beta-turns have traditionally been classified into various types based on the backbone dihedral angles (phi2, psi2, phi3 and psi3). Indeed, 57-68% of beta-turns are currently classified into 8 different backbone families (Type I, Type II, Type I', Type II', Type VIII, Type VIa1, Type VIa2 and Type VIb and Type IV which represents unclassified beta-turns). Although this classification of beta-turns has been useful, the resulting beta-turn types are not ideal for the design of beta-turn mimetics as they do not reflect topological features of the recognition elements, the side chains. To overcome this, we have extracted beta-turns from a data set of non-homologous and high-resolution protein crystal structures. The side chain positions, as defined by C(alpha)-C(beta) vectors, of these turns have been clustered using the kth nearest neighbor clustering and filtered nearest centroid sorting algorithms. Nine clusters were obtained that cluster 90% of the data, and the average intra-cluster RMSD of the four C(alpha)-C(beta) vectors is 0.36. The nine clusters therefore represent the topology of the side chain scaffold architecture of the vast majority of beta-turns. The mean structures of the nine clusters are useful for the development of beta-turn mimetics and as biological descriptors for focusing combinatorial chemistry towards biologically relevant topological space.

Using steric hindrance to design new inhibitors of class C beta-lactamases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trehan, Indi; Morandi, F.; Blaszczak, L.C.

{beta}-lactamases confer resistance to {beta}-lactam antibiotics such as penicillins and cephalosporins. However, {beta}-lactams that form an acyl-intermediate with the enzyme but subsequently are hindered from forming a catalytically competent conformation seem to be inhibitors of {beta}-lactamases. This inhibition may be imparted by specific groups on the ubiquitous R1 side chain of {beta}-lactams, such as the 2-amino-4-thiazolyl methoxyimino (ATMO) group common among third-generation cephalosporins. Using steric hindrance of deacylation as a design guide, penicillin and carbacephem substrates were converted into effective {beta}-lactamase inhibitors and antiresistance antibiotics. To investigate the structural bases of inhibition, the crystal structures of the acyl-adducts of themore » penicillin substrate amoxicillin and the new analogous inhibitor ATMO-penicillin were determined. ATMO-penicillin binds in a catalytically incompetent conformation resembling that adopted by third-generation cephalosporins, demonstrating the transferability of such sterically hindered groups in inhibitor design.« less

Characterization and membrane organization of beta 1----3- and beta 1----4-galactosyltransferases from human colonic adenocarcinoma cell lines Colo 205 and SW403: basis for preferential synthesis of type 1 chain lacto-series carbohydrate structures.

PubMed

Holmes, E H

1989-05-01

Evidence indicates that activation of a beta 1----3N-acetylglucosaminyltransferase is responsible for accumulation of large quantities of lacto-series tumor-associated antigens in human colonic adenocarcinomas. Expression of type 1 and 2 core chain derivatives characterize human colonic adenocarcinomas, whereas normal adult colonic epithelial cells express detectable quantities of only type 1 chain derivatives. The basis for preferential synthesis of type 1 chain lacto-series carbohydrate structures characteristic of normal colonic mucosa and human colonic adenocarcinoma Colo 205 cells has been studied. The beta 1----3- and beta 1----4galactosyltransferase enzymes associated with synthesis of type 1 and 2 core chain structures, respectively, have been separated from a Triton X-100 solubilized membrane fraction of Colo 205 cells by chromatography on an alpha-lactalbumin-Sepharose column and their properties studied. Optimal transfer of beta 1----3-linked galactose to acceptor Lc3 occurred in the presence of 0.1% Triton CF-54 with Triton X-100 providing 75% of maximal activity. The enzyme was active over a broad pH range from 6.5 to 7.5 and had a near absolute requirement for Mn2+. The Km values for donor UDPgalactose and acceptor Lc3 were determined to be 48 and 13 microM, respectively. In contrast, the beta 1----4galactosyltransferase required taurodeoxycholate for maximal activity and the Km for Lc3 was found to be 20-fold higher than that for the beta 1----3-specific enzyme under the same assay conditions. Studies with membrane-bound beta 1----3- and beta 1----4galactosyltransferases as found in Golgi-rich membrane fractions of SW403 and Colo 205 adenocarcinoma cells showed that preferential synthesis of type 1 chain structures occurs under conditions similar to those in vivo for biosynthesis of lacto-series core chains. The results suggest that both the higher affinity of the beta 1----3galactosyltransferase for acceptor Lc3 and the membrane organizational features result in preferential synthesis of type 1 chain structures.

The J beta segment of the T cell receptor contributes to the V beta-specific T cell expansion caused by staphylococcal enterotoxin B and Urtica dioica superantigens.

PubMed

Musette, P; Galelli, A; Truffa-Bachi, P; Peumans, W; Kourilsky, P; Gachelin, G

1996-03-01

We have used a new polymerase chain reaction-based technique to analyze at the clonal level the CDR3 diversity and the J beta usage associated with the V beta-dependent T cell receptor (TCR) recognition of two superantigens: the staphylococcal enterotoxin B and the Urtica dioica agglutinin. Our results show that subset of J beta elements is preferentially expanded in a given V beta family, independently of the nature of the superantigen. By contrast, the CDR3 loop does not contribute significantly to the T cell expansion induced by the superantigens. We conclude that the J beta segment of the TCR beta chain, but not the CDR3 region, participates in superantigen binding, presumably by influencing the quaternary structure of the TCR beta chain.

Hb Molfetta [beta126(H4)Val-->Leu, GTG-->CTG]: a new, silent, neutral beta chain variant found in an Italian woman.

PubMed

Qualtieri, Antonio; Le, Pera Maria; Pedace, Vera; Magariello, Angela; Brancati, Carlo

2002-02-01

We have identified a new neutral hemoglobin variant in a pregnant Italian woman, that resulted from a GTG-->CTG replacement at codon 126 of the beta chain, corresponding to a Val-->Leu amino acid change at position beta126(H4). Thermal and isopropanol stability tests were normal and there were no abnormal clinical features. Routine electrophoretic and ion exchange chromatographic methods for hemoglobin separation failed to show this variant, but reversed phase high performance liquid chromatography revealed an abnormal peak eluting near the normal beta chain. No abnormal tryptic peptide was revealed on the high performance liquid chromatographic elution pattern of the total globin digest. The mutation was determined at the DNA level by amplification of the three beta exons by polymerase chain reaction and direct sequencing of one exon that showed an abnormal migration on single strand conformational polymorphism analysis.

Contributions of arginines-43 and -94 of human choriogonadotropin. beta. to receptor binding and activation as determined by oligonucleotide-based mutagenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang Chen; Puett, D.

1991-10-22

Members of the glycoprotein hormone family contain a common {alpha} subunit and a hormone-specific {beta} subunit. Human choriogonadotropin (hCG) {beta} is a 145 amino acid residue protein glycosylated at 6 positions (2 N-linked and 4 O-linked oligosaccharides). In an effort to elucidate receptor determinants on hCG{beta}, the authors have used site-directed mutagenesis to prepare and express several mutant cDNAs with replacements at arginines-43 and -94. Arg-43 is invariant in all known mammalian CG/lutropin {beta} amino acid sequences, and Arg-94 is conserved in 10 of the 12 sequences. Moreover, various studies involving synthetic peptides and enzymatic digestions of intact {beta} chainsmore » suggest that these residues may be important in hCG receptor binding. Point mutants were made in which these two arginines were replaced with the corresponding residues in human follitropin {beta}, Leu-43 and Asp-94. The wild-type and mutant {beta} chains were expressed in CHO cells containing a stably integrated gene for bovine {alpha}, and heterodimer formation occurred. These heterologous gonadotropins were active in assays using transformed Leydig cells, competitive binding with standard {sup 125}I-hCG, and cAMP and progesterone production, but the potency was considerably less than that associated with the hCG{beta} wild-type-containing gonadotropin. The double-mutant protein Arg-43 to Leu/Arg-94 to Asp also associated with bovine {alpha}, but the resultant heterodimer exhibited only low activity. Replacement but the Lys-43-containing {beta} chain appeared to exhibit a low degree of subunit association or reduced stability relative to the expressed hCG{beta} wild type. These results demonstrate that arginines-43 and -94 contribute to receptor binding through a positive charge.« less

Synthesis and studies of polypeptide materials: Enantioselective polymerization of gamma-benzyl glutamate-N-carboxyanhydride and synthesis of optically active poly(beta-peptides)

NASA Astrophysics Data System (ADS)

Cheng, Jianjun

A class of zero-valent transition metal complexes have been developed by Deming et al for the controlled polymerization of alpha-aminoacid-N-carboxyanhydrides (alpha-NCAs). This discovery provided a superior starting point for the development of enantioselective polymerizations of racemic alpha-NCAs. Bidentate chiral ligands were synthesized and tested for their abilities to induce enantioselective polymerization of gamma-benzyl-glutamate NCA (Glu NCA) when they were coordinated to zero-valent nickel complexes. When optically active 2-pyridinyl oxazoline ligands were mixed with bis(1,5-cyclooctadiene)nickel in THF, chiral nickel complexes were formed that selectively polymerized one enantiomer of Glu NCA over the other. The highest selectivity was observed with the nickel complex of (S)-4-tert-butyl-2-pyridinyl oxazoline, which gave a ratio of enantiomeric polymerization rate constants (kD/kL) of 5.2. It was found that subtle modification of this ligand by incorporation of additional substituents had a substantial impact on initiator enantioselectivities. In separate efforts, methodology was developed for the general synthesis of optically active beta-aminoacid-N-carboxyanhydrides (beta-NCAs) via cyclization of Nbeta-Boc- or Nbeta-Cbz-beta-amino acids using phosphorus tribromide. The beta-NCA molecules could be polymerized in good yields using strong bases or transition metal complexes to give optically active poly(beta-peptides) bearing proteinogenic side chains. The resulting poly(beta-peptides), which have moderate molecular weights, adopt stable helical conformations in solution. Poly(beta-homoglutamate and poly(beta-homolysine), the side-chain deprotected polymers, were found to display pH dependent helix-coil conformation transitions in aqueous solution, similar to their alpha-analogs. A novel method for poly(beta-aspartate) synthesis was developed via the polymerization of L-aspartate alkyl ester beta lactams using metal-amido complexes. Poly(beta-aspartates) bearing short ethylene glycol side chains were obtained with controlled molecular weights and narrow molecular weight distributions when Sc(N(TMS)2)3 was used as initiator for the beta-lactam polymerizations. Polymer chain lengths could be controlled by both stoichiometry and monomer conversion, characteristic of a living polymerization system. Di- and tri-block copoly(beta-peptides) with desired chain lengths were also synthesized using this method. It was found that these techniques were generally applicable for the synthesis of poly(beta-peptides), bearing other proteinogetic side chains. Synthesis and studies of polypeptide materials were extended to unexplored areas by incorporation of both alpha- and beta-amino acid residues into single polymer chains. Two sequence specific polypeptides bearing alternating beta-alpha, or beta-alpha-alpha amino acid residues were synthesized. Both polymers were found to adopt unprecedented stable conformations in solution.

Translation and assembly of HLA-DR antigens in Xenopus oocytes injected with mRNA from a human B-cell line.

PubMed Central

Long, E O; Gross, N; Wake, C T; Mach, J P; Carrel, S; Accolla, R; Mach, B

1982-01-01

HLA-DR antigens are polymorphic cell surface glycoproteins, expressed primarily in B lymphocytes and macrophages, which are thought to play an important role in the immune response. Two polypeptide chains, alpha and beta, are associated at the cell surface, and a third chain associates with alpha and beta intracellularly. RNA isolated from the human B-cell line Raji was injected in Xenopus laevis oocytes. Immunoprecipitates of translation products with several monoclonal antibodies revealed the presence of HLA-DR antigens similar to those synthesized in Raji cells. One monoclonal antibody was able to bind the beta chain after dissociation of the three polypeptide chains with detergent. The presence of all three chains was confirmed by two-dimensional gel electrophoresis. The glycosylation pattern of the three chains was identical to that observed in vivo, as evidenced in studies using tunicamycin, an inhibitor of N-linked glycosylation. The presence of alpha chains assembled with beta chains in equimolar ratio was further demonstrated by amino-terminal sequencing. An RNA fraction enriched for the three mRNAs, encoding alpha, beta, and intracellular chains, was isolated. This translation-assembly system and the availability of monoclonal antibodies make it possible to assay for mRNA encoding specific molecules among the multiple human Ia-like antigens. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:6821356

Differential effects of Phe19 and Phe20 on fibril formation by amyloidogenic peptide A beta 16-22 (Ac-KLVFFAE-NH2).

PubMed

Inouye, Hideyo; Gleason, Katherine A; Zhang, Dong; Decatur, Sean M; Kirschner, Daniel A

2010-08-01

The sequence KLVFFAE (A beta 16-22) in Alzheimer's beta-amyloid is thought to be a core beta-structure that could act as a template for folding other parts of the polypeptide or molecules into fibrillar assemblies rich in beta-sheet. To elucidate the mechanism of the initial folding process, we undertook combined X-ray fiber/powder diffraction and infrared (IR) spectroscopy to analyze lyophilized A beta 16-22 and solubilized/dried peptide containing nitrile probes at F19 and/or F20. Solubilized/dried wild-type (WT) A beta 16-22 and the peptide containing cyanophenylalanine at F19 (19CN) or at F20 (20CN) gave fiber patterns consistent with slab-like beta-crystallites that were cylindrically averaged around the axis parallel to the polypeptide chain direction. The WT and 19CN assemblies showed 30-A period arrays arising from the stacking of the slabs along the peptide chain direction, whereas the 20CN assemblies lacked any such stacking. The electron density projection along the peptide chain direction indicated similar side-chain dispositions for WT and 20CN, but not for 19CN. These X-ray results and modeling imply that in the assembly of WT A beta 16-22 the F19 side chain is localized within the intersheet space and is involved in hydrophobic contact with amino acids across the intersheet space, whereas the F20 side chain localized near the slab surface is less important for the intersheet interaction, but involved in slab stacking. IR observations for the same peptides in dilute solution showed a greater degree of hydrogen bonding for the nitrile groups in 20CN than in 19CN, supporting this interpretation. (c) 2010 Wiley-Liss, Inc.

Identification of functional domains within the alpha and beta subunits of beta-hexosaminidase A through the expression of alpha-beta fusion proteins.

PubMed

Tse, R; Wu, Y J; Vavougios, G; Hou, Y; Hinek, A; Mahuran, D J

1996-08-20

There are three human beta-hexosaminidase isozymes which are composed of all possible dimeric combinations of an alpha and/or a beta subunit; A (alpha beta), and B (beta beta), and S (alpha alpha). The amino acid sequences of the two subunits are 60% identical. The homology between the two chains varies with the middle > the carboxy-terminal > > the amino-terminal portions. Although dimerization is required for activity, each subunit contains its own active site and differs in its substrate specificity and thermal stability. The presence of the beta subunit in hexosaminidase A also influences the substrate specificity of the alpha subunit; e.g., in vivo only the A heterodimer can hydrolyze GM2 ganglioside. In this report, we localize functional regions in the two subunits by cellular expression of alpha/beta fusion proteins joined at adjacently aligned residues. First, a chimeric alpha/beta chain was made by replacing the least well-conserved amino-terminal section of the beta chain with the corresponding alpha section. The biochemical characteristics of this protein were nearly identical to hexosaminidase B. Therefore, the most dissimilar regions in the subunits are not responsible for their dissimilar biochemical properties. A second fusion protein was made that also included the more homologous middle section of the alpha chain. This protein expressed the substrate specificity unique to isozymes containing an alpha subunit (A and S). We conclude that the region responsible for the ability of the alpha subunit to bind negatively charged substrates is located within residues alpha 132-283. Interestingly, the remaining carboxy-terminal section from the beta chain, beta 316-556, was sufficient to allow this chimera to hydrolyze GM2 ganglioside with 10% the specific activity of heterodimeric hexosaminidase A. Thus, the carboxy-terminal section of each subunit is likely involved in subunit-subunit interactions.

Preferential V beta gene usage and lack of junctional sequence conservation among human T cell receptors specific for a tetanus toxin- derived peptide: evidence for a dominant role of a germline-encoded V region in antigen/major histocompatibility complex recognition

PubMed Central

1992-01-01

To investigate the structural and genetic basis of the T cell response to defined peptide/major histocompatibility (MHC) class II complexes in humans, we established a large panel of T cell clones (61) from donors of different HLA-DR haplotypes and reactive with a tetanus toxin- derived peptide (tt830-844) recognized in association with most DR molecules (universal peptide). By using a bacterial enterotoxin-based proliferation assay and cDNA sequencing, we found preferential use of a particular V beta region gene segment, V beta 2.1, in three of the individuals studied (64%, n = 58), irrespective of whether the peptide was presented by the DR6wcI, DR4w4, or DRw11.1 and DRw11.2 alleles, demonstrating that shared MHC class II antigens are not required for shared V beta gene use by T cell receptors (TCRs) specific for this peptide. V alpha gene use was more heterogeneous, with at least seven different V alpha segments derived from five distinct families encoding alpha chains able to pair with V beta 2.1 chains to form a tt830-844/DR- specific binding site. Several cases were found of clones restricted to different DR alleles that expressed identical V beta and (or very closely related) V alpha gene segments and that differed only in their junctional sequences. Thus, changes in the putative complementary determining region 3 (CDR3) of the TCR may, in certain cases, alter MHC specificity and maintain peptide reactivity. Finally, in contrast to what has been observed in other defined peptide/MHC systems, a striking heterogeneity was found in the junctional regions of both alpha and beta chains, even for TCRs with identical V alpha and/or V beta gene segments and the same restriction. Among 14 anti-tt830-844 clones using the V beta 2.1 gene segment, 14 unique V beta-D-J beta junctions were found, with no evident conservation in length and/or amino acid composition. One interpretation for this apparent lack of coselection of specific junctional sequences in the context of a common V element, V beta 2.1, is that this V region plays a dominant role in the recognition of the tt830-844/DR complex. PMID:1371303

Prediction of beta-turns from amino acid sequences using the residue-coupled model.

PubMed

Guruprasad, K; Shukla, S

2003-04-01

We evaluated the prediction of beta-turns from amino acid sequences using the residue-coupled model with an enlarged representative protein data set selected from the Protein Data Bank. Our results show that the probability values derived from a data set comprising 425 protein chains yielded an overall beta-turn prediction accuracy 68.74%, compared with 94.7% reported earlier on a data set of 30 proteins using the same method. However, we noted that the overall beta-turn prediction accuracy using probability values derived from the 30-protein data set reduces to 40.74% when tested on the data set comprising 425 protein chains. In contrast, using probability values derived from the 425 data set used in this analysis, the overall beta-turn prediction accuracy yielded consistent results when tested on either the 30-protein data set (64.62%) used earlier or a more recent representative data set comprising 619 protein chains (64.66%) or on a jackknife data set comprising 476 representative protein chains (63.38%). We therefore recommend the use of probability values derived from the 425 representative protein chains data set reported here, which gives more realistic and consistent predictions of beta-turns from amino acid sequences.

Molecular analysis of Hb Q-H disease and Hb Q-Hb E in a Singaporean family.

PubMed

Tan, J; Tay, J S; Wong, Y C; Kham, S K; Bte Abd Aziz, N; Teo, S H; Wong, H B

1995-01-01

Hb Q (alpha 74Asp-His) results from a mutation in the alpha-gene such that abnormal alpha Q-chains are synthesized. The alpha Q-chains combine with the normal Beta A-chains to form abnormal Hb alpha 2Q beta 2A (Hb Q). Hb Q-H disease is rare, and has been reported only in the Chinese. We report here a Chinese family, were the mother diagnosed with Hb Q-H disease and the father with Hb E heterozygosity and a child with Hb Q-E-thalassemia. Thalassemia screening of the mother's blood revealed a Hb level of 6.8g/dl with low MCV and MCH. Her blood film was indicative of thalassemia. Cellulose acetate electrophoresis showed Hb H and Hb Q with the absence of Hb A. Globin chain biosynthesis was carried out and alpha Q- and beta-chains were detected. Normal alpha- chains were absent. Digestion of the mother's DNA with Bam HI and Bgl II followed by hybridization with the 1.5 kb alpha-Pst probe showed a two alpha-gene deletion on one chromosome and the -alpha Q chain mutant with the -alpha 4.2 defect on the other chromosome. DNA amplification studies indicated the two-gene deletion to be of the -SEA/ defect. The patient was concluded to possess Hb Q-H disease (--SEA/-alpha 4.2Q). Cellulose acetate electrophoresis of the father's blood showed the presence of Hb A, F and E. Molecular analysis of the father's DNA confirmed an intact set of alpha-genes (alpha alpha/alpha alpha). Globin chain biosynthesis of fetal blood of their child showed gamma, beta A, beta E, alpha A and alpha Q-chains. Molecular analysis of the child's DNA showed one alpha-gene deletion, thus giving a genotype of alpha alpha/-alpha 4.2Q beta beta E.

V(D)J recombination and allelic exclusion of a TCR beta-chain minilocus occurs in the absence of a functional promoter.

PubMed

Alvarez, J D; Anderson, S J; Loh, D Y

1995-08-01

Transcriptional activation of rearranging Ag receptor gene segments has been hypothesized to regulate their accessibility to V(D)J recombination. We analyzed the role of a functional promoter in the rearrangement of the murine TCR beta-chain locus using two transgenic minilocus constructs. These miniloci each contain an unrearranged V beta 8.3 gene. One has a wild-type V beta 8.3 gene, but the other has a V beta 8.3 gene with a promoter mutation that was previously shown to abrogate transcription in tissue culture. FACS analysis of thymus and lymph node cells from transgenic mouse lines showed that only the lines with the wild-type V beta 8.3 gene promoter express an 8.3 TCR beta-chain. Consistent with the protein expression data, V beta 8.3 gene transcripts were found only in the transgenic lines with the wild-type promoter. Using a quantitative PCR-based assay, it was shown that both types of transgenic lines recombine the V beta 8.3 gene at similar levels. Rearrangement of the transgenes was normal with respect to thymic development and junctional reading frame. Interestingly, both types of miniloci also underwent allelic exclusion in that recombination was blocked by the expression of a rearranged TCR beta-chain transgene. We conclude that a functional V beta gene promoter is not necessary for proper V(D)J recombination to occur.

Significance of two distinct types of tryptophan synthase beta chain in Bacteria, Archaea and higher plants.

PubMed

Xie, Gary; Forst, Christian; Bonner, Carol; Jensen, Roy A

2002-01-01

Tryptophan synthase consists of two subunits, alpha and beta. Two distinct subgroups of beta chain exist. The major group (TrpEb_1) includes the well-studied beta chain of Salmonella typhimurium. The minor group of beta chain (TrpEb_2) is most frequently found in the Archaea. Most of the amino-acid residues important for catalysis are highly conserved between both TrpE subfamilies. Conserved amino-acid residues of TrpEb_1 that make allosteric contact with the TrpEa subunit (the alpha chain) are absent in TrpEb_2. Representatives of Archaea, Bacteria and higher plants all exist that possess both TrpEb_1 and TrpEb_2. In those prokaryotes where two trpEb genes coexist, one is usually trpEb_1 and is adjacent to trpEa, whereas the second is trpEb_2 and is usually unlinked with other tryptophan-pathway genes. TrpEb_1 is nearly always partnered with TrpEa in the tryptophan synthase reaction. However, by default at least six lineages of the Archaea are likely to use TrpEb_2 as the functional beta chain, as TrpEb_1 is absent. The six lineages show a distinctive divergence within the overall TrpEa phylogenetic tree, consistent with the lack of selection for amino-acid residues in TrpEa that are otherwise conserved for interfacing with TrpEb_1. We suggest that the standalone function of TrpEb_2 might be to catalyze the serine deaminase reaction, an established catalytic capability of tryptophan synthase beta chains. A coincident finding of interest is that the Archaea seem to use the citramalate pathway, rather than threonine deaminase (IlvA), to initiate the pathway of isoleucine biosynthesis.

Antiproliferative properties of toremifene on AIDS-related Kaposi's sarcoma cells.

PubMed

Hong, Angela; Leigh, Bryan R

2002-12-01

Kaposi's sarcoma (KS) is the most common neoplastic apoptosis manifestation of acquired immunodeficiency syndrome. Toremifene is known to upregulate transforming growth factor beta-1 (TGF-beta1), which is a growth-inhibitory factor for KS. We investigated the in vitro effect of toremifene on KS cells. MTT assay was used to measure the growth of four KS cell lines and a human umbilical vein endothelial (HUVE) cell line after incubation with toremifene. Reverse transcription polymerase chain reaction and ELISA were used to measure the level of TGF-beta1. The IC(50) for the KS cells ranged from 2.2 to 3.2 microM, and 80% of the growth inhibition occurred within 24 h. Toremifene enhanced TGF-beta1 mRNA expression, and the level of TGF-beta1 increased from 103 to 473 pg/ml after 48 h of incubation. Toremifene had no effect on the growth of HUVE cells. Toremifene has a specific antiproliferative effect on KS cells. The stimulation of TGF-beta1 production may play a role in the antiproliferative process. Copyright 2002 S. Karger AG, Basel

Properties of polyproline II, a secondary structure element implicated in protein-protein interactions.

PubMed

Cubellis, M V; Caillez, F; Blundell, T L; Lovell, S C

2005-03-01

The polyproline II (PPII) conformation of protein backbone is an important secondary structure type. It is unusual in that, due to steric constraints, its main-chain hydrogen-bond donors and acceptors cannot easily be satisfied. It is unable to make local hydrogen bonds, in a manner similar to that of alpha-helices, and it cannot easily satisfy the hydrogen-bonding potential of neighboring residues in polyproline conformation in a manner analogous to beta-strands. Here we describe an analysis of polyproline conformations using the HOMSTRAD database of structurally aligned proteins. This allows us not only to determine amino acid propensities from a much larger database than previously but also to investigate conservation of amino acids in polyproline conformations, and the conservation of the conformation itself. Although proline is common in polyproline helices, helices without proline represent 46% of the total. No other amino acid appears to be greatly preferred; glycine and aromatic amino acids have low propensities for PPII. Accordingly, the hydrogen-bonding potential of PPII main-chain is mainly satisfied by water molecules and by other parts of the main-chain. Side-chain to main-chain interactions are mostly nonlocal. Interestingly, the increased number of nonsatisfied H-bond donors and acceptors (as compared with alpha-helices and beta-strands) makes PPII conformers well suited to take part in protein-protein interactions. Copyright 2005 Wiley-Liss, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rouyer-Fessard, P.; Garel, M.C.; Domenget, C.

The soluble pool of alpha hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, beta A hemoglobin chain labeled with ({sup 3}H)N-ethylmaleimide. This pool of soluble alpha chains was 0.067 {plus minus} 0.017% of hemoglobin in blood of normal adult, 0.11 {plus minus} 0.03% in heterozygous beta thalassemia and ranged from 0.26 to 1.30% in homozygous beta thalassemia intermedia. This elevated pool of soluble alpha chains observed in human beta thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% inmore » the most dense red blood cells. The amount of insoluble alpha chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In beta thalassemia intermedia the amount of insoluble alpha chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using ({sup 3}H)N-ethylmaleimide added to membrane ghosts prior to urea and Triton X-100 electrophoresis. Spectrin and ankyrin were the major proteins with the most important decrease of thiol groups.« less

Overproduction of alpha chains provides a proton-insensitive component to the bluefish hemoglobin system.

PubMed

Bonaventura, Celia; Godette, Gerald; Stevens, Robert; Brenowitz, Michael; Henkens, Robert

2005-12-09

Expression of alpha and beta chains and their post-translational assembly into alpha(2)beta(2) tetramers is fundamental to the formation and function of most vertebrate hemoglobins. There is a strong evolutionary bias that favors expression of equal amounts of the two types of chains, because cooperativity, pH sensitivity, and anionic control of function occurs only for the alpha(2)beta(2) tetramers. Remarkably, an over-production of alpha chains, as in the pathological condition known as beta thalassemia in humans, is adaptive rather than pathological in the bluefish hemoglobin system. The thalassemia of the bluefish is a novel means of providing for oxygen uptake and delivery when low pH conditions incapacitate the highly pH-sensitive Root effect hemoglobins of the fish. Although fish often have pH-insensitive along with highly pH-sensitive hemoglobins, having pH-insensitive alpha chain monomers in circulation is an unusual structural variation. The role of bluefish alpha chains in oxygen transport is enabled by their remarkably lower oxygen affinity relative to human alpha chains. This is the first reported case of a thalassemic condition that is maintained in a species as an adaptive advantage.

O-linked oligosaccharides on insulin receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collier, E.; Gorden, P.

1991-02-01

The insulin receptor, an integral membrane glycoprotein, is synthesized as a single-chain precursor that is cleaved to produce two mature subunits, both of which contain N-linked oligosaccharide chains and covalently linked fatty acids. We report that the beta-subunit also contains O-linked oligosaccharides. The proreceptor, alpha-subunit, and beta-subunit were labeled with (3H)mannose and (3H)galactose in the presence or absence of an inhibitor of O-linked glycosylation. Tryptic peptides from each component were separated by reverse-phase high-performance liquid chromatography. N- and O-linked oligosaccharide chains were identified on these peptides by specific enzymatic digestions. The proreceptor and alpha-subunit contained only N-linked oligosaccharides, whereas themore » beta-subunit contained both N- and O-linked oligosaccharides. The O-linked oligosaccharide chains were attached to a single tryptic fraction of the beta-subunit, which also contained N-linked chains. This fraction was further localized to the NH2-terminal tryptic peptide of the beta-subunit by specific immunoprecipitation with an anti-peptide antibody with specificity for this region. Binding of insulin and autophosphorylation of the beta-subunit were not dependent on O-linked glycosylation, because cells grown in the presence of the inhibitor exhibited a normal dose response to insulin. Therefore, the insulin receptor contains O-linked oligosaccharides on the NH2-terminal tryptic peptide of the beta-subunit, and these O-linked oligosaccharides are not necessary to the binding or autophosphorylation function of the receptor.« less

Purification and characterization of an amidohydrolase for N4-long-chain fatty acyl derivatives of 1-beta-D-arabinofuranosylcytosine from mouse liver microsomes.

PubMed

Hori, K; Tsuruo, T; Tsukagoshi, S; Sakurai, Y

1984-03-01

N4-Long-chain fatty acyl-1-beta-D-arabinofuranosylcytosine amidohydrolase, a metabolizing enzyme for N4-acyl derivatives of 1-beta-D-arabinofuranosylcytosine with long-chain fatty acids, was purified from mouse liver microsomes. The purification was accomplished by solubilization of liver microsomes with Triton X-100, diethylaminoethyl cellulose chromatography, gel filtrations, hydroxyapatite chromatography, and concanavalin A:Sepharose chromatography. On sodium dodecyl sulfate:polyacrylamide gel electrophoresis, the purified enzyme preparation produced a single protein band with a molecular weight of 54,000. The enzyme had an optimal pH of 9.0, and the Michaelis constant for N4-palmitoyl-1-beta-D-arabinofuranosylcytosine was 67 microM. The thiols such as dithiothreitol or 2-mercaptoethanol stabilized the enzyme and stimulated its activity. p-Chloromercuribenzoate, N-ethylmaleimide, diisopropylfluorophosphate, and phenylmethylsulfonyl fluoride strongly inhibited the reaction. Bovine serum albumin markedly stimulated the enzyme activity, whereas detergents such as Triton X-100, deoxycholate, and sodium dodecyl sulfate had little effect. The enzyme did not require monovalent or divalent cations. Among the series of N4-acyl derivatives of 1-beta-D-arabinofuranosylcytosine with different chain lengths of acyl residues, the purified enzyme preferentially hydrolyzed the derivatives with long-chain fatty acids (C12 to C18), and N4-palmitoyl-1-beta-D-arabinofuranosylcytosine was the most susceptible. The purified enzyme was inactive on various N-acylamino acids, amides, oligopeptides, proteins, N-acylsphingosines (ceramides), triglyceride, lecithin, and lysolecithin. These results suggest that N4-long-chain fatty acyl-1-beta-D-arabinofuranosylcytosine amidohydrolase may be a new type of linear amidase.

Inferring High-Confidence Human Protein-Protein Interactions

DTIC Science & Technology

2012-01-01

comprised proteins that had the same specific func- tion or were subunits of the same protein complex, such as branched chain keto acid E1 alpha (BCKDHA...and branched chain keto acid E1 beta (BCKDHB) [3,29], and dynein cytoplasmic 2 intermediate chain 1 (D2LIC) and dynein cytoplasmic 2 heavy chain 1...474.3 28.0 1337.0 BCKDHA 5 Branched chain keto acid dehydro. E1, alpha BCKDHB 4 Branched chain keto acid dehydro. E1, beta 4 471.4 29.0 1337.5 ARTN 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patrick, N.; Miyakawa, F.; Hunt, J.A.

The distribution of {beta}-thalassemia [{beta}{sup Th}] mutations is unique to each ethnic group. Most mutations affect one or a few bases; large deletions have been rare. Among families screened in Hawaii, [{beta}{sup Th}] heterozygotes were diagnosed by microcytosis, absence of abnormal hemoglobins on isoelectric focusing, and raised Hb A{sub 2} by chromatography. Gene frequency for {beta}{sup Th} was 0.02 in Filipinos. In Filipinos, polymerase chain reaction [PCR] with denaturing gradient gel electrophoresis for {beta}{sup Th} mutations detected a mutation in only 6 of 42 {beta}{sup Th} heterozygotes; an IVS2-666 C/T polymorphism showed non-heterozygosity in 37 and heterozygosity in only 5more » of these {beta}{sup Th} heterozygotes. One {beta}{sup Th}/{beta}{sup Th} major patient and his mother had no mutation detected by allele-specific oligomer hybridization; PCR failed to amplify any DNA from his {beta}-globin gene. After a total {beta}-globin gene deletion [{beta}{sup Del}] was found in a Filipino family in Ontario, specific PCR amplification for {beta}{sup Del} detected this in 43 of 53 {beta}{sup Th} Filipino samples tested; the above {beta}{sup Th}/{beta}{sup Th} patient was a ({beta}{sup Del}/{beta}{sup Del}) homozygote. The {beta}{sup Del} may account for over 60% of all {beta}{sup Th} alleles in Filipinos; this is the highest proportion of a deletion {beta}{sup Th} mutation reported from any population. Most but not all {beta}{sup Del} heterozygotes had high Hb F [5.13 {plus_minus} 3.94 mean {plus_minus} 1 s.d.] compared to the codon 41/42 four base deletion common in Chinese [2.30 {plus_minus} 0.86], or to {beta}{sup Th} heterozygotes with normal {alpha}-globin genes [2.23 {plus_minus} 0.80].« less

The Val192Leu mutation in the alpha-subunit of beta-hexosaminidase A is not associated with the B1-variant form of Tay-Sachs disease.

PubMed Central

Hou, Y.; Vavougios, G.; Hinek, A.; Wu, K. K.; Hechtman, P.; Kaplan, F.; Mahuran, D. J.

1996-01-01

Substitution mutations adversely affecting the alpha-subunit of beta-hexosaminidase A (alphabeta) (EC 3.2.1.52) result in Tay-Sachs disease. The majority affect the initial folding of the pro-alpha chain in the endoplasmic reticulum, resulting in its retention and degradation. A much less common occurrence is a mutation that specifically affects an "active-site" residue necessary for substrate binding and/or catalysis. In this case, hexosaminidase A is present in the lysosome, but it lacks all alpha-specific activity. This biochemical phenotype is referred to as the "B1-variant form" of Tay-Sachs disease. Kinetic analysis of suspected B1-variant mutations is complex because hexosaminidase A is heterodimeric and both subunits possess similar active sites. In this report, we examine a previously identified B1-variant mutation, alpha-Val192Leu. Chinese hamster ovary cells were permanently cotransfected with an alpha-cDNA-construct encoding the substitution and a mutant beta-cDNA (beta-Arg211Lys), encoding a beta-subunit that is inactive but normal in all other respects. We were surprised to find that the Val192Leu substitution, produced a pro-alpha chain that did not form alpha-beta dimers and was not transported to the lysosome. Finally, we reexamined the hexosaminidase activity and protein levels in the fibroblasts from the original patient. These data were also not consistent with the biochemical phenotype of the B1 variant of Tay-Sachs disease previously reported to be present. Thus, we conclude that the Val192Leu substitution does not specifically affect the alpha-active site. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8659543

The Val192Leu mutation in the alpha-subunit of beta-hexosaminidase A is not associated with the B1-variant form of Tay-Sachs disease.

PubMed

Hou, Y; Vavougios, G; Hinek, A; Wu, K K; Hechtman, P; Kaplan, F; Mahuran, D J

1996-07-01

Substitution mutations adversely affecting the alpha-subunit of beta-hexosaminidase A (alphabeta) (EC 3.2.1.52) result in Tay-Sachs disease. The majority affect the initial folding of the pro-alpha chain in the endoplasmic reticulum, resulting in its retention and degradation. A much less common occurrence is a mutation that specifically affects an "active-site" residue necessary for substrate binding and/or catalysis. In this case, hexosaminidase A is present in the lysosome, but it lacks all alpha-specific activity. This biochemical phenotype is referred to as the "B1-variant form" of Tay-Sachs disease. Kinetic analysis of suspected B1-variant mutations is complex because hexosaminidase A is heterodimeric and both subunits possess similar active sites. In this report, we examine a previously identified B1-variant mutation, alpha-Val192Leu. Chinese hamster ovary cells were permanently cotransfected with an alpha-cDNA-construct encoding the substitution and a mutant beta-cDNA (beta-Arg211Lys), encoding a beta-subunit that is inactive but normal in all other respects. We were surprised to find that the Val192Leu substitution, produced a pro-alpha chain that did not form alpha-beta dimers and was not transported to the lysosome. Finally, we reexamined the hexosaminidase activity and protein levels in the fibroblasts from the original patient. These data were also not consistent with the biochemical phenotype of the B1 variant of Tay-Sachs disease previously reported to be present. Thus, we conclude that the Val192Leu substitution does not specifically affect the alpha-active site.

A GMCSF and IL-15 fusokine leads to paradoxical immunosuppression in vivo via asymmetrical JAK/STAT signaling through the IL-15 receptor complex.

PubMed

Rafei, Moutih; Wu, Jian Hui; Annabi, Borhane; Lejeune, Laurence; François, Moïra; Galipeau, Jacques

2007-03-01

We hypothesized that a granulocyte macrophage colony-stimulating factor (GMCSF) and interleukin 15 (IL-15) fusokine (GIFT15) would possess greater immune-stimulatory properties than their combined use. Unexpectedly, tumor cells engineered to secrete GIFT15 protein led to suppression of natural killer (NK) and NKT-cell recruitment in vivo, suggesting an unanticipated immune-suppressive effect. We found GIFT15 to have pleiotropic effects on an array of immune-competent cells. Among these, macrophages treated with GIFT15 secrete de novo the tissue inhibitor of metalloproteinase-2 (TIMP-2); activated matrix metalloproteinase-2 (MMP-2); transforming growth factor-beta (TGF-beta); as well as vascular endothelial growth factor (VEGF). We show that the GIFT15 fusokine has increased affinity for the alpha chain component of the IL-15R, leading to aberrant signaling through the beta chain manifested by the hyperphosphorylation of STAT3 both in macrophages and splenocytes. Suppression of common gamma chain-mediated STAT5 phosphorylation and blockade of the IL-15-dependent IFN-gamma response in mouse splenocytes were also observed. We tested GIFT15 as an immunosuppressor and demonstrated that it allowed engraftment of allogeneic B16F0 and human xenograft U87GM glioma cells in immunocompetent mice. Thus, GIFT15 defines a new class of fusokine that mediates proangiogenic and immunosuppressive effects via aberrant signaling by the IL-15R in lymphomyeloid cells.

Conformation of methyl beta-lactoside bound to the ricin B-chain: Interpretation of transferred nuclear Overhauser effects facilitated by spin simulation and selective deuteration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bevilacqua, V.L.; Thomson, D.S.; Prestegard, J.H.

1990-06-12

Spin simulation and selective deuteration have been used to aid in the interpretation of 1D transferred nuclear Overhauser effect (TRNOE) NMR experiments on ricin B-chain/ligand systems. Application of these methods has revealed a change in the conformation of deuterated methyl beta-lactoside upon binding to the ricin B-chain which results in a slight change in glycosidic torsional angels which appear to dominate in the solution conformation. The combination of simulation and experiment also shows an important sensitivity of TRNOE magnitudes to dissociation rate constants and available spin-diffusion pathways for the ricin B-chain/ligand systems under study. The sensitivity to dissociation rates allowsmore » determination of rate constants for methyl beta-lactoside and methyl beta-galactoside of 50 and 300 s-1, respectively.« less

Coexistence of Southeast Asian ovalocytosis and beta-thalassemia: a molecular and hematological analysis.

PubMed

Fucharoen, Goonnapa; Fucharoen, Supan; Singsanan, Sanita; Sanchaisuriya, Kanokwan

2007-05-01

We describe hematological and molecular characterization of a Thai female who had Southeast Asian ovalocytosis (SAO) associated with beta+-thalassemia trait. The proband had mild microcytosis with Hb 12.9 g/dl, Hct 35.8%, MCV 74.4 fl, MCH 26.8 pg, MCHC 36.0 g/dl, and elevated Hb A2 (5.6%), characteristics of beta-thalassemia trait. Peripheral blood film examination revealed prominent ovalocytosis. However, a one-tube osmotic fragility (OF) test commonly used for thalassemia screening was negative and a normal OF curve was observed. Further polymerase chain reaction (PCR) analyses identified the beta(-28A-G) mutation in the beta-globin gene and a 27 bp deletion in erythrocyte band 3 protein gene, indicating a genetically compound heterozygote. Hematological data of the proband was comparatively presented with those of eight female and 15 male carriers of pure beta-thalassemia with the same mutation. The finding demonstrates that although the association of the SAO and beta-thalassemia does not produce a more severe clinical picture, this could lead to a mis-screening of beta-thalassemia using an OF test as a primary screening test. Additional blood film examination followed by PCR could help in the detection of this unusual genetic interaction in the region. (c) 2006 Wiley-Liss, Inc.

Normal T lymphocytes can express two different T cell receptor beta chains: implications for the mechanism of allelic exclusion

PubMed Central

1995-01-01

We have examined the extent of allelic exclusion at the T cell receptor (TCR) beta locus using monoclonal antibodies specific for V beta products. A small proportion (approximately 1%) of human peripheral blood T cells express two V beta as determined by flow cytometric analysis, isolation of representative clones, and sequencing of the corresponding V beta chains. Dual beta T cells are present in both the CD45R0+ and CD45R0- subset. These results indicate that dual beta expression is compatible with both central and peripheral selection. They also suggest that the substantial degree of TCR beta allelic exclusion is dependent only on asynchronous rearrangements at the beta locus, whereas the role of the pre-TCR is limited to signaling the presence of at least one functional beta protein. PMID:7699339

Absence of Fc epsilonRI alpha chain results in upregulation of Fc gammaRIII-dependent mast cell degranulation and anaphylaxis. Evidence of competition between Fc epsilonRI and Fc gammaRIII for limiting amounts of FcR beta and gamma chains.

PubMed Central

Dombrowicz, D; Flamand, V; Miyajima, I; Ravetch, J V; Galli, S J; Kinet, J P

1997-01-01

In mouse mast cells, both Fc epsilonRI and Fc gammaRIII are alpha beta gamma2 tetrameric complexes in which different alpha chains confer IgE or IgG ligand recognition while the signaling FcR beta and gamma chains are identical. We used primarily noninvasive techniques (changes in body temperature, dye extravasation) to assess systemic anaphylactic responses in nonanesthetized wild-type, Fc epsilonRI alpha chain -/- and FcR gamma chain -/- mice. We confirm that systemic anaphylaxis in mice can be mediated largely through IgG1 and Fc gammaRIII and we provide direct evidence that these responses reflect activation of Fc gammaRIII rather than Fc gammaRI. Furthermore, we show that Fc gammaRIII-dependent responses are more intense in normal than in congenic mast cell-deficient KitW/KitW-v mice, indicating that Fc gammaRIII responses have mast cell-dependent and -independent components. Finally, we demonstrate that the upregulation of cell surface expression of Fc gammaRIII seen in Fc epsilonRI alpha chain -/- mice corresponds to an increased association of Fc gammaRIII alpha chains with FcR beta and gamma chains and is associated with enhanced Fc gammaRIII-dependent mast cell degranulation and systemic anaphylactic responses. Therefore, the phenotype of the Fc epsilonRI alpha chain -/- mice suggests that expression of Fc epsilonRI and Fc gammaRIII is limited by availability of the FcR beta and gamma chains and that, in normal mice, changes in the expression of one receptor (Fc epsilonRI) may influence the expression of functional responses dependent on the other (Fc gammaRIII). PMID:9062349

beta-Methyl-15-p-iodophenylpentadecanoic acid metabolism and kinetics in the isolated rat heart.

PubMed

DeGrado, T R; Holden, J E; Ng, C K; Raffel, D M; Gatley, S J

1989-01-01

The use of 15-p-iodophenyl-beta-methyl-pentadecanoic acid (beta Me-IPPA) as an indicator of long chain fatty acid (LCFA) utilization in nuclear medicine studies was evaluated in the isolated, perfused, working rat heart. Time courses of radioactivity (residue curves) were obtained following bolus injections of both beta Me-IPPA and its straight chain counterpart 15-p-iodophenyl-pentadecanoic acid (IPPA). IPPA kinetics clearly indicated flow independent impairment of fatty acid oxidation caused by the carnitine palmitoyltransferase I inhibitor 2[5(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA). In contrast, beta Me-IPPA kinetics were insensitive to changes in fatty acid oxidation rate and net utilization of long chain fatty acid. Analysis of radiolabeled species in coronary effluent and heart homogenates showed the methylated fatty acid to be readily incorporated into complex lipids but a poor substrate for oxidation. POCA did not significantly alter metabolism of the tracer, suggesting that the tracer is poorly metabolized beyond beta Me-IPPA-CoA in the oxidative pathway.

Identification and comparative expression analysis of interleukin 2/15 receptor B chain in chickens infected with E. tenella

USDA-ARS?s Scientific Manuscript database

Background: Interleukin (IL) 2 and IL15 receptor beta chain (IL2/15Receptor beta, CD122) play critical roles in signal transduction for the biological activities of IL2 and IL15. Increased knowledge of non-mammalian IL2/15Receptor beta will enhance the understanding of IL2 and IL15 functions. Meth...

Alternative splicing produces transcripts coding for alpha and beta chains of a hetero-dimeric phosphagen kinase.

PubMed

Ellington, W Ross; Yamashita, Daisuke; Suzuki, Tomohiko

2004-06-09

Glycocyamine kinase (GK) catalyzes the reversible phosphorylation of glycocyamine (guanidinoacetate), a reaction central to cellular energy homeostasis in certain animals. GK is a member of the phosphagen kinase enzyme family and appears to have evolved from creatine kinase (CK) early in the evolution of multi-cellular animals. Prior work has shown that GK from the polychaete Neanthes (Nereis) diversicolor exits as a hetero-dimer in vivo and that the two polypeptide chains (termed alpha and beta) are coded for by unique transcripts. In the present study, we demonstrate that the GK from a congener Nereis virens is also hetero-dimeric and is coded for by alpha and beta transcripts, which are virtually identical to the corresponding forms in N. diversicolor. The GK gene from N. diversicolor was amplified by PCR. Sequencing of the PCR products showed that the alpha and beta chains are the result of alternative splicing of the GK primary mRNA transcript. These results also strongly suggest that this gene underwent an early tandem exon duplication event. Full-length cDNAs for N. virens GKalpha and GKbeta were individually ligated into expression vectors and the resulting constructs used to transform Escherichia coli expression hosts. Regardless of expression conditions, minimal GK activity was observed in both GKalpha and GKbeta constructs. Inclusion bodies for both were harvested, unfolded in urea and alpha chains, beta chains and mixtures of alpha and beta chains were refolded by sequential dialysis. Only modest amounts of GK activity were observed when alpha and beta were refolded individually. In contrast, when refolded the alpha and beta mixture yielded highly active hetero-dimers, as validated by size exclusion chromatography, electrophoresis and mass spectrometry, with a specific activity comparable to that of natural GK. The above evidence suggests that there is a preference for hetero-dimer formation in the GKs from these two polychaetes. The evolution of the alternate splicing and an additional exon in these GKs, producing alpha and beta transcripts, can be viewed as a possible compensation for a mutation(s) in the original gene, which most likely coded for a homo-dimeric protein.

Clinical and molecular epidemiology of beta-hemolytic streptococcal infections in India.

PubMed

Mathur, Purva; Bhardwaj, Nidhi; Mathur, Kushal; Behera, Bijayini; Gupta, Gunjan; Kapil, Arti; Singh, Sarman; Misra, Mahesh Chandra

2014-03-13

Beta-hemolytic streptococci (βHS) cause a diverse array of human infections. Despite the high number of cases of streptococcal carriers and diseases, studies discerning the molecular epidemiology of βHS in India are limited. This study reports the molecular and clinical epidemiology of beta-hemolytic streptococcal infections from two geographically distinct regions of India. A total of 186 isolates of βHS from north and south India were included. The isolates were identified to species level and subjected to antimicrobial susceptibility testing. Polymerase chain reaction (PCR) was done to detect exotoxin genes, and emm types of group A streptococci (GAS) strains were ascertained by sequencing. GAS was the most common isolate (71.5%), followed by group G streptococci (GGS) (21%). A large proportion of GAS produced speB (97%), smeZ (89%), speF (91%), and speG (84%). SmeZ was produced by 21% and 50% of GGS and GGS, respectively. A total of 45 different emm types/subtypes were seen in GAS, with emm 11 being the most common. Resistance to tetracycline (73%) and erythromycin (34.5%) was commonly seen in GAS. A high diversity of emm types was seen in Indian GAS isolates with high macrolide and tetracycline resistance. SpeA was less commonly seen in Indian GAS isolates. There was no association between disease severity and exotoxin gene production.

Structural basis for the mechanism and substrate specificity of glycocyamine kinase, a phosphagen kinase family member

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, Kap; Pullalarevu, Sadhana; Surabian, Karen Talin

2010-03-12

Glycocyamine kinase (GK), a member of the phosphagen kinase family, catalyzes the Mg{sup 2+}-dependent reversible phosphoryl group transfer of the N-phosphoryl group of phosphoglycocyamine to ADP to yield glycocyamine and ATP. This reaction helps to maintain the energy homeostasis of the cell in some multicelullar organisms that encounter high and variable energy turnover. GK from the marine worm Namalycastis sp. is heterodimeric, with two homologous polypeptide chains, {alpha} and {beta}, derived from a common pre-mRNA by mutually exclusive N-terminal alternative exons. The N-terminal exon of GK{beta} encodes a peptide that is different in sequence and is 16 amino acids longermore » than that encoded by the N-terminal exon of GK{alpha}. The crystal structures of recombinant GK{alpha}{beta} and GK{beta}{beta} from Namalycastis sp. were determined at 2.6 and 2.4 {angstrom} resolution, respectively. In addition, the structure of the GK{beta}{beta} was determined at 2.3 {angstrom} resolution in complex with a transition state analogue, Mg{sup 2+}-ADP-NO{sub 3}{sup -}-glycocyamine. Consistent with the sequence homology, the GK subunits adopt the same overall fold as that of other phosphagen kinases of known structure (the homodimeric creatine kinase (CK) and the monomeric arginine kinase (AK)). As with CK, the GK N-termini mediate the dimer interface. In both heterodimeric and homodimeric GK forms, the conformations of the two N-termini are asymmetric, and the asymmetry is different than that reported previously for the homodimeric CKs from several organisms. The entire polypeptide chains of GK{alpha}{beta} are structurally defined, and the longer N-terminus of the {beta} subunit is anchored at the dimer interface. In GK{beta}{beta} the 24 N-terminal residues of one subunit and 11 N-terminal residues of the second subunit are disordered. This observation is consistent with a proposal that the GK{alpha}{beta} amino acids involved in the interface formation were optimized once a heterodimer emerged as the physiological form of the enzyme. As a consequence, the homodimer interface (either solely {alpha} or solely {beta} chains) has been corrupted. In the unbound state, GK exhibits an open conformation analogous to that observed with ligand-free CK or AK. Upon binding the transition state analogue, both subunits of GK undergo the same closure motion that clasps the transition state analogue, in contrast to the transition state analogue complexes of CK, where the corresponding transition state analogue occupies only one subunit, which undergoes domain closure. The active site environments of the GK, CK, and AK at the bound states reveal the structural determinants of substrate specificity. Despite the equivalent binding in both active sites of the GK dimer, the conformational asymmetry of the N-termini is retained. Thus, the coupling between the structural asymmetry and negative cooperativity previously proposed for CK is not supported in the case of GK.« less

Structural relationships between human erythrocyte sialoglycoproteins beta and gamma and abnormal sialoglycoproteins found in certain rare human erythrocyte variants lacking the Gerbich blood-group antigen(s).

PubMed Central

Reid, M E; Anstee, D J; Tanner, M J; Ridgwell, K; Nurse, G T

1987-01-01

The human erythrocyte membrane sialoglycoproteins beta and gamma are important for the maintenance of the discoid shape of the normal erythrocyte. In this paper we show that the human erythrocyte sialoglycoproteins beta and gamma (hereafter called beta and gamma) are structurally related. Rabbit antisera produced against purified beta and beta 1 and rendered specific to the cytoplasmic portion of these proteins also react with the cytoplasmic portion of gamma. Some human anti-Gerbich (Ge) sera react with the extracellular portion of both beta and gamma. This reactivity is shown to be directed towards a common epitope on beta and gamma. However, most anti-Ge sera do not react with beta, but react with an extracellular epitope only present on gamma. All individuals who lack the Ge antigens lack beta and gamma. In some cases abnormal sialoglycoproteins are present in the erythrocytes, and these are shown to be structurally related to beta and gamma. Rabbit antisera raised against the purified abnormal sialoglycoprotein from a Ge-negative erythrocyte type reacted with the cytoplasmic portion of both beta and gamma. Unlike normal beta and gamma, the abnormal sialoglycoproteins found in Ge-negative erythrocytes migrate as a diffuse band on SDS/polyacrylamide-gel electrophoresis. Studies using endoglycosidases suggest that the diffuse nature of these bands results from carbohydrate heterogeneity and that the abnormal sialoglycoproteins contain N-glycosidically linked oligosaccharides with repeating lactosamine units. Such polylactosamine chains are not present on normal beta or gamma. Images Fig. 1. Fig. 2. Fig. 3. PMID:2444210

The Promiscuity of [beta]-Strand Pairing Allows for Rational Design of [beta]-Sheet Face Inversion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Makabe, Koki; Koide, Shohei

2009-06-17

Recent studies suggest the dominant role of main-chain H-bond formation in specifying {beta}-sheet topology. Its essentially sequence-independent nature implies a large degree of freedom in designing {beta}-sheet-based nanomaterials. Here we show rational design of {beta}-sheet face inversions by incremental deletions of {beta}-strands from the single-layer {beta}-sheet of Borrelia outer surface protein A. We show that a {beta}-sheet structure can be maintained when a large number of native contacts are removed and that one can design large-scale conformational transitions of a {beta}-sheet such as face inversion by exploiting the promiscuity of strand-strand interactions. High-resolution X-ray crystal structures confirmed the success ofmore » the design and supported the importance of main-chain H-bonds in determining {beta}-sheet topology. This work suggests a simple but effective strategy for designing and controlling nanomaterials based on {beta}-rich peptide self-assemblies.« less

The Val{sup 192}Leu mutation in the {alpha}-subunit of {beta}-hexosaminidase A is not associated with the B1-variant form of Tay-Sachs disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hou, Y.; Vavougios, G.; Hinek, A.

1996-07-01

Substitution mutations adversely affecting the {alpha}-subunit of {beta}-hexosaminidase A ({alpha}{beta}) (EC 3.2.1.52) result in Tay-Sachs disease. The majority affect the initial folding of the pro-{alpha} chain in the endoplasmic reticulum, resulting in its retention and degradation. A much less common occurrence is a mutation that specifically affects an {open_quotes}active-site{close_quotes} residue necessary for substrate binding and/or catalysis. In this case, hexosaminidase A is present in the lysosome, but it lacks all {alpha}-specific activity. This biochemical phenotype is referred to as the {open_quotes}B1-variant form{close_quotes} of Tay-Sachs disease. Kinetic analysis of suspected B1-variant mutations is complex because hexosaminidase A is heterodimeric and bothmore » subunits possess similar active sites. In this report, we examine a previously identified B1-variant mutation, {alpha}-Val{sup 192}Leu. Chinese hamster ovary cells were permanently cotransfected with an {alpha}-cDNA-construct encoding the substitution and a mutant {beta}-cDNA ({beta}-Arg{sup 211}Lys), encoding a {beta}-subunit that is inactive but normal in all other respects. We were surprised to find that the Val{sup 192}Leu substitution produced a pro-{alpha} chain that did not form {alpha}-{beta} dimers and was not transported to the lysosome. Finally, we reexamined the hexosaminidase activity and protein levels in the fibroblasts from the original patient. These data were also not consistent with the biochemical phenotype of the B1 variant of Tay-Sachs disease previously reported to be present. Thus, we conclude that the Val{sup 192}Leu substitution does not specifically affect the {alpha}-active site. 23 refs., 4 figs., 2 tabs.« less

Transcription Analysis of the Beta-Glucosidase Precursor in Wild-Type and l-4i Mutant Bombyx mori (Lepidoptera: Bombycidae).

PubMed

Kang, Lequn; Huang, Fei; Wu, Fan; Zhao, Qiaoling

2015-01-01

Lethal fourth-instar larvae (l-4i) mutant of Bombyx mori, a recently discovered novel mutant, die from energy depletion due to genetic mutation. Beta-glucosidase is a common digestive enzyme that hydrolyzes cellulose in the diet to provide energy. In this study, the mRNA expression profiles of B. mori beta-glucosidase precursor (BmpreBG) were characterized by reverse transcription polymerase chain reaction and quantitative real-time polymerase chain reaction. The transcription level of BmpreBG varied in different tissues and developmental stages, except in the pupa and moth, which are the no-diet period. Remarkably, the mRNA expression level of BmpreBG was sharply reduced in l-4i but not in the wild type, which suggested that the digestive function of the mutant was severely damaged. This was consistent with the l-4i phenotypic traits of not eating mulberries, lack of energy, and ultimate death. 5'-rapid amplification of cDNA ends showed, for the first time, that BmpreBG has a 160-bp 5'-untranslated region. These findings suggested that B. mori β-glucosidase precursor was involved in the death process of l-4i mutant larvae. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor.

PubMed

Jouvin, M H; Adamczewski, M; Numerof, R; Letourneur, O; Vallé, A; Kinet, J P

1994-02-25

Nonreceptor tyrosine kinases such as the newly described 70-kDa (ZAP-70/Syk) and Src-related tyrosine kinases are coupled to a variety of receptors, including the antigen receptors on B- and T-cells and the Fc receptors for IgE (Fc epsilon RI) and IgG (Fc gamma RI, Fc gamma RIII/CD16). Various subunits of these receptors contain homologous activation motifs which appear capable of autonomously triggering cell activation. Two forms of this motif are present in the Fc epsilon RI multimeric complex: one in the beta chain and one in the gamma chain. Here we show that each of the two tyrosine kinases known to be involved in Fc epsilon RI signaling is controlled by a distinct motif-containing chain. Lyn associates with the nonactivated beta chain, whereas gamma promotes the activation of Syk. We also show that neither the beta nor the gamma motif alone can account for the full signaling capacity of the entire receptor. We propose that, upon triggering of the tetrameric receptor, Lyn already bound to beta becomes activated and phosphorylates beta and gamma; the phosphorylation of gamma induces the association of Syk with gamma and also the activation of Syk, resulting in the phosphorylation and activation of phospholipase C gamma 1. Cooperative recruitment of specific kinases by the various signaling chains found in this family of antigen receptors could represent a way to achieve the full signaling capacity of the multimeric complexes.

Penicillin and beta-lactam allergy: epidemiology and diagnosis.

PubMed

Macy, Eric

2014-11-01

Penicillin is the most common beta-lactam antibiotic allergy and the most common drug class allergy, reported in about 8% of individuals using health care in the USA. Only about 1% of individuals using health care in the USA have a cephalosporin allergy noted in their medical record, and other specific non-penicillin, non-cephalosporin beta-lactam allergies are even rarer. Most reported penicillin allergy is not associated with clinically significant IgE-mediated reactions after penicillin rechallenge. Un-verified penicillin allergy is a significant and growing public health problem. Clinically significant IgE-mediated penicillin allergy can be safely confirmed or refuted using skin testing with penicilloyl-poly-lysine and native penicillin G and, if skin test is negative, an oral amoxicillin challenge. Acute tolerance of an oral therapeutic dose of a penicillin class antibiotic is the current gold standard test for a lack of clinically significant IgE-mediated penicillin allergy. Cephalosporins and other non-penicillin beta-lactams are widely, safely, and appropriately used in individuals, even with confirmed penicillin allergy. There is little, if any, clinically significant immunologic cross-reactivity between penicillins and other beta-lactams. Routine cephalosporin skin testing should be restricted to research settings. It is rarely needed clinically to safely manage patients and has unclear predictive value at this time. The use of alternative cephalosporins, with different side chains, is acceptable in the setting of a specific cephalosporin allergy. Carbapenems and monobactams are also safely used in individuals with confirmed penicillin allergy. A certain predictable, but low, rate of adverse reactions will occur with all beta-lactam antibiotic use both pre- and post-beta-lactam allergy evaluations.

Motion of spin label side chains in cellular retinol-binding protein: correlation with structure and nearest-neighbor interactions in an antiparallel beta-sheet.

PubMed

Lietzow, Michael A; Hubbell, Wayne L

2004-03-23

A goal in the development of site-directed spin labeling in proteins is to correlate the motion of a nitroxide side chain with local structure, interactions, and dynamics. Significant progress toward this goal has been made using alpha-helical proteins of known structure, and the present study is the first step in a similar exploration of a beta-sheet protein, cellular retinol-binding protein (CRBP). Nitroxide side chains were introduced along both interior and edge strands. At sites in interior strands, the side-chain motion is strongly influenced by interactions with side chains of neighboring strands, giving rise to a rich variety of dynamic modes (weakly ordered, strongly ordered, immobilized) and complex electron paramagnetic resonance spectra that are modulated by strand twist. The interactions giving rise to the dynamic modes are explored using mutagenesis, and the results demonstrate the particular importance of the non-hydrogen-bonded neighbor residue in giving rise to highly ordered states. Along edge strands of the beta-sheet, the motion of the side chain is simple and weakly ordered, resembling that at solvent-exposed surfaces of an alpha-helix. A simple working model is proposed that can account for the wide variety of dynamic modes encountered. Collectively, the results suggest that the nitroxide side chain is an effective probe of side-chain interactions, and that site-directed spin labeling should be a powerful means of monitoring conformational changes that involve changes in beta-sheet topology.

Analysis of common transforming growth factor beta-1 gene polymorphisms in gastric and duodenal ulcer disease: pilot study.

PubMed

Polonikov, Alexey V; Ivanov, Vladimir P; Belugin, Dmitry A; Khoroshaya, Irina V; Kolchanova, Inessa O; Solodilova, Mariya A; Tutochkina, Margarita P; Stepchenko, Alexander A

2007-04-01

Transforming growth factor-beta1 (TGF-beta1) has been shown to be an important cytokine that plays a role in cell proliferation, differentiation, tissue injury repair and ulcer healing. The purpose of this pilot study was to investigate if common polymorphisms Leu10Pro, Arg25Pro and C-509T within the TGF-beta1 gene are associated with susceptibility to gastric and duodenal ulcer disease in Russians. Blood samples from 377 unrelated patients with gastric and duodenal ulcer disease and 226 sex- and age-matched healthy controls were used to determine TGF-beta1 gene polymorphisms by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Leu10Pro substitution in the signal peptide of TGF-beta1 has been found to be associated with susceptibility to gastric ulcer (odds ratio [OR] 1.76, 95% confidence interval [CI] 1.12-2.77). A genotype combination of 10Leu/Leu x 25Arg/Arg x -509C/C was also associated with susceptibility to gastric ulcer disease (OR 1.81, P = 0.01). In addition, the frequency of a combination of genotypes 10Pro/Pro x 25Arg/Pro x -509C/T was statistically lower in patients with duodenal ulcer than in controls (OR 0.42, P = 0.05). A significant difference (P = 0.04) in the distribution of rare haplotypes of the TGF-beta1 gene between patients with duodenal ulcer and healthy controls has been found. Polymorphism Leu10Pro was in positive linkage disequilibrium with C-509T polymorphism (coefficient D = 0.191; P < 0.0001). These findings indicate that the Leu10Pro and C-509T polymorphisms may be involved in the modulation of expression of the TGF-beta1 gene, and therefore a predisposition to peptic ulcer disease could be linked to particular alleles of this gene. In particular, a possible role of TGF-beta1 in the pathogenesis of gastric ulcer disease is discussed.

Correlation of beta-catenin localization with cyclooxygenase-2 expression and CpG island methylator phenotype (CIMP) in colorectal cancer.

PubMed

Kawasaki, Takako; Nosho, Katsuhiko; Ohnishi, Mutsuko; Suemoto, Yuko; Kirkner, Gregory J; Dehari, Reiko; Meyerhardt, Jeffrey A; Fuchs, Charles S; Ogino, Shuji

2007-07-01

The WNT/beta-catenin (CTNNB1) pathway is commonly activated in the carcinogenic process. Cross-talks between the WNT and cyclooxygenase-2 (COX-2 or PTGS2)/prostaglandin pathways have been suggested. The relationship between beta-catenin activation and microsatellite instability (MSI) in colorectal cancer has been controversial. The CpG island methylator phenotype (CIMP or CIMP-high) with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer, which is associated with MSI-high. However, no study has examined the relationship between beta-catenin activation and CIMP status. Using 832 population-based colorectal cancer specimens, we assessed beta-catenin localization by immunohistochemistry. We quantified DNA methylation in eight CIMP-specific promoters [CACNA1G, CDKN2A(p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1] by real-time polymerase chain reaction (MethyLight). MSI-high, CIMP-high, and BRAF mutation were associated inversely with cytoplasmic and nuclear beta-catenin expressions (i.e., beta-catenin activation) and associated positively with membrane expression. The inverse relation between beta-catenin activation and CIMP was independent of MSI. COX-2 overexpression correlated with cytoplasmic beta-catenin expression (even after tumors were stratified by CIMP status), but did not correlate significantly with nuclear or membrane expression. In conclusion, beta-catenin activation is inversely associated with CIMP-high independent of MSI status. Cytoplasmic beta-catenin is associated with COX-2 overexpression, supporting the role of cytoplasmic beta-catenin in stabilizing PTGS2 (COX-2) mRNA.

Pattern of cytokine receptors expressed by human dendritic cells migrated from dermal explants.

PubMed Central

Larregina, A T; Morelli, A E; Kolkowski, E; Sanjuan, N; Barboza, M E; Fainboim, L

1997-01-01

Different reasons account for the lack of information about the expression of cytokine receptors on human dendritic cells (DC): (a) DC are a trace population; (b) the proteolytic treatment used to isolate DC may alter enzyme-sensitive epitopes; and (c) low numbers of receptors per cell. In the present work the expression of cytokine receptors was analysed by flow cytometry on the population of dermal DC (DDC) that spontaneously migrate from short-term culture dermal explants. DDC obtained after dermal culture were CD1alow, CD1b+, CD1c+, human leucocyte antigen (HLA)-DR+, CD11chigh, CD11b+ and CD32+. The DC lineage was confirmed by ultrastructural analysis. DDC expressed interleukin (IL)-1R type 1 (monoclonal antibody (mAb) hIL-1R1-M1; and 6B5); IL-1R type 2 (mAb hIL-1R2-M22); IL-2R alpha chain (mAb anti-Tac; and hIL-2R-M1) and IL-2R gamma chain (mAb 3B5; and AG14C). DDC did not stain for IL-2R beta chain using four mAbs recognizing two different epitopes of IL-2R beta (mAb 2R-B; Mik-beta 1; and CF1; Mik-beta 3, respectively). DDC were also positive for the cytokine binding chains (alpha chains) of IL-3R (mAb 9F5); IL-4R (mAb hIL-4R-M57; and S456C9); and IL-7R (mAb hIL-7R-M20; and R3434). DDC showed low levels of IL-6R alpha chain (mAb B-F19; B-R6; and B-E23) and its signal transducer gp130 (mAb A2; and B1). DDC strongly expressed interferon-gamma receptor (IFN-gamma R) (mAb GIR-208) and were negative for IL-8R (mAb B-G20; and B-F25). All DDC were highly positive for granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) alpha chain (mAb hGM-CSFR-M1; SC06; SC04, and 8G6) and to a lesser extent for the common beta chain of GM-CSFR, IL-3R and IL-5R (mAb 3D7). On the other hand, reactivity was not found for granulocyte colony-stimulating factor receptor (G-CSFR) (mAb hGCSFR-M1) nor macrophage colony-stimulating factor receptor (M-CSFR) (mAb 7-7A3-17) confirming the DC lineage of DDC. As previously reported for lymphoid DC, DDC expressed tumour necrosis factor receptort (TNFR) 75000 MW (mAb utr-1; hTNFR-M1; and MR2-1) but lacked TNFR 55000 MW (mAb htr-9; MR1-1; and MR1-2). In summary, DDC express receptors for a broad panel of cytokines, even receptors for cytokines whose effects on DC are still unknown (i.e. IL-2R alpha gamma; IL-6R alpha/gp 130; IL-7R alpha gamma). Images Figure 1 PMID:9227332

Light-induced yellowing of selectively 13C-enriched dehydrogenation polymers (DHPs). Part 1, Side-chain 13C-enriched DHP ([alpha], [beta], and [gamma]-13C)

Treesearch

Jim Parkas; Magnus Paulsson; Terashima Noritsugu; Ulla Westermark; Sally Ralph

2004-01-01

Light-induced yellowing has been studied using side-chain ([alpha], [beta], and [gamma]) 13C-enriched DHP (dehydrogenation polymer) and quantitative solution state 13C NMR spectroscopy. The DHP was formed from 13C-enriched coniferin using an enzymatic system consisting of [beta]-glucosidase, glucose oxidase, and peroxidase in a pH 6 buffer solution. The DHP was applied...

Increased myosin heavy chain-beta with atrial expression of ventricular light chain-2 in canine cardiomyopathy.

PubMed

Fuller, Geraldine A; Bicer, Sabahattin; Hamlin, Robert L; Yamaguchi, Mamoru; Reiser, Peter J

2007-10-01

Dilated cardiomyopathy is a naturally occurring disease in humans and dogs. Human studies have shown increased levels of myosin heavy chain (MHC)-beta in failing ventricles and the left atria (LA) and of ventricular light chain (VLC)-2 in the right atria in dilated cardiomyopathy. This study evaluates the levels of MHC-beta in all heart chambers in prolonged canine right ventricular pacing. In addition, we determined whether levels of VLC2 were altered in these hearts. Failing hearts demonstrated significantly increased levels of MHC-beta in the right atria, right atrial appendage, LA, left atrial appendage (LAA), and right ventricle compared with controls. Significant levels of VLC2 were detected in the right atria of paced hearts. Differences in MHC-beta expression were observed between the LA and the LAA of paced and control dogs. MHC-beta expression was significantly greater in the LA of paced and control dogs compared with their respective LAA. The cardiac myosin isoform shifts in this study were similar to those observed in end-stage human heart failure and more severe than those reported in less prolonged pacing models, supporting the use of this model for further study of end-stage human heart failure. The observation of consistent differences between sampling sites, especially LA versus LAA, indicates the need for rigorous sampling consistency in future studies.

Use of beta-methylphenylalanine (beta MeF) residues to probe the nature of the interaction of substance P with its receptor: effects of beta MeF-containing substance P analogs on rabbit iris smooth muscle contraction.

PubMed

Birney, D M; Cole, D C; Crosson, C E; Kahl, B F; Neff, B W; Reid, T W; Ren, K; Walkup, R D

1995-06-23

The effects of substituting (2S,3S)-beta-methylphenylalanine (S-beta MeF) or (2S,3R)-beta-methylphenylalanine (R-beta MeF) for the Phe7 and/or Phe8 residues of the tachykinin substance P (SP, RPKPQQFFGLM-NH2) upon the ability of SP to stimulate contraction of the rabbit iris smooth muscle were investigated. The eight beta MeF-containing SP analogs (four monosubstituted analogs, four disubstituted analogs) 1-8 were synthesized and found to be agonsts of SP in the smooth muscle contraction assay, having EC50 values ranging from 0.15 to 10.0 nM. Three analogs are significantly more active than SP [8R-(beta MeF)SP (4), 7S,8S-(beta MeF)2SP (5), and 7R,8S-(beta MeF)2SP (6)], three analogs are approximately equipotent with SP [7S-(beta MeF)SP (1), 7R-(beta MeF)SP (2), and 7S,8R-(beta MeF)2SP (8)], and two analogs are significantly less active than SP [8S-(beta MeF)SP (3) and 7R,8R-(beta MeF)2SP (7)]. The effects of the beta MeF substitutions upon the activity of SP are not additive and cannot be explained using simple conformational models which focus only on the side chain conformations of the beta MeF residues. It is postulated that the beta MeF residues induce minor distortions in the peptide backbone with resultant consequences upon peptide-receptor binding which are not dictated soley by the side chain conformations. This idea is consistent with 1H-NMR data for the monosubstituted analogs 1-4, which imply that the beta MeF substitutions cause slight distortions in the peptide backbone and that the beta MeF side chains are assuming trans or gauche(-) conformations.

A novel mutation of the beta myosin heavy chain gene responsible for familial hypertrophic cardiomyopathy.

PubMed

Wang, Juan; Xu, Shi-Jie; Zhou, Hua; Wang, Li-Jie; Hu, Bo; Fang, Fang; Zhang, Xu-Min; Luo, Yi-Wei; He, Xiao-Yan; Zhuang, Shao-Wei; Li, Xin-Ming; Liu, Zhong-Ming; Hu, Da-Yi

2009-09-01

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disorder and shows high variability in genetic heterogeneity and phenotypic characteristics. The genetic etiology responsible for HCM in many individuals remains unclear. This instigation was sought to identify novel genetic determinants for familial hypertrophic cardiomyopathy. Six unrelated Chinese families with HCM were studied. For each of the 13 established HCM-susceptibility genes, 3 to 5 microsatellite markers were selected to perform genotyping and haplotype analysis. The linked genes were sequenced. Haplotype analyses on candidate genetic loci revealed cosegregation of the gene beta-myosin heavy chain (MYH7) with HCM in a single family. A novel double heterozygous missense mutation of Ala26Val plus Arg719Trp in MYH7 was subsequently identified by sequencing in this family and was associated with a severe phenotype of HCM. The novel double mutation of Ala26Val plus Arg719Trp in MYH7 identified in a Chinese family highlights the remarkable genetic heterogeneity of HCM, which provides important information for genetic counseling, accurate diagnosis, prognostic evaluation, and appropriate clinical management. Copyright 2009 Wiley Periodicals, Inc.

Three-dimensional crystal structure of recombinant murine interferon-beta.

PubMed Central

Senda, T; Shimazu, T; Matsuda, S; Kawano, G; Shimizu, H; Nakamura, K T; Mitsui, Y

1992-01-01

The crystal structure of recombinant murine interferon-beta (IFN-beta) has been solved by the multiple isomorphous replacement method and refined to an R-factor of 20.5% against 2.6 A X-ray diffraction data. The structure shows a variant of the alpha-helix bundle with a new chain-folding topology, which seems to represent a basic structural framework of all the IFN-alpha and IFN-beta molecules belonging to the type I family. Functionally important segments of the polypeptide chain, as implied through numerous gene manipulation studies carried out so far, are spatially clustered indicating the binding site(s) to the receptor(s). Comparison of the present structure with those of other alpha-helical cytokine proteins, including porcine growth hormone, interleukin 2 and interferon gamma, indicated either a topological similarity in chain folding or a similar spatial arrangement of the alpha-helices. Images PMID:1505514

Analysis of Novel Prostate Cancer Biomarkers and their Predictive Utility in an Active Surveillance Protocol

DTIC Science & Technology

2014-05-01

Shock Protein, Annexin A3, Sorbitol Dehydrogenase, Fibrinogen Beta Chain Precursor, Creatine Kinase B-Type, Annexin A1, Cystatin B, and AZI. We have...Shock Protein, Annexin A3, Sorbitol Dehydrogenase, Fibrinogen Beta Chain Precursor, and Creatine Kinase B-Type. After testing these candidates with

In silico study of full-length amyloid beta 1-42 tri- and penta-oligomers in solution.

PubMed

Masman, Marcelo F; Eisel, Ulrich L M; Csizmadia, Imre G; Penke, Botond; Enriz, Ricardo D; Marrink, Siewert Jan; Luiten, Paul G M

2009-08-27

Amyloid oligomers are considered to play causal roles in the pathogenesis of amyloid-related degenerative diseases including Alzheimer's disease. Using MD simulation techniques, we explored the contributions of the different structural elements of trimeric and pentameric full-length Abeta1-42 aggregates in solution to their stability and conformational dynamics. We found that our models are stable at a temperature of 310 K, and converge toward an interdigitated side-chain packing for intermolecular contacts within the two beta-sheet regions of the aggregates: beta1 (residues 18-26) and beta2 (residues 31-42). MD simulations reveal that the beta-strand twist is a characteristic element of Abeta-aggregates, permitting a compact, interdigitated packing of side chains from neighboring beta-sheets. The beta2 portion formed a tightly organized beta-helix, whereas the beta1 portion did not show such a firm structural organization, although it maintained its beta-sheet conformation. Our simulations indicate that the hydrophobic core comprising the beta2 portion of the aggregate is a crucial stabilizing element in the Abeta aggregation process. On the basis of these structure-stability findings, the beta2 portion emerges as an optimal target for further antiamyloid drug design.

Partial deletion of beta9 loop in pancreatic lipase-related protein 2 reduces enzyme activity with a larger effect on long acyl chain substrates.

PubMed

Dridi, Kaouthar; Amara, Sawsan; Bezzine, Sofiane; Rodriguez, Jorge A; Carrière, Frédéric; Gaussier, Hélène

2013-07-01

Structural studies on pancreatic lipase have revealed a complex architecture of surface loops surrounding the enzyme active site and potentially involved in interactions with lipids. Two of them, the lid and beta loop, expose a large hydrophobic surface and are considered as acyl chain binding sites based on their interaction with an alkyl phosphonate inhibitor. While the role of the lid in substrate recognition and selectivity has been extensively studied, the implication of beta9 loop in acyl chain stabilization remained hypothetical. The characterization of an enzyme with a natural deletion of the lid, guinea pig pancreatic lipase-related protein 2 (GPLRP2), suggests however an essential contribution of the beta9 loop in the stabilization of the acyl enzyme intermediate formed during the lipolysis reaction. A GPLRP2 mutant with a seven-residue deletion of beta9 loop (GPLRP2-deltabeta9) was produced and its enzyme activity was measured using various substrates (triglycerides, monoglycerides, galactolipids, phospholipids, vinyl esters) with short, medium and long acyl chains. Whatever the substrate tested, GPLRP2-deltabeta9 activity is drastically reduced compared to that of wild-type GPLRP2 and this effect is more pronounced as the length of substrate acyl chain increases. Changes in relative substrate selectivity and stereoselectivity remained however weak. The deletion within beta9 loop has also a negative effect on the rate of enzyme inhibition by alkyl phosphonates. All these findings indicate that the reduced enzyme turnover observed with GPLRP2-deltabeta9 results from a weaker stabilization of the acyl enzyme intermediate due to a loss of hydrophobic interactions.

INS-gene mutations: from genetics and beta cell biology to clinical disease.

PubMed

Liu, Ming; Sun, Jinhong; Cui, Jinqiu; Chen, Wei; Guo, Huan; Barbetti, Fabrizio; Arvan, Peter

2015-04-01

A growing list of insulin gene mutations causing a new form of monogenic diabetes has drawn increasing attention over the past seven years. The mutations have been identified in the untranslated regions of the insulin gene as well as the coding sequence of preproinsulin including within the signal peptide, insulin B-chain, C-peptide, insulin A-chain, and the proteolytic cleavage sites both for signal peptidase and the prohormone convertases. These mutations affect a variety of different steps of insulin biosynthesis in pancreatic beta cells. Importantly, although many of these mutations cause proinsulin misfolding with early onset autosomal dominant diabetes, some of the mutant alleles appear to engage different cellular and molecular mechanisms that underlie beta cell failure and diabetes. In this article, we review the most recent advances in the field and discuss challenges as well as potential strategies to prevent/delay the development and progression of autosomal dominant diabetes caused by INS-gene mutations. It is worth noting that although diabetes caused by INS gene mutations is rare, increasing evidence suggests that defects in the pathway of insulin biosynthesis may also be involved in the progression of more common types of diabetes. Collectively, the (pre)proinsulin mutants provide insightful molecular models to better understand the pathogenesis of all forms of diabetes in which preproinsulin processing defects, proinsulin misfolding, and ER stress are involved. Copyright © 2014 Elsevier Ltd. All rights reserved.

INS-gene mutations: From genetics and beta cell biology to clinical disease

PubMed Central

Liu, Ming; Sun, Jinhong; Cui, Jinqiu; Chen, Wei; Guo, Huan; Barbetti, Fabrizio; Arvan, Peter

2015-01-01

A growing list of insulin gene mutations causing a new form of monogenic diabetes has drawn increasing attention over the past seven years. The mutations have been identified in the untranslated regions of the insulin gene as well as the coding sequence of preproinsulin including within the signal peptide, insulin B-chain, C-peptide, insulin A-chain, and the proteolytic cleavage sites both for signal peptidase and the prohormone convertases. These mutations affect a variety of different steps of insulin biosynthesis in pancreatic beta cells. Importantly, although many of these mutations cause proinsulin misfolding with early onset autosomal dominant diabetes, some of the mutant alleles appear to engage different cellular and molecular mechanisms that underlie beta cell failure and diabetes. In this article, we review the most recent advances in the field and discuss challenges as well as potential strategies to prevent/delay the development and progression of autosomal dominant diabetes caused by INS-gene mutations. It is worth noting that although diabetes caused by INS gene mutations is rare, increasing evidence suggests that defects in the pathway of insulin biosynthesis may also be involved in the progression of more common types of diabetes. Collectively, the (pre)proinsulin mutants provide insightful molecular models to better understand the pathogenesis of all forms of diabetes in which preproinsulin processing defects, proinsulin misfolding, and ER stress are involved. PMID:25542748

Hepatic beta-oxidation and carnitine palmitoyltransferase I in neonatal pigs after dietary treatments of clofibric acid, isoproterenol, and medium-chain triglycerides.

PubMed

Peffer, Pasha Lyvers; Lin, Xi; Odle, Jack

2005-06-01

A suckling piglet model was used to study nutritional and pharmacologic means of stimulating hepatic fatty acid beta-oxidation. Newborn pigs were fed milk diets containing either long- or medium-chain triglycerides (LCT or MCT). The long-chain control diet was supplemented further with clofibric acid (0.5%) or isoproterenol (40 ppm), and growth was monitored for 10-12 days. Clofibrate increased rates of hepatic peroxisomal and mitochondrial beta-oxidation of [1-(14)C]-palmitate by 60 and 186%, respectively. Furthermore, malonyl-CoA sensitive carnitine palmitoyltransferase (CPT I) activity increased 64% (P < 0.05) in pigs receiving clofibrate. Increased CPT I activity was not congruent with changes in message, as elevated abundance of CPT I mRNA was not detected (P = 0.16) when assessed by qRT-PCR. Neither rates of beta-oxidation nor CPT activities were affected by dietary MCT or by isoproterenol treatment (P > 0.1). Collectively, these findings indicate that clofibrate effectively induced hepatic CPT activity concomitant with increased fatty acid beta-oxidation.

The conformational requirements for the mechanical precipitation of hemoglobin S and other mutants.

PubMed

Roth, E F; Elbaum, D; Bookchin, R M; Nagel, R L

1976-08-01

The mechanical stability of human hemoglobin mutants was studied for the specific effects of single and double amino acid substitutions, the ligand state of each chain, and the effect of hybrids between oxy and cyanmet partners on precipitability. It was found that the beta6Glu leads to Val and the beta73 Asp leads to Asn mutations increased the degree of mechanical precipitation in the liganded but not in the deoxy form. When these mutations occurred on the same chain, the effects were approximately additive. Heat labile mutants such as Hb Gun Hill and Hb Leiden exhibited mechanical instability, but probably through a different mechanism, as very little dependence on ligand state was apparent. Studies with valency hybrids of HbS(alpha2 betas2-and-alpha2 betas2 where = cyanmet) revealed that instability was primarily determined by the state of the betas chain, which must be liganded to confer instability on the tetramer. A good agreement between surface activity and mechanical precipitability of these mutants has been found.

Cross-Reactivity among Beta-Lactams.

PubMed

Romano, Antonino; Gaeta, Francesco; Arribas Poves, Maria Francisca; Valluzzi, Rocco Luigi

2016-03-01

Penicillins and cephalosporins are the major classes of beta-lactam (BL) antibiotics in use today and one of the most frequent causes of hypersensitivity reactions to drugs. Monobactams, carbapenems, oxacephems, and beta-lactamase inhibitors constitute the four minor classes of BLs. This review takes into account mainly the prospective studies which evaluated cross-reactivity among BLs in subjects with a well-demonstrated hypersensitivity to a certain class of BLs by performing allergy tests with alternative BLs and, in case of negative results, administering them. In subjects with either IgE-mediated or T-cell-mediated hypersensitivity, cross-reactivity among BLs, particularly among penicillins and among cephalosporins, as well as between penicillins and cephalosporins, seems to be mainly related to structural similarities among their side-chain determinants. Specifically, in penicillin-allergic subjects, cross-reactivity between penicillins and cephalosporins may exceed 30% when they are administered cephalosporins with identical side chains to those of responsible penicillins. In these subjects, a few prospective studies have demonstrated a rate of cross-reactivity between penicillins and both carbapenems and aztreonam lower than 1%. With regard to subjects with an IgE-mediated hypersensitivity to cephalosporins, in a single study, about 25% of the 98 subjects with such hypersensitivity had positive results to penicillins, 3% to aztreonam, 2% to imipenem/cilastatin, and 1% to meropenem. The cross-reactivity related to the selective recognition of the BL ring by IgE or T lymphocytes, which entails positive responses to all BLs tested, appears to be exceptional. Some studies concerning cross-reactivity among BLs have found patterns of allergy-test positivity which cannot be explained by either the common BL ring or by similar or identical side chains, thus indicating the possibility of coexisting sensitivities to different BLs because of prior exposures to them.

Mechanistic insights into phosphoprotein-binding FHA domains.

PubMed

Liang, Xiangyang; Van Doren, Steven R

2008-08-01

[Structure: see text]. FHA domains are protein modules that switch signals in diverse biological pathways by monitoring the phosphorylation of threonine residues of target proteins. As part of the effort to gain insight into cellular avoidance of cancer, FHA domains involved in the cellular response to DNA damage have been especially well-characterized. The complete protein where the FHA domain resides and the interaction partners determine the nature of the signaling. Thus, a key biochemical question is how do FHA domains pick out their partners from among thousands of alternatives in the cell? This Account discusses the structure, affinity, and specificity of FHA domains and the formation of their functional structure. Although FHA domains share sequence identity at only five loop residues, they all fold into a beta-sandwich of two beta-sheets. The conserved arginine and serine of the recognition loops recognize the phosphorylation of the threonine targeted. Side chains emanating from loops that join beta-strand 4 with 5, 6 with 7, or 10 with 11 make specific contacts with amino acids of the ligand that tailor sequence preferences. Many FHA domains choose a partner in extended conformation, somewhat according to the residue three after the phosphothreonine in sequence (pT + 3 position). One group of FHA domains chooses a short carboxylate-containing side chain at pT + 3. Another group chooses a long, branched aliphatic side chain. A third group prefers other hydrophobic or uncharged polar side chains at pT + 3. However, another FHA domain instead chooses on the basis of pT - 2, pT - 3, and pT + 1 positions. An FHA domain from a marker of human cancer instead chooses a much longer protein fragment that adds a beta-strand to its beta-sheet and that presents hydrophobic residues from a novel helix to the usual recognition surface. This novel recognition site and more remote sites for the binding of other types of protein partners were predicted for the entire family of FHA domains by a bioinformatics approach. The phosphopeptide-dependent dynamics of an FHA domain, SH2 domain, and PTB domain suggest a common theme: rigid, preformed binding surfaces support van der Waals contacts that provide favorable binding enthalpy. Despite the lack of pronounced conformational changes in FHA domains linked to binding events, more subtle adjustments may be possible. In the one FHA domain tested, phosphothreonine peptide binding is accompanied by increased flexibility just outside the binding site and increased rigidity across the beta-sandwich. The folding of the same FHA domain progresses through near-native intermediates that stabilize the recognition loops in the center of the phosphoprotein-binding surface; this may promote rigidity in the interface and affinity for targets phosphorylated on threonine.

Allele related mutation specific-polymerase chain reaction for rapid diagnosis of Hb New York (beta 113 (G15) Val-->Glu, beta(CD113 GTG-->GAG)).

PubMed

Viprakasit, Vip; Tachavanich, Kalaya; Suwantol, Lerlugsn; Pung-Amritt, Parichat; Chinchang, Worawut; Tanphaichitr, Voravarn S

2002-08-01

Hemoglobin New York (beta 113 (G15) Val-->Glu), a beta-globin variant, was first reported in a Chinese family living in New York. Subsequently, this abnormal hemoglobin was reported in many Chinese descendants from several groups and it was also known as Hb Kaohsiung. The subtle change in alpha1beta1 contact region apart from the heme group connecting area by Val-->Glu substitution has minor changes in both the electrophoretic mobility and stability making this hemoglobin variant difficult to distinguish from Hb A using routine hemoglobin analysis. The authors described a case of heterozygosity of Hb New York diagnosed by a molecular technique and revealed a mutation in beta(CD113 GTG-->GAG). A novel Allele Related Mutation Specific-Polymerase Chain Reaction (ARMS-PCR) for rapid diagnosis of this mutation has been proposed.

Microvascular pericytes express platelet-derived growth factor-beta receptors in human healing wounds and colorectal adenocarcinoma.

PubMed Central

Sundberg, C.; Ljungström, M.; Lindmark, G.; Gerdin, B.; Rubin, K.

1993-01-01

The expression of platelet-derived growth factor- beta (PDGF-beta) receptors in the microvasculature of human healing wounds and colorectal adenocarcinoma was investigated. Frozen sections were subjected to double immunofluorescence staining using monoclonal antibodies (MAbs) specific for pericytes (MAb 225.28 recognizing the high-molecular weight-melanoma-associated antigen, expressed by activated pericytes during angiogenesis), endothelial cells (MAb PAL-E), laminin, as well as PDGF-beta receptors (MAb PDGFR-B2) and its ligand PDGF-B chain (MAb PDGF 007). Stained sections were analyzed by computer-aided imaging processing that allowed for a numerical quantification of the degree of colocalization of the investigated antigens. An apparent background colocalization, varying between 23 and 35%, between markers for cells not expected to co-localize was recorded. This background could be due to limitations of camera resolution, to out-of-focus fluorescence, and to interdigitations of the investigated structures. In all six tumor specimens, co-localization of PDGF-beta receptors and PAL-E was not different from the background co-localization, whereas that of PDGF-beta receptors and high-molecular weight-melanoma-associated antigen was significantly higher with mean values between 57 and 71%. Qualitatively, the same pattern was obtained in the two investigated healing wounds. PDGF-B chain did not co-localize with either PAL-E or high-molecular weight-melanoma-associated antigen, but PDGF-B chain-expressing cells were, however, frequently found juxtaposed to the microvasculature. The expression of PDGF-beta receptors on pericytes in activated microvessels and the presence of PDGF-B chain-expressing cells in close proximity to the microvasculature of healing wounds and colorectal adenocarcinoma is compatible with a role for PDGF in the physiology of the microvasculature in these conditions. Images Figure 1 p1381-a Figure 3 Figure 4 PMID:8238254

Hb L'Aquila [beta106(G8)Leu-->Val, CTG-->GTG]: a novel thalassemic hemoglobin variant.

PubMed

Amato, Antonio; Cappabianca, Maria Pia; Ponzini, Donatella; Rinaldi, Silvana; Biagio, Paola Di; Foglietta, Enrica; Grisanti, Paola; Mastropietro, Fabrizio

2007-01-01

A new beta-globin variant at codon 106 (CTG-->GTG), and which we named Hb L'Aquila [beta106(G8)Leu-->Val], was detected by DNA analysis. The proband and her father presented with the features of a mild beta(+)-thalassemia (thal), confirmed by their alpha/beta-globin chain biosynthesis ratios.

Molecular cloning and characterization of adult Sparus aurata hemoglobin genes.

PubMed

Campo, Salvatore; Nastasi, Giancarlo; Fedeli, Donatella; D'Ascola, Angela; Campo, Giuseppe M; Avenoso, Angela; Ferlazzo, Alida; Calatroni, Alberto; Falcioni, Giancarlo

2010-04-01

Among Teleosts, Sparus aurata occupies a prominent place in the gastronomic and economic fields of the Mediterranean basin and other geographic districts. The knowledge of its molecular structures and functional features, such as hemoglobin, may be helpful to understand the adaptive biochemical mechanisms that allow this fish to live under extreme conditions, including fish farming. In Sparus aurata red blood cells two different alpha and one beta hemoglobin genes have been identified. The alpha1 gene codifies a putative protein of 144 amino acids, the alpha2 gene produces a protein of 143 amino acids, and the beta gene encodes a chain of 148 amino acids. Comparative analysis of various hemoglobins indicates that allosteric regulation can be modified by the substitution of one or a few key residues. The comparison of the percentage sequence differences for alpha and beta chains in fishes indicates that evolutionary relationships between different species may be helpful to understand the mechanisms of their differentiation from other vertebrates. Hemoglobin alpha and beta chains of about 50 teleostean temperate and Antarctic fishes were analyzed to build phylogenetic trees using different algorithms: the neighbor-joining method, the maximum likelihood approach, and the Bayesian inference computation. Sparus aurata alpha chains are positioned in a paraphyletic cluster, which includes the same subunit of Chrysophrys auratus and Seriola quinqueradiata, whereas the beta chain is on an homophyletic branch with that of Chrysophrys auratus. Therefore, the phylogenetic approach suggests that both Sparus aurata hemoglobin alpha genes are paralogues and may have derived from a duplication event.

Atomistic Model for the Polyamide Formation from β-Lactam Catalyzed by Candida Antarctica Lipase B

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baum, Iris; Elsasser, Brigitta M.; Schwab, Leendert

2011-04-01

Candida antarctica lipase B (CALB) is an established biocatalyst for a variety of transesterification, amidation, and polymerization reactions. In contrast to polyesters, polyamides are not yet generally accessible via enzymatic polymerization. In this regard, an enzyme-catalyzed ring-opening polymerization of {beta}-lactam (2-azetidinone) using CALB is the first example of an enzymatic polyamide formation yielding unbranched poly({beta}-alanine), nylon 3. The performance of this polymerization, however, is poor, considering the maximum chain length of 18 monomer units with an average length of 8, and the molecular basis of the reaction so far is not understood. We have employed molecular modeling techniques using dockingmore » tools, molecular dynamics, and QM/MM procedures to gain insight into the mechanistic details of the various reaction steps involved. As a result, we propose a catalytic cycle for the oligomerization of {beta}-lactam that rationalizes the activation of the monomer, the chain elongation by additional {beta}-lactam molecules, and the termination of the polymer chain. In addition, the processes leading to a premature chain termination are studied. Particularly, the QM/MM calculation enables an atomistic description of all eight steps involved in the catalytic cycle, which features an in situ-generated {beta}-alanine as the elongating monomer and which is compatible with the experimental findings.« less

Maple syrup urine disease: The E1{beta} gene of human branched-chain {alpha}-ketoacid dehydrogenase complex has 11 rather than 10 exons, and the 3{prime} UTR in one of the two E1{beta} mRNAs arises from intronic sequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chuang, J.L.; Chuang, D.T.; Cox, R.P.

1996-06-01

Maple syrup urine disease (MSUD) or branched-chain ketoaciduria is caused by a deficiency in the mitochondrial branched-chain {alpha}-ketoacid dehydrogenase (BCKAD) complex. The clinical manifestations are characterized by accumulation of branched chain amino and {alpha}-ketoacids, which leads to severe cerebral edema with seizures, ketoacidosis, and mental retardation. The BCKAD complex comprises three catalytic components, i.e., a decarboxylase (E1) consisting of two E1{alpha} (M{sub r} = 46,000) and two E1{Beta} (M{sub r} = 37,500) subunits, a transacylase (E2) that contains 24 lipoic acid-bearing subunits, and a dehydrogenase (E3), which is a homodimeric flavoprotein. MSUD is genetically heterogeneous, since mutations in the E1{alpha}more » subunit (type IA MSUD), the E1{Beta} subunit (type IB), the E2 subunit (type II) and the E3 subunit (type III) have been described. The functional consequences of certain mutations in the BCKAD complex have been studied. 23 refs., 3 figs.« less

Flomoxef, a new oxacephem antibiotic, does not cause hemostatic defects.

PubMed

Cazzola, M; Brancaccio, V; De Giglio, C; Paternò, E; Matera, M G; Rossi, F

1993-03-01

Antibiotics of the beta-lactam class may cause coagulation defects and bleeding. It has been suggested that N-methyltetrazolethiol (NMTT), a common side chain group at the 3'-position of the cephem or 1-oxacephem frame, could be responsible for the hypoprothrombinemic effect of the antibiotics and that it could inhibit the liver vitamin K-epoxide reductase activity. Flomoxef (6315-S) is a new oxacephem antibiotic which differs from latamoxef because it has [1-(2-hydroxethyl)-1H-tetrazol-5-yl] thiomethyl (HTT) as a side chain at the 3'-position of cephem group instead of NMTT and an extensive modification of 7 beta-acylamino side chain. The present study was carried out to study its effects on vitamin K-dependent blood coagulation parameters in human volunteers. Ten adult patients (6 men and 4 women), suffering from chronic bronchitis, entered into the study. Each patient received ten 1 g i.m. injections of flomoxef at 12-hourly intervals. Apparently, the treatment with this oxacephem antibiotic had no significant effect. PT, PTT and fibrinogen remained in the normal range in all patients and factors II+VII+X, protein C, protein S and AT III were not depleted. The trend was similar both in men and women. Based on the results of the present study, we conclude that flomoxef is an antibiotic that does not exhibit an effect on blood coagulation, even in males.(ABSTRACT TRUNCATED AT 250 WORDS)

Three-dimensional structure of holo 3 alpha,20 beta-hydroxysteroid dehydrogenase: a member of a short-chain dehydrogenase family.

PubMed Central

Ghosh, D; Weeks, C M; Grochulski, P; Duax, W L; Erman, M; Rimsay, R L; Orr, J C

1991-01-01

The x-ray structure of a short-chain dehydrogenase, the bacterial holo 3 alpha,20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53), is described at 2.6 A resolution. This enzyme is active as a tetramer and crystallizes with four identical subunits in the asymmetric unit. It has the alpha/beta fold characteristic of the dinucleotide binding region. The fold of the rest of the subunit, the quaternary structure, and the nature of the cofactor-enzyme interactions are, however, significantly different from those observed in the long-chain dehydrogenases. The architecture of the postulated active site is consistent with the observed stereospecificity of the enzyme and the fact that the tetramer is the active form. There is only one cofactor and one substrate-binding site per subunit; the specificity for both 3 alpha- and 20 beta-ends of the steroid results from the binding of the steroid in two orientations near the same cofactor at the same catalytic site. Images PMID:1946424

Three mouse models of human thalassemia.

PubMed Central

Martinell, J; Whitney, J B; Popp, R A; Russell, L B; Anderson, W F

1981-01-01

Three types of mice with globin gene mutations, called 352HB, 27HB, and Hbath-J, appear to be true animal models of human thalassemia. Expression of the alpha-globin genes in three stocks of mice, each one heterozygous for one of the alpha-globin mutations, was examined at the polypeptide, RNA, and DNA levels. alpha-Globin polypeptide chains, relative to beta-globin chains in heterozygous thalassemic mice, are present at approximately 80% of normal. The ratios of alpha-globin to beta-globin RNA sequences are also 75-80% of normal, exactly reflecting the alpha-globin to beta-globin chain ratios. In the case of mutant 352HB, at least one alpha-globin gene is deleted. Thalassemic mouse erythroid cells appear to compensate partially for the loss of half of their alpha-globin genes. Images PMID:6946454

Use of cephalosporins in patients with immediate penicillin hypersensitivity: cross-reactivity revisited.

PubMed

Lee, Q U

2014-10-01

A 10% cross-reactivity rate is commonly cited between penicillins and cephalosporins. However, this figure originated from studies in the 1960s and 1970s which included first-generation cephalosporins with similar side-chains to penicillins. Cephalosporins were frequently contaminated by trace amount of penicillins at that time. The side-chain hypothesis for beta-lactam hypersensitivity is supported by abundant scientific evidence. Newer generations of cephalosporins possess side-chains that are dissimilar to those of penicillins, leading to low cross-reactivity. In the assessment of cross-reactivity between penicillins and cephalosporins, one has to take into account the background beta-lactam hypersensitivity, which occurs in up to 10% of patients. Cross-reactivity based on skin testing or in-vitro test occurs in up to 50% and 69% of cases, respectively. Clinical reactivity and drug challenge test suggest an average cross-reactivity rate of only 4.3%. For third- and fourth-generation cephalosporins, the rate is probably less than 1%. Recent international guidelines are in keeping with a low cross-reactivity rate. Despite that, the medical community in Hong Kong remains unnecessarily skeptical. Use of cephalosporins in patients with penicillin hypersensitivity begins with detailed history and physical examination. Clinicians can choose a cephalosporin with a different side-chain. Skin test for penicillin is not predictive of cephalosporin hypersensitivity, while cephalosporin skin test is not sensitive. Drug provocation test by experienced personnel remains the best way to exclude or confirm the diagnosis of drug hypersensitivity and to find a safe alternative for future use. A personalised approach to cross-reactivity is advocated.

Calorimetric and spectroscopic studies of the thermotropic phase behavior of lipid bilayer model membranes composed of a homologous series of linear saturated phosphatidylserines.

PubMed Central

Lewis, R N; McElhaney, R N

2000-01-01

The thermotropic phase behavior of lipid bilayer model membranes composed of the even-numbered, N-saturated 1,2-diacyl phosphatidylserines was studied by differential scanning calorimetry and by Fourier-transform infrared and (31)P-nuclear magnetic resonance spectroscopy. At pH 7.0, 0.1 M NaCl and in the absence of divalent cations, aqueous dispersions of these lipids, which have not been incubated at low temperature, exhibit a single calorimetrically detectable phase transition that is fully reversible, highly cooperative, and relatively energetic, and the transition temperatures and enthalpies increase progressively with increases in hydrocarbon chain length. Our spectroscopic observations confirm that this thermal event is a lamellar gel (L(beta))-to-lamellar liquid crystalline (L(alpha)) phase transition. However, after low temperature incubation, the L(beta)/L(alpha) phase transition of dilauroyl phosphatidylserine is replaced by a higher temperature, more enthalpic, and less cooperative phase transition, and an additional lower temperature, less enthalpic, and less cooperative phase transition appears in the longer chain phosphatidylserines. Our spectroscopic results indicate that this change in thermotropic phase behavior when incubated at low temperatures results from the conversion of the L(beta) phase to a highly ordered lamellar crystalline (L(c)) phase. Upon heating, the L(c) phase of dilauroyl phosphatidylserine converts directly to the L(alpha) phase at a temperature slightly higher than that of its original L(beta)/L(alpha) phase transition. Calorimetrically, this process is manifested by a less cooperative but considerably more energetic, higher-temperature phase transition, which replaces the weaker L(beta)/L(alpha) phase transition alluded to above. However, with the longer chain compounds, the L(c) phase first converts to the L(beta) phase at temperatures some 10-25 degrees C below that at which the L(beta) phase converts to the L(alpha) phase. Our results also suggest that shorter chain homologues form L(c) phases that are structurally related to, but more ordered than, those formed by the longer chain homologues, but that these L(c) phases are less ordered than those formed by other phospholipids. These studies also suggest that polar/apolar interfaces of the phosphatidylserine bilayers are more hydrated than those of other glycerolipid bilayers, possibly because of interactions between the polar headgroup and carbonyl groups of the fatty acyl chains. PMID:11023908

Microbial degradation of [C14C]polystyrene and 1,3-diphenylbutane.

PubMed

Sielicki, M; Focht, D D; Martin, J P

1978-07-01

Microbial degradation of [beta-14C]polystyrene and 1,3-diphenylbutane, a compound structurally representing the smallest repeating unit of styrene (dimer), was investigated in soil and liquid enrichment cultures. Degradation rates in soil, as determined by 14CO2 evolution from applied [14C]polystyrene, varied from 1.5 to 3.0% for a 4-month period. Although relatively low, these percentages were 15 to 30 times greater than values previously reported. Enrichment cultures, containing 1,3-diphenylbutane as the only carbon souce, were used to determine the mechanisms of microbial oxidation of the polymer chain ends. Metabolism of 1,3-diphenylbutane appeared to involve the attack by a monooxygenease to form 2-phenyl-4-hydroxyphenylbutane followed by a further oxidation and subsequent fission of the benzene ring to yield 4-phenylvaleric acid and an unidentified 5-carbon fragment via the classic meta-fission pathway. Phenylacetic acid was probably formed from 4-phenylvaleric acid by subsequent beta-oxidation of the side chain, methyl-oxidation and decarboxylation. An initial examination of the population of microorganisms in the diphenylbutane enrichment cultures indicated that these oxidative reactions are carried out by common soil microorganism of the genera Bacillus, Pseudomonas, Micrococcus, and Nocardia.

Characterization of T cell repertoire changes in acute Kawasaki disease

PubMed Central

1993-01-01

Kawasaki disease (KD) is an acute multisystem vasculitis of unknown etiology that is associated with marked activation of T cells and monocyte/macrophages. Using a quantitative polymerase chain reaction (PCR) technique, we recently found that the acute phase of KD is associated with the expansion of T cells expressing the V beta 2 and V beta 8.1 gene segments. In the present work, we used a newly developed anti-V beta 2 monoclonal antibody (mAb) and studied a new group of KD patients to extend our previous PCR results. Immunofluorescence analysis confirmed that V beta 2-bearing T cells are selectively increased in patients with acute KD. The increase occurred primarily in the CD4 T cell subset. The percentages of V beta 2+ T cells as determined by mAb reactivity and flow cytometry correlated linearly with V beta expression as quantitated by PCR. However, T cells from acute KD patients appeared to express proportionately higher levels of V beta 2 transcripts per cell as compared with healthy controls or convalescent KD patients. Sequence analysis of T cell receptor beta chain genes of V beta 2 and V beta 8.1 expressing T cells from acute KD patients showed extensive junctional region diversity. These data showing polyclonal expansion of V beta 2+ and V beta 8+ T cells in acute KD provide additional insight into the immunopathogenesis of this disease. PMID:8094737

Molecular analysis of the beta-thalassemia phenotype associated with inheritance of hemoglobin E (alpha 2 beta2(26)Glu leads to Lys).

PubMed Central

Benz, E J; Berman, B W; Tonkonow, B L; Coupal, E; Coates, T; Boxer, L A; Altman, A; Adams, J G

1981-01-01

Inheritance of the gene for betaE-globin is associated with hypochromia and microcytosis, reminiscent of typical heterozygous beta-thalassemia. Patients with hemoglobin (Hb)E-beta-thalassemia exhibit clinical phenotypes of severe beta-thalassemia, a circumstance not encountered in other compound heterozygous states for structural beta-chain mutations and beta-thalassemia. We have analyzed the kinetics of globin synthesis and the levels of globin messenger (m) RNA accumulation in patients with Hb E-beta-thalassemia and Hb E trait. The initial rate of beta-globin synthesis (betaE/alpha=0.20-0.34) was less than expected on the basis of gene dosage, or comparable studies of other compound heterozygous states for beta-thalassemia and structurally abnormal beta-chains. betaE-globin synthesis was not only reduced during short-term incubations (1-5 min), but also remained relatively unchanged during long-term pulse or chase incubations up to 5h. Analysis of globin mRNA by cell-free translation and molecular hybridization confirmed that the unexpectedly low levels of betaE-globin synthesis were associated with comparable reduction in the levels of beta-globin mRNA. In Hb E-beta-thalassemia the betaA + betaE (alpha globin nRNA ratio observed were substantially lower than those obtained from reticulocytes of patients with heterozygous beta-thalassemia, or Hb S-betaO-thalassemia, while in Hb E trait, the betaA + betaE/alpha mRNA ratio was in the ranged observed for beta-thalassemia trait. The betaE-globin gene specifies reduced accumulation of betaE-globin mRNA, a property characteristic of other forms of beta-thalassemia. The beta-thalassemia phenotype associated with inheritance of Hb E is thus determined at the level of beta-globin mRNA metabolism. PMID:6166632

DOE Office of Scientific and Technical Information (OSTI.GOV)

Popp, R.A.; Enlow, M.K.

The clinical hematologic change in 2 groups of progeny from mice carrying radiation-induced strain SEC ..cap alpha..-chain deficiencies was found to be similar to the hematologic alterations in persons with ..cap alpha..-thalassemia. The heterozygous deletion or inactivation of the ..cap alpha..-chain gene in mice caused an anemia similar to ..cap alpha..-thalassemina minor in persons. The ..cap alpha..-chain deficiency in mice created an erythrocytosis, reticulocytosis, and microcytic, hypochromic anemia comparable with the changes in human ..cap alpha..-thalassemia minor resulting from deletion of the ..cap alpha..-chain gene. These mouse mutants are the only known animal models of human thalassemia. A comparison ofmore » hematologic values obtained from progeny possessing an ..cap alpha..-chain gene deficiency and from progeny possessing a ..beta..-chain duplication suggested that the deficiency of ..cap alpha..-chain synthesis, rather than a simple imbalance between the amounts of ..cap alpha..- and ..beta..-chains produced, was primarily responsible for the altered hematologic characteristics in these ..cap alpha..-thalassemic mice.« less

Unprecedented {sup 1}/{sub {infinity}}[{beta}-Mo{sub 8}O{sub 26}]{sup 4-} polymeric chains and four novel organic-inorganic hybrids based on Mo-POMs and azaheterocycles templates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du Haijuan; Zunzhe Shu; Niu Yunyin, E-mail: niuyy@zzu.edu.cn

2012-06-15

Abstrct: Four novel organic-inorganic hybrid materials based on Mo-POMs and organic templates, namely [DEB] [{beta}-Mo{sub 8}O{sub 26}] [NH{sub 4}]{sub 2} (1), [BMIM] [{beta}-Mo{sub 8}O{sub 26}]{sub 0.5}{center_dot}H{sub 2}O (2), [BMIM] [1D-Mo{sub 8}O{sub 26}]{sub 0.5} (3) and {l_brace}3D-[Cu(DIE){sub 2}] [1D-Mo{sub 8}O{sub 26}]{sub 0.5}{r_brace}{sub {infinity}} (4) [DEB= 1,1 Prime -diethyl-4,4 Prime -bipyridinium, BMIM=1,1 Prime -bis(1-methylimidazolium)methylene, DIE=1,2-diimidazoloethane] have been hydrothermally synthesized and characterized by elemental analyses, IR spectroscopy, thermal gravimetric analysis(TGA) and single-crystal X-ray diffraction. Both compounds 1 and 2 are POMs-based supramolecular compounds consisted of independent [{beta}-Mo{sub 8}O{sub 26}]{sup 4-} anions and [DEB]{sup 2+} or [BMIM]{sup 2+} organic cations. Compound 3 is themore » first external template example of Mo-POMs-based supramolecular network incorporated with novel {sup 1}/{sub {infinity}}[{beta}-Mo{sub 8}O{sub 26}]{sup 4-} polymeric chains. Compound 4 is a rare supramolecular structure that contains octamolybdate {sup 1}/{sub {infinity}}[{beta}-Mo{sub 8}O{sub 26}]{sup 4-} polymeric chains interconnected via DIE ligands to form a 3D net. Moreover, it was indicated that these polyacid compounds had definite catalytic activities on the probe reaction of acetaldehyde oxidation to acetic acid with H{sub 2}O{sub 2}. - Graphical abstract: Four novel organic templated polyoxometalates comprising of 0D, 1D and 3D supramolecular frameworks together with the catalytic activities on the acetaldehyde oxidation to acetic acid were reported. Highlights: Using cation templated self-assembly four novel polyoxometalates were prepared. Compounds 1 and 2 consisted of independent [{beta}-Mo{sub 8}O{sub 26}]{sup 4-} anions and organic cations. Compound 3 is the first external template-assisted POMs with {sup 1}/{sub {infinity}}[{beta}-Mo{sub 8}O{sub 26}]{sup 4-} chain. Compound 4 is a rare 3D net containing {sup 1}/{sub {infinity}}[{beta}-Mo{sub 8}O{sub 26}]{sup 4-} 1D chain and DIE ligands. These compounds had definite catalytic activities on the acetaldehyde oxidation.« less

The three-dimensional structure of a T-cell antigen receptor V alpha V beta heterodimer reveals a novel arrangement of the V beta domain.

PubMed Central

Housset, D; Mazza, G; Grégoire, C; Piras, C; Malissen, B; Fontecilla-Camps, J C

1997-01-01

The crystal structure of a mouse T-cell antigen receptor (TCR) Fv fragment complexed to the Fab fragment of a specific anti-clonotypic antibody has been determined to 2.6 A resolution. The polypeptide backbone of the TCR V alpha domain is very similar to those of other crystallographically determined V alphas, whereas the V beta structure is so far unique among TCR V beta domains in that it displays a switch of the c" strand from the inner to the outer beta-sheet. The beta chain variable region of this TCR antigen-binding site is characterized by a rather elongated third complementarity-determining region (CDR3beta) that packs tightly against the CDR3 loop of the alpha chain, without leaving any intervening hydrophobic pocket. Thus, the conformation of the CDR loops with the highest potential diversity distinguishes the structure of this TCR antigen-binding site from those for which crystallographic data are available. On the basis of all these results, we infer that a significant conformational change of the CDR3beta loop found in our TCR is required for binding to its cognate peptide-MHC ligand. PMID:9250664

ICI 182,780-regulated gene expression in DU145 prostate cancer cells is mediated by estrogen receptor-beta/NFkappaB crosstalk.

PubMed

Leung, Yuet-Kin; Gao, Ying; Lau, Kin-Mang; Zhang, Xiang; Ho, Shuk-Mei

2006-04-01

Estrogen receptor (ER)-beta is the predominant ER subtype in prostate cancer (PCa). We previously demonstrated that ICI 182,780 (ICI), but not estrogens, exerted dose-dependent growth inhibition on DU145 PCa cells by an ER-beta-mediated pathway. Transcriptional profiling detected a greater than three-fold upregulation of seven genes after a 12-hour exposure to 1 microM ICI. Semiquantitative reverse transcriptase polymerase chain reaction confirmed the upregulation of four genes by ICI: interleukin-12alpha chain, interleukin-8, embryonic growth/differentiation factor, and RYK tyrosine kinase. Treatment with an ER-beta antisense oligonucleotide reduced cellular ER-beta mRNA and induced loss of expression of these genes. Sequence analysis revealed the presence of consensus NFkappaB sites, but not estrogen-responsive elements, in promoters of all four genes. Reporter assay and chromatin immunoprecipitation experiments demonstrated that ICI-induced gene expression could be mediated by crosstalk between ER-beta and the NFkappaB signaling pathway, denoting a novel mechanism of ER-beta-mediated ICI action. Therefore, combined therapies targeting ER-beta and NFkappaB signaling may be synergistic as treatment for PCa.

Myocardium expression of connexin 43, SERCA2a, and myosin heavy chain isoforms are preserved in nitrofen-induced congenital diaphragmatic hernia rat model.

PubMed

Baptista, Maria João; Recamán, Mónica; Melo-Rocha, Gustavo; Nogueira-Silva, Cristina; Roriz, José-Mário; Soares-Fernandes, João; Gonzaga, Silvia; Santos, Marta; Leite-Moreira, Adelino; Areias, José Carlos; Correia-Pinto, Jorge

2006-09-01

Previous morphological studies had produced controversial results with regard to heart development in congenital diaphragmatic hernia (CDH), whereas a few publications investigated cardiac function and myocardial maturation. Myocardium maturation is associated with age-dependent increasing of gene expression of gap junction protein connexin 43 (Cx43), adenosine triphosphatase of the sarcoplasmic reticulum (SERCA2a), as well as switching of myosin heavy chains (MHCs) from beta to alpha isoforms. Our aim was to evaluate myocardium maturity in nitrofen-induced CDH rat model. Fetuses from dated pregnant Sprague-Dawley rats were assigned to 3 experimental groups: control, nitrofen (exposed to nitrofen, without CDH), and CDH (exposed to nitrofen, with CDH). Myocardial samples collected from left ventricle free wall were processed to (i) quantification of messenger RNA (mRNA) of Cx43, SERCA2a, alpha and beta MHC isoforms, as well as beta-actin (housekeeping gene); and (ii) separation of MHC isoforms (alpha and beta isoforms) by sodium dodecyl sulfate polyacrylamide gel electrophoresis silver stained. We demonstrated that there is no difference in myocardial gene expression of Cx43 (control, 1.0 +/- 0.1; nitrofen, 1.1 +/- 0.2; CDH, 1.3 +/- 0.2) and SERCA2a (control, 1.0 +/- 0.1; nitrofen, 0.9 +/- 0.1; CDH, 1.0 +/- 0.2). Myocardial gene expressions of alpha and beta mRNA of MHC isoforms were slightly decreased both in nitrofen and CDH fetuses when compared with control fetuses, but evaluation of the alpha-to-beta ratios of MHC isoforms at protein level revealed no significant differences between CDH and control (control, 16.9 +/- 2.5; CDH, 17.0 +/- 2.0). Myocardial quantification of Cx43 and SERCA2a mRNA, as well as the expression pattern of MHC isoforms at protein levels, was similar in all studied groups. We predict, therefore, that acute heart failure commonly observed in CDH infants might be attributed predominantly to cardiac overload secondary to severe pulmonary hypertension rather than to myocardial immaturity.

Direct binding of F actin to the cytoplasmic domain of the alpha 2 integrin chain in vitro

NASA Technical Reports Server (NTRS)

Kieffer, J. D.; Plopper, G.; Ingber, D. E.; Hartwig, J. H.; Kupper, T. S.

1995-01-01

The transmembrane integrins have been shown to interact with the cytoskeleton via noncovalent binding between cytoplasmic domains (CDs) of integrin beta chains and various actin binding proteins within the focal adhesion complex. Direct or indirect integrin alpha chain CD binding to the actin cytoskeleton has not been reported. We show here that actin, as an abundant constituent of focal adhesion complex proteins isolated from fibroblasts, binds strongly and specifically to alpha 2 CD, but not to alpha 1 CD peptide. Similar specific binding to alpha 2 CD peptide was seen for highly purified F actin, free of putative actin-binding proteins. The bound complex of actin and peptide was visualized directly by coprecipitation, and actin binding was abrogated by removal of a five amino acid sequence from the alpha 2 CD peptide. Our findings may explain the earlier observation that, while integrins alpha 2 beta 1 and alpha 1 beta 1 both bind to collagen, only alpha 2 beta 1 can mediate contraction of extracellular collagen matrices.

Stabilization of non-productive conformations underpins rapid electron transfer to electron-transferring flavoprotein.

PubMed

Toogood, Helen S; van Thiel, Adam; Scrutton, Nigel S; Leys, David

2005-08-26

Crystal structures of protein complexes with electron-transferring flavoprotein (ETF) have revealed a dual protein-protein interface with one region serving as anchor while the ETF FAD domain samples available space within the complex. We show that mutation of the conserved Glu-165beta in human ETF leads to drastically modulated rates of interprotein electron transfer with both medium chain acyl-CoA dehydrogenase and dimethylglycine dehydrogenase. The crystal structure of free E165betaA ETF is essentially identical to that of wild-type ETF, but the crystal structure of the E165betaA ETF.medium chain acyl-CoA dehydrogenase complex reveals clear electron density for the FAD domain in a position optimal for fast interprotein electron transfer. Based on our observations, we present a dynamic multistate model for conformational sampling that for the wild-type ETF. medium chain acyl-CoA dehydrogenase complex involves random motion between three distinct positions for the ETF FAD domain. ETF Glu-165beta plays a key role in stabilizing positions incompatible with fast interprotein electron transfer, thus ensuring high rates of complex dissociation.

Proteomic investigation of protein profile changes and amino acid residue-level modification in cooked lamb longissimus thoracis et lumborum: The effect of roasting.

PubMed

Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

2016-09-01

Protein modifications of meat cooked by typical dry-heat methods (e.g., roasting) are currently not well understood. The present study utilised a shotgun proteomic approach to examine the molecular-level effect of roasting on thin lamb longissimus thoracis et lumborum patties, in terms of changes to both the protein profile and amino acid residue side-chain modifications. Cooking caused aggregation of actin, myosin heavy chains and sarcoplasmic proteins. Longer roasting time resulted in significantly reduced protein extractability as well as protein truncation involving particularly a number of myofibrillar and sarcoplasmic proteins, e.g., 6-phosphofructokinase, beta-enolase, l-lactate dehydrogenase A chain, alpha-actinin-3, actin and possibly myosin heavy chains. Modifications that have potential influence on nutritional properties, including carboxyethyllysine and a potentially glucose-derived N-terminal Amadori compound, were observed in actin and myoglobin after roasting. This study provided new insights into molecular changes resulting from the dry-heat treatment of meat, such as commonly used in food preparation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Sequence data - Magnitude and implications of some ambiguities.

NASA Technical Reports Server (NTRS)

Holmquist, R.; Jukes, T. H.

1972-01-01

A stochastic model is applied to the divergence of the horse-pig lineage from a common ansestor in terms of the alpha and beta chains of hemoglobin and fibrinopeptides. The results are compared with those based on the minimum mutation distance model of Fitch (1972). Buckwheat and cauliflower cytochrome c sequences are analyzed to demonstrate their ambiguities. A comparative analysis of evolutionary rates for various proteins of horses and pigs shows that errors of considerable magnitude are introduced by Glx and Asx ambiguities into evolutionary conclusions drawn from sequences of incompletely analyzed proteins.

Spatial and Time Coincidence Detection of the Decay Chain of Short-Lived Radioactive Nuclei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Granja, Carlos; Jakubek, Jan; Platkevic, Michal

The quantum counting position sensitive pixel detector Timepix with per-pixel energy and time resolution enables to detect radioactive ions and register the consecutive decay chain by simultaneous position-and time-correlation. This spatial and timing coincidence technique in the same sensor is demonstrated by the registration of the decay chain {sup 8}He{yields}{sup {beta} 8}Li and {sup 8}Li{yields}{sup {beta}-} {sup 8}Be{yields}{alpha}+{alpha} and by the measurement of the {beta} decay half-lives. Radioactive ions, selectively obtained from the Lohengrin fission fragment spectrometer installed at the High Flux Reactor of the ILL Grenoble, are delivered to the Timepix silicon sensor where decays of the implanted ionsmore » and daughter nuclei are registered and visualized. We measure decay lifetimes in the range {>=}{mu}s with precision limited just by counting statistics.« less

Lipid composition and chemotaxonomy of Pseudomonas putrefaciens (Alteromonas putrefaciens).

PubMed

Wilkinson, S G; Caudwell, P F

1980-06-01

The major polar lipids in cells of Pseudomonas putrefaciens NCIB 10472 grown on nutrient agar were phosphatidylethanolamine, phoisphatidylglycerol, a glucosyldiacylglycerol, a glucuronosyldiacylglycerol and an ornithine amide lipid. An additional phospholipid, tentatively identified as acyl phosphatidylglycerol or bis-phosphatidic acid, was a trace component of the wall lipids from broth cultures, which lacked the glycolipids and the ornithine amide lipid. The wall lipids from broth cultures of three further strains of P. putrefaciens (NCIB 10471, NCIB 11156 and NCTC 10737) contained all of the above lipids, and in two cases (strains NCIB 10471 and NCIB 11156) had an unusually high content of free fatty acid. Fatty acid compositions of the extractable lipids were qualitatively similar for all four strains: the major components were iso-pentadecanoic acid, pentadecanoic acid, a cis-heptadecenoic acid and a cis-hexadecenoic acid. Anteiso fatty acids were minor components in strain NCIB 10472. Lipid mixtures in which the ornithine amide lipid was present also contained small amounts of beta-hydroxy fatty acids: in strain NCIB 10472 the major ones were the straight-chain and iso-branched C16 acids. Lipopolysaccharides from all four strains had similar, complex fatty acid compositions. The major non-hydroxy acids were the straight-chain and iso-branched C13 acids. beta-Hydroxy acids common to all strains included the straight-chain C11, C12, C13, C14 and C15 acids, together with branched-chain C13 and C15 acids probably belonging to the iso series. The lipopolysaccharide from strains NCIB 10472 also contained C12 and C14 hydroxy acids of the same series, and small amounts of C13 and C15 beta-hydroxy acids probably belonging to the anteiso series. The close resemblance in both polar lipid and fatty acid compositions between strains of P. putrefaciens and Pseudomonas rubescens is further evidence that these species are synonymous. Significant differences between the lipids and fatty acids of P. putrefaciens and those reported for a strain of Alteromonas haloplanktis do not harmonize with a proposal to transfer the former organism to the genus Alteromonas.

Molecular basis of beta-thalassemia in the Maldives.

PubMed

Furuumi, H; Firdous, N; Inoue, T; Ohta, H; Winichagoon, P; Fucharoen, S; Fukumaki, Y

1998-03-01

We have systematically analyzed beta-thalassemia genes using polymerase chain reaction-related techniques, dot-blot hybridization with oligonucleotide probes, allele specific-polymerase chain reaction, and sequencing of amplified DNA fragments from 41 unrelated patients, including 37 beta-thalassemia homozygotes, three with beta-thalassemia/Hb E, and one with beta-thalassemia/Hb S. Four different beta-thalassemia mutations were detected in 78 alleles. These are the IVS-I-5 (G-->C), codon 30 (AGG-->ACG) [also indicated as IVS-I (-1)], IVS-I-1 (G-->A), and codons 41/42 (-TTCT) mutations. The distribution of the beta-thalassemia mutations in the Maldives is 58 alleles (74.3%) with the IVS-I-5 (G-->C) mutation, 12 (15.4%) with the codon 30 (AGG-->ACG) mutation, seven (9%) with the IVS-I-1 (G-->A) mutation, and one with the codons 41/42 (-TTCT) mutation. The first three mutations account for 98.7% of the total number of beta-thalassemia chromosomes studied. These mutations are clustered in the region spanning 6 bp around the junction of exon 1 and the first intervening sequence of the beta-globin gene. These observations have significant implications for setting up a thalassemia prevention and control program in the Maldives. Analysis of haplotypes and frameworks of chromosomes bearing each beta-thalassemia mutation suggested that the origin and spread of these mutations were reflected by the historical record.

Expression and function of CD8 alpha/beta chains on rat and human mast cells.

PubMed

Kim, Mi-Sun; Kim, Sung-Hoon; Lee, Hye-Jung; Kim, Hyung-Min

2004-03-01

The expression and functional role of CD8 glycoprotein, a marker of cytotoxic/suppressor T lymphocytes and NK cells, were not studied on freshly isolated connective tissue type rat peritoneal mast cells, a rat mucosal type mast cell line (RBL 2H3), or human mast cell line (HMC-1). We used the reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, immunohistochemistry and enzyme-linked immunosorbent assay. RT-PCR and Western blot analysis identified the presence of CD8 alpha/beta chains on the mast cells, and immunohistochemistry confirmed CD8alpha expression on rat or human mast cells. Functional studies demonstrated that stimulation of CD8 alpha/beta chains on rat mast cells induced the secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), which are regarded as important mediators during infection. However, co-stimulation with stem cell factor had no effect on CD8-induced mediator secretion. Our findings demonstrate novel biological roles of CD8 molecules in mast cells.

The common inhalation anesthetic isoflurane induces caspase activation and increases amyloid beta-protein level in vivo.

PubMed

Xie, Zhongcong; Culley, Deborah J; Dong, Yuanlin; Zhang, Guohua; Zhang, Bin; Moir, Robert D; Frosch, Matthew P; Crosby, Gregory; Tanzi, Rudolph E

2008-12-01

An estimated 200 million patients worldwide have surgery each year. Anesthesia and surgery have been reported to facilitate emergence of Alzheimer's disease. The commonly used inhalation anesthetic isoflurane has previously been reported to induce apoptosis, and to increase levels and aggregation of Alzheimer's disease-associated amyloid beta-protein (Abeta) in cultured cells. However, the in vivo relevance has not been addressed. We therefore set out to determine effects of isoflurane on caspase activation and levels of beta-site amyloid precursor protein-cleaving enzyme (BACE) and Abeta in naive mice, using Western blot, immunohistochemistry, and reverse transcriptase polymerase chain reaction. Here we show for the first time that a clinically relevant isoflurane anesthesia (1.4% isoflurane for 2 hours) leads to caspase activation and modest increases in levels of BACE 6 hours after anesthesia in mouse brain. Isoflurane anesthesia induces caspase activation, and increases levels of BACE and Abeta up to 24 hours after anesthesia. Isoflurane may increase BACE levels by reducing BACE degradation. Moreover, the Abeta aggregation inhibitor, clioquinol, was able to attenuate isoflurane-induced caspase-3 activation in vivo. Given that transient insults to brain may lead to long-term brain damage, these findings suggest that isoflurane may promote Alzheimer's disease neuropathogenesis and, as such, have implications for use of isoflurane in humans, pending human study confirmation.

Serotypes, antimicrobial susceptibility, and beta-lactam resistance mechanisms of clinical Haemophilus influenzae isolates from Bulgaria in a pre-vaccination period.

PubMed

Setchanova, Lena Petrova; Kostyanev, Tomislav; Markovska, Rumyana; Miloshev, George; Mitov, Ivan Gergov

2013-02-01

To determine the serotypes, antimicrobial susceptibility, and beta-lactam resistance mechanisms of Haemophilus influenzae strains isolated from invasive and respiratory tract infections (RTIs) prior to the introduction of Haemophilus influenzae type b (Hib) vaccination in Bulgaria. A total of 259 isolates were serotyped by polymerase chain reaction. Susceptibility to antibiotics and beta-lactamase production were determined, and DNA sequencing of the ftsI gene was performed for ampicillin non-susceptible strains. The invasive H. influenzae infections in children were mainly due to serotype b (94.5% in meningitis and 88.9% in other invasive cases). Non-typeable strains (97.4%) were the most frequently found H. influenzae strains in RTIs both in children and adults. Non-susceptibility to ampicillin occurred in 22% of all strains. Ceftriaxone and levofloxacin were the most active agents tested. Ampicillin resistance occurred in 34.4% of invasive strains, and beta-lactamase production was the only mechanism found. Among respiratory tract isolates, ampicillin non-susceptible strains (18%) were classified into the following groups: beta-lactamase-positive, ampicillin-resistant (BLPAR) strains (7.2%); beta-lactamase-negative, ampicillin-non-susceptible (BLNAR) strains (8.2%); and beta- lactamase-positive, amoxicillin-clavulanate-resistant (BLPACR) strains (2.6%). Among 21 BLNAR and BLPACR strains there were 9 different patterns of multiple-amino acid substitutions in penicillin-binding protein 3. Of these, most isolates (81.0%) belonged to group II, defined by the Asn526Lys substitution. Beta-lactamase production was more common among invasive strains than in respiratory isolates. BLNAR and BLPACR H. influenzae were found only among respiratory tract isolates.

Predicting beta-turns and their types using predicted backbone dihedral angles and secondary structures.

PubMed

Kountouris, Petros; Hirst, Jonathan D

2010-07-31

Beta-turns are secondary structure elements usually classified as coil. Their prediction is important, because of their role in protein folding and their frequent occurrence in protein chains. We have developed a novel method that predicts beta-turns and their types using information from multiple sequence alignments, predicted secondary structures and, for the first time, predicted dihedral angles. Our method uses support vector machines, a supervised classification technique, and is trained and tested on three established datasets of 426, 547 and 823 protein chains. We achieve a Matthews correlation coefficient of up to 0.49, when predicting the location of beta-turns, the highest reported value to date. Moreover, the additional dihedral information improves the prediction of beta-turn types I, II, IV, VIII and "non-specific", achieving correlation coefficients up to 0.39, 0.33, 0.27, 0.14 and 0.38, respectively. Our results are more accurate than other methods. We have created an accurate predictor of beta-turns and their types. Our method, called DEBT, is available online at http://comp.chem.nottingham.ac.uk/debt/.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pirastu, M.; Galanello, R.; Doherty, M.A.

The predominant ..beta..-thalassemia in Sardinia is the ..beta../sup 0/ type in which no ..beta..-globin chains are synthesized in the homozygous state. The authors determined the ..beta..-thalassemia mutations in this population by the oligonucleotide-probe method and defined the chromosome haplotypes on which the mutation resides. The same ..beta../sup 39(CAG..-->..TAG)/ nonsense mutation was found on nine different chromosome haplotypes. Although this mutation may have arisen more than once, the multiple haplotypes could also be generated by crossing over and gene conversion events. These findings underscore the frequency of mutational events in the ..beta..-globin gene region.

BetaSCPWeb: side-chain prediction for protein structures using Voronoi diagrams and geometry prioritization

PubMed Central

Ryu, Joonghyun; Lee, Mokwon; Cha, Jehyun; Laskowski, Roman A.; Ryu, Seong Eon; Kim, Deok-Soo

2016-01-01

Many applications, such as protein design, homology modeling, flexible docking, etc. require the prediction of a protein's optimal side-chain conformations from just its amino acid sequence and backbone structure. Side-chain prediction (SCP) is an NP-hard energy minimization problem. Here, we present BetaSCPWeb which efficiently computes a conformation close to optimal using a geometry-prioritization method based on the Voronoi diagram of spherical atoms. Its outputs are visual, textual and PDB file format. The web server is free and open to all users at http://voronoi.hanyang.ac.kr/betascpweb with no login requirement. PMID:27151195

Structural and functional characterization of a novel homodimeric three-finger neurotoxin from the venom of Ophiophagus hannah (king cobra).

PubMed

Roy, Amrita; Zhou, Xingding; Chong, Ming Zhi; D'hoedt, Dieter; Foo, Chun Shin; Rajagopalan, Nandhakishore; Nirthanan, Selvanayagam; Bertrand, Daniel; Sivaraman, J; Kini, R Manjunatha

2010-03-12

Snake venoms are a mixture of pharmacologically active proteins and polypeptides that have led to the development of molecular probes and therapeutic agents. Here, we describe the structural and functional characterization of a novel neurotoxin, haditoxin, from the venom of Ophiophagus hannah (King cobra). Haditoxin exhibited novel pharmacology with antagonism toward muscle (alphabetagammadelta) and neuronal (alpha(7), alpha(3)beta(2), and alpha(4)beta(2)) nicotinic acetylcholine receptors (nAChRs) with highest affinity for alpha(7)-nAChRs. The high resolution (1.5 A) crystal structure revealed haditoxin to be a homodimer, like kappa-neurotoxins, which target neuronal alpha(3)beta(2)- and alpha(4)beta(2)-nAChRs. Interestingly however, the monomeric subunits of haditoxin were composed of a three-finger protein fold typical of curaremimetic short-chain alpha-neurotoxins. Biochemical studies confirmed that it existed as a non-covalent dimer species in solution. Its structural similarity to short-chain alpha-neurotoxins and kappa-neurotoxins notwithstanding, haditoxin exhibited unique blockade of alpha(7)-nAChRs (IC(50) 180 nm), which is recognized by neither short-chain alpha-neurotoxins nor kappa-neurotoxins. This is the first report of a dimeric short-chain alpha-neurotoxin interacting with neuronal alpha(7)-nAChRs as well as the first homodimeric three-finger toxin to interact with muscle nAChRs.

Mutation of Arg723Gly in beta-myosin heavy chain gene in five Chinese families with hypertrophic cardiomyopathy.

PubMed

Yang, Jun-hua; Zheng, Dong-dong; Dong, Ning-zheng; Yang, Xiang-jun; Song, Jian-ping; Jiang, Ting-bo; Cheng, Xu-jie; Li, Hong-xia; Zhou, Bing-yuan; Zhao, Cai-ming; Jiang, Wen-ping

2006-11-05

Hypertrophic cardiomyopathy (HCM) is a form of cardiomyopathy with an autosomal dominant inherited disease, which is caused by mutations in at least one of the sarcomeric protein genes. Mutations in the beta-myosin heavy chain (beta-MHC) are the most common cause of HCM. This study was to reveal the disease-causing gene mutations in Chinese population with HCM, and to analyze the correlation between the genotype and phenotype. The exons 3 to 26 of MYH7 were amplified by PCR, and the PCR products were sequenced in five non-kin HCM patients. A 17-year-old patient was detected to be an Arg723Gly mutation carrier. Then his family was gene-screened, and the correlation between genotype and phenotype was analyzed. The mutation of Arg723Gly in a Chinese family with HCM was detected for the first time. With a C-G transversion in nucleotide 13,619 of the MYH7 gene, located at the essential light chain interacting region in S1, the replacement of arginine by glycine took place at amino acid residue 723. A two-dimensional echocardiogram showed moderate asymmetrical septal hypertrophy with left atria enlargement. There was no obstruction in the left ventricular outflow tract. In his family, a total of 13 individuals were diagnosed HCM and 5 of them were dead of congestive heart failure at a mean age of 66-year-old. Eight living members were all detected to carry the mutation, in which 3 developed progressive heart failure. Moreover, the heart function of the people evidently deteriorates when their age are older than 50. The mutation and the disease show co-separated. The Arg723Gly mutation is a malignant type. In Chinese the mutation has the similar characters to the former report but has low degree malignant.

Structural features of water-soluble novel polysaccharide components from the leaves of Tridax procumbens Linn.

PubMed

Raju, T S; Davidson, E A

1994-05-20

Two water-soluble polysaccharide fractions, WSTP-IA and WSTP-IB were purified from the leaves of Tridax procumbens Linn. with graded ethanol precipitation followed by mild delignification and size-exclusion chromatography. WSTP-IA contained L-Araf and D-Galp in approximately 1:3 molar proportions, and WSTP-IB contained only D-Galp as the major sugar component. The results of methylation linkage analysis, and 1H and 13C NMR studies on the native and modified polysaccharides, indicated that WSTP-IA is an L-arabino-D-galactan with a beta-(1-->6)-D-galactan main chain in which at least one in every two D-Galp residues carries single residues of either L-Araf (alpha-/beta-) or beta-D-Galp end-group as substituents at O-3. WSTP-IB is a linear beta-(1-->6)-D-galactan. This is the first report of polysaccharides containing a beta-(1-->6)-D-galactan main chain isolated from plant sources.

Beta-hairpin formation in aqueous solution and in the presence of trifluoroethanol: a (1)H and (13)C nuclear magnetic resonance conformational study of designed peptides.

PubMed

Santiveri, Clara M; Pantoja-Uceda, David; Rico, Manuel; Jiménez, M Angeles

2005-10-15

In order to check our current knowledge on the principles involved in beta-hairpin formation, we have modified the sequence of a 3:5 beta-hairpin forming peptide with two different purposes, first to increase the stability of the formed 3:5 beta-hairpin, and second to convert the 3:5 beta-hairpin into a 2:2 beta-hairpin. The conformational behavior of the designed peptides was investigated in aqueous solution and in 30% trifluoroethanol (TFE) by analysis of the following nuclear magnetic resonance (NMR) parameters: nuclear Overhauser effect (NOE) data, and C(alpha)H, (13)C(alpha), and (13)C(beta) conformational shifts. From the differences in the ability to adopt beta-hairpin structures in these peptides, we have arrived to the following conclusions: (i) beta-Hairpin population increases with the statistical propensity of residues to occupy each turn position. (ii) The loop length, and in turn, the beta-hairpin type, can be modified as a function of the type of turn favored by the loop sequence. These two conclusions reinforce previous results about the importance of beta-turn sequence in beta-hairpin folding. (iii) Side-chain packing on each face of the beta-sheet may play a major role in beta-hairpin stability; hence simplified analysis in terms of isolated pair interactions and intrinsic beta-sheet propensities is insufficient. (iv) Contributions to beta-hairpin stability of turn and strand sequences are not completely independent. (v) The burial of hydrophobic surface upon beta-hairpin formation that, in turn, depends on side-chain packing also contributes to beta-hairpin stability. (vi) As previously observed, TFE stabilizes beta-hairpin structures, but the extent of the contribution of different factors to beta-hairpin formation is sometimes different in aqueous solution and in 30% TFE. (c) 2005 Wiley Periodicals, Inc. Biopolymers 79: 150-162, 2005.

A new common mutation in the cardiac beta-myosin heavy chain gene in Finnish patients with hypertrophic cardiomyopathy.

PubMed

Jääskeläinen, Pertti; Heliö, Tiina; Aalto-Setälä, Katriina; Kaartinen, Maija; Ilveskoski, Erkki; Hämäläinen, Liisa; Melin, John; Kärkkäinen, Satu; Peuhkurinen, Keijo; Nieminen, Markku S; Laakso, Markku; Kuusisto, Johanna

2014-09-01

In the nationwide FinHCM Study including 306 Finnish patients with hypertrophic cardiomyopathy (HCM), we have previously identified two founder mutations in the alpha-tropomyosin (TPM1-D175N) and myosin-binding protein C (MYBPC3-Q1061X) genes, accounting for 18% of all cases. Objective. To screen additional mutations, previously identified in eastern Finnish cohorts with HCM, in the FinHCM Study population. Ten mutations in the beta-myosin heavy chain gene (MYH7), TPM1, and MYBPC3 were screened. MYH7-R1053Q was found in 17 of 306 patients (5.6%). No carriers of MYH7-R719W or N696S were found. A novel TPM1-D175G mutation was found in a single patient. MYBPC3 mutations were found in 14 patients: IVS5-2A-C in two, IVS14-13G-A in two, K811del in six, and A851insT in four patients. Altogether, a HCM-causing mutation was identified in 32 patients, accounting for 10.5% of all cases. In addition, two MYBPC3 variants R326Q and V896M with uncertain pathogenicity were found in eight and in 10 patients, respectively. Combining the present findings with our previous results, a causative mutation was identified in 28% of the FinHCM cohort. MYH7-R1053Q was the third most common mutation, and should be screened in all new cases of HCM in Finland.

Axolotl hemoglobin: cDNA-derived amino acid sequences of two alpha globins and a beta globin from an adult Ambystoma mexicanum.

PubMed

Shishikura, Fumio; Takeuchi, Hiro-aki; Nagai, Takatoshi

2005-11-01

Erythrocytes of the adult axolotl, Ambystoma mexicanum, have multiple hemoglobins. We separated and purified two kinds of hemoglobin, termed major hemoglobin (Hb M) and minor hemoglobin (Hb m), from a five-year-old male by hydrophobic interaction column chromatography on Alkyl Superose. The hemoglobins have two distinct alpha type globin polypeptides (alphaM and alpham) and a common beta globin polypeptide, all of which were purified in FPLC on a reversed-phase column after S-pyridylethylation. The complete amino acid sequences of the three globin chains were determined separately using nucleotide sequencing with the assistance of protein sequencing. The mature globin molecules were composed of 141 amino acid residues for alphaM globin, 143 for alpham globin and 146 for beta globin. Comparing primary structures of the five kinds of axolotl globins, including two previously established alpha type globins from the same species, with other known globins of amphibians and representatives of other vertebrates, we constructed phylogenetic trees for amphibian hemoglobins and tetrapod hemoglobins. The molecular trees indicated that alphaM, alpham, beta and the previously known alpha major globin were adult types of globins and the other known alpha globin was a larval type. The existence of two to four more globins in the axolotl erythrocyte is predicted.

Soluble donor HLA class I and beta 2m-free heavy chain in serum of lung transplant recipients: steady-state levels and increases in patients with recurrent CMV infection, acute rejection episodes, and poor outcome.

PubMed

DeVito-Haynes, L D; Jankowska-Gan, E; Meyer, K C; Cornwell, R D; Zeevi, A; Griffith, B; Dauber, J; Iacono, A; Burlingham, W J; Love, R B

2000-12-01

We determined the concentration of donor sHLA/beta(2)m and total beta(2)m-free heavy chain (HC) in the serum of lung transplant recipients with ELISA assays. While we were unable to detect specific donor beta(2)m-free HCs due to a lack of available antibodies, we could determine if events that led to an increase in the release of beta(2)m-free HC also led to an increase in the release of donor sHLA/beta(2)m, particularly the 36 kDa, proteolytically cleaved form. We found that lung transplants constituitively release donor sHLA/beta(2)m at ng/ml levels. The levels (both of donor sHLA/beta(2)m and total beta(2)m-free HC) were significantly increased in CMV-sero-negative recipients (but not in CMV-sero-positive recipients) at the onset of post-transplant CMV disease. Acute rejection episodes were also associated with an increased release of donor sHLA/beta(2)m, but not of beta(2)m-free HC. However, in patients with particularly poor outcome (i.e., graft loss within 1 year) there was a significant release of beta(2)m-free HC. Analysis of one such patient showed a predominance of 36 kDa forms of donor-sHLA/beta(2)m. Our data are consistent with the hypothesis that the metalloproteinase that cleaves beta(2)m-free HC is active during uncontrolled CMV infection and acute rejection. However, recall responses to CMV and controlled immune responses to donor may result in little or no activation of sHLA class I release.

Expression of a mutation causing hypertrophic cardiomyopathy disrupts sarcomere assembly in adult feline cardiac myocytes.

PubMed

Marian, A J; Yu, Q T; Mann, D L; Graham, F L; Roberts, R

1995-07-01

Mutations in the beta-myosin heavy chain (beta MyHC) induce hypertrophic cardiomyopathy (HCM), cardiac hypertrophy, and sarcomere disarray, with the latter being the characteristic hallmark. Thus, we sought to determine whether expression of mutant beta MyHC in adult feline cardiac myocytes, a species known to develop HCM with a phenotype identical to that in humans, induces sarcomere disarray. A full-length beta MyHC cDNA was cloned from a human heart cDNA library, and an HCM-causing mutation (Arg403Gln) was induced in the beta MyHC cDNA by site-directed mutagenesis using polymerase chain reaction (PCR). The normal and mutant beta MyHC cDNAs were cloned into p delta E1spIB shuttle vector, downstream from a cytomegalovirus (CMV) promoter. Replication-deficient recombinant adenoviral constructs (Ad5/CMV/beta MyHC-N and Ad5/CMV/beta MyHC-403) were generated through homologous recombination of p delta E1spIB/CMV/beta MyHC-N or Ad5/CMV/beta MyHC-403 and pBHG10 after cotransfection in 293 host cells. Infection of COS-1 cells with the beta MyHC construct resulted in the expression of a full-length myosin protein. Efficiency of infection of isolated adult cardiac myocytes was > 95%. Expression of the beta MyHC constructs into mRNA at 48 hours after infection of feline cardiac myocytes was confirmed by reverse transcription-PCR. The net total protein and beta-myosin synthesis were determined by using the amount of incorporation of [3H]phenylalanine into total protein and beta-myosin, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Testa, U.; Hinard, N.; Beuzard, Y.

During incubation of reticulocytes from patients with beta-thalassemia, after labeling of the hemoglobin with radioactive amino acids, the excess alpha chains are gradually lost from the cells. The aim of this study was to investigate the mechanism of this phenomenon. A system was developed in which reticulocytes from beta-thalassemia patients are labeled with (3H)leucine, washed several times in nonradioactive medium, and then incubated in the same medium containing puromycin added in order to stop further protein synthesis. The results have clearly shown that excess alpha chains are gradually degraded by proteolysis. N-ethylmaleimide or epsilon-aminocaproic acid inhibited the proteolysis of freemore » alpha chains. The addition of either ATP or hemin did not change the rate of alpha chain degradation. The time required to degrade 50% of the pool of free alpha chains was directly dependent on the initial value of this pool. This finding suggests the absence of a significant individual variation in the ability to proteolyse free alpha chains.« less

KM+, a mannose-binding lectin from Artocarpus integrifolia: amino acid sequence, predicted tertiary structure, carbohydrate recognition, and analysis of the beta-prism fold.

PubMed Central

Rosa, J. C.; De Oliveira, P. S.; Garratt, R.; Beltramini, L.; Resing, K.; Roque-Barreira, M. C.; Greene, L. J.

1999-01-01

The complete amino acid sequence of the lectin KM+ from Artocarpus integrifolia (jackfruit), which contains 149 residues/mol, is reported and compared to those of other members of the Moraceae family, particularly that of jacalin, also from jackfruit, with which it shares 52% sequence identity. KM+ presents an acetyl-blocked N-terminus and is not posttranslationally modified by proteolytic cleavage as is the case for jacalin. Rather, it possesses a short, glycine-rich linker that unites the regions homologous to the alpha- and beta-chains of jacalin. The results of homology modeling implicate the linker sequence in sterically impeding rotation of the side chain of Asp141 within the binding site pocket. As a consequence, the aspartic acid is locked into a conformation adequate only for the recognition of equatorial hydroxyl groups on the C4 epimeric center (alpha-D-mannose, alpha-D-glucose, and their derivatives). In contrast, the internal cleavage of the jacalin chain permits free rotation of the homologous aspartic acid, rendering it capable of accepting hydrogen bonds from both possible hydroxyl configurations on C4. We suggest that, together with direct recognition of epimeric hydroxyls and the steric exclusion of disfavored ligands, conformational restriction of the lectin should be considered to be a new mechanism by which selectivity may be built into carbohydrate binding sites. Jacalin and KM+ adopt the beta-prism fold already observed in two unrelated protein families. Despite presenting little or no sequence similarity, an analysis of the beta-prism reveals a canonical feature repeatedly present in all such structures, which is based on six largely hydrophobic residues within a beta-hairpin containing two classic-type beta-bulges. We suggest the term beta-prism motif to describe this feature. PMID:10210179

Diving behaviour and haemoglobin function: the primary structure of the alpha- and beta-chains of the sea turtle (Caretta caretta) and its functional implications.

PubMed

Petruzzelli, R; Aureli, G; Lania, A; Galtieri, A; Desideri, A; Giardina, B

1996-06-15

The amino acid sequence of the alpha- and beta-chains of haemoglobin (Hb) from the loggerhead sea turtle (Caretta caretta) has been determined. Comparison with that of human Hb shows differences in several residues involved in both alpha 1 beta 1 and alpha 1 beta 2 packing contacts. On the whole, in spite of the mutations, the essential characteristics of both interfaces seem to be maintained. The functional properties of the sea turtle Hb have been investigated at different temperatures and as a function of proton, chloride and organic phosphate concentrations. In addition to overall similarities shared with most of the vertebrate Hbs previously described, this molecule shows significant differences which could be related to the life behaviour of the turtle. In fact, while the shape of the Bohr-effect curve is well adapted for gas exchange during prolonged dives, the very small enthalpy change for O2 binding ensures that O2 delivery becomes essentially insensitive to the temperature changes of the environment. Moreover, and similarly to the case of emperor penguin Hb, the small alkaline Bohr effect appears to be only choride-linked, since the pH dependence of the O2 affinity is abolished in the absence of this ion. These functional characteristics are discussed on the basis of the primary structure of alpha- and beta-chains.

Intrinsic folding of small peptide chains: spectroscopic evidence for the formation of beta-turns in the gas phase.

PubMed

Chin, Wutharath; Dognon, Jean-Pierre; Piuzzi, François; Tardivel, Benjamin; Dimicoli, Iliana; Mons, Michel

2005-01-19

Laser desorption of model peptides coupled to laser spectroscopic techniques enables the gas-phase observation of genuine secondary structures of biology. Spectroscopic evidence for the formation of beta-turns in gas-phase peptide chains containing glycine and phenylalanine residues establishes the intrinsic stability of these forms and their ability to compete with other stable structures. The precise characterization of local minima on the potential energy surface from IR spectroscopy constitutes an acute assessment for the state-of-the-art quantum mechanical calculations also presented. The observation of different types of beta-turns depending upon the residue order within the sequence is found to be consistent with the residue propensities in beta-turns of proteins, which suggests that the prevalence of glycine in type II and II' turns stems essentially from an energetic origin, already at play under isolated conditions.

Sequence inversion and phenylalanine surrogates at the beta-turn enhance the antibiotic activity of gramicidin S.

PubMed

Solanas, Concepción; de la Torre, Beatriz G; Fernández-Reyes, María; Santiveri, Clara M; Jiménez, M Angeles; Rivas, Luis; Jiménez, Ana I; Andreu, David; Cativiela, Carlos

2010-05-27

A series of gramicidin S (GS) analogues have been synthesized where the Phe (i + 1) and Pro (i + 2) residues of the beta-turn have been swapped while the respective chiralities (D-, L-) at each position are preserved, and Phe is replaced by surrogates with aromatic side chains of diverse size, orientation, and flexibility. Although most analogues preserve the beta-sheet structure, as assessed by NMR, their antibiotic activities turn out to be highly dependent on the bulkiness and spatial arrangement of the aromatic side chain. Significant increases in microbicidal potency against both Gram-positive and Gram-negative pathogens are observed for several analogues, resulting in improved therapeutic profiles. Data indicate that seemingly minor replacements at the GS beta-turn can have significant impact on antibiotic activity, highlighting this region as a hot spot for modulating GS plasticity and activity.

GammaM23K, gammaM232K, and gammaL77K single substitutions in the TF1-ATPase lower ATPase activity by disrupting a cluster of hydrophobic side chains.

PubMed

Bandyopadhyay, Sanjay; Allison, William S

2004-07-27

In crystal structures of the bovine F(1)-ATPase (MF(1)), the side chains of gammaMet(23), gammaMet(232), and gammaLeu(77) interact in a cluster. Substitution of the corresponding residues in the alpha(3)beta(3)gamma subcomplex of TF(1) with lysine lowers the ATPase activity to 2.3, 11, and 15%, respectively, of that displayed by wild-type. In contrast, TF(1) subcomplexes containing the gammaM(23)C, gammaM(232)C, and gammaL(77)C substitutions display 36, 36, and 130%, respectively, of the wild-type ATPase activity. The ATPase activity of the gammaM(23)C/gammaM(232)C double mutant subcomplex is 36% that of the wild-type subcomplex before and after cross-linking the introduced cysteines, whereas the ATPase activity of the gammaM(23)C/L(77)C double mutant increased from 50 to 85% that of wild-type after cross-linking the introduced cysteines. Only beta-beta cross-links formed when the alpha(3)(betaE(395)C)(3)gammaM(23)C double mutant was inactivated with CuCl(2). The overall results suggest that the attenuated ATPase of the mutant subcomplexes containing the gammaM(23)K, gammaL(77)K, and gammaM(232)K substitutions is caused by disruption of the cluster of hydrophobic amino acid side chains and that the midregion of the coiled-coil comprised of the amino- and carboxyl-terminal alpha helices of the gamma subunit does not undergo unwinding or major displacement from the side chain of gammaLeu(77) during ATP-driven rotation of the gamma subunit.

BetaSCPWeb: side-chain prediction for protein structures using Voronoi diagrams and geometry prioritization.

PubMed

Ryu, Joonghyun; Lee, Mokwon; Cha, Jehyun; Laskowski, Roman A; Ryu, Seong Eon; Kim, Deok-Soo

2016-07-08

Many applications, such as protein design, homology modeling, flexible docking, etc. require the prediction of a protein's optimal side-chain conformations from just its amino acid sequence and backbone structure. Side-chain prediction (SCP) is an NP-hard energy minimization problem. Here, we present BetaSCPWeb which efficiently computes a conformation close to optimal using a geometry-prioritization method based on the Voronoi diagram of spherical atoms. Its outputs are visual, textual and PDB file format. The web server is free and open to all users at http://voronoi.hanyang.ac.kr/betascpweb with no login requirement. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Endocytosis of lysosomal acid phosphatase; involvement of mannose receptor and effect of lectins.

PubMed

Imai, K; Yoshimura, T

1994-08-01

Acid phosphatase and beta-glucosidase are unique among lysosomal enzymes in that they have both high mannose and complex type sugasr chains, whereas oligosaccharide chains of lysosomal enzymes in matrix are of high mannose type. We have previously shown that beta-glucosidase was endocytosed into macrophages via an unidentified receptor different from a mannose/fucose receptor (K. Imai, Cell Struct. Funct. 13, 325-332, 1988). Here, we show that uptake of acid phosphatase purified from rat liver lysosomes into rat macrophages was inhibited by ligands for a mannose/fucose receptor and was mediated via an apparently single binding site with Kuptake of 24.7 nM. These results indicate that acid phosphatase and beta-glucosidase recognize different types of receptors even if they have similar sugar chains. Polyvalent concanavalin A which binds both to the enzyme and to macrophages specifically stimulated the uptake in a dose dependent manner, whereas wheat germ agglutinin and phytohaemagglutinin did not.

Genetic modulation of sickle cell anemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steinberg, M.H.

1995-05-01

Sickle cell anemia, a common disorder associated with reduced life span of the red blood cell and vasoocclusive events, is caused by a mutation in the {Beta}-hemoglobin gene. Yet, despite this genetic homogeneity, the phenotype of the disease is heterogeneous. This suggests the modulating influence of associated inherited traits. Some of these may influence the accumulation of fetal hemoglobin, a hemoglobin type that interferes with the polymerization of sickle hemoglobin. Another inherited trait determines the accumulation of {alpha}-globin chains. This review focuses on potential genetic regulators of the phenotype of sickle cell anemia. 125 refs., 6 figs., 3 tabs.

In vivo regulation of the beta-myosin heavy chain gene in soleus muscle of suspended and weight-bearing rats

NASA Technical Reports Server (NTRS)

Giger, J. M.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

2000-01-01

In the weight-bearing hindlimb soleus muscle of the rat, approximately 90% of muscle fibers express the beta-myosin heavy chain (beta-MHC) isoform protein. Hindlimb suspension (HS) causes the MHC isoform population to shift from beta toward the fast MHC isoforms. Our aim was to establish a model to test the hypothesis that this shift in expression is transcriptionally regulated through specific cis elements of the beta-MHC promoter. With the use of a direct gene transfer approach, we determined the activity of different length beta-MHC promoter fragments, linked to a firefly luciferase reporter gene, in soleus muscle of control and HS rats. In weight-bearing rats, the relative luciferase activity of the longest beta-promoter fragment (-3500 bp) was threefold higher than the shorter promoter constructs, which suggests that an enhancer sequence is present in the upstream promoter region. After 1 wk of HS, the reporter activities of the -3500-, -914-, and -408-bp promoter constructs were significantly reduced ( approximately 40%), compared with the control muscles. However, using the -215-bp construct, no differences in promoter activity were observed between HS and control muscles, which indicates that the response to HS in the rodent appears to be regulated within the -408 and -215 bp of the promoter.

Using support vector machine to predict beta- and gamma-turns in proteins.

PubMed

Hu, Xiuzhen; Li, Qianzhong

2008-09-01

By using the composite vector with increment of diversity, position conservation scoring function, and predictive secondary structures to express the information of sequence, a support vector machine (SVM) algorithm for predicting beta- and gamma-turns in the proteins is proposed. The 426 and 320 nonhomologous protein chains described by Guruprasad and Rajkumar (Guruprasad and Rajkumar J. Biosci 2000, 25,143) are used for training and testing the predictive model of the beta- and gamma-turns, respectively. The overall prediction accuracy and the Matthews correlation coefficient in 7-fold cross-validation are 79.8% and 0.47, respectively, for the beta-turns. The overall prediction accuracy in 5-fold cross-validation is 61.0% for the gamma-turns. These results are significantly higher than the other algorithms in the prediction of beta- and gamma-turns using the same datasets. In addition, the 547 and 823 nonhomologous protein chains described by Fuchs and Alix (Fuchs and Alix Proteins: Struct Funct Bioinform 2005, 59, 828) are used for training and testing the predictive model of the beta- and gamma-turns, and better results are obtained. This algorithm may be helpful to improve the performance of protein turns' prediction. To ensure the ability of the SVM method to correctly classify beta-turn and non-beta-turn (gamma-turn and non-gamma-turn), the receiver operating characteristic threshold independent measure curves are provided. (c) 2008 Wiley Periodicals, Inc.

Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis

NASA Technical Reports Server (NTRS)

Donelan, Matthew J.; Morfini, Gerardo; Julyan, Richard; Sommers, Scott; Hays, Lori; Kajio, Hiroshi; Briaud, Isabelle; Easom, Richard A.; Molkentin, Jeffery D.; Brady, Scott T.;

The Role of Oxidative Stress-Induced Epigenetic Alterations in Amyloid-β Production in Alzheimer's Disease

PubMed Central

Best, Thomas M.

2015-01-01

An increasing number of studies have proposed a strong correlation between reactive oxygen species (ROS)-induced oxidative stress (OS) and the pathogenesis of Alzheimer's disease (AD). With over five million people diagnosed in the United States alone, AD is the most common type of dementia worldwide. AD includes progressive neurodegeneration, followed by memory loss and reduced cognitive ability. Characterized by the formation of amyloid-beta (Aβ) plaques as a hallmark, the connection between ROS and AD is compelling. Analyzing the ROS response of essential proteins in the amyloidogenic pathway, such as amyloid-beta precursor protein (APP) and beta-secretase (BACE1), along with influential signaling programs of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and c-Jun N-terminal kinase (JNK), has helped visualize the path between OS and Aβ overproduction. In this review, attention will be paid to significant advances in the area of OS, epigenetics, and their influence on Aβ plaque assembly. Additionally, we aim to discuss available treatment options for AD that include antioxidant supplements, Asian traditional medicines, metal-protein-attenuating compounds, and histone modifying inhibitors. PMID:26543520

The Role of Oxidative Stress-Induced Epigenetic Alterations in Amyloid-β Production in Alzheimer's Disease.

PubMed

Zuo, Li; Hemmelgarn, Benjamin T; Chuang, Chia-Chen; Best, Thomas M

2015-01-01

An increasing number of studies have proposed a strong correlation between reactive oxygen species (ROS)-induced oxidative stress (OS) and the pathogenesis of Alzheimer's disease (AD). With over five million people diagnosed in the United States alone, AD is the most common type of dementia worldwide. AD includes progressive neurodegeneration, followed by memory loss and reduced cognitive ability. Characterized by the formation of amyloid-beta (Aβ) plaques as a hallmark, the connection between ROS and AD is compelling. Analyzing the ROS response of essential proteins in the amyloidogenic pathway, such as amyloid-beta precursor protein (APP) and beta-secretase (BACE1), along with influential signaling programs of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and c-Jun N-terminal kinase (JNK), has helped visualize the path between OS and Aβ overproduction. In this review, attention will be paid to significant advances in the area of OS, epigenetics, and their influence on Aβ plaque assembly. Additionally, we aim to discuss available treatment options for AD that include antioxidant supplements, Asian traditional medicines, metal-protein-attenuating compounds, and histone modifying inhibitors.

ENU mutagenesis identifies mice with cardiac fibrosis and hepatic steatosis caused by a mutation in the mitochondrial trifunctional protein beta-subunit.

PubMed

Kao, Hsiao-Jung; Cheng, Ching-Feng; Chen, Yen-Hui; Hung, Shuen-Iu; Huang, Cheng-Chih; Millington, David; Kikuchi, Tateki; Wu, Jer-Yuarn; Chen, Yuan-Tsong

2006-12-15

Using the metabolomics-guided screening coupled to N-ethyl-N-nitrosourea-mediated mutagenesis, we identified mice that exhibited elevated levels of long-chain acylcarnitines. Whole genome homozygosity mapping with 262 SNP markers mapped the disease gene to chromosome 5 where candidate genes Hadha and Hadhb, encoding the mitochondria trifunctional protein (MTP) alpha- and beta-subunits, respectively, are located. Direct sequencing revealed a normal alpha-subunit, but detected a nucleotide T-to-A transversion in exon 14 (c.1210T>A) of beta-subunit (Hadhb) which resulted in a missense mutation of methionine to lysine (M404K). Western blot analysis showed a significant reduction of both the alpha- and beta-subunits, consistent with reduced enzyme activity in both the long-chain 3-hydroxyacyl-CoA dehydrogenase and the long-chain 3-ketoacyl-CoA thiolase activities. These mice had a decreased weight gain and cardiac arrhythmias which manifested from a prolonged PR interval to a complete atrio-ventricular dissociation, and died suddenly between 9 and 16 months of age. Histopathological studies showed multifocal cardiac fibrosis and hepatic steatosis. This mouse model will be useful to further investigate the mechanisms underlying arrhythmogenesis relating to lipotoxic cardiomyopathy and to investigate pathophysiology and treatment strategies for human MTP deficiency.

A molecular view of the role of chirality in charge-driven polypeptide complexation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoffmann, K. Q.; Perry, S. L.; Leon, L.

Polyelectrolyte molecules of opposite charge are known to form stable complexes in solution. Depending on the system conditions, such complexes can be solid or liquid. The latter are known as complex coacervates, and they appear as a second liquid phase in equilibrium with a polymer-dilute aqueous phase. This work considers the complexation between poly(glutamic acid) and poly(lysine), which is of particular interest because it enables examination of the role of chirality in ionic complexation, without changes to the overall chemical composition. Systematic atomic-level simulations are carried out for chains of poly(glutamic acid) and poly(lysine) with varying combinations of chirality alongmore » the backbone. Achiral chains form unstructured complexes. In contrast, homochiral chains lead to formation of stable beta-sheets between molecules of opposite charge, and experiments indicate that beta-sheet formation is correlated with the formation of solid precipitates. Changes in chirality along the peptide backbone are found to cause "kinks" in the beta-sheets. These are energetically unfavorable and result in irregular structures that are more difficult to pack together. Taken together, these results provide new insights that may be of use for the development of simple yet strong bioinspired materials consisting of beta-rich domains and amorphous regions.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rahman, Shaikh M., E-mail: rmizanoor@hotmail.com; Choudhury, Mahua; Janssen, Rachel C.

Highlights: Black-Right-Pointing-Pointer LXR agonist activation increases liver TG accumulation by increasing lipogenesis. Black-Right-Pointing-Pointer C/EBP{beta}{sup -/-} mouse prevents LXR activation-mediated induction of hepatic lipogenesis. Black-Right-Pointing-Pointer C/EBP{beta} deletion increases mitochondrial transport chain function. Black-Right-Pointing-Pointer Beneficial effects of LXR activation on liver cholesterol metabolism did not change. Black-Right-Pointing-Pointer C/EBP{beta} inhibition might have important therapeutic potential. -- Abstract: Drugs designed specifically to activate liver X receptors (LXRs) have beneficial effects on lowering cholesterol metabolism and inflammation but unfortunately lead to severe hepatic steatosis. The transcription factor CCAAT/enhancer binding protein beta (C/EBP{beta}) is an important regulator of liver gene expression but little is known aboutmore » its involvement in LXR-based steatosis and cholesterol metabolism. The present study investigated the role of C/EBP{beta} expression in LXR agonist (T0901317)-mediated alteration of hepatic triglyceride (TG) and lipogenesis in mice. C/EBP{beta} deletion in mice prevented LXR agonist-mediated induction of lipogenic gene expression in liver in conjunction with significant reduction of liver TG accumulation. Surprisingly, C/EBP{beta}{sup -/-} mice showed a major increase in liver mitochondrial electron chain function compared to WT mice. Furthermore, LXR activation in C/EBP{beta}{sup -/-} mice increased the expression of liver ATP-binding cassette transporter ABCG1, a gene implicated in cholesterol efflux and reducing blood levels of total and LDL-cholesterol. Together, these findings establish a central role for C/EBP{beta} in the LXR-mediated steatosis and mitochondrial function, without impairing the influence of LXR activation on lowering LDL and increasing HDL-cholesterol. Inactivation of C/EBP{beta} might therefore be an important therapeutic strategy to prevent LXR activation-mediated adverse effects on liver TG metabolism without disrupting its beneficial effects on cholesterol metabolism.« less

Beta-ketoacyl-acyl carrier protein synthase IV: a key enzyme for regulation of medium-chain fatty acid synthesis in Cuphea lanceolata seeds.

PubMed

Schütt, Burkhardt Siegfried; Abbadi, Amine; Loddenkötter, Brigitte; Brummel, Monika; Spener, Friedrich

2002-09-01

With the aim of elucidating the mechanisms involved in the biosynthesis of medium-chain fatty acids in Cuphea lanceolata Ait., a crop accumulating up to 90% decanoic acid in seed triacylglycerols, cDNA clones of a beta-ketoacyl-acyl carrier protein (ACP) synthase IV (clKAS IV, EC 2.3.1.41) were isolated from C. lanceolata seed embryos. The amino acid sequence deduced from clKAS IV cDNA showed 80% identity to other plant KAS II-type enzymes, 55% identity towards plant KAS I and over 90% towards other Cuphea KAS IV-type sequences. Recombinant clKAS IV was functionally overexpressed in Escherichia coli, and substrate specificity of purified enzyme showed strong preference for elongation of short-chain and medium-chain acyl-ACPs (C4- to C10-ACP) with nearly equal activity. Further elongation steps were catalysed with distinctly less activity. Moreover, short- and medium-chain acyl-ACPs exerted a chain-length-specific and concentration-dependent substrate inhibition of clKAS IV. Based on these findings a regulatory mechanism for medium-chain fatty acid synthesis in C. lanceolata is presented.

A Logical OR Redundancy within the Asx-Pro-Asx-Gly Type 1 {Beta}-Turn Motif

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jihun; Dubey, Vikash Kumar; Longo, Lian M.

2008-04-19

Turn secondary structure is essential to the formation of globular protein architecture. Turn structures are, however, much more complex than either {alpha}-helix or {beta}-sheet, and the thermodynamics and folding kinetics are poorly understood. Type I {beta}-turns are the most common type of reverse turn, and they exhibit a statistical consensus sequence of Asx-Pro-Asx-Gly (where Asx is Asp or Asn). A comprehensive series of individual and combined Asx mutations has been constructed within three separate type I 3:5 G1 bulge {beta}-turns in human fibroblast growth factor-1, and their effects on structure, stability, and folding have been determined. The results show amore » fundamental logical OR relationship between the Asx residues in the motif, involving H-bond interactions with main-chain amides within the turn. These interactions can be modulated by additional interactions with residues adjacent to the turn at positions i + 4 and i + 6. The results show that the Asx residues in the turn motif make a substantial contribution to the overall stability of the protein, and the Asx logical OR relationship defines a redundant system that can compensate for deleterious point mutations. The results also show that the stability of the turn is unlikely to be the prime determinant of formation of turn structure in the folding transition state.« less

Reactive Secondary Sequence Oxidative Pathology Polymer Model and Antioxidant Tests

PubMed Central

Petersen, Richard C.

2014-01-01

Aims To provide common Organic Chemistry/Polymer Science thermoset free-radical crosslinking Sciences for Medical understanding and also present research findings for several common vitamins/antioxidants with a new class of drugs known as free-radical inhibitors. Study Design Peroxide/Fenton transition-metal redox couples that generate free radicals were combined with unsaturated lipid oils to demonstrate thermoset-polymer chain growth by crosslinking with the α-β-unsaturated aldehyde acrolein into rubbery/adhesive solids. Further, Vitamin A and beta carotene were similarly studied for crosslink pathological potential. Also, free-radical inhibitor hydroquinone was compared for antioxidant capability with Vitamin E. Place and Duration of Study Department of Materials Science and Engineering and Department of Biomaterials, University of Alabama at Birmingham, between June 2005 and August 2012. Methodology Observations were recorded for Fenton free-radical crosslinking of unsaturated lipids and vitamin A/beta carotene by photography further with weight measurements and percent-shrinkage testing directly related to covalent crosslinking of unsaturated lipids recorded over time with different concentrations of acrolein. Also, hydroquinone and vitamin E were compared at concentrations from 0.0–7.3wt% as antioxidants for reductions in percent-shrinkage measurements, n = 5. Results Unsaturated lipid oils responded to Fenton thermoset-polymer reactive secondary sequence reactions only by acrolein with crosslinking into rubbery-type solids and different non-solid gluey products. Further, molecular oxygen crosslinking was demonstrated with lipid peroxidation and acrolein at specially identified margins. By peroxide/Fenton free-radical testing, both vitamin A and beta-carotene demonstrated possible pathology chemistry for chain-growth crosslinking. During lipid/acrolein testing over a 50 hour time period at 7.3wt% antioxidants, hydroquinone significantly reduced percent shrinkage greatly compared to the standard antioxidant vitamin E, %shrinkage at 11.6 ±1.3 for hydroquinone and 27.8 ±2.2 for vitamin E, P = .001. Conclusion Free radicals crosslinked unsaturated lipid fatty acids into thermoset polymers through Fenton reactions when combined with acrolein. Further, hydroquinone was a superior antioxidant to vitamin E. PMID:25909053

Impact of fatty acyl composition and quantity of triglycerides on bioaccessibility of dietary carotenoids.

PubMed

Huo, Tianyao; Ferruzzi, Mario G; Schwartz, Steven J; Failla, Mark L

2007-10-31

A carotenoid-rich salad meal with varying amounts and types of triglycerides (TG) was digested using simulated gastric and small intestinal conditions. Xanthophylls (lutein and zeaxanthin) and carotenes (alpha-carotene, beta-carotene, and lycopene) in chyme and micelle fraction were quantified to determine digestive stability and efficiency of micellarization (bioaccessibility). Micellarization of lutein (+zeaxanthin) exceeded that of alpha- and beta-carotenes, which was greater than that of lycopene for all test conditions. Micellarization of carotenes, but not lutein (+zeaxanthin), was enhanced (P < 0.05) by addition of TG (2.5% v/w) to the meal and was dependent on fatty acyl chain length in structured TG (c18:1 > c8:0 > c4:0). The degree of unsaturation of c18 fatty acyl chains in TG added to the salad purée did not significantly alter the efficiency of micellarization of carotenoids. Relatively low amounts of triolein and canola oil (0.5-1%) were required for maximum micellarization of carotenes, but more oil (approximately 2.5%) was required when TG with medium chain saturated fatty acyl groups (e.g., trioctanoin and coconut oil) was added to the salad. Uptake of lutein and beta-carotene by Caco-2 cells also was examined by exposing cells to micelles generated during the simulated digestion of salad purée with either triolein or trioctanoin. Cell accumulation of beta-carotene was independent of fatty acyl composition of micelles, whereas lutein uptake was slightly, but significantly, increased from samples with digested triolein compared to trioctanoin. The results show that the in vitro transfer of alpha-carotene, beta-carotene, and lycopene from chyme to mixed micelles during digestion requires minimal (0.5-1%) lipid content in the meal and is affected by the length of fatty acyl chains but not the degree of unsaturation in TG. In contrast, fatty acyl chain length has limited if any impact on carotenoid uptake by small intestinal epithelial cells. These data suggest that the amount of TG in a typical meal does not limit the bioaccessibility of carotenoids.

Recurrence of 49-base decamers, nonomers, and octamers within mouse C mu gene of Ig heavy chain and its primordial building block.

PubMed Central

Yazaki, A; Ohno, S

1983-01-01

Within the published 2,168-base-long mouse C mu gene of Ig heavy chain consisting of four coding and four noncoding segments, 2 base decamers, 8 nonomers, and 39 octamers recurred. Recurring base heptamers (about 100) and hexamers (about 350) were simply too numerous to merit individual identification. In spite of extensive overlaps between these recurring base decamers to hexamers, they occupied nearly the entire length of mouse Ig C mu gene. As with other genes of the beta-sheet-forming beta 2-microglobulin family, the Ig C mu gene (flanking and intervening noncoding sequences included) is not a unique sequence but rather it is degenerate repeats of the 45-base-long primordial building-block sequence uniquely its own. This primordial building block must originally have specified the 15-amino-acid-residue-long primordial arm of beta-sheet-forming loops, the characteristics of the beta 2-microglobulin family of polypeptides. PMID:6403948

Beta-and gamma-turns in proteins revisited: a new set of amino acid turn-type dependent positional preferences and potentials.

PubMed

Guruprasad, K; Rajkumar, S

2000-06-01

The number of beta-turns in a representative set of 426 protein three-dimensional crystal structures selected from the recent Protein Data Bank has nearly doubled and the number of gamma-turns in a representative set of 320 proteins has increased over seven times since the previous analysis. Beta-turns (7153) and gamma-turns (911) extracted from these proteins were used to derive a revised set of type-dependent amino acid positional preferences and potentials. Compared with previous results, the preference for proline, methionine and tryptophan has increased and the preference for glutamine, valine, glutamic acid and alanine has decreased for beta-turns. Certain new amino acid preferences were observed for both turn types and individual amino acids showed turn-type dependent positional preferences. The rationale for new amino acid preferences are discussed in the light of hydrogen bonds and other interactions involving the turns. Where main-chain hydrogen bonds of the type NH(i + 3) --> CO(i) were not observed for some beta-turns, other main-chain hydrogen bonds or solvent interactions were observed that possibly stabilize such beta-turns. A number of unexpected isolated beta-turns with proline at i + 2 position were also observed. The NH(i + 2) --> CO(i) hydrogen bond was observed for almost all gamma-turns. Nearly 20% classic gamma-turns and 43% inverse gamma-turns are isolated turns.

Intermediates of peroxisomal beta-oxidation. A study of the fatty acyl-CoA esters which accumulate during peroxisomal beta-oxidation of [U-14C]hexadecanoate.

PubMed Central

Bartlett, K; Hovik, R; Eaton, S; Watmough, N J; Osmundsen, H

1990-01-01

1. 14C-labelled fatty acyl-CoA esters resulting from beta-oxidation of [U-14C]hexadecanoate by peroxisomal fractions isolated from rats treated with clofibrate showed the presence of the full range of saturated intermediates down to acetyl-CoA. 2. The pattern of intermediates generated was fairly constant. At low concentrations of [U-14C]hexadecanoate (50 microM), decanoyl-CoA was present in lowest amounts. At higher concentrations of [U-14C]hexadecanoate (greater than 100 microM), all intermediates of chain length shorter than 12 carbon atoms (except acetyl-CoA) were present at similar low concentrations; the process of beta-oxidation now resembling chain-shortening of hexadecanoate by two cycles of beta-oxidation. 3. In the absence of an NAD(+)-regenerating system [pyruvate and lactate dehydrogenase (EC 1.1.1.28)] 2-enoyl- and 3-hydroxyacyl-CoA esters were generated, suggesting that re-oxidation of NADH is essential for optimal rates of peroxisomal beta-oxidation in vitro. 4. At high concentrations of [U-14C]hexadecanoate (greater than 100 microM), 3-oxohexadecanoyl-CoA was produced, suggesting that thiolase (acetyl-CoA acetyltransferase; EC 2.3.1.9) can become rate-limiting for peroxisomal beta-oxidation. Images Fig. 2. Fig. 3. Fig. 4. PMID:2396977

Cykotine mRNA expression in mouse retina after laser injury by reverse transcriptase-polymerase chain reaction (RT-PCR)

NASA Astrophysics Data System (ADS)

Schuschereba, Steven T.; Bowman, Phillip D.; Ujimore, Veronica; Hoxie, Stephen W.; Pizarro, Jose M.; Cross, Michael E.; Lund, David J.

1996-04-01

The purpose of this study was to identify cytokines produced by the retina after laser injury. With the aid of a scanning laser ophthalmoscope (SLO), right eyes of mice received lesions from a continuous wave argon laser. Left eyes served as unirradiated controls. At 2, 4, 6, 12, 24, and 48 hr after laser irradiation groups of 3 mice were euthanized and retinas fixed for histology or isolated for RNA. Messenger RNA (mRNA) was reverse-transcribed into complementary DNA (cDNA) and subjected to polymerase chain reaction for the following cytokines: tumor necrosis factor-(alpha) (TNF-(alpha) ), interleukin-1(alpha) /(Beta) (IL- 1(alpha) /(Beta) ), interleukin-6 (IL-6), transforming growth factor-(Beta) 1 (TGF- (Beta) 1), macrophage colony stimulating factor (M-CSF), inducible nitric oxide synthase (iNOS), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH). Histologically, lesions were confined to the photoreceptors, retinal pigment epithelium, and choroid. In laser-injured retinas, mRNA levels were elevated for IL-1(alpha) , TGF-(Beta) 1, iNOS, and G3PDH, but not TNF-(alpha) , IL-1(Beta) , or IL-6. It appears that the retina, in response to laser injury, upregulates a select number of cytokines in a time-course dependent fashion.

Alkyl gallates, intensifiers of beta-lactam susceptibility in methicillin-resistant Staphylococcus aureus.

PubMed

Shibata, Hirofumi; Kondo, Kyoko; Katsuyama, Ryo; Kawazoe, Kazuyoshi; Sato, Yoichi; Murakami, Kotaro; Takaishi, Yoshihisa; Arakaki, Naokatu; Higuti, Tomihiko

2005-02-01

We found that ethyl gallate purified from a dried pod of tara (Caesalpinia spinosa) intensified beta-lactam susceptibility in methicillin-resistant and methicillin-sensitive strains of Staphylococcus aureus (MRSA and MSSA strains, respectively). This compound and several known alkyl gallates were tested with MRSA and MSSA strains to gain new insights into their structural functions in relation to antimicrobial and beta-lactam susceptibility-intensifying activities. The maximum activity of alkyl gallates against MRSA and MSSA strains occurred at 1-nonyl and 1-decyl gallate, with an MIC at which 90% of the isolates tested were inhibited of 15.6 microg/ml. At concentrations lower than the MIC, alkyl gallates synergistically elevated the susceptibility of MRSA and MSSA strains to beta-lactam antibiotics. Such a synergistic activity of the alkyl gallates appears to be specific for beta-lactam antibiotics, because no significant changes were observed in the MICs of other classes of antibiotics examined in this study. The length of the alkyl chain was also associated with the modifying activity of the alkyl gallates, and the optimum length was C5 to C6. The present work clearly demonstrates that the length of the alkyl chain has a key role in the elevation of susceptibility to beta-lactam antibiotics.

Investigating the principles of recrystallization from glyceride melts.

PubMed

Windbergs, Maike; Strachan, Clare J; Kleinebudde, Peter

2009-01-01

Different lipids were melted and resolidified as model systems to gain deeper insight into the principles of recrystallization processes in lipid-based dosage forms. Solid-state characterization was performed on the samples with differential scanning calorimetry and X-ray powder diffraction. Several recrystallization processes could be identified during storage of the lipid layers. Pure triglycerides that generally crystallize to the metastable alpha-form from the melt followed by a recrystallization process to the stable beta-form with time showed a chain-length-dependent behavior during storage. With increasing chain length, the recrystallization to the stable beta-form was decelerated. Partial glycerides exhibited a more complex recrystallization behavior due to the fact that these substances are less homogenous. Mixtures of a long-chain triglyceride and a partial glyceride showed evidence of some interaction between the two components as the partial glyceride hindered the recrystallization of the triglyceride to the stable beta-form. In addition, the extent of this phenomenon depended on the amount of partial glyceride in the mixture. Based on these results, changes in solid dosage forms based on glycerides during processing and storage can be better understood.

Localization of functional receptor epitopes on the structure of ciliary neurotrophic factor indicates a conserved, function-related epitope topography among helical cytokines.

PubMed

Panayotatos, N; Radziejewska, E; Acheson, A; Somogyi, R; Thadani, A; Hendrickson, W A; McDonald, N Q

1995-06-09

By rational mutagenesis, receptor-specific functional analysis, and visualization of complex formation in solution, we identified individual amino acid side chains involved specifically in the interaction of ciliary neurotrophic factor (CNTF) with CNTFR alpha and not with the beta-components, gp130 and LIFR. In the crystal structure, the side chains of these residues, which are located in helix A, the AB loop, helix B, and helix D, are surface accessible and are clustered in space, thus constituting an epitope for CNTFR alpha. By the same analysis, a partial epitope for gp130 was also identified on the surface of helix A that faces away from the alpha-epitope. Superposition of the CNTF and growth hormone structures showed that the location of these epitopes on CNTF is analogous to the location of the first and second receptor epitopes on the surface of growth hormone. Further comparison with proposed binding sites for alpha- and beta-receptors on interleukin-6 and leukemia inhibitory factor indicated that this epitope topology is conserved among helical cytokines. In each case, epitope I is utilized by the specificity-conferring component, whereas epitopes II and III are used by accessory components. Thus, in addition to a common fold, helical cytokines share a conserved order of receptor epitopes that is function related.

Interaction between two discontiguous chain segments from the beta-sheet of Escherichia coli thioredoxin suggests an initiation site for folding.

PubMed

Tasayco, M L; Fuchs, J; Yang, X M; Dyalram, D; Georgescu, R E

2000-09-05

The approach of comparing folding and folding/binding processes is exquisitely poised to narrow down the regions of the sequence that drive protein folding. We have dissected the small single alpha/beta domain of oxidized Escherichia coli thioredoxin (Trx) into three complementary fragments (N, residues 1-37; M, residues 38-73; and C, residues 74-108) to study them in isolation and upon recombination by far-UV CD and NMR spectroscopy. The isolated fragments show a minimum of ellipticity of ca. 197 nm in their far-UV CD spectra without concentration dependence, chemical shifts of H(alpha) that are close to the random coil values, and no medium- and long-range NOE connectivities in their three-dimensional NMR spectra. These fragments behave as disordered monomers. Only the far-UV CD spectra of binary or ternary mixtures that contain N- and C-fragments are different from the sum of their individual spectra, which is indicative of folding and/or binding of these fragments. Indeed, the cross-peaks corresponding to the rather hydrophobic beta(2) and beta(4) regions of the beta-sheet of Trx disappear from the (1)H-(15)N HSQC spectra of isolated labeled N- and C-fragments, respectively, upon addition of the unlabeled complementary fragments. The disappearing cross-peaks indicate interactions between the beta(2) and beta(4) regions, and their reappearance at lower temperatures indicates unfolding and/or dissociation of heteromers that are predominantly held by hydrophobic forces. Our results argue that the folding of Trx begins by zippering two discontiguous and rather hydrophobic chain segments (beta(2) and beta(4)) corresponding to neighboring strands of the native beta-sheet.

The spatial distribution of fixed mutations within genes coding for proteins

NASA Technical Reports Server (NTRS)

Holmquist, R.; Goodman, M.; Conroy, T.; Czelusniak, J.

1983-01-01

An examination has been conducted of the extensive amino acid sequence data now available for five protein families - the alpha crystallin A chain, myoglobin, alpha and beta hemoglobin, and the cytochromes c - with the goal of estimating the true spatial distribution of base substitutions within genes that code for proteins. In every case the commonly used Poisson density failed to even approximate the experimental pattern of base substitution. For the 87 species of beta hemoglobin examined, for example, the probability that the observed results were from a Poisson process was the minuscule 10 to the -44th. Analogous results were obtained for the other functional families. All the data were reasonably, but not perfectly, described by the negative binomial density. In particular, most of the data were described by one of the very simple limiting forms of this density, the geometric density. The implications of this for evolutionary inference are discussed. It is evident that most estimates of total base substitutions between genes are badly in need of revision.

Biochemistry of snake venom neurotoxins and their application to the study of synapse. [Neurotoxins isolated from venom of the Formosan banded krait

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hanley, M.R.

1978-11-01

The crude venom of the Formosan banded krait, Bungarus multicinctus, was separated into eleven lethal protein fractions. Nine fractions were purified to final homogeneous toxins, designated ..cap alpha..-bungarotoxin, ..beta..-bungarotoxin, and toxins 7, 8, 9A, 11, 12, 13, and 14. Three of the toxins, ..cap alpha..-bungarotoxin, 7, and 8, were identified as post-synaptic curarimimetic neurotoxins. The remaining toxins were identified as pre-synaptic neurotoxins. ..cap alpha..-Bungarotoxin, toxin 7, and toxin 8 are all highly stable basic polypeptides of approx. 8000 daltons molecular weight. The pre-synaptic toxins fell into two structural groups: toxin 9A and 14 which were single basic chains of approx.more » 14,000 daltons, and ..beta..-bungarotoxin, and toxins 11 thru 13 which were composed of two chains of approx. 8000 and approx. 13,000 daltons covalently linked by disulfides. All the pre-synaptic neurotoxins were shown to have intrinsic calcium-dependent phospholipase A activities. Under certain conditions, intact synaptic membranes were hydrolyzed more rapidly than protein-free extracted synaptic-lipid liposomes which, in turn, were hydrolyzed more rapidly than any other tested liposomes. It was speculated that cell-surface arrays of phosphatidyl serine/glycolipids created high affinity target sites for ..beta..-bungarotoxin. Single-chain toxins were found to be qualitatively different from the two-chain toxins in their ability to block the functioning of acetylcholine receptors, and were quantitatively different in their enzymatic and membrane disruptive activities. ..beta..-Bungarotoxin was shown to be an extremely potent neuronal lesioning agent. There was no apparent selectivity for cholinergic over non-cholinergic neurons, nor for nerve terminals over cell bodies. It was suggested that ..beta..-bungarotoxin can be considered a useful new histological tool, which may exhibit some regional selectivity.« less

Natural polypeptide scaffolds: beta-sheets, beta-turns, and beta-hairpins.

PubMed

Rotondi, Kenneth S; Gierasch, Lila M

2006-01-01

This paper provides an introduction to fundamental conformational states of polypeptides in the beta-region of phi,psi space, in which the backbone is extended near to its maximal length, and to more complex architectures in which extended segments are linked by turns and loops. There are several variants on these conformations, and they comprise versatile scaffolds for presentation of side chains and backbone amides for molecular recognition and designed catalysts. In addition, the geometry of these fundamental folds can be readily mimicked in peptidomimetics. Copyright 2005 Wiley Periodicals, Inc.

Molecular analysis of abnormal hemoglobins in beta chain in Aegean region of Turkey and first reports of hemoglobin Andrew-Minneapolis and Hb Hinsdale from Turkey.

PubMed

Aykut, Ayça; Onay, Hüseyin; Durmaz, Asude; Karaca, Emin; Vergin, Canan; Aydınok, Yeşim; Özkınay, Ferda

2015-07-01

The Agean is one of the regions in Turkey where thalassemias and abnormal hemoglobins (Hbs) are prevalent. Combined heterozygosity of thalassemia mutations with a variety of structural Hb variants lead to an extremely wide spectrum of clinical and hematological phenotypes which is of importance for prenatal diagnosis. One hundred and seventeen patients and carriers diagnosed by hemoglobin electrophoresis (HPLC), at risk for abnormal hemoglobinopathies were screened for mutational analysis of the beta-globin gene. The full coding the 5' UTR, and the 3' UTR sequences of beta-globin gene (GenBank accession no. U01317) were amplified and sequenced. In this study, a total of 118 (12.24%) structural Hb variant alleles were identified in 1341 mutated beta-chain alleles in Medical Genetics Department of Ege University between January 2006 and November 2013. Here, we report the mutation spectrum of abnormal Hbs associated with the beta-globin gene in Aegean region of Turkey. In the present study, the Hb Hinsdale and Hb Andrew-Minneapolis variants are demonstrated for the first time in the Turkish population.

Secondary reduction of alpha7B integrin in laminin alpha2 deficient congenital muscular dystrophy supports an additional transmembrane link in skeletal muscle.

PubMed

Cohn, R D; Mayer, U; Saher, G; Herrmann, R; van der Flier, A; Sonnenberg, A; Sorokin, L; Voit, T

1999-03-01

The integrins are a large family of heterodimeric transmembrane cellular receptors which mediate the association between the extracellular matrix (ECM) and cytoskeletal proteins. The alpha7beta1 integrin is a major laminin binding integrin in skeletal and cardiac muscle and is thought to be involved in myogenic differentiation and migration processes. The main binding partners of the alpha7 integrin are laminin-1 (alpha1-beta1-gamma1), laminin-2 (alpha2-beta1-gamma1) and laminin-4 (alpha2-beta2-gamma1). Targeted deletion of the gene for the alpha7 integrin subunit (ITGA7) in mice leads to a novel form of muscular dystrophy. In the present study we have investigated the expression of two alternative splice variants, the alpha7B and beta1D integrin subunits, in normal human skeletal muscle, as well as in various forms of muscular dystrophy. In normal human skeletal muscle the expression of the alpha7 integrin subunit appeared to be developmentally regulated: it was first detected at 2 years of age. In contrast, the beta1D integrin could be detected in immature and mature muscle in the sarcolemma of normal fetal skeletal muscle at 18 weeks gestation. The expression of alpha7B integrin was significantly reduced at the sarcolemma in six patients with laminin alpha2 chain deficient congenital muscular dystrophy (CMD) (age >2 years). However, this reduction was not correlated with the amount of laminin alpha2 chain expressed. In contrast, the expression of the laminin alpha2 chain was not altered in the skeletal muscle of the alpha7 knock-out mice. These data argue in favor that there is not a tight correlation between the expression of the alpha7 integrin subunit and that of the laminin alpha2 chain in either human or murine dystrophic muscle. Interestingly, in dystrophinopathies (Duchenne and Becker muscular dystrophy; DMD/BMD) expression of alpha7B was upregulated irrespective of the level of dystrophin expression as shown by a strong sarcolemmal staining pattern even in young boys (age <2 years). The expression of the beta1D integrin subunit was not altered in any of our patients with different types of muscular dystrophy. In contrast, sarcolemmal expression of beta1D integrin was significantly reduced in the alpha7 integrin knock-out mice, whereas the expression of the components of the DGC was not altered. The secondary loss of alpha7B in laminin alpha2 chain deficiency defines a biochemical change in the composition of the plasma membrane resulting from a primary protein deficiency in the basal lamina. These findings, in addition to the occurrence of a muscular dystrophy in alpha7 deficient mice, implies that the alpha7B integrin is an important laminin receptor within the plasma membrane which plays a significant role in skeletal muscle function and stability.

Hamiltonian Markov Chain Monte Carlo Methods for the CUORE Neutrinoless Double Beta Decay Sensitivity

NASA Astrophysics Data System (ADS)

Graham, Eleanor; Cuore Collaboration

2017-09-01

The CUORE experiment is a large-scale bolometric detector seeking to observe the never-before-seen process of neutrinoless double beta decay. Predictions for CUORE's sensitivity to neutrinoless double beta decay allow for an understanding of the half-life ranges that the detector can probe, and also to evaluate the relative importance of different detector parameters. Currently, CUORE uses a Bayesian analysis based in BAT, which uses Metropolis-Hastings Markov Chain Monte Carlo, for its sensitivity studies. My work evaluates the viability and potential improvements of switching the Bayesian analysis to Hamiltonian Monte Carlo, realized through the program Stan and its Morpho interface. I demonstrate that the BAT study can be successfully recreated in Stan, and perform a detailed comparison between the results and computation times of the two methods.

Differences among the cell wall galactomannans from Aspergillus wentii and Chaetosartorya chrysella and that of Aspergillus fumigatus.

PubMed

Gómez-Miranda, Begoña; Prieto, Alicia; Leal, Juan Antonio; Ahrazem, Oussama; Jiménez-Barbero, Jesús; Bernabé, Manuel

2004-01-01

The alkali extractable and water-soluble cell wall polysaccharides F1SS from Aspergillus wentii and Chaetosartorya chrysella have been studied by methylation analysis, 1D- and 2D-NMR, and MALDI-TOF analysis. Their structures are almost identical, corresponding to the following repeating unit: [--> 3)-beta-D-Gal f -(1 --> 5)-beta-D-Gal f-(1 -->]n --> mannan core. The structure of this galactofuranose side chain differs from that found in the pathogenic fungus Aspergillus fumigatus, in other Aspergillii and members of Trichocomaceae: [--> 5)-beta-D-Gal f-(1 -->]n --> mannan core. The mannan cores have also been investigated, and are constituted by a (1 --> 6)-alpha-mannan backbone, substituted at positions 2 by chains from 1 to 7 residues of (1 --> 2) linked alpha-mannopyranoses. Copyright 2004 Kluwer Academic Publishers

Insights into Strand Exchange in BTB Domain Dimers from the Crystal Structures of FAZF and Miz1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stogios, Peter J.; Cuesta-Seijo, Jose Antonio; Chen, Lu

2010-09-22

The BTB domain is a widely distributed protein-protein interaction motif that is often found at the N-terminus of zinc finger transcription factors. Previous crystal structures of BTB domains have revealed tightly interwound homodimers, with the N-terminus from one chain forming a two-stranded anti-parallel {beta}-sheet with a strand from the other chain. We have solved the crystal structures of the BTB domains from Fanconi anemia zinc finger (FAZF) and Miz1 (Myc-interacting zinc finger 1) to resolutions of 2.0 {angstrom} and 2.6 {angstrom}, respectively. Unlike previous examples of BTB domain structures, the FAZF BTB domain is a nonswapped dimer, with each N-terminalmore » {beta}-strand associated with its own chain. As a result, the dimerization interface in the FAZF BTB domain is about half as large as in the domain-swapped dimers. The Miz1 BTB domain resembles a typical swapped BTB dimer, although it has a shorter N-terminus that is not able to form the interchain sheet. Using cysteine cross-linking, we confirmed that the promyelocytic leukemia zinc finger (PLZF) BTB dimer is strand exchanged in solution, while the FAZF BTB dimer is not. A phylogenic tree of the BTB fold based on both sequence and structural features shows that the common ancestor of the BTB domain in BTB-ZF (bric a brac, tramtrack, broad-complex zinc finger) proteins was a domain-swapped dimer. The differences in the N-termini seen in the FAZF and Miz1 BTB domains appear to be more recent developments in the structural evolution of the domain.« less

Efficient recombinant expression and secretion of a thermostable GH26 mannan endo-1,4-beta-mannosidase from Bacillus licheniformis in Escherichia coli.

PubMed

Songsiriritthigul, Chomphunuch; Buranabanyat, Bancha; Haltrich, Dietmar; Yamabhai, Montarop

2010-04-11

Mannans are one of the key polymers in hemicellulose, a major component of lignocellulose. The Mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannanase (EC 3.2.1.78), commonly named beta-mannanase, is an enzyme that can catalyze random hydrolysis of beta-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans. The enzyme has found a number of applications in different industries, including food, feed, pharmaceutical, pulp/paper industries, as well as gas well stimulation and pretreatment of lignocellulosic biomass for the production of second generation biofuel. Bacillus licheniformis is a Gram-positive endospore-forming microorganism that is generally non-pathogenic and has been used extensively for large-scale industrial production of various enzymes; however, there has been no previous report on the cloning and expression of mannan endo-1,4-beta-mannosidase gene (manB) from B. licheniformis. The mannan endo-1,4-beta-mannosidase gene (manB), commonly known as beta-mannanase, from Bacillus licheniformis strain DSM13 was cloned and overexpressed in Escherichia coli. The enzyme can be harvested from the cell lysate, periplasmic extract, or culture supernatant when using the pFLAG expression system. A total activity of approximately 50,000 units could be obtained from 1-l shake flask cultures. The recombinant enzyme was 6 x His-tagged at its C-terminus, and could be purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The specific activity of the purified enzyme when using locust bean gum as substrate was 1672 +/- 96 units/mg. The optimal pH of the enzyme was between pH 6.0 - 7.0; whereas the optimal temperature was at 50 - 60 degrees C. The recombinant beta-mannanase was stable within pH 5 - 12 after incubation for 30 min at 50 degrees C, and within pH 6 - 9 after incubation at 50 degrees C for 24 h. The enzyme was stable at temperatures up to 50 degrees C with a half-life time of activity (tau1/2) of approximately 80 h at 50 degrees C and pH 6.0. Analysis of hydrolytic products by thin layer chromatography revealed that the main products from the bioconversion of locus bean gum and mannan were various manno-oligosaccharide products (M2 - M6) and mannose. Our study demonstrates an efficient expression and secretion system for the production of a relatively thermo- and alkali-stable recombinant beta-mannanase from B. licheniformis strain DSM13, suitable for various biotechnological applications.

Identification and quantification of metabolites common to 17alpha-methyltestosterone and mestanolone in horse urine.

PubMed

Yamada, Masayuki; Aramaki, Sugako; Okayasu, Toshimasa; Hosoe, Tomoo; Kurosawa, Masahiko; Kijima-Suda, Isao; Saito, Koichi; Nakazawa, Hiroyuki

2007-09-21

Anabolic steroids with the 17alpha-methyl,17beta-hydroxyl group, which were developed as oral formulations for therapeutic purposes, have been abused in the field of human sports. These anabolic steroids are also used to enhance racing performance in racehorses. In humans, structurally related 17alpha-methyltestosterone (MTS) and mestanolone (MSL), which are anabolic steroids with the 17alpha-methyl,17beta-hydroxyl group, have metabolites in common. The purpose of this study was to determine metabolites common to these two steroids in horses, which may serve as readily available screening targets for the doping test of these steroids in racehorses. Urine sample collected after administering MTS and MSL to horses was treated to obtain unconjugated steroid, glucuronide, and sulfate fractions. The fractions were subjected to gas chromatography/mass spectrometry (GC/MS), and 17alpha-methyl-5alpha-androstan-3beta,17beta-diol, 17alpha-hydroxymethyl-5alpha-androstan-3beta,17beta-diol, 17alpha-methyl-5alpha-androstan-3beta,16beta,17beta-triol, and 17alpha-methyl-5alpha-androstan-3beta,16alpha,17beta-triol were detected as the common metabolites by comparison with synthesized reference standards. The urinary concentrations of these metabolites after dosing were determined by GC/MS. 17Alpha-methyl-5alpha-androstan-3beta,16beta,17beta-triol was mainly detected in the sulfate fractions of urine samples after administration. This compound was consistently detected for the longest time in the urine samples after dosing with both steroids. The results suggest that 17alpha-methyl-5alpha-androstan-3beta,16beta,17beta-triol is a very useful screening target for the doping test of MTS and MSL in racehorses.

Determining Beta Sheet Crystallinity in Fibrous Proteins by Thermal Analysis and Infrared Spectroscopy

NASA Astrophysics Data System (ADS)

Hu, Xiao; Kaplan, David; Cebe, Peggy

2007-03-01

We report a study of self-assembled beta pleated sheets in Bombyx mori silk fibroin films using thermal analysis and infrared spectroscopy. Crystallization of beta pleated sheets was effected either by heating the films above the glass transition temperature (Tg) and holding isothermally, or by exposure to methanol. The fractions of secondary structural components including random coils, alpha helices, beta pleated sheets, turns, and side chains, were evaluated using Fourier self-deconvolution (FSD) of the infrared absorbance spectra. As crystalline beta sheets form, the heat capacity increment from the TMDSC trace at Tg is systematically decreased and is linearly well correlated with beta sheet content determined from FSD. This analysis of beta sheet content can serve as an alternative to X-ray methods and may have wide applicability to other crystalline beta sheet forming proteins.

Murine Ia-associated invariant chain's processing to complex oligosaccharide forms and its dissociation from the I-Ak complex.

PubMed

Holt, G D; Swiedler, S J; Freed, J H; Hart, G W

1985-07-01

The processing of murine invariant chain (Ii) to a cell surface form bearing complex N-linked oligosaccharides has been demonstrated in the B cell lymphoma, AKTB-1b. In addition, the rate of processing of pulse-labeled Ii has been determined relative to its rate of dissociation from the alpha/beta complex of I-Ak. Ii, alpha-, and beta-chains were immunoprecipitated with anti-I-Ak or anti-Ii monoclonal antibodies. The heretofore uncharacterized complex oligosaccharide form of Ii (Ii-c) was identified in gel-purified immunoprecipitates by peptide mapping with reverse-phase HPLC. Ii-c is resistant to deglycosylation by Endo H, which is specific for high-mannose N-linkages, but can be digested with Endo F, a glycosidase capable of cleaving both complex and high-mannose N-linked oligosaccharides. Immunoprecipitation of surface iodinated cells indicates that Ii-c is expressed on the plasma membrane. Pulse-chase metabolic labeling data show that the processing of Ii to Ii-c occurs with a t1/2 of about 120 min. In contrast, the processing of both alpha- and beta-chains of I-Ak to complex forms occurs with a t1/2 of 15 to 20 min. Our data show that Ii-hm begins to dissociate rapidly from the I-Ak complex after 100 to 120 min of chase. Only a small amount (less than 5% on a per mole basis) of Ii-c was found associated with the I-Ak complexes after 300 min of continuous metabolic labeling. These results are consistent with Ii serving as a carrier for Ia antigens as they are transported to the cell surface. In addition, they suggest that the processing of Ii to Ii-c, or a late processing event of the alpha- and beta-chains, such as their sialylation, may be a possible mechanism for inducing the dissociation of Ii from the I-Ak complex.

Lewis type 1 antigen synthase (beta3Gal-T5) is transcriptionally regulated by homeoproteins.

PubMed

Isshiki, Soichiro; Kudo, Takashi; Nishihara, Shoko; Ikehara, Yuzuru; Togayachi, Akira; Furuya, Akiko; Shitara, Kenya; Kubota, Tetsuro; Watanabe, Masahiko; Kitajima, Masaki; Narimatsu, Hisashi

2003-09-19

The type 1 carbohydrate chain, Galbeta1-3GlcNAc, is synthesized by UDP-galactose:beta-N-acetylglucosamine beta1,3-galactosyltransferase (beta3Gal-T). Among six beta3Gal-Ts cloned to date, beta3Gal-T5 is an essential enzyme for the synthesis of type 1 chain in epithelium of digestive tracts or pancreatic tissue. It forms the type 1 structure on glycoproteins produced from such tissues. In the present study, we found that the transcriptional regulation of the beta3Gal-T5 gene is controlled by homeoproteins, i.e. members of caudal-related homeobox protein (Cdx) and hepatocyte nuclear factor (HNF) families. We found an important region (-151 to -121 from the transcription initiation site), named the beta3Gal-T5 control element (GCE), for the promoter activity. GCE contained the consensus sequences for members of the Cdx and HNF families. Mutations introduced into this sequence abolished the transcriptional activity. Four factors, Cdx1, Cdx2, HNF1alpha, and HNF1beta, could bind to GCE and transcriptionally activate the beta3Gal-T5 gene. Transcriptional regulation of the beta3Gal-T5 gene was consistent with that of members of the Cdx and HNF1 families in two in vivo systems. 1) During in vitro differentiation of Caco-2 cells, transcriptional up-regulation of beta3Gal-T5 was observed in correlation with the increase in transcripts for Cdx2 and HNF1alpha. 2) Both transcript and protein levels of beta3Gal-T5 were determined to be significantly reduced in colon cancer. This down-regulation was correlated with the decrease of Cdx1 and HNF1beta expression in cancer tissue. This is the first finding that a glycosyltransferase gene is transcriptionally regulated under the control of homeoproteins in a tissue-specific manner. beta3Gal-T5, controlled by the intestinal homeoproteins, may play an important role in the specific function of intestinal cells by modifying the carbohydrate structure of glycoproteins.

Clostridium perfringens enterotoxin is a superantigen reactive with human T cell receptors V beta 6.9 and V beta 22

PubMed Central

1992-01-01

Candidate superantigens were screened for their ability to induce lysis of human histocompatibility leukocyte antigen class II-positive targets by human CD8+ influenza-specific cytotoxic T cell (CTL) lines. Clostridium perfringens enterotoxin (CPET) induced major histocompatibility complex unrestricted killing by some but not all CTL lines. Using "anchored" polymerase chain reactions, CPET was shown to selectively stimulate peripheral blood lymphocytes bearing T cell receptor V beta 6.9 and V beta 22 in five healthy donors. V beta 24, V beta 21, V beta 18, V beta 5, and V beta 6.1-5 appeared to be weakly stimulated. Antigen processing was not required for CPET to induce proliferation. Like the staphylococcal enterotoxins, CPET is a major cause of food poisoning. These data suggest that superantigenic and enterotoxigenic properties may be closely linked. PMID:1512551

Laboratory investigation of hemoglobinopathies and thalassemias: review and update.

PubMed

Clarke, G M; Higgins, T N

2000-08-01

Structural hemoglobin (Hb) variants typically are based on a point mutation in a globin gene that produce a single amino acid substitution in a globin chain. Although most are of limited clinical significance, a few important subtypes have been identified with some frequency. Homozygous Hb C and Hb S (sickle cell disease) produce significant clinical manifestations, whereas Hb E and Hb D homozygotes may be mildly symptomatic. Although heterozygotes for these variants are typically asymptomatic, diagnosis may be important for genetic counseling. Thalassemia, in contrast, results from quantitative reductions in globin chain synthesis. Those with diminished beta-globin chains are termed beta-thalassemias, whereas those with decreased alpha-chain production are called alpha-thalassemias. Severity of clinical manifestations in these disorders relates to the amount of globin chain produced and the stability of residual chains present in excess. The thalassemia minor syndromes are characterized clinically by mild anemia with persistent microcytosis. Thalassemia intermedia (i.e., Hb H disease) is typified by a moderate, variably compensated hemolytic anemia that may present with clinical symptoms during a period of physiologic stress such as infection, pregnancy, or surgery. The thalassemia major syndromes produce severe, life-threatening anemia. alpha-Thalassemia major usually is incompatible with extrauterine life; beta-thalassemia major presents in infancy and requires life-long transfusion therapy and/or bone marrow transplantation for successful control of the disease. Double heterozygosity for certain structural variants and/or thalassemia syndromes may also lead to severe clinical disease. Several guidelines have been published that outline the required steps for hemoglobinopathy and thalassemia investigation. The availability of HPLC has streamlined many of these requirements, allowing an efficient stepwise diagnostic strategy for these complex disorders.

The effect of smoking cessation pharmacotherapies on pancreatic beta cell function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woynillowicz, Amanda K.; Raha, Sandeep; Nicholson, Catherine J.

The goal of our study was to evaluate whether drugs currently used for smoking cessation (i.e., nicotine replacement therapy, varenicline [a partial agonist at nicotinic acetylcholine receptors (nAChR)] and bupropion [which acts in part as a nAChR antagonist]) can affect beta cell function and determine the mechanism(s) of this effect. INS-1E cells, a rat beta cell line, were treated with nicotine, varenicline and bupropion to determine their effects on beta cell function, mitochondrial electron transport chain enzyme activity and cellular/oxidative stress. Treatment of INS-1E cells with equimolar concentrations (1 μM) of three test compounds resulted in an ablation of normalmore » glucose-stimulated insulin secretion by the cells. This disruption of normal beta cell function was associated with mitochondrial dysfunction since all three compounds tested significantly decreased the activity of mitochondrial electron transport chain enzyme activity. These results raise the possibility that the currently available smoking cessation pharmacotherapies may also have adverse effects on beta cell function and thus glycemic control in vivo. Therefore whether or not the use of nicotine replacement therapy, varenicline and bupropion can cause endocrine changes which are consistent with impaired pancreatic function warrants further investigation. -- Highlights: ► Smoking cessation drugs have the potential to disrupt beta cell function in vitro. ► The effects of nicotine, varenicline and bupropion are similar. ► The impaired beta cell function is mediated by mitochondrial dysfunction. ► If similar effects are seen in vivo, these drugs may increase the risk of diabetes.« less

Integrins (alpha7beta1) in muscle function and survival. Disrupted expression in merosin-deficient congenital muscular dystrophy.

PubMed Central

Vachon, P H; Xu, H; Liu, L; Loechel, F; Hayashi, Y; Arahata, K; Reed, J C; Wewer, U M; Engvall, E

1997-01-01

Mutations in genes coding for dystrophin, for alpha, beta, gamma, and delta-sarcoglycans, or for the alpha2 chain of the basement membrane component merosin (laminin-2/4) cause various forms of muscular dystrophy. Analyses of integrins showed an abnormal expression and localization of alpha7beta1 isoforms in myofibers of merosin-deficient human patients and mice, but not in dystrophin-deficient or sarcoglycan-deficient humans and animals. It was shown previously that skeletal muscle fibers require merosin for survival and function (Vachon, P.H., F. Loechel, H. Xu, U.M. Wewer, and E. Engvall. 1996. J. Cell Biol. 134:1483-1497). Correction of merosin deficiency in vitro through cell transfection with the merosin alpha2 chain restored the normal localization of alpha7beta1D integrins as well as myotube survival. Overexpression of the apoptosis-suppressing molecule Bcl-2 also promoted the survival of merosin-deficient myotubes, but did not restore a normal expression of alpha7beta1D integrins. Blocking of beta1 integrins in normal myotubes induced apoptosis and severely reduced their survival. These findings (a) identify alpha7beta1D integrins as the de facto receptors for merosin in skeletal muscle; (b) indicate a merosin dependence for the accurate expression and membrane localization of alpha7beta1D integrins in myofibers; (c) provide a molecular basis for the critical role of merosin in myofiber survival; and (d) add new insights to the pathogenesis of neuromuscular disorders. PMID:9312189

Carbohydrate binding specificity of immobilized Psathyrella velutina lectin.

PubMed

Endo, T; Ohbayashi, H; Kanazawa, K; Kochibe, N; Kobata, A

1992-01-15

The carbohydrate binding specificity of Psathyrella velutina lectin (PVL) was thoroughly investigated by analyzing the behavior of various complex-type oligosaccharides and human milk oligosaccharides on a PVL-Affi-Gel 10 column. Basically, the lectin interacts with the nonreducing terminal beta-N-acetylglucosamine residue, but does not show any affinity for the nonreducing terminal N-acetylgalactosamine or N-acetylneuraminic acid residue. Substitution of the terminal N-acetylglucosamine residues of oligosaccharides by galactose completely abolishes their affinity to the column. GlcNAc beta 1----3Gal beta 1----4sorbitol binds to the column, but GlcNAc beta 1----6Gal beta 1----4sorbitol is only retarded in the column. The behavior of degalactosylated N-linked oligosaccharides is quite interesting. Although all degalactosylated monoantennary sugar chain isomers are retarded in the column, those with the GlcNAc beta 1----2Man group interact more strongly with the column than those with the GlcNAc beta 1----4Man group or the GlcNAc beta 1----6Man group. The degalactosylated bi- and triantennary sugar chains bind to the column, but the tetraantennary ones are only retarded in the column. These results indicated that the binding affinity is not simply determined by the number of terminal N-acetylglucosamine residues. Addition of the bisecting N-acetylglucosamine residue reduces the affinity of oligosaccharides to the column, but addition of an alpha-fucosyl residue at the C-6 position of the proximal N-acetylglucosamine residue does not affect the behavior of oligosaccharides in the column. These results indicated that the binding specificity of PVL is quite different from those of other N-acetylglucosamine-binding lectins from higher plants, which interact preferentially with the GlcNAc beta 1----4 residue.

Structure-activity relationship studies of (+/-)-terbutaline and (+/-)-fenoterol on beta3-adrenoceptors in the guinea pig gastric fundus.

PubMed

Horinouchi, T; Nakagawa, Y; Wakabayashi, M; Koike, K

2001-08-01

(+/-)-Terbutaline and (+/-)-fenoterol are both arylethanolamine analogs that have tertbutyl and aryliso-propyl substituents respectively at the a position on the nitrogen of the ethanolamine side chain. In the present study, we have investigated the structure-activity relationships of (+/-)-terbutaline and (+/-)-fenoterol as beta3-adrenoceptor agonists in the guinea pig gastric fundus. (+/-)-Terbutaline and (+/-)-fenoterol induced concentration-dependent relaxation of the precontracted gastric fundus with pD2 values of 4.45+/-0.10 and 5.90+/-0.09, and intrinsic activities of 1.00+/-0.03 and 0.99+/-0.01 respectively. The combination of the selective beta1-adrenoceptor antagonist (+/-)-atenolol (100 microM), and the selective beta2-adrenoceptor antagonist (+/-)-butoxamine (100 microM), produced a 2 and 6 fold rightward shift of the concentration-response curves for (+/-)-terbutaline and (+/-)-fenoterol respectively, without depressing the maximal responses. The order of potency of these agonists was (pD2 value): (+/-)-fenoterol (5.09+/-0.10) > (+/-)-terbutaline (4.13+/-0.08). In the presence of (+/-)-atenolol and (+/-)-butoxamine, however, the non-selective beta1, beta2- and beta3-adrenoceptor antagonist (+/-)-bupranolol caused a concentration-dependent rightward shift of the concentration-response curves for (+/-)-terbutaline and (+/-)-fenoterol. Schild plot analyses of the effects of (+/-)-bupranolol against these agonists gave pA2 values of 6.21+/-0.07 ((+/-)-terbutaline) and 6.37+/-0.06 ((+/-)-fenoterol) respectively, and the slopes of the Schild plot were not significantly different from unity (p>0.05). These results suggest that the relaxant responses to (+/-)-terbutaline and (+/-)-fenoterol are mainly mediated through beta3-adrenoceptors in the guinea pig gastric fundus. The beta3-adrenoceptor agonist potencies of arylethanolamine analogs depend on the size of the end of the alkylamine side chain.

Crystal Structure of the Catalytic Domain of Drosophila [beta]1,4-Galactosyltransferase-7

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramakrishnan, Boopathy; Qasba, Pradman K.

2010-11-03

The {beta}1,4-galactosyltransferase-7 ({beta}4Gal-T7) enzyme, one of seven members of the {beta}4Gal-T family, transfers in the presence of manganese Gal from UDP-Gal to an acceptor sugar (xylose) that is attached to a side chain hydroxyl group of Ser/Thr residues of proteoglycan proteins. It exhibits the least protein sequence similarity with the other family members, including the well studied family member {beta}4Gal-T1, which, in the presence of manganese, transfers Gal from UDP-Gal to GlcNAc. We report here the crystal structure of the catalytic domain of {beta}4Gal-T7 from Drosophila in the presence of manganese and UDP at 1.81 {angstrom} resolution. In the crystalmore » structure, a new manganese ion-binding motif (HXH) has been observed. Superposition of the crystal structures of {beta}4Gal-T7 and {beta}4Gal-T1 shows that the catalytic pocket and the substrate-binding sites in these proteins are similar. Compared with GlcNAc, xylose has a hydroxyl group (instead of an N-acetyl group) at C2 and lacks the CH{sub 2}OH group at C5; thus, these protein structures show significant differences in their acceptor-binding site. Modeling of xylose in the acceptor-binding site of the {beta}4Gal-T7 crystal structure shows that the aromatic side chain of Tyr{sup 177} interacts strongly with the C5 atom of xylose, causing steric hindrance to any additional group at C5. Because Drosophila Cd7 has a 73% protein sequence similarity to human Cd7, the present crystal structure offers a structure-based explanation for the mutations in human Cd7 that have been linked to Ehlers-Danlos syndrome.« less

The effect of TGF-beta2 on MMP-2 production and activity in highly metastatic human bladder carcinoma cell line 5637.

PubMed

Dehnavi, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram; Ataei, Zahra; Aryan, Hajar

2009-06-01

Transforming growth factor-beta (TGF-beta) superfamily regulates matrix metalloproteinases (MMP), which intrinsically regulate various cell behaviors leading to metastasis. We investigated the effect of TGF-beta(2) on MMP-2 regulation in human bladder carcinoma cell line 5637. Zymography, ELISA, and real-time polymerase chain reaction revealed that TGF-beta(2) stimulated MMP-2 production, but the transcription of its gene remained unchanged. Wortmannin could not inhibit MMP-2 secretion and activity and conversely the amount of the protein and its enzymatic activity were increased. These data suggest that TGF-beta(2) increased MMP-2 at the posttranscriptional level and this upregulation was independent of phosphatidylinositol 3-kinase signaling pathway.

Three exopolysaccharides of the beta-(1-->6)-D-glucan type and a beta-(1-->3;1-->6)-D-glucan produced by strains of Botryosphaeria rhodina isolated from rotting tropical fruit.

PubMed

Vasconcelos, Ana Flora D; Monteiro, Nilson K; Dekker, Robert F H; Barbosa, Aneli M; Carbonero, Elaine R; Silveira, Joana L M; Sassaki, Guilherme L; da Silva, Roberto; de Lourdes Corradi da Silva, Maria

2008-09-22

Four exopolysaccharides (EPS) obtained from Botryosphaeria rhodina strains isolated from rotting tropical fruit (graviola, mango, pinha, and orange) grown on sucrose were purified on Sepharose CL-4B. Total acid hydrolysis of each EPS yielded only glucose. Data from methylation analysis and (13)C NMR spectroscopy indicated that the EPS from the graviola isolate consisted of a main chain of glucopyranosyl (1-->3) linkages substituted at O-6 as shown in the putative structure below: [carbohydrate structure: see text]. The EPS of the other fungal isolates consisted of a linear chain of (1-->6)-linked glucopyranosyl residues of the following structure: [carbohydrate structure: see text]. FTIR spectra showed one band at 891 cm(-1), and (13)C NMR spectroscopy showed that all glucosidic linkages were of the beta-configuration. Dye-inclusion studies with Congo Red indicated that each EPS existed in a triple-helix conformational state. beta-(1-->6)-d-Glucans produced as exocellular polysaccharides by fungi are uncommon.

Comparative characterization of short monomeric polyglutamine peptides by replica exchange molecular dynamics simulation.

PubMed

Nakano, Miki; Watanabe, Hirofumi; Rothstein, Stuart M; Tanaka, Shigenori

2010-05-27

Polyglutamine (polyQ) diseases are caused by an abnormal expansion of CAG repeats. While their detailed structure remains unclear, polyQ peptides assume beta-sheet structures when they aggregate. To investigate the conformational ensemble of short, monomeric polyQ peptides, which consist of 15 glutamine residues (Q(15)), we performed replica exchange molecular dynamics (REMD) simulations. We found that Q(15) can assume multiple configurations due to all of the residues affecting the formation of side-chain hydrogen bonds. Analysis of the free energy landscape reveals that Q(15) has a basin for random-coil structures and another for alpha-helix or beta-turn structures. To investigate properties of aggregated polyQ peptides, we performed multiple molecular dynamics (MMD) simulations for monomeric and oligomeric Q(15). MMD revealed that the formation of oligomers stabilizes the beta-turn structure by increasing the number of hydrogen bonds between the main chains.

Structural characterization of novel L-galactose-containing oligosaccharide subunits of jojoba seed xyloglucans.

PubMed

Hantus, S; Pauly, M; Darvill, A G; Albersheim, P; York, W S

1997-10-28

Jojoba seed xyloglucan was shown to be a convenient source of biologically active xyloglucan oligosaccharides that contain both L- and D-galactosyl residues [E. Zablackis et al., Science, 272 (1996) 1808-1810]. Oligosaccharides were isolated by liquid chromatography of the mixture of oligosaccharides generated by treating jojoba seed xyloglucan with a beta-(1-->4)-endoglucanase. The purified oligosaccharides were reduced with NaBH4, converting them to oligoglycosyl alditol derivatives that were structurally characterized by a combination of mass spectrometry and 2-dimensional NMR spectroscopy. This analysis established that jojoba xyloglucan oligosaccharides contain the novel side-chain [alpha-L-Gal p-(1-->2)-beta-D-Galp-(1-->2)-alpha-D-Xyl p-(1-->6)-], which is structurally homologous to the fucose-containing side-chain [alpha-L-Fucp-(1-->2)-beta-D-Galp-(1-->2)-alpha-D-Xyl p-(1-->6)-] found in other biologically active xyloglucan oligosaccharides.

Enzymatic preparation of. cap alpha. - and. beta. -deuterated or tritiated amino acids with l-methionine. gamma. -lyase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Esaki, N.; Sawada, S.; Tanaka, H.

L-Methionine ..gamma..-lyase catalyzes the exchange of ..cap alpha..- and ..beta..-hydrogens of L-methionine and S-methyl-L-cysteine with deuterium or tritium of solvents. The rate of ..cap alpha..-hydrogen exchange with deuterium was about 40 times faster than that of the elimination reactions. The deuterium and tritium were exchanged also with the ..cap alpha..- and ..beta..-hydrogens of the straight-chain amino acids which do not undergo the elimination: L-alanine, L-..cap alpha..-aminobutyrate, L-norvaline, and L-norleucine. No exchange occurs for the D-isomers, acidic L-amino acids, basic L-amino acids, and branched-chain L-amino acids, although ..cap alpha..-hydrogen of glycine, L-trypotophan, and L-phenylalanine is exchanged slowly. These enzymatic hydrogen-exchange reactionsmore » facilitate specific labeling of the L-amino acids with deuterium and tritium.« less

A Tc-99m-labeled long chain fatty acid derivative for myocardial imaging.

PubMed

Magata, Yasuhiro; Kawaguchi, Takayoshi; Ukon, Misa; Yamamura, Norio; Uehara, Tomoya; Ogawa, Kazuma; Arano, Yasushi; Temma, Takashi; Mukai, Takahiro; Tadamura, Eiji; Saji, Hideo

2004-01-01

C-11- and I-123-labeled long chain fatty acid derivatives have been reported as useful radiopharmaceuticals for the estimation of myocardial fatty acid metabolism. We have reported that Tc-99m-labeled N-[[[(2-mercaptoethyl)amino]carbonyl]methyl]-N-(2-mercaptoethyl)-6-aminohexanoic acid ([(99m)Tc]MAMA-HA), a medium chain fatty acid derivative, is metabolized by beta-oxidation in the liver and that the MAMA ligand is useful for attaching to the omega-position of fatty acid derivatives as a chelating group for Tc-99m. On the basis of these findings, we focused on developing a Tc-99m-labeled long chain fatty acid derivative that reflected fatty acid metabolism in the myocardium. In this study, we synthesized a dodecanoic acid derivative, MAMA-DA, and a hexadecanoic acid derivative, MAMA-HDA, and performed radiolabeling and biodistribution studies. [(99m)Tc]MAMA-DA and [(99m)Tc]MAMA-HDA were prepared using a ligand-exchange reaction. Biodistribution studies were carried out in normal mice and rats. Then, a high initial uptake of Tc-99m was observed, followed by a rapid clearance from the heart. The maximum heart/blood ratio was 3.6 at 2 min postinjection of [(99m)Tc]MAMA-HDA. These kinetics were similar to those with postinjection of p-[(125)I]iodophenylpentadecanoic acid. Metabolite analysis showed [(99m)Tc]MAMA-HDA was metabolized by beta-oxidation in the body. In conclusion, [(99m)Tc]MAMA-HDA is a promising compound as a long chain fatty acid analogue for estimating beta-oxidation of fatty acid in the heart.

Hb taradale [beta82(EF6)Lys-->Arg]: a novel mutation at a 2,3-diphosphoglycerate binding site.

PubMed

Brennan, Stephen O; Sheen, Campbell; Chan, Tim; George, Peter M

2005-01-01

Hb Taradale [beta82(EF6)Lys-->Arg] was initially detected as a split Hb A0 peak on Hb A1c, monitoring. Red cell parameters, hemoglobin (Hb) electrophoresis and stability tests were normal. Mass spectrometry (ms) clearly identified a variant beta chain with a mass increase of 28 Da and peptide mapping located the mutation site to peptide betaT-9. DNA sequencing confirmed the presence of a novel beta82(EF6)Lys-->Arg mutation. This conservative substitution at a 2,3-diphosphoglycerate (2,3-DPG) binding site did not, however, appear to affect the P50 for oxygen binding.

Tubulin Beta-3 Chain as a New Candidate Protein Biomarker of Human Skin Aging: A Preliminary Study

PubMed Central

2017-01-01

Skin aging is a complex process, and a lot of efforts have been made to identify new and specific targets that could help to diagnose, prevent, and treat skin aging. Several studies concerning skin aging have analyzed the changes in gene expression, and very few investigations have been performed at the protein level. Moreover, none of these proteomic studies has used a global quantitative labeled proteomic offgel approach that allows a more accurate description of aging phenotype. We applied such an approach on human primary keratinocytes obtained from sun-nonexposed skin biopsies of young and elderly women. A total of 517 unique proteins were identified, and 58 proteins were significantly differentially expressed with 40 that were downregulated and 18 upregulated with aging. Gene ontology and pathway analysis performed on these 58 putative biomarkers of skin aging evidenced that these dysregulated proteins were mostly involved in metabolism and cellular processes such as cell cycle and signaling pathways. Change of expression of tubulin beta-3 chain was confirmed by western blot on samples originated from several donors. Thus, this study suggested the tubulin beta-3 chain has a promising biomarker in skin aging. PMID:28626498

Spontaneous healing and growth of locked magnetic island chains in toroidal plasmas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fitzpatrick, R.; Waelbroeck, F. L.

2012-11-15

Recent experiments have demonstrated that locked magnetic island chains in stellarator plasmas spontaneously heal under certain conditions, and spontaneously grow under others. A formalism initially developed to study magnetic island dynamics in tokamak plasmas is employed to investigate this phenomenon. It is found that island healing/growth transitions can be caused either by a breakdown in torque balance in the vicinity of the island chain, or by an imbalance between the various terms in the island width evolution equation. The scaling of the healing/growth thresholds with the standard dimensionless plasma parameters {beta}, {nu}{sub *}, and {rho}{sub *} is determined. In accordancemore » with the experimental data, it is found that island healing generally occurs at high {beta} and low {nu}{sub *}, and island growth at low {beta} and high {nu}{sub *}. In further agreement, it is found that island healing is accompanied an ion poloidal velocity shift in the electron diamagnetic direction, and island growth by a velocity shift in the ion diamagnetic direction. Finally, it is found that there is considerable hysteresis in the healing/growth cycle, as is also seen experimentally.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Driscoll, P.C.; Gronenborn, A.M.; Beress, L.

The three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata has been determined on the basis of 489 interproton and 24 hydrogen-bonding distance restraints supplemented by 23 {phi} backbone and 21 {sub {chi}1} side-chain torsion angle restraints derived from nuclear magnetic resonance (NMR) measurements. A total of 42 structures is calculated by a hybrid metric matrix distance geometry-dynamical simulated annealing approach. Both the backbone and side-chain atom positions are well defined. The average atomic rms difference between the 42 individual SA structures and the mean structure obtained by averaging their coordinates is 0.67more » {plus minus} 0.12 {angstrom} for the backbone atoms and 0.90 {plus minus} 0.17 {angstrom} for all atoms. The core of the protein is formed by a triple-stranded antiparallel {beta}-sheet composed of residues 14-16 (strand 1), 30-34 (strand 2), and 37-41 (strand 3) with an additional mini-antiparallel {beta}-sheet at the N-terminus (residues 6-9). The first and second strands of the triple-stranded antiparallel {beta}-sheet are connected by a long exposed loop. A number of side-chain interactions are discussed in light of the structure.« less

Tubulin Beta-3 Chain as a New Candidate Protein Biomarker of Human Skin Aging: A Preliminary Study.

PubMed

Lehmann, Sylvia G; Bourgoin-Voillard, Sandrine; Seve, Michel; Rachidi, Walid

2017-01-01

Skin aging is a complex process, and a lot of efforts have been made to identify new and specific targets that could help to diagnose, prevent, and treat skin aging. Several studies concerning skin aging have analyzed the changes in gene expression, and very few investigations have been performed at the protein level. Moreover, none of these proteomic studies has used a global quantitative labeled proteomic offgel approach that allows a more accurate description of aging phenotype. We applied such an approach on human primary keratinocytes obtained from sun-nonexposed skin biopsies of young and elderly women. A total of 517 unique proteins were identified, and 58 proteins were significantly differentially expressed with 40 that were downregulated and 18 upregulated with aging. Gene ontology and pathway analysis performed on these 58 putative biomarkers of skin aging evidenced that these dysregulated proteins were mostly involved in metabolism and cellular processes such as cell cycle and signaling pathways. Change of expression of tubulin beta-3 chain was confirmed by western blot on samples originated from several donors. Thus, this study suggested the tubulin beta-3 chain has a promising biomarker in skin aging.

Study of a large Anglo-Saxon family with beta-thalassaemia trait.

PubMed

Raik, E; Powell, E; Gordon, S

1976-01-01

Study of a large Anglo-Saxon family with beta-thalassaemia trait revealed evidence of consanguinity, moreover both branches of the family shared a Spanish ancestor. The manifestations of the disorder were varied in severity and yet the degree of severity appeared to breed true within any individual part of the family. Our explanation for the inheritance pattern observed in the family was to postulate the existence of two non-allelic genes influencing the rate of beta-chain synthesis.

Differentiating Amino Acid Residues and Side Chain Orientations in Peptides Using Scanning Tunneling Microscopy

PubMed Central

Claridge, Shelley A.; Thomas, John C.; Silverman, Miles A.; Schwartz, Jeffrey J.; Yang, Yanlian; Wang, Chen; Weiss, Paul S.

2014-01-01

Single-molecule measurements of complex biological structures such as proteins are an attractive route for determining structures of the large number of important biomolecules that have proved refractory to analysis through standard techniques such as X-ray crystallography and nuclear magnetic resonance. We use a custom-built low-current scanning tunneling microscope to image peptide structure at the single-molecule scale in a model peptide that forms β sheets, a structural motif common in protein misfolding diseases. We successfully differentiate between histidine and alanine amino acid residues, and further differentiate side chain orientations in individual histidine residues, by correlating features in scanning tunneling microscope images with those in energy-optimized models. Beta sheets containing histidine residues are used as a model system due to the role histidine plays in transition metal binding associated with amyloid oligomerization in Alzheimer’s and other diseases. Such measurements are a first step toward analyzing peptide and protein structures at the single-molecule level. PMID:24219245

Problem-Solving Test: The Mechanism of Protein Synthesis

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2009-01-01

Terms to be familiar with before you start to solve the test: protein synthesis, ribosomes, amino acids, peptides, peptide bond, polypeptide chain, N- and C-terminus, hemoglobin, [alpha]- and [beta]-globin chains, radioactive labeling, [[to the third power]H] and [[to the fourteenth power]C]leucine, cytosol, differential centrifugation, density…

Harnessing the theoretical foundations of the exponential and beta-Poisson dose-response models to quantify parameter uncertainty using Markov Chain Monte Carlo.

PubMed

Schmidt, Philip J; Pintar, Katarina D M; Fazil, Aamir M; Topp, Edward

2013-09-01

Dose-response models are the essential link between exposure assessment and computed risk values in quantitative microbial risk assessment, yet the uncertainty that is inherent to computed risks because the dose-response model parameters are estimated using limited epidemiological data is rarely quantified. Second-order risk characterization approaches incorporating uncertainty in dose-response model parameters can provide more complete information to decisionmakers by separating variability and uncertainty to quantify the uncertainty in computed risks. Therefore, the objective of this work is to develop procedures to sample from posterior distributions describing uncertainty in the parameters of exponential and beta-Poisson dose-response models using Bayes's theorem and Markov Chain Monte Carlo (in OpenBUGS). The theoretical origins of the beta-Poisson dose-response model are used to identify a decomposed version of the model that enables Bayesian analysis without the need to evaluate Kummer confluent hypergeometric functions. Herein, it is also established that the beta distribution in the beta-Poisson dose-response model cannot address variation among individual pathogens, criteria to validate use of the conventional approximation to the beta-Poisson model are proposed, and simple algorithms to evaluate actual beta-Poisson probabilities of infection are investigated. The developed MCMC procedures are applied to analysis of a case study data set, and it is demonstrated that an important region of the posterior distribution of the beta-Poisson dose-response model parameters is attributable to the absence of low-dose data. This region includes beta-Poisson models for which the conventional approximation is especially invalid and in which many beta distributions have an extreme shape with questionable plausibility. © Her Majesty the Queen in Right of Canada 2013. Reproduced with the permission of the Minister of the Public Health Agency of Canada.

Genetic basis of human complement C8[beta] deficiency

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaufmann, T.; Rittner, C.; Schneider, P.M.

1993-06-01

The eighth component of human complement (c8) is a serum protein consisting of three chains ([alpha], [beta], and [gamma]) and encoded by three different genes, C8A, C8B, and C8G. C8A and C8B are closely linked on chromosome 1p, whereas C8G is located on chromosome 9q. In the serum the [beta] subunit is non-covalently bound to the disulfide-linked [alpha]-[gamma] subunit. Patients with C8[beta] deficiency suffer from recurrent neisserial infections such as meningitis. Exon-specific polymerase chain reaction (PCR) amplification with primer pairs from the flanking intron sequences was used to amplify all 12 C8B exons separately. No difference regarding the exon sizesmore » was observed in a C8[beta]-deficient patient compared with a normal person. Therefore, direct sequence analysis of all exon-specific PCR products from normal and C8[beta]-deficient individuals was carried out. As a cause for C8[beta] deficiency, we found a single C-T exchange in exon 9 leading to a stop codon. An allele-specific PCR system was designed to detect the normal and the deficiency allele simultaneously. Using this approach as well as PCR typing of the Taql polymorphism located in intron 11, five families with 7 C8[beta]-deficient members were investigated. The mutation was not found to be restricted to one of the two Taql RFLP alleles. The mutant allele was observed in all families investigated and can therefore be regarded as a major cause of C8[beta] deficiency in the Caucasian population. In addition, two C8[beta]-deficient patients were found to be heterozygous for the C-T exchange. The molecular basis of the alleles without this point mutation also causing deficiency has not yet been defined. 23 refs., 4 figs., 3 tabs.« less

TGF{beta}1 polymorphisms and late clinical radiosensitivity in patients treated for gynecologic tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ruyck, Kim de; Van Eijkeren, Marc; Claes, Kathleen

2006-07-15

Purpose: To investigate the association between six transforming growth factor {beta}1 gene (TGF{beta}1) polymorphisms (-1.552delAGG, -800G>A, -509C>T, Leu10Pro, Arg25Pro, Thr263Ile) and the occurrence of late normal tissue reactions after gynecologic radiotherapy (RT). Methods and Materials: Seventy-eight women with cervical or endometrial cancer and 140 control individuals were included in the study. According to the Common Terminology Criteria for Adverse Events version 3.0 (CTCAEv3.0) scale, 25 patients showed late adverse RT reactions (CTC2+), of whom 11 had severe complications (CTC3+). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), single base extension and genotyping assays were performed to examine the polymorphic sites inmore » TGF{beta}1. Results: Homozygous variant -1.552delAGG, -509TT, and 10Pro genotypes were associated with the risk of developing late severe RT reactions. Triple (variant) homozygous patients had a 3.6 times increased risk to develop severe RT reactions (p = 0.26). Neither the -800A allele, nor the 25Pro allele or the 263Ile allele were associated with clinical radiosensitivity. There was perfect linkage disequilibrium (LD) between the -1.552delAGG and the -509C>T polymorphisms, and tight LD between the -1.552/-509 and the Leu10Pro polymorphisms. Haplotype analysis revealed two major haplotypes but could not distinguish radiosensitive from nonradiosensitive patients. Conclusions: The present study shows that homozygous variant TGF{beta}1 -1.552delAGG, -509TT, and 10Pro genotypes may be associated with severe clinical radiosensitivity after gynecologic RT.« less

"Fast" and "slow" skeleto-fusimotor innervation in cat tenuissimus spindles; a study with the glycogen-depletion method.

PubMed

Jami, L; Lan-Couton, D; Malmgren, K; Petit, J

1978-07-01

The glycogen-depletion method was used to investigate the motor supply to tenuissimus with respect to the presence of fast beta axons and to assess the total proportion of both fast and slow beta-innervated spindles in this muscle. In a first series of 5 expts., groups of motor axons with conduction velocities higher than 85 m/s were repetitively stimulated so as to produce glycogen depletion in the muscle fibres they innervated. The whole muscle was then quick-frozen, serially cut, stained to demonstrate glycogen and examined for intrafusal glycogen depletion. Zones of glycogen depletion were found in 16 of the 46 examined spindles; they were most frequently located in the longest of the chain intrafusal muscle fibres. Since it is known that there are no purely fusimotor axons to tenuissimus with conduction velocities above 50 m/s, it was concluded that beta axons are present among the fastest axons to this muscle. In a second series of 5 expts. as many motor axons as possible with conduction velocities above 60 m/s were stimulated. Zones of glycogen depletion were found in 19 of the 47 examined spindles. They affected chain fibres in about half of the instances and bag1 fibers in the others. As this latter location is characteristic of slow dynamic beta axons, it was concluded that both slow and fast beta axons occur regularly in the motor supply to tenuissimus. beta-innervation is present in at least 40% of tenuissimus spindles with almost no convergence of fast and slow beta axons onto the same spindle.

Effects of high-fat diets on hepatic fatty acid oxidation in the rat. Isolation of rat liver peroxisomes by vertical-rotor centrifugation by using a self-generated, iso-osmotic, Percoll gradient.

PubMed Central

Neat, C E; Thomassen, M S; Osmundsen, H

1981-01-01

1. Rat liver peroxisomal fractions were isolated in iso-osmotic Percoll gradients by using vertical-rotor centrifugation. The fractions obtained with rats given various dietary treatments were characterized. 2. The effect on peroxisomal beta-oxidation of feeding 15% by wt. of dietary fat for 3 weeks was investigated. High-fat diets caused induction of peroxisomal beta-oxidation, but diets rich in very-long-chain mono-unsaturated fatty acids produced a more marked induction. 3. Peroxisomal beta-oxidation induced by diets rich in very-long-chain mono-unsaturated fatty acids can oxidize such acids. Trans-isomers of mono-unsaturated fatty acids are oxidized at rates that are faster than, or similar to, those obtained with corresponding cis-isomers. 4. Rates of oxidation of [14-14C]erucic acid by isolated rat hepatocytes isolated from rats fed on high-fat diets increased with the time on those diets in a fashion very similar to that previously reported for peroxisomal beta-oxidation [see Neat, Thomassen & Osmundsen (1980) Biochem, J. 186, 369-371]. 5. Total liver capacities for peroxisomal beta-oxidation (expressed as acetyl groups produced per min) were estimated to range from 10 to 30% of mitochondrial capacities, depending on dietary treatment and fatty acid substrate. A role is proposed for peroxisomal beta-oxidation in relation to the metabolism of fatty acids that are poorly oxidized by mitochondrial beta-oxidation, and, in general, as regards oxidation of fatty acids during periods of sustained high hepatic influx of fatty acids. PMID:6272750

Synthesis and evaluation of N-alkyl-beta-D-glucosylamines on the growth of two wood fungi, Coriolus versicolor and Poria placenta.

PubMed

Muhizi, Théoneste; Coma, Véronique; Grelier, Stéphane

2008-09-22

Various glucosylamines were synthesized from glucose and different alkyl amine compounds. These amino compounds are beta-D-glucopyranosylamine (GPA), N-ethyl-beta-D-glucopyranosylamine (EtGPA), N-butyl-beta-D-glucopyranosylamine (BuGPA), N-hexyl-beta-D-glucopyranosylamine (HeGPA), N-octyl-beta-D-glucopyranosylamine (OcGPA), N-dodecyl-beta-D-glucopyranosylamine (DoGPA), N-(2-hydroxyethyl)-beta-D-glucopyranosylamine (HEtGPA) and N,N-di(2-hydroxyethyl)-beta-D-glucopyranosylamine (DHEtGPA). They were tested for their antifungal activity against the growth of Coriolus versicolor and Poria placenta. An improvement of the antifungal activity with the increase of alkyl chain length was observed. DoGPA exhibited the best antifungal activity against both strains. It completely inhibited the fungal growth at 0.01x10(-3)molmL(-1) and 0.0075x10(-3)molmL(-1) for C. versicolor and P. placenta, respectively. For other glucosylamines higher concentrations were needed for complete inhibition of fungi.

[Characterization of microstructure of ibuprofen-hydroxypropyl-beta-cyclodextrin and ibuprofen-beta-cyclodextrin by atomic force microscope].

PubMed

Wang, Li-juan; Zhu, Zhao-jing; Che, Ke-ke; Ju, Feng-ge

2008-09-01

The microstructures of ibuprofen-hydroxypropyl-bets-cyclodextrin (IBU-HP-beta-CyD) and ibuprofen-beta-cyclodextrin (IBU-beta-CyD) were observed by atomic force microscope (AFM). The high resolving capability of AFM has the tungsten filament probe with the spring constant of 0.06 N x m(-1). Samples were observed in a small scale scanning area of 10.5 nm x 10.5 nm and 800 x 800 pixels. The original scanning images were gained by tapping mode at room temperature. Their three-dimensional reconstruction of microstructure was performed by G3DR software. The outer diameters of HP-beta-CyD and beta-CyD are 1.53 nm. The benzene diameter of IBU is 0.62 nm, fitting to the inner diameters of HP-beta-CyD and beta-CyD. The benzene and hydrophobic chain of IBU enter into the hole of cyclodextrin at 1:1 ratio. The results were evidenced by IR, X-ray diffraction and the phase solubility.

Limits on uranium and thorium bulk content in GERDA Phase I detectors

NASA Astrophysics Data System (ADS)

Collaboration, Gerda; Agostini, M.; Allardt, M.; Bakalyarov, A. M.; Balata, M.; Barabanov, I.; Baudis, L.; Bauer, C.; Becerici-Schmidt, N.; Bellotti, E.; Belogurov, S.; Belyaev, S. T.; Benato, G.; Bettini, A.; Bezrukov, L.; Bode, T.; Borowicz, D.; Brudanin, V.; Brugnera, R.; Caldwell, A.; Cattadori, C.; Chernogorov, A.; D'Andrea, V.; Demidova, E. V.; di Vacri, A.; Domula, A.; Doroshkevich, E.; Egorov, V.; Falkenstein, R.; Fedorova, O.; Freund, K.; Frodyma, N.; Gangapshev, A.; Garfagnini, A.; Grabmayr, P.; Gurentsov, V.; Gusev, K.; Hakemüller, J.; Hegai, A.; Heisel, M.; Hemmer, S.; Hofmann, W.; Hult, M.; Inzhechik, L. V.; Janicskó Csáthy, J.; Jochum, J.; Junker, M.; Kazalov, V.; Kihm, T.; Kirpichnikov, I. V.; Kirsch, A.; Kish, A.; Klimenko, A.; Kneißl, R.; Knöpfle, K. T.; Kochetov, O.; Kornoukhov, V. N.; Kuzminov, V. V.; Laubenstein, M.; Lazzaro, A.; Lebedev, V. I.; Lehnert, B.; Liao, H. Y.; Lindner, M.; Lippi, I.; Lubashevskiy, A.; Lubsandorzhiev, B.; Lutter, G.; Macolino, C.; Majorovits, B.; Maneschg, W.; Medinaceli, E.; Mingazheva, R.; Misiaszek, M.; Moseev, P.; Nemchenok, I.; Palioselitis, D.; Panas, K.; Pandola, L.; Pelczar, K.; Pullia, A.; Riboldi, S.; Rumyantseva, N.; Sada, C.; Salamida, F.; Salathe, M.; Schmitt, C.; Schneider, B.; Schönert, S.; Schreiner, J.; Schütz, A.-K.; Schulz, O.; Schwingenheuer, B.; Selivanenko, O.; Shevchik, E.; Shirchenko, M.; Simgen, H.; Smolnikov, A.; Stanco, L.; Stepaniuk, M.; Vanhoefer, L.; Vasenko, A. A.; Veresnikova, A.; von Sturm, K.; Wagner, V.; Walter, M.; Wegmann, A.; Wester, T.; Wiesinger, C.; Wojcik, M.; Yanovich, E.; Zhitnikov, I.; Zhukov, S. V.; Zinatulina, D.; Zuber, K.; Zuzel, G.

2017-05-01

Internal contaminations of 238U, 235U and 232Th in the bulk of high purity germanium detectors are potential backgrounds for experiments searching for neutrinoless double beta decay of 76Ge. The data from GERDA Phase I have been analyzed for alpha events from the decay chain of these contaminations by looking for full decay chains and for time correlations between successive decays in the same detector. No candidate events for a full chain have been found. Upper limits on the activities in the range of a few nBq/kg for 226Ra, 227Ac and 228Th, the long-lived daughter nuclides of 238U, 235U and 232Th, respectively, have been derived. With these upper limits a background index in the energy region of interest from 226Ra and 228Th contamination is estimated which satisfies the prerequisites of a future ton scale germanium double beta decay experiment.

The core structure of ginsenan PA, a phagocytosis-activating polysaccharide from the root of Panax ginseng.

PubMed

Tomoda, M; Hirabayashi, K; Shimizu, N; Gonda, R; Ohara, N

1994-09-01

Controlled Smith degradation and limited hydrolysis of ginsenan PA, the main phagocytosis-activating polysaccharide isolated from the root of Panax ginseng C. A. Meyer, were performed. The reticuloendothelial system-potentiating and anti-complementary activities of the degradation products were investigated. Methylation analysis of the primary and secondary Smith degradation products indicated that the core structural features of ginsenan PA include a backbone chain mainly composed of beta-1,3-linked D-galactose. Almost half of the galactose units in the backbone carry side-chains composed of beta-1,6-linked D-galactosyl residues at position 6. Further 3,6-branching of D-galactose units was observed in a part of the side-chains. alpha-L-Arabinose units are connected mainly to the core galactose moieties via position 6. Removal of most of the arabinose units had a considerable effect on immunological activity.

Expression of TGF-beta1, osteonectin, and BMP-4 in mandibular distraction osteogenesis with compression stimulation: reverse transcriptase-polymerase chain reaction study and biomechanical test.

PubMed

Kim, Uk-Kyu; Park, Seong-Jin; Seong, Wook-Jin; Heo, Jun; Hwang, Dae-Seok; Kim, Yong-Deok; Shin, Sang-Hun; Kim, Gyoo-Cheon

2010-09-01

This study compared the levels of transforming growth factor-beta1 (TGF-beta1), osteonectin, and bone morphogenetic protein-4 (BMP-4) expression in regenerated bone in a rabbit mandible that had undergone conventional distraction osteogenesis (DO) with those in regenerated bone from a modified DO technique with compression stimulation. A total of 42 rabbits were used in this reverse transcriptase-polymerase chain reaction study. In the control group, distraction was performed at 1 mm/day for 8 days. In the experimental group, overdistraction was performed for 10 days, followed by a 3-day latency period and 2 days of compression to achieve the same amount of DO. Three rabbits per subgroup were killed at 0, 5, 13, 20, 27, 34, and 41 days after the initial osteotomy. The levels of TGF-beta1, osteonectin, and BMP-4 in the bone regenerates were measured by reverse transcriptase-polymerase chain reaction. A biomechanical microhardness test was also performed in 8 rabbits as a separate experiment. Reverse transcriptase-polymerase chain reaction revealed a greater level of TGF-beta1 in the experimental group immediately after applying the compression force that continued for 2 weeks. The level then decreased to that of the control group at 3 weeks. The greater level of osteonectin in the experimental group after compression than that in the control group continued for 3 weeks. In the experimental group, the level of BMP-4 increased immediately after compression. However, the level in the control group decreased. The microhardness ratio of distracted bone to normal bone on the cortex was statistically different at 0.47 in the control group and 0.80 in the experimental group (P = .049) at 55 days after osteotomy. The effectiveness of the new DO technique with compression stimulation was confirmed by the gene expression study and the biomechanical test findings. Copyright 2010 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

Strong linkage disequilibrium of a HbE variant with the (AT)9(T)5 repeat in the BP1 binding site upstream of the beta-globin gene in the Thai population.

PubMed

Ohashi, Jun; Naka, Izumi; Patarapotikul, Jintana; Hananantachai, Hathairad; Brittenham, Gary; Looareesuwan, Sornchai; Clark, Andrew G; Tokunaga, Katsushi

2005-01-01

A binding site for the repressor protein BP1, which contains a tandem (AT)x(T)y repeat, is located approximately 530 bp 5' to the human beta-globin gene (HBB). There is accumulating evidence that BP1 binds to the (AT)9(T)5 allele more strongly than to other alleles, thereby reducing the expression of HBB. In this study, we investigated polymorphisms in the (AT)x(T)y repeat in 57 individuals living in Thailand, including three homozygotes for the hemoglobin E variant (HbE; beta26Glu-->Lys), 22 heterozygotes, and 32 normal homozygotes. We found that (AT)9(T)5 and (AT)7(T)7 alleles were predominant in the studied population and that the HbE variant is in strong linkage disequilibrium with the (AT)9(T)5 allele, which can explain why the betaE chain is inefficiently synthesized compared to the normal betaA chain. Moreover, the mildness of the HbE disease compared to other hemoglobinopathies in Thai may be due, in part, to the presence of the (AT)9(T)5 repeat on the HbE chromosome. In addition, a novel (AC)n polymorphism adjacent to the (AT)x(T)y repeat (i.e., (AC)3(AT)7(T)5) was found through the variation screening in this study.

Crystal structure and novel recognition motif of rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis.

PubMed

Han, S; Arvai, A S; Clancy, S B; Tainer, J A

2001-01-05

Clostridium botulinum C3 exoenzyme inactivates the small GTP-binding protein family Rho by ADP-ribosylating asparagine 41, which depolymerizes the actin cytoskeleton. C3 thus represents a major family of the bacterial toxins that transfer the ADP-ribose moiety of NAD to specific amino acids in acceptor proteins to modify key biological activities in eukaryotic cells, including protein synthesis, differentiation, transformation, and intracellular signaling. The 1.7 A resolution C3 exoenzyme structure establishes the conserved features of the core NAD-binding beta-sandwich fold with other ADP-ribosylating toxins despite little sequence conservation. Importantly, the central core of the C3 exoenzyme structure is distinguished by the absence of an active site loop observed in many other ADP-ribosylating toxins. Unlike the ADP-ribosylating toxins that possess the active site loop near the central core, the C3 exoenzyme replaces the active site loop with an alpha-helix, alpha3. Moreover, structural and sequence similarities with the catalytic domain of vegetative insecticidal protein 2 (VIP2), an actin ADP-ribosyltransferase, unexpectedly implicates two adjacent, protruding turns, which join beta5 and beta6 of the toxin core fold, as a novel recognition specificity motif for this newly defined toxin family. Turn 1 evidently positions the solvent-exposed, aromatic side-chain of Phe209 to interact with the hydrophobic region of Rho adjacent to its GTP-binding site. Turn 2 evidently both places the Gln212 side-chain for hydrogen bonding to recognize Rho Asn41 for nucleophilic attack on the anomeric carbon of NAD ribose and holds the key Glu214 catalytic side-chain in the adjacent catalytic pocket. This proposed bipartite ADP-ribosylating toxin turn-turn (ARTT) motif places the VIP2 and C3 toxin classes into a single ARTT family characterized by analogous target protein recognition via turn 1 aromatic and turn 2 hydrogen-bonding side-chain moieties. Turn 2 centrally anchors the catalytic Glu214 within the ARTT motif, and furthermore distinguishes the C3 toxin class by a conserved turn 2 Gln and the VIP2 binary toxin class by a conserved turn 2 Glu for appropriate target side-chain hydrogen-bonding recognition. Taken together, these structural results provide a molecular basis for understanding the coupled activity and recognition specificity for C3 and for the newly defined ARTT toxin family, which acts in the depolymerization of the actin cytoskeleton. This beta5 to beta6 region of the toxin fold represents an experimentally testable and potentially general recognition motif region for other ADP-ribosylating toxins that have a similar beta-structure framework. Copyright 2001 Academic Press.

Structural re-alignment in an immunologic surface region of ricin A chain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zemla, A T; Zhou, C E

2007-07-24

We compared structure alignments generated by several protein structure comparison programs to determine whether existing methods would satisfactorily align residues at a highly conserved position within an immunogenic loop in ribosome inactivating proteins (RIPs). Using default settings, structure alignments generated by several programs (CE, DaliLite, FATCAT, LGA, MAMMOTH, MATRAS, SHEBA, SSM) failed to align the respective conserved residues, although LGA reported correct residue-residue (R-R) correspondences when the beta-carbon (Cb) position was used as the point of reference in the alignment calculations. Further tests using variable points of reference indicated that points distal from the beta carbon along a vector connectingmore » the alpha and beta carbons yielded rigid structural alignments in which residues known to be highly conserved in RIPs were reported as corresponding residues in structural comparisons between ricin A chain, abrin-A, and other RIPs. Results suggest that approaches to structure alignment employing alternate point representations corresponding to side chain position may yield structure alignments that are more consistent with observed conservation of functional surface residues than do standard alignment programs, which apply uniform criteria for alignment (i.e., alpha carbon (Ca) as point of reference) along the entirety of the peptide chain. We present the results of tests that suggest the utility of allowing user-specified points of reference in generating alternate structural alignments, and we present a web server for automatically generating such alignments.« less

Medium-chain, triglyceride-containing lipid emulsions increase human neutrophil beta2 integrin expression, adhesion, and degranulation.

PubMed

Wanten, G J; Geijtenbeek, T B; Raymakers, R A; van Kooyk, Y; Roos, D; Jansen, J B; Naber, A H

2000-01-01

To test the hypothesis that lipid emulsions with different triglyceride structures have distinct immunomodulatory properties, we analyzed human neutrophil adhesion and degranulation after lipid incubation. Neutrophils, isolated from the blood of 10 healthy volunteers, were incubated in medium or physiologic (2.5 mmol/L) emulsions containing long-chain (LCT), medium-chain (MCT), mixed LCT/MCT, or structured (SL) triglycerides. Expression of adhesion molecules and degranulation markers was evaluated by flow cytometry. Also, functional adhesion was investigated by means of a flow cytometric assay using fluorescent beads coated with the integrin ligand intercellular adhesion molecule (ICAM)-1. Although LCT and SL had no effect, LCT/MCT significantly increased expression of the beta2 integrins lymphocyte-function-associated antigen 1 (+18%), macrophage antigen 1 (+387%), p150,95 (+82%), and (alphaDbeta2 (+230%). Degranulation marker expression for azurophilic (CD63, +210%) and specific granules (CD66b, +370%) also significantly increased, whereas L-selectin (CD62L, -70%) decreased. The effects of LCT/MCT were mimicked by the MCT emulsion. ICAM-1 adhesion (% beads bound) was increased by LCT/MCT (34% +/- 4%), whereas LCT (19% +/-3%) and SL (20% +/- 2%) had no effect compared with medium (17% +/- 3%). LCT/MCT and MCT, contrary to LCT and SL emulsions, increased neutrophil beta2 integrin expression, adhesion, and degranulation. Apart from other emulsion constituents, triglyceride chain length might therefore be a key feature in the interaction of lipid emulsions and the phagocyte immune system.

Micro-power dissipation device described

NASA Astrophysics Data System (ADS)

Mao, X.; Zhou, L.; Zhou, J.

1985-11-01

The common-emitter current gain beta of a common two-pole transistor is generally below 250. They are referred to as high-beta or high gain transistors when the beta of such transistors exceeds 300. When the beta of a transistor is higher than 1,000, it is called a super-beta transistor (SBT) or supergain transistor. The micropower dissipation type has the widest applications among the high-beta. Micropower dissipation high-beta means that there is a high gain or a superhigh gain under a microcurrent. The device is widely used in small signal-detection systems and stereo audio equipment because of their characteristics of high gain, low frequency and low noise under small signals.

K(2)MM'(3)Se(6) (M = Cu, Ag; M' = Ga, In), A new series of metal chalcogenides with chain-sublayer-chain slabs: (infinity)(1)[M'Se(4)]-(infinity)(2)[(MSe(4))(M'Se(4))]-(infinity)(1)[M'Se(4)].

PubMed

Ma, Hong-Wei; Guo, Guo-Cong; Wang, Ming-Sheng; Zhou, Guo-Wei; Lin, Shan-Hou; Dong, Zhen-Chao; Huang, Jin-Shun

2003-02-24

A new series of novel isostructural metal chalcogenides, K(2)CuIn(3)Se(6) (1), K(2)CuGa(3)Se(6) (2), and K(2)AgIn(3)Se(6) (3), were obtained by a reactive flux technique and structurally characterized. Compounds 1, 2, and 3 crystallize in the space group C2/c of the monoclinic system with eight formula units in a cell: a = 11.445(2) A, b = 11.495(2) A, c = 21.263(4) A, beta = 97.68(3) degrees, V = 2772(1) A(3), R1/wR2 = 0.0676/0.1652 for 1; a = 11.031(2) A, b = 11.050(4) A, c = 20.808(7) A, beta = 97.71(2) degrees, V = 2513(1) A(3), R1/wR2 = 0.0301/0.0511 for 2; and a = 11.633(1) A, b = 11.587(1) A, c = 21.355(1) A, beta = 98.010(8) degrees, V = 2850.4(4) A(3), R1/wR2 = 0.0471/0.0732 for 3. These isostructural compounds are characterized by a chain-sublayer-chain slab structure. The sublayer, composed of alternative corner-sharing mixed-metal tetrahedra, is sandwiched by parallel corner-sharing tetrahedral chains. Optical absorption spectra of compounds 1, 2, and 3 reveal the presence of a sharp optical gap of 1.68, 1.72, and 1.64 eV, respectively, suggesting that these materials are semiconductors and suitable for efficient absorption of solar radiation in solar cell applications. IR spectra show no obvious absorption in the range 800-4000 cm(-)(1).

Crystallographic studies of bovine beta2-microglobulin.

PubMed Central

Becker, J W; Ziffer, J A; Edelman, G M; Cunningham, B A

1977-01-01

Crystals of the bovine milk protein lactollin yield x-ray diffraction data extending to a resolution of 2.8 A. Lactollin is a bovine analogue of beta2-microglobulin, a protein that is homologous in amino acid sequence to the constant domains of immunoglobulins and is the light chain of the human and murine major histocompatability antigens. The protein crystallizes in the orthorhombic space group P2(1)2(1)2(1) with a = 77.4, b = 47.9, and c = 34.3 A. The unit cell parameters and physical chemical solution studies indicate that the molecule exists in the crystal and in solution as a single polypeptide chain of 12,000 daltons. Images PMID:71731

Automated main-chain model building by template matching and iterative fragment extension.

PubMed

Terwilliger, Thomas C

2003-01-01

An algorithm for the automated macromolecular model building of polypeptide backbones is described. The procedure is hierarchical. In the initial stages, many overlapping polypeptide fragments are built. In subsequent stages, the fragments are extended and then connected. Identification of the locations of helical and beta-strand regions is carried out by FFT-based template matching. Fragment libraries of helices and beta-strands from refined protein structures are then positioned at the potential locations of helices and strands and the longest segments that fit the electron-density map are chosen. The helices and strands are then extended using fragment libraries consisting of sequences three amino acids long derived from refined protein structures. The resulting segments of polypeptide chain are then connected by choosing those which overlap at two or more C(alpha) positions. The fully automated procedure has been implemented in RESOLVE and is capable of model building at resolutions as low as 3.5 A. The algorithm is useful for building a preliminary main-chain model that can serve as a basis for refinement and side-chain addition.

In vitro biosynthesis of galactans by membrane-bound galactosyltransferase from radish ( Raphanus sativus L.) seedlings.

PubMed

Kato, Hideaki; Takeuchi, Yoshimi; Tsumuraya, Yoichi; Hashimoto, Yohichi; Nakano, Hirofumi; Kovác, Pavol

2003-06-01

We investigated a galactosyltransferase (GalT) involved in the synthesis of the carbohydrate portion of arabinogalactan-proteins (AGPs), which consist of a beta-(1-->3)-galactan backbone from which consecutive (1-->6)-linked beta-Gal p residues branch off. A membrane preparation from 6-day-old primary roots of radish ( Raphanus sativus L.) transferred [(14)C]Gal from UDP-[(14)C]Gal onto a beta-(1-->3)-galactan exogenous acceptor. The reaction occurred maximally at pH 5.9-6.3 and 30 degrees C in the presence of 15 mM Mn(2+) and 0.75% Triton X-100. The apparent K(m) and V(max) values for UDP-Gal were 0.41 mM and 1,000 pmol min(-1) (mg protein)(-1), respectively. The reaction with beta-(1-->3)-galactan showed a bi-phasic kinetic character with K(m) values of 0.43 and 2.8 mg ml(-1). beta-(1-->3)-Galactooligomers were good acceptors and enzyme activity increased with increasing polymerization of Gal residues. In contrast, the enzyme was less efficient on beta-(1-->6)-oligomers. The transfer reaction for an AGP from radish mature roots was negligible but could be increased by prior enzymatic or chemical removal of alpha- l-arabinofuranose (alpha- l-Ara f) residues or both alpha- l-Ara f residues and (1-->6)-linked beta-Gal side chains. Digestion of radiolabeled products formed from beta-(1-->3)-galactan and the modified AGP with exo-beta-(1-->3)-galactanase released mainly radioactive beta-(1-->6)-galactobiose, indicating that the transfer of [(14)C]Gal occurred preferentially onto consecutive (1-->3)-linked beta-Gal chains through beta-(1-->6)-linkages, resulting in the formation of single branching points. The enzyme produced mainly a branched tetrasaccharide, Galbeta(1-->3)[Galbeta(1-->6)] Galbeta(1-->3)Gal, from beta-(1-->3)-galactotriose by incubation with UDP-Gal, confirming the preferential formation of the branching linkage. Localization of the GalT in the Golgi apparatus was revealed on a sucrose density gradient. The membrane preparation also incorporated [(14)C]Gal into beta-(1-->4)-galactan, indicating that the membranes contained different types of GalT isoform catalyzing the synthesis of different types of galactosidic linkage.

Integrins beta 5, beta 3 and alpha v are apically distributed in endometrial epithelium.

PubMed

Aplin, J D; Spanswick, C; Behzad, F; Kimber, S J; Vićovac, L

1996-07-01

Several adhesion molecules have been shown to occur at the surface of endometrial cells. One of these is the integrin alpha v subunit which associates with various beta chains including beta 5. We demonstrate the presence of integrin beta 5 polypeptide in human endometrial epithelial cells throughout the menstrual cycle using immunocytochemistry with monospecific antibodies, and at the mRNA level by thermal amplification from endometrial cDNA. Integrin beta 5 is also found in a population of bone marrow-derived cells. A notable feature of the distribution of the beta 5 subunit in the glandular and luminal epithelium is its apical localization, which may suggest an involvement in implantation. However, no evidence was found for regulated expression of epithelial beta 5. In mouse, the beta 5 subunit is found at both the apical and basal surface of epithelial cells and expression is essentially oestrous cycle-independent. Comparisons are made in both species with the distribution of the alpha v and beta 3 subunits which also localize to the apical epithelium.

[Advances in research and application of beta-tricalcium phosphate, collagen and beta-tricalcium phosphate/collagen composite in bone tissue engineering].

PubMed

Han, Xiang-Yong; Fu, Yuan-Fei; Zhang, Fu-Qiang

2007-02-01

Bone defects in oral and maxillofacial region was a common problem. To repair the defect, bone grafts including autograft, allograft and artificial bone graft were used in clinic despite of their disadvantages. Nowadays, bone tissue engineering has become a commonly used method to repair bone defect. This paper reviewed the application of beta-TCP, collagen and beta-TCP/collagen composite in bone tissue engineering. It was concluded that beta-TCP/collagen composite was a promising materials in bone tissue engineering.

Insights into the phosphoregulation of beta-secretase sorting signal by the VHS domain of GGA1.

PubMed

Shiba, Tomoo; Kametaka, Satoshi; Kawasaki, Masato; Shibata, Masahiro; Waguri, Satoshi; Uchiyama, Yasuo; Wakatsuki, Soichi

2004-06-01

BACE (beta-site amyloid precursor protein cleaving enzyme, beta-secretase) is a type-I membrane protein which functions as an aspartic protease in the production of beta-amyloid peptide, a causative agent of Alzheimer's disease. Its cytoplasmic tail has a characteristic acidic-cluster dileucine motif recognized by the VHS domain of adaptor proteins, GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-interacting). Here we show that BACE is colocalized with GGAs in the trans-Golgi network and peripheral structures, and phosphorylation of a serine residue in the cytoplasmic tail enhances interaction with the VHS domain of GGA1 by about threefold. The X-ray crystal structure of the complex between the GGA1-VHS domain and the BACE C-terminal peptide illustrates a similar recognition mechanism as mannose 6-phosphate receptors except that a glutamine residue closes in to fill the gap created by the shorter BACE peptide. The serine and lysine of the BACE peptide point their side chains towards the solvent. However, phosphorylation of the serine affects the lysine side chain and the peptide backbone, resulting in one additional hydrogen bond and a stronger electrostatic interaction with the VHS domain, hence the reversible increase in affinity.

Digestion by fungal glycanases of arabinoxylans with different feruloylated side-chains.

PubMed

Wende, G; Fry, S C

1997-07-01

Alcohol-insoluble residues (AIRs) from Festuca and Zea cell cultures contained 7.4 and 35 nmol esterified ferulate mg-1, respectively. Driselase solubilised 79% of the feruloylated material from both AIRs. Of the feruloyl esters solubilised from Festuca and Zea AIRs, 72 and 56% respectively were small enough to be mobile on paper chromatography. The major feruloylated product of Zea AIR was the known 5-O-feruloyl-alpha-L-Araf-(1-->3)-beta-D-Xylp-(1-->4)- D-Xyl (Fer-Ara-Xyl-Xyl). In contrast, the smallest major feruloylated product of Festuca AIR was a feruloyl pentasaccharide (3) containing 3 Xyl, 1 Ara and 1 non-pentose residue (NPR). The Ara and two of the three Xyl groups of 3 were resistant to NaIO4. Mild acid hydrolysis of 3 gave xylobiose, a feruloyl trisaccharide and beta-D-Xylp-(1-->2)-(5-O-feruloyl)-L-Ara. Compound 3 was therefore NPR-(1-->3)-beta-D-Xylp-(1-->2)-(5-O-feruloyl)-alpha-L-Ar af-(1-->3)-beta-D-Xylp-(1-->4)-D-Xyl. We conclude that the complex feruloyl oligosaccharide side-chains of Festuca arabinoxylan do not protect the polysaccharide against hydrolysis by the fungal glycanases present in Driselase.

Extensive esterification of adrenal C19-delta 5-sex steroids to long-chain fatty acids in the ZR-75-1 human breast cancer cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poulin, R.; Poirier, D.; Merand, Y.

1989-06-05

Estrogen-sensitive human breast cancer cells (ZR-75-1) were incubated with the 3H-labeled adrenal C19-delta 5-steroids dehydroepiandrosterone (DHEA) and its fully estrogenic derivative, androst-5-ene-3 beta,17 beta-diol (delta 5-diol) for various time intervals. When fractionated by solvent partition, Sephadex LH-20 column chromatography and silica gel TLC, the labeled cell components were largely present (40-75%) in three highly nonpolar, lipoidal fractions. Mild alkaline hydrolysis of these lipoidal derivatives yielded either free 3H-labeled DHEA or delta 5-diol. The three lipoidal fractions cochromatographed with the synthetic DHEA 3 beta-esters, delta 5-diol 3 beta (or 17 beta)-monoesters and delta 5-diol 3 beta,17 beta-diesters of long-chain fatty acids.more » DHEA and delta 5-diol were mainly esterified to saturated and mono-unsaturated fatty acids. For delta 5-diol, the preferred site of esterification of the fatty acids is the 3 beta-position while some esterification also takes place at the 17 beta-position. Time course studies show that ZR-75-1 cells accumulate delta 5-diol mostly (greater than 95%) as fatty acid mono- and diesters while DHEA is converted to delta 5-diol essentially as the esterified form. Furthermore, while free C19-delta 5-steroids rapidly diffuse out of the cells after removal of the precursor (3H)delta 5-diol, the fatty acid ester derivatives are progressively hydrolyzed, and DHEA and delta 5-diol thus formed are then sulfurylated prior to their release into the culture medium. The latter process however is rate-limited, since new steady-state levels of free steroids and fatty acid esters are rapidly reached and maintained for extended periods of time after removal of precursor, thus maintaining minimal concentrations of intracellular steroids.« less

[Trofosides A and B and other cytostatic steroid-derived compounds from the Far East starfish Trofodiscus über].

PubMed

Levina, E V; Kalinovskiĭ, A I; Andriiashchenko, P V; Menzorova, N I; Dmitrenok, P S

2007-01-01

Three new polar steroids identified as trofoside A, (20R,24S)-24-O-(3-O-methyl-beta-D-xylopyranosyl)-3beta,6alpha,8,15beta,24-pentahydroxy-5alpha-cholestane, its 22(23)-dehydro derivative (trofoside B), and 15-sulfoxy-(20R,24S)-5alpha-cholestane-3beta,6beta,8,15alpha,24-pentaol sodium salt, were isolated from Trofodiscus uber starfish extracts collected in the Sea of Okhotsk. Two known compounds, trofoside A aglycone, (20R,24S)-3beta,6alpha,8,15beta,24-pentahydroxy-5alpha-cholestane, and triseramide, (20R,24R,25S,22E)-24-methyl-3beta,6alpha,8,15beta-tetrahydroxy-5alpha-cholest-22-en-27-oic acid (2-sulfoethyl)amide sodium salt, were also found. The structures of the isolated polyoxysteroids were established from their spectra. Minimal concentrations causing degradation of unfertilized egg-cells of the sea-urchin Strongylocentrotus intermedius (C(min)) and terminating the cell division at the stage of the first division (C(min) embr.), as well as the concentrations causing 50% immobilization of sperm cells (ImC50) and inhibiting their ability to fertilize egg-cells by 50% (IC50) were determined for the isolated compounds. Of three compounds highly toxic in embryos and sea-urchin sperm cells, the polyol with a sulfo group in the steroid core was the most active; two glycosides with monosaccharide chains located at C3 and C24 atoms were less toxic. Note that all the compounds with the spermiotoxic activities differently affected the embryo development. The positions of monosaccharide residues in the core considerably influence the compound activity. For example, both mono- and double chained glycosides with the monosaccharide fragment at C3 and C24 atoms are active against sea-urchin sperm cells and embryos, whereas the C24 glycosylated trofoside A does not affect embryos and displays a poor spermiotoxicity.

Evaluation of pharmacological induction of fatty acid beta-oxidation in X-linked adrenoleukodystrophy.

PubMed

McGuinness, M C; Zhang, H P; Smith, K D

2001-01-01

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurometabolic disorder associated with elevated levels of saturated unbranched very-long-chain fatty acids (VLCFA; C > 22:0) in plasma and tissues, and reduced VLCFA beta-oxidation in fibroblasts, white blood cells, and amniocytes from X-ALD patients. The X-ALD gene (ABCD1) at Xq28 encodes the adrenoleukodystrophy protein (ALDP) that is related to the peroxisomal ATP-binding cassette (ABCD) transmembrane half-transporter proteins. The function of ALDP is unknown and its role in VLCFA accumulation unresolved. Previously, our laboratory has shown that sodium 4-phenylbutyrate (4PBA) treatment of X-ALD fibroblasts results in increased peroxisomal VLCFA beta-oxidation activity and increased expression of the X-ALD-related protein, ALDRP, encoded by the ABCD2 gene. In this study, the effect of various pharmacological agents on VLCFA beta-oxidation in ALD mouse fibroblasts is tested. 4PBA, styrylacetate and benzyloxyacetate (structurally related to 4PBA), and trichostatin A (functionally related to 4PBA) increase both VLCFA (peroxisomal) and long-chain fatty acid [LCFA (peroxisomal and mitochondrial)] beta-oxidation. Isobutyrate, zaprinast, hydroxyurea, and 5-azacytidine had no effect on VLCFA or LCFA beta-oxidation. Lovastatin had no effect on fatty acid beta-oxidation under normal tissue culture conditions but did result in an increase in both VLCFA and LCFA beta-oxidation when ALD mouse fibroblasts were cultured in the absence of cholesterol. The effect of trichostatin A on peroxisomal VLCFA beta-oxidation is shown to be independent of an increase in ALDRP expression, suggesting that correction of the biochemical abnormality in X-ALD is not dependent on pharmacological induction of a redundant gene (ABCD2). These studies contribute to a better understanding of the role of ALDP in VLCFA accumulation and may lead to the development of more effective pharmacological therapies. Copyright 2001 Academic Press.

Quantification of antibody (IgY) titers in hen eggs following immunization and their use in detecting cell surface molecules on nitrocellulose membranes.

PubMed

Ruan, Guang-Ping; Ma, Li; Meng, Xiao-Jing; Meng, Min-Jie; Wang, Xiao-Ning; Lin, Ying; Wu, Zheng-Qiang; He, Xiao-Wei; Wang, Ju-Fang; Zhu, Yong

2007-01-01

HLA-A*0201 alpha chain and beta2m were expressed from a prokaryotic system, and after refolding and purification, the alpha chain and beta2m were used to immunize eight laying hens. The titer of egg yolk antibody against alpha chain increased from 10(2) to 10(5.3) The titer of egg yolk antibody against beta2m increased from 10(1) to 10(4.7). The extent of titer increase is similar between the two antigens. An average of 135 mg purified polyclonal antibody (IgY) can be easily obtained from one egg yolk. The use of egg collection rather than serum collection is compatible with modern animal protection regulations. An average of 28 eggs were obtained from a laying hen every month, with a total amount of 3780 mg immunoglobulin extracted from one immunized hen every month, which would be equivalent to 630 mL of serum or 1260 mL of blood per month. Chickens are an optimal host for the production of polyclonal antibodies with high titer and high yield. Purified IgY was labeled with horseradish peroxidase and reacted with PBMC on nitrocellulose membranes indicating that the antibody can bind to the native conformation of class I HLA molecule on PBMC.

New ordered metastable phases between the gel and subgel phases in hydrated phospholipids.

PubMed Central

Tenchov, B; Koynova, R; Rapp, G

2001-01-01

Formation of low-temperature ordered gel phases in several fully hydrated phosphatidylethanolamines (PEs) and phosphatidylcholines (PCs) with saturated chains as well as in dipalmitoylphosphatidylglycerol (DPPG) was observed by synchrotron x-ray diffraction, microcalorimetry, and densitometry. The diffraction patterns recorded during slow cooling show that the gel-phase chain reflection cooperatively splits into two reflections, signaling a transformation of the usual gel phase into a more ordered phase, with an orthorhombic chain packing (the Y-transition). This transition is associated with a small decrease (2-4 microl/g) or inflection of the partial specific volume. It is fully reversible with the temperature and displays in heating direction as a small (0.1-0.7 kcal/mol) endothermic event. We recorded a Y-transition in distearoyl PE, dipalmitoyl PE (DPPE), mono and dimethylated DPPE, distearoyl PC, dipalmitoyl PC, diC(15)PC, and DPPG. No such transition exists in dimyristoyl PE and dilauroyl PE where the gel L(beta) phase transforms directly into subgel L(c) phase, as well as in the unsaturated dielaidoyl PE. The PE and PC low-temperature phases denoted L(R1) and SGII, respectively, have different hydrocarbon chain packing. The SGII phase is with tilted chains, arranged in an orthorhombic lattice of two-nearest-neighbor type. Except for the PCs, it was also registered in ionized DPPG. In the L(R1) phase, the chains are perpendicular to the bilayer plane and arranged in an orthorhombic lattice of four-nearest-neighbor type. It was observed in PEs and in protonated DPPG. The L(R1) and SGII phases are metastable phases, which may only be formed by cooling the respective gel L(beta) and L(beta') phases, and not by heating the subgel L(c) phase. Whenever present, they appear to represent an indispensable intermediate step in the formation of the latter phase. PMID:11259300

Primary structure of the hemoglobins from Sphenodon (Sphenodon punctatus, Tuatara, Rynchocephalia). Evidence for the expression of alpha D-gene.

PubMed

Abbasi, A; Wells, R M; Brittain, T; Braunitzer, G

1988-08-01

Sphenodon is the sole representative of the "beakhead" reptiles which were widely distributed during the Triassic period before the spectacular rise of dinosaurs. Sphenodon punctatus is the only survivor ("living fossil") of this period. The morphological features of Sphenodon are remarkably conservative and differ little from reptiles living 200 million years ago. In the present paper the determination of the primary structure of the tetrameric hemoglobins is described: three components are identified: hemoglobin A' (alpha A2 beta II2), hemoglobin A (alpha A2 beta I2) and hemoglobin D (alpha D2 beta II2). The components were characterized electrophoretically, the four different peptide chains were characterized by Triton electrophoresis as well as by high-performance liquid chromatography. The hemoglobins and--under dissociating conditions--also the chains, were isolated on columns of cellulose ion exchangers. Sequence determination was carried out after cleavage of the individual chains with trypsin and after a specific chemical cleavage of the Asp-Pro bond. For sequence determination the film technique and gas-phase method were employed. The data are compared with the sequence of the human hemoglobin, and interpretations of the amino-acid sequences are given. Particularly notable is the evidence of hemoglobin D: this hemoglobin (alpha D2 beta II2) is found only in birds, and in two cases in turtles. However, this component is not found in other reptiles. The results make possible an interpretation of the relatively high oxygen affinity and explain the lack of cooperativity (myoglobin properties) of these tetrameric hemoglobins.

Higher-order molecular packing in amyloid-like fibrils constructed with linear arrangements of hydrophobic and hydrogen-bonding side-chains.

PubMed

Saiki, Masatoshi; Honda, Shinya; Kawasaki, Kazunori; Zhou, Deshan; Kaito, Akira; Konakahara, Takeo; Morii, Hisayuki

2005-05-13

Various mutants of the protein fragment, barnase module-1 (1-24) were investigated in order to reveal the structural principle of amyloid-like fibrils. By means of circular dichroism spectroscopy, X-ray diffraction, electron microscopy, and thioflavin T binding assay, we found that the molecules containing two beta-strands and an intervening turn structure are assembled to form a cross-beta structure. Stabilization by both the hydrophobic interactions and hydrogen bonding between the respective paired side-chains on the coupled beta-strands was essential for fibril formation. These two types of interaction can also arrange the corresponding residues in lines on both sheet surfaces of protofilaments with a cross-beta structure. This leads to the most probable fibril structure constructed with the line-matching interactions between protofilaments. Consideration of the geometrical symmetry resulted in our finding that a limited number of essential models for molecular packing in fibril structure are stable, which would rationally explain the occurrence of two or three morphologies from an identical molecular species. The ribbon-like fibrils exhibited striped texture along the axis, which was assigned to a stacked two-sheet repeat as a structural unit. The comprehensively proposed structural model, that is, the sheet-sheet interaction between left-handed cross-beta structures, results in a slightly right-handed twist of beta-sheet stacking, which reasonably elucidates the intrinsic sizes of the fibril width and its helical period along the fibril axis, as the bias in the orientation of the hydrogen-bonded beta-strand pair at the lateral edge is larger than that at the central protofilament.

Molecular cloning of a small prostate protein, known as beta-microsemenoprotein, PSP94 or beta-inhibin, and demonstration of transcripts in non-genital tissues.

PubMed

Ulvsbäck, M; Lindström, C; Weiber, H; Abrahamsson, P A; Lilja, H; Lundwall, A

1989-11-15

In order to study the gene expression of the seminal plasma protein beta-microseminoprotein, also known as PSP94 and beta-inhibin, clones encoding this protein were isolated from a cDNA library constructed in lambda gt11. Nucleotide sequencing confirmed the structure of a previously cloned cDNA. By northern blot analysis identical sized transcripts were demonstrated in the prostate, the respiratory (tracheal, bronchial and lung) tissues and the antrum part of the gastric mucosa. Thus, the protein is not primarily associated with male reproductive function. Although probably of no physiological significance, a slight structural similarity to the ovarian inhibin beta-chains was identified in the C-terminal half of the molecule.

Specific reaction of alpha,beta-unsaturated carbonyl compounds such as 6-shogaol with sulfhydryl groups in tubulin leading to microtubule damage.

PubMed

Ishiguro, Kazuhiro; Ando, Takafumi; Watanabe, Osamu; Goto, Hidemi

2008-10-15

6-Shogaol and 6-gingerol are ginger components with similar chemical structures. However, while 6-shogaol damages microtubules, 6-gingerol does not. We have investigated the molecular mechanism of 6-shogaol-induced microtubule damage and found that the action of 6-shogaol results from the structure of alpha,beta-unsaturated carbonyl compounds. alpha,beta-Unsaturated carbonyl compounds such as 6-shogaol react with sulfhydryl groups of cysteine residues in tubulin, and impair tubulin polymerization. The reaction with sulfhydryl groups depends on the chain length of alpha,beta-unsaturated carbonyl compounds. In addition, alpha,beta-unsaturated carbonyl compounds are more reactive with sulfhydryl groups in tubulin than in 2-mercaptoethanol, dithiothreitol, glutathione and papain, a cysteine protease.

Hb Alesha [beta67(E11)Val-->Met, GTG-->ATG] in an Argentinean girl.

PubMed

Eberle, Silvia Eandi; Noguera, Nélida I; Sciuccati, Gabriela; Bonduel, Mariana; Díaz, Lilian; Staciuk, Raquel; Targovnik, Héctor M; Feliu-Torres, Aurora

2007-01-01

Hb Alesha is caused by a GTG-->ATG mutation at codon 67 of the beta-globin gene, resulting in abnormal beta-globin chains in which the normal beta67(E11) valine is changed to methionine. This hemoglobin (Hb) is also known as Hb Bristol, the first unstable Hb described, since in a fraction of the variant the methionine is modified into an aspartic acid by a posttranslational modification. This replacement disrupts the apolar bonds between the valine and the heme group, producing an unstable Hb and severe hemolysis. We have identified this rare hemoglobinopathy in an Argentinean girl with severe hemolytic anemia, splenomegaly and frequent requirement for red blood cell transfusions.

Expanding the remarkable structural diversity of uranyl tellurites: hydrothermal preparation and structures of K[UO(2)Te(2)O(5)(OH)], Tl(3)[(UO(2))(2)[Te(2)O(5)(OH)](Te(2)O(6))].2H(2)O, beta-Tl(2)[UO(2)(TeO(3))(2)], and Sr(3)[UO(2)(TeO(3))(2)](TeO(3))(2).

PubMed

Almond, Philip M; Albrecht-Schmitt, Thomas E

2002-10-21

The reactions of UO(2)(C(2)H(3)O(2))(2).2H(2)O with K(2)TeO(3).H(2)O, Na(2)TeO(3) and TlCl, or Na(2)TeO(3) and Sr(OH)(2).8H(2)O under mild hydrothermal conditions yield K[UO(2)Te(2)O(5)(OH)] (1), Tl(3)[(UO(2))(2)[Te(2)O(5)(OH)](Te(2)O(6))].2H(2)O (2) and beta-Tl(2)[UO(2)(TeO(3))(2)] (3), or Sr(3)[UO(2)(TeO(3))(2)](TeO(3))(2) (4), respectively. The structure of 1 consists of tetragonal bipyramidal U(VI) centers that are bound by terminal oxo groups and tellurite anions. These UO(6) units span between one-dimensional chains of corner-sharing, square pyramidal TeO(4) polyhedra to create two-dimensional layers. Alternating corner-shared oxygen atoms in the tellurium oxide chains are protonated to create short/long bonding patterns. The one-dimensional chains of corner-sharing TeO(4) units found in 1 are also present in 2. However, in 2 there are two distinct chains present, one where alternating corner-shared oxygen atoms are protonated, and one where the chains are unprotonated. The uranyl moieties in 2 are bound by five oxygen atoms from the tellurite chains to create seven-coordinate pentagonal bipyramidal U(VI). The structures of 3 and 4 both contain one-dimensional [UO(2)(TeO(3))(2)](2-) chains constructed from tetragonal bipyramidal U(VI) centers that are bridged by tellurite anions. The chains differ between 3 and 4 in that all of the pyramidal tellurite anions in 3 have the same orientation, whereas the tellurite anions in 4 have opposite orientations on each side of the chain. In 4, there are also additional isolated TeO(3)(2-) anions present. Crystallographic data: 1, orthorhombic, space group Cmcm, a = 7.9993(5) A, b = 8.7416(6) A, c = 11.4413(8) A, Z = 4; 2, orthorhombic, space group Pbam, a = 10.0623(8) A, b = 23.024(2) A, c = 7.9389(6) A, Z = 4; 3, monoclinic, space group P2(1)/n, a = 5.4766(4) A, b = 8.2348(6) A, c = 20.849(3) A, beta = 92.329(1) degrees, Z = 4; 4, monoclinic, space group C2/c, a = 20.546(1) A, b = 5.6571(3) A, c = 13.0979(8) A, beta = 94.416(1) degrees, Z = 4.

Mixed isotype class II antigen expression. A novel class II molecule is expressed on a murine B cell lymphoma

PubMed Central

1989-01-01

The structures of Ia molecules expressed by two BALB/c B cell lymphoma lines, A20-1.11 (A20) and 2PK3, were analyzed in an effort to explain the differences in antigen-presenting capacity displayed by these cells. Alloreactive T cell hybridomas specific for I-Ad and antigen- specific, I-Ad-restricted T cells responded well to A20 as the APC. The same alloreactive T cell hybridomas responded weakly or not at all to 2PK3 and the responses of the antigen-specific, I-Ad-restricted T cells were consistently lower to antigen presented by 2PK3 as compared with A20. T cells restricted to I-Ed responded equally well to either A20 or 2PK3 as APC. Additionally 2PK3, but not A20, stimulated a strong syngeneic mixed lymphocyte response. Structural analyses of the Ia antigens revealed that I-A and I-E molecules were expressed by A20, whereas an I-E and a novel I-A-like molecule were expressed by 2PK3. The novel class II molecule was affinity purified from 2PK3 cells using an mAb specific for Ad beta (MK-D6), and this molecule was subsequently shown by an RIA to react with an E alpha-specific mAb (14-4-4S) as well. Chain-specific polyclonal antisera raised against I-A and I-E alpha and beta chains indicated that the 2PK3 "I-A" alpha chain reacted in immunoblot with E alpha-specific and not A alpha-specific antisera, whereas the beta chain reacted with A beta- and not E beta-specific antisera. Peptide map and partial amino acid sequence analyses indicated that the "I-A" molecule expressed by 2PK3 represented a mixed isotype structure resulting from the pairing of Ed alpha with Ad beta. By immunofluorescence staining analysis, 2PK3 did not react with an mAb specific for Ad alpha. 2PK3 was capable of limited antigen presentation through the mixed isotype molecule to I-Ad-restricted OVA-specific T cell hybridomas, although the responses induced were low compared with presentation through I-A on A20. Previous descriptions of the expression of mixed isotype class II molecules in the mouse have resulted primarily from DNA-mediated gene transfer experiments. The results presented indicate that a mixed isotype class II molecule can be expressed naturally. PMID:2647893

Genetic heterogeneity of Beta thalassemia in Lebanon reflects historic and recent population migration.

PubMed

Makhoul, N J; Wells, R S; Kaspar, H; Shbaklo, H; Taher, A; Chakar, N; Zalloua, P A

2005-01-01

Beta thalassemia is an autosomal recessive disorder characterized by reduced (beta(+)) or absent (beta(0)) beta-globin chain synthesis. In Lebanon it is the most predominant genetic defect. In this study we investigated the religious and geographic distribution of the beta-thalassemia mutations identified in Lebanon, and traced their precise origins. A total of 520 beta-globin chromosomes from patients of different religious and regional backgrounds was studied. Beta thalassemia mutations were identified using Amplification Refractory Mutation System (ARMS) PCR or direct gene sequencing. Six (IVS-I-110, IVS-I-1, IVS-I-6, IVS-II-1, cd 5 and the C > T substitution at cd 29) out of 20 beta-globin defects identified accounted for more than 86% of the total beta-thalassemia chromosomes. Sunni Muslims had the highest beta-thalassemia carrier rate and presented the greatest heterogeneity, with 16 different mutations. Shiite Muslims followed closely with 13 mutations, whereas Maronites represented 11.9% of all beta-thalassemic subjects and carried 7 different mutations. RFLP haplotype analysis showed that the observed genetic diversity originated from both new mutational events and gene flow from population migration. This study provides information about the types and distribution of beta-thalassemia mutations within each religious group and geographic region, which is essential for the implementation of screening and prevention programs.

Knocking out the MFE-2 gene of Candida bombicola leads to improved medium-chain sophorolipid production.

PubMed

Van Bogaert, Inge N A; Sabirova, Julia; Develter, Dirk; Soetaert, Wim; Vandamme, Erick J

2009-06-01

The nonpathogenic yeast Candida bombicola synthesizes sophorolipids. These biosurfactants are composed of the disaccharide sophorose linked to a long-chain hydroxy fatty acid and have potential applications in the food, pharmaceutical, cosmetic and cleaning industries. In order to expand the range of application, a shift of the fatty acid moiety towards medium-chain lengths would be recommendable. However, the synthesis of medium-chain sophorolipids by C. bombicola is a challenging objective. First of all, these sophorolipids can only be obtained by fermentations on unconventional carbon sources, which often have a toxic effect on the cells. Furthermore, medium-chain substrates are partially metabolized in the beta-oxidation pathway. In order to redirect unconventional substrates towards sophorolipid synthesis, the beta-oxidation pathway was blocked on the genome level by knocking out the multifunctional enzyme type 2 (MFE-2) gene. The total gene sequence of the C. bombicola MFE-2 (6033 bp) was cloned (GenBank accession number EU371724), and the obtained nucleotide sequence was used to construct a knock-out cassette. Several knock-out mutants with the correct geno- and phenotype were evaluated in a fermentation on 1-dodecanol. All mutants showed a 1.7-2.9 times higher production of sophorolipids, indicating that in those strains the substrate is redirected towards the sophorolipid synthesis.

Complementary DNA sequences encoding the multimammate rat MHC class II DQ alpha and beta chains and cross-species sequence comparison in rodents.

PubMed

de Bellocq, J Goüy; Leirs, H

2009-09-01

Sequences of the complete open reading frame (ORF) for rodents major histocompatibility complex (MHC) class II genes are rare. Multimammate rat (Mastomys natalensis) complementary DNA (cDNA) encoding the alpha and beta chains of MHC class II DQ gene was cloned from a rapid amplifications of cDNA Emds (RACE) cDNA library. The ORFs consist of 801 and 771 bp encoding 266 and 256 amino acid residues for DQB and DQA, respectively. The genomic structure of Mana-DQ genes is globally analogous to that described for other rodents except for the insertion of a serine residue in the signal peptide of Mana-DQB, which is unique among known rodents.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Popp, R.A.; Marsh, C.L.; Skow, L.C.

Hemoglobins of mouse embryos at 11.5 through 16.5 days of gestation were separated by electrophoresis on cellulose acetate and quantitated by a scanning densitometer to study the effects of two radiation-induced mutations on the expression of embryonic hemoglobin genes in mice. Normal mice produce three kinds of embryonic hemoglobins. In heterozygous ..cap alpha..-thalassemic embryos, expression of EI (x/sub 2/y/sub 2/) and EII (..cap alpha../sub 2/y/sub 2/) is deficient because the x- and ..cap alpha..-globin genes of one of the allelic pairs of Hba on chromosome 11 was deleted or otherwise inactivated by X irradiation. Simultaneous inactivation of the x- andmore » ..cap alpha..-globin genes indicates that these genes must be closely linked. Reduced x- and ..cap alpha..-chain synthesis results in an excess of y chains that associate as homotetramers. This unique y/sub 4/ hemoglobin also appears in ..beta..-duplication embryos where excess y chains are produced by the presence of three rather than two functional alleles of y- and ..beta..-globin genes. In double heterozygotes, which have a single functional allele of x- and ..cap alpha..-globin genes and three functional alleles of y- and ..beta..-globin genes, synthesis of ..cap alpha.. and non-..cap alpha.. chains is severely imbalanced and half of the total hemoglobin is y/sub 4/. Mouse y/sub 4/ has a high affinity for oxygen, P/sub 50/ of less than 10 mm Hg, but it lacks cooperativity so is inefficient for oxygen transport. The death of double heterozygotes in late fetal or neonatal life may be in large part to oxygen deprivation to the tissues.« less

Characterization of beta-turn and Asx-turns mimicry in a model peptide: stabilization via C--H . . . O interaction.

PubMed

Thakur, A K; Kishore, R

2006-04-15

The chemical synthesis and single-crystal X-ray diffraction analysis of a model peptide, Boc-Thr-Thr-NH2 (1) comprised of proteinogenic residues bearing an amphiphilic Cbeta -stereogenic center, has been described. Interestingly, the analysis of its molecular structure revealed the existence of a distinct conformation that mimics a typical beta-turn and Asx-turns, i.e., the two Thr residues occupy the left- and right-corner positions. The main-chain torsion angles of the N- and C-terminal residues i.e., semiextended: phi = -68.9 degrees , psi = 128.6 degrees ; semifolded: phi = -138.1 degrees , psi = 2.5 degrees conformations, respectively, in conjunction with a gauche- disposition of the obligatory C-terminus Thr CgammaH3 group, characterize the occurrence of the newly described beta-turn- and Asx-turns-like topology. The preferred molecular structure is suggested to be stabilized by an effective nonconventional main-chain to side-chain Ci=O . . . H--Cgamma(i+2)-type intraturn hydrogen bond. Noteworthy, the observed topology of the resulting 10-membered hydrogen-bonded ring is essentially similar to the one perceived for a classical beta-turn and the Asx-turns, stabilized by a conventional intraturn hydrogen bond. Considering the signs as well as magnitudes of the backbone torsion angles and the orientation of the central peptide bond, the overall mimicked topology resembles the type II beta-turn or type II Asx-turns. An analysis of Xaa-Thr sequences in high-resolution X-ray elucidated protein structures revealed the novel topology prevalence in functional proteins (unpublished). In view of indubitable structural as well as functional importance of nonconventional interactions in bioorganic and biomacromolecules, we intend to highlight the participation of Thr CgammaH in the creation of a short-range C=O . . . H--Cgamma -type interaction in peptides and proteins. Copyright 2006 Wiley Periodicals, Inc.

Tolerability to beta-blocker therapy among heart failure patients in clinical practice.

PubMed

Butler, Javed; Khadim, Ghazanfar; Belue, Rhonda; Chomsky, Don; Dittus, Robert S; Griffin, Marie; Wilson, John R

2003-06-01

Although beta-blockers were well-tolerated by heart failure (HF) patients in clinical trials, tolerability of these drugs in a general population of HF patients is not well-described. We studied a total of 308 encounters with beta-blockers therapy in 268 ambulatory HF patients. Side effects and frequency and predictors of discontinuation of therapy were studied. Independent predictors of discontinuation were assessed. Weight gain (59%), fatigue (56%), dizziness (41%), and dyspnea (29%) were the most common side effects. Fifty-one patients (19%) were discontinued on therapy with any 1 particular beta-blocker. Fatigue (30%) and hypotension (28%) were the most common reasons for discontinuation. Forty (78%) of these were given a trial with a different beta-blocker. Of these, 22 (55%) attempts with a different beta-blocker were tolerated. Thus the overall absolute discontinuation rate was only 7% for patients who were given a trial with different beta-blockers or 11% for the entire study population. Independent predictors of discontinuation of therapy included advanced symptoms, nonischemic etiology, history of pulmonary disease, and higher diuretic doses. Side effects with beta-blockers in a general population of HF patients are common; however, with changes in medical management, most patients can tolerate them eventually. In case of intolerance to one kind, a trial with a different beta-blocker is indicated.

Spectroscopic evidence for gas-phase formation of successive beta-turns in a three-residue peptide chain.

PubMed

Chin, Wutharath; Compagnon, Isabelle; Dognon, Jean-Pierre; Canuel, Clélia; Piuzzi, François; Dimicoli, Iliana; von Helden, Gert; Meijer, Gerard; Mons, Michel

2005-02-09

We report the first gas-phase spectroscopic study of a three-residue model of a peptide chain, Ac-Phe-Gly-Gly-NH2 (Ac = acetyl), using the IR/UV double resonance technique. The existence of at least five different conformers under supersonic expansion conditions is established, most of them exhibiting rather strong intramolecular H-bonds. One of the most populated conformers, however, exhibits a different H-bonding network characterized by two weak H-bonds. Comparison of the amide A and I/II experimental data with density functional theory calculations carried out on a series of selected conformations enables us to assign this conformer to two successive beta-turns along the peptide chain, the two H-bonds being of C10 type, i.e., each of them closing a 10-atom ring in the molecule. The corresponding form is found to be more stable than the 310 helix secondary structure (not observed), presumably because of specific effects due to the glycine residues.

Reactivity of long chain alkylamines to lignin moieties: implications on hydrophobicity of lignocellulose materials.

PubMed

Kudanga, Tukayi; Prasetyo, Endry Nugroho; Sipilä, Jussi; Guebitz, Georg M; Nyanhongo, Gibson S

2010-08-20

Enzymatic processes provide new perspectives for modification of lignocellulose materials. In the current study, laccase catalyzed coupling of long chain alkylamines to lignin model molecules and lignocellulose was investigated. Up to two molecules of dodecylamine (DA) and dihexylamine (DHA) were successfully coupled with lignin monomers (guaiacol, catechol and ferulic acid) while coupling onto complex lignin model compounds (syringylglycerol beta-guaiacyl ether, guaiacylglycerol beta-guaiacyl ether and dibenzodioxocin) yielded 1:1 coupling products. Surface analysis of beech veneers enzymatically grafted with DA showed an increase in nitrogen content of 3.18% compared to 0.71% in laccase only treated controls while the O/C ratio decreased from 0.52 to 0.46. Concomitantly the grafting of DHA or DA onto beech veneers resulted in a 53.8% and 84.2% increase in hydrophobicity, respectively when compared to simple adsorption. Therefore, laccase-mediated grafting of long chain alkylamines onto lignocellulose materials can be potentially exploited for improving their hydrophobicity. Copyright 2010 Elsevier B.V. All rights reserved.

The Beta-Delayed Proton and Gamma Decay of 27P for Nuclear Astrophysics

NASA Astrophysics Data System (ADS)

McCleskey, E.; Banu, A.; McCleskey, M.; Roeder, B.; Saastamoinen, A.; Spiridon, A.; Trache, L.; Tribble, R. E.; Davinson, T.; Doherty, D.; Lotay, G. J.; Wallace, J.; Woods, P. J.

2013-10-01

The main creation site of 26Al is currently under debate. The reactions for its creation or destruction are also not completely known. When 26Al is created in novae, the reaction chain is: 24Mg(p,γ)25Al(β + v)25Mg(p,γ)26Al, but this chain can be by-passed by another chain: 25Al(p,γ)26Si(p,γ)27P and it can also be destroyed directly. Another way to by-pass it is through 26mAl(p,γ)27Si* which is dominated by resonant capture. Using the Momentum Achromat Recoil Spectrometer (MARS) at the Texas A&M Cyclotron Institute and inverse kinematics, this destruction reaction was studied by the beta-delayed proton and gamma decay of 27P. Due to selection rules, states populated above the proton threshold in the compound system (27Si*) can decay to 26mAl, which are the states of interest for the capture reaction. James Madison University, VA, USA.

Alpha-beta T cells provide protection against lethal encephalitis in the murine model of VEEV infection

PubMed Central

Paessler, Slobodan; Yun, Nadezhda E.; Judy, Barbara M.; Dziuba, Natallia; Zacks, Michele A.; Grund, Anna H.; Frolov, Ilya; Campbell, Gerald A.; Weaver, Scott C.; Estes, D. Mark

2007-01-01

We evaluated the safety and immunogenicity of a chimeric alphavirus vaccine candidate in mice with selective immunodeficiencies. This vaccine candidate was highly attenuated in mice with deficiencies in the B and T cell compartments, as well as in mice with deficient gamma-interferon responsiveness. However, the level of protection varied among the strains tested. Wild type mice were protected against lethal VEEV challenge. In contrast, alpha/beta (αβ) TCR-deficient mice developed lethal encephalitis following VEEV challenge, while mice deficient in gamma/delta ( γδ) T cells were protected. Surprisingly, the vaccine potency was diminished by 50% in animals lacking interferon-gamma receptor alpha chain (R1)-chain and a minority of vaccinated immunoglobulin heavy chain-deficient (μMT) mice survived challenge, which suggests that neutralizing antibody may not be absolutely required for protection. Prolonged replication of encephalitic VEEV in the brain of pre-immunized mice is not lethal and adoptive transfer experiments indicate that CD3+ T cells are required for protection. PMID:17610927

Functional overload increases beta-MHC promoter activity in rodent fast muscle via the proximal MCAT (betae3) site.

PubMed

Giger, Julia M; Haddad, Fadia; Qin, Anqi X; Baldwin, Kenneth M

2002-03-01

Functional overload (OL) of the rat plantaris muscle by the removal of synergistic muscles induces a shift in the myosin heavy chain (MHC) isoform expression profile from the fast isoforms toward the slow type I, or, beta-MHC isoform. Different length rat beta-MHC promoters were linked to a firefly luciferase reporter gene and injected in control and OL plantaris muscles. Reporter activities of -3,500, -914, -408, and -215 bp promoters increased in response to 1 wk of OL. The smallest -171 bp promoter was not responsive to OL. Mutation analyses of putative regulatory elements within the -171 and -408 bp region were performed. The -408 bp promoters containing mutations of the betae1, distal muscle CAT (MCAT; betae2), CACC, or A/T-rich (GATA), were still responsive to OL. Only the proximal MCAT (betae3) mutation abolished the OL response. Gel mobility shift assays revealed a significantly higher level of complex formation of the betae3 probe with nuclear protein from OL plantaris compared with control plantaris. These results suggest that the betae3 site functions as a putative OL-responsive element in the rat beta-MHC gene promoter.

Novel Humanized mice to test Therapeutics for Human Type 1 Diabetes

DTIC Science & Technology

2014-01-06

performed in non- obese diabetic (NOD) mice, the closest animal model for human T1D, have identified different immune cells involved in pancreatic β-cell...periphery. Each MHC class II chain contributes to the formation of a groove where the peptide is embedded (78). 17 The polygenetic factor underlying...diabetes mellitus in non- obese diabetic mice by transgenes encoding modified I-A beta-chain or normal I-E alpha-chain. Nature 345:727-9 63. Marek

Synthesis and cytotoxic analysis of some disodium 3beta,6beta-dihydroxysterol disulfates.

PubMed

Cui, Jianguo; Wang, Hui; Huang, Yanmin; Xin, Yi; Zhou, Aimin

2009-01-01

Disodium 3beta,6beta-dihydroxy-5alpha-cholestane disulfate (1) was synthesized in 4 steps with a high overall yield from cholesterol. First, cholesterol (4a) was converted to cholest-4-en-3,6-dione (5a) via oxidation with pyridinium chlorochromate (PCC) and then 5a was reduced by NaBH(4) in the presence of NiCl(2) to produce cholest-3beta,6beta-diol (6a). The reaction of 6a with the triethylamine-sulfur trioxide complex generated diammonium 3beta,6beta-dihydroxy-5alpha-cholestane disulfate (7a) and the treatment of 7a by cation exchange resin 732 (sodium form)(Na(+)) yielded the target steroid 1. Disodium 24-ethyl-3beta,6beta-dihydroxycholest-22-ene disulfate (2) and disodium 24-ethyl-3beta,6beta-dihydroxycholestane disulfate (3) were synthesized using a similar method. The cytotoxicity of these compounds against Sk-Hep-1 (human liver carcinoma cell line), H-292 (human lung carcinoma cell line), PC-3 (human prostate carcinoma cell line) and Hey-1B (human ovarian carcinoma cell line) cells was investigated. Our results indicate that presence of a cholesterol-type side chain at position 17 is necessary for their biological activity.

Electrostatic persistence length.

PubMed

Fixman, Marshall

2010-03-11

The persistence length is calculated for polyelectrolyte chains with fixed bond lengths and bond angles (pi-theta), and a potential energy consisting of the screened Coulomb interaction between beads, potential wells alpha phi(i)2 for the dihedral angles phi(i), and coupling terms beta phi(i) phi(i+/-1). This model defines a librating chain that reduces in appropriate limits to the freely rotating or wormlike chains, it can accommodate local crumpling or extreme stiffness, and it is easy to simulate. A planar-quadratic (pq), analytic approximation is based on an expansion of the electrostatic energy in eigenfunctions of the quadratic form that describes the backbone energy, and on the assumption that the quadratic form not only is positive but also adequately confines the chain in an infinite phase space of dihedral angles to the physically unique part with all |phi(i)| < pi. The pq approximation is available under these weak constraints, but the simulations confirm its quantitative accuracy only under the expected condition that alpha is large, that is, for very stiff chains. Stiff chains can also be simulated with small alpha and small theta and compared to an OSF approximation suitably generalized to chains with finite rather than vanishing theta, and increasing agreement with OSF is found the smaller is theta. The two approximations, one becoming exact as alpha --> infinity with fixed theta, the other as theta --> 0 with fixed alpha, are quantitatively similar in behavior, both giving a persistence length P = P0 + aD2 for stiff chains, where D is the Debye length. However, the coefficient apq is about twice the value of aOSF. Under other conditions the simulations show that P may or not be linear in D2 at small or moderate D, depending on the magnitudes of alpha, beta, theta, and the charge density but always becomes linear at large D. Even at a moderately low charge density, corresponding to fewer than 20% of the beads being charged, and with strong crumpling induced by large beta, increasing D dissolves blobs and recovers a linear dependence of P on D2, although a lower power of D gives an adequate fit at moderate D. For the class of models considered, it is concluded that the only universal feature is the asymptotic linearity of P in D2, regardless of flexibility or stiffness.

Decursin suppresses human androgen-independent PC3 prostate cancer cell proliferation by promoting the degradation of beta-catenin.

PubMed

Song, Gyu-Yong; Lee, Jee-Hyun; Cho, Munju; Park, Byeoung-Soo; Kim, Dong-Eun; Oh, Sangtaek

2007-12-01

Alterations in the Wnt/beta-catenin pathway are associated with the development and progression of human prostate cancer. Decursin, a pyranocoumarin isolated from the Korean Angelica gigas root, inhibits the growth of androgen-independent human prostate cancer cells, but little is known about its mechanism of action. Using a cell-based screen, we found that decursin attenuates the Wnt/beta-catenin pathway. Decursin antagonized beta-catenin response transcription (CRT), which was induced with Wnt3a-conditioned medium and LiCl, by promoting the degradation of beta-catenin. Furthermore, decursin suppressed the expression of cyclin D1 and c-myc, which are downstream target genes of beta-catenin and thus inhibited the growth of PC3 prostate cancer cells. In contrast, decursinol, in which the (CH3)2-C=CH-COO- side chain of decursin is replaced with -OH, had no effect on CRT, the level of intracellular beta-catenin, or PC3 cell proliferation. Our findings suggest that decursin exerts its anticancer activity in prostate cancer cells via inhibition of the Wnt/beta-catenin pathway.

Rapid model building of beta-sheets in electron-density maps.

PubMed

Terwilliger, Thomas C

2010-03-01

A method for rapidly building beta-sheets into electron-density maps is presented. beta-Strands are identified as tubes of high density adjacent to and nearly parallel to other tubes of density. The alignment and direction of each strand are identified from the pattern of high density corresponding to carbonyl and C(beta) atoms along the strand averaged over all repeats present in the strand. The beta-strands obtained are then assembled into a single atomic model of the beta-sheet regions. The method was tested on a set of 42 experimental electron-density maps at resolutions ranging from 1.5 to 3.8 A. The beta-sheet regions were nearly completely built in all but two cases, the exceptions being one structure at 2.5 A resolution in which a third of the residues in beta-sheets were built and a structure at 3.8 A in which under 10% were built. The overall average r.m.s.d. of main-chain atoms in the residues built using this method compared with refined models of the structures was 1.5 A.

Polymorphism in 'L' shaped lipids: structure of N-, O-diacylethanolamines with mixed acyl chains.

PubMed

Tarafdar, Pradip K; Swamy, Musti J

2009-11-01

Although solid state polymorphism in lipids has been established by spectroscopic and calorimetric studies long ago, only in a few cases crystal structures of different polymorphs of the same compound have been reported, possibly due to difficulties in obtaining high quality single crystals of individual polymorphs. Recent studies show that N-, O-diacylethanolamines (DAEs) can be derived by the O-acylation of the stress-related lipids, the N-acylethanolamines under physiological conditions. In this study, two DAEs with mixed acyl chains, namely N-palmitoyl, O-octanoylethanolamine and N-palmitoyl, O-decanoylethanolamine have been synthesized and their three-dimensional structures were determined. Both the compounds were found to adopt 'L' shaped structures and exist in two polymorphic forms, alpha and beta. In the alpha form a mixed-type chain packing has been observed whereas in the beta form the chain packing is symmetric. Similar polymorphic forms are likely to exist in other 'L' shaped lipids such as 1,3-diacylglycerols and ceramides, where polymorphism has been detected earlier, but three-dimensional structures - which can give precise information about the packing at atomic resolution - have not been reported.

Enhanced production of longer side-chain polyhydroxyalkanoic acid with omega-aromatic group substitution in phaZ-disrupted Pseudomonas fluorescens BM07 mutant through unrelated carbon source cometabolism and salicylic acid beta-oxidation inhibition.

PubMed

Choi, Mun Hwan; Xu, Ju; Rho, Jong Kook; Zhao, Xu Ping; Yoon, Sung Chul

2010-06-01

The deletion of the intracellular polyhydroxyalkanoate (PHA) depolymerase gene (phaZ) in Pseudomonas fluorescens BM07 was found to increase more efficiently the levels of longer medium-chain-length (MCL) omega-aromatic monomer-units than in the wild-type strain when the cells were grown with a mixture of fructose and MCL omega-aromatic fatty acid in the presence of salicylic acid that is known as a beta-oxidation inhibitor in BM07 strain. When 11-phenoxyundecanoic acid was used as co-carbon source, the longest monomer-unit 3-hydroxy-11-phenoxyundecanoate, not reported in literature yet, was incorporated into the polymer chain up to approximately 10 mol%. An advantage of salicylic acid inhibition technique is that salicylic acid is not metabolized in BM07 strain, thus, the effective concentration of the inhibitor remaining constant throughout the cultivation. In conclusion, this new technique could be exploited for the enhanced production of side-chain modulated functional MCL-PHA with improved physicochemical properties in P. fluorescens BM07. (c) 2010 Elsevier Ltd. All rights reserved.

Long-chain aliphatic beta-diketones from epicuticular wax of Vanilla bean species. Synthesis of nervonoylacetone.

PubMed

Ramaroson-Raonizafinimanana, B; Gaydou, E M; Bombarda, I

2000-10-01

Analysis of the neutral lipids from Vanilla fragrans and Vanilla tahitensis (Orchidaceae) without saponification resulted in the isolation and identification of a new product family in this plant: beta-dicarbonyl compounds. The compound structures are composed of a long aliphatic chain with 2,4-dicarbonyl carbons and a cis double bond at the n-9 position. They represent approximately 28% of the neutral lipids, that is, 1.5%, in immature beans, and approximately 10% of the neutral lipids, that is, 0.9%, in mature beans. Using retention indices, gas chromatography-mass spectrometry, derivatization reactions, and chemical degradation, five beta-dicarbonyl compounds have been identified including 16-pentacosene-2,4-dione, 18-heptacosene-2,4-dione, 20-nonacosene-2, 4-dione, 22-hentriacontene-2,4-dione, and 24-tritriacontene-2, 4-dione. Among them (Z)-18-heptacosene-2,4-dione, or nervonoylacetone, has been synthesized in two steps starting from nervonic acid. The major constituent, nervonoylacetone, represented 74.5% of the beta-dicarbonyl fraction. The range of these compounds has been studied in relation with bean maturity for V. fragrans and V. tahitensis species. This compound family has not been found in the leaves or stems of any of the three vanilla species studied and is markedly absent in the beans of V. madagascariensis.

Biosynthetic processing of the oligosaccharide chains of cellular fibronectin.

PubMed

Olden, K; Hunter, V A; Yamada, K M

1980-10-15

We have examined the maturation or processing of the oligosaccharides of cellular fibronectin in cultured chick embryo fibroblasts. Fibronectin was pulse-labeled with [2-3H]mannose of [35S]methionine, and the turnover rates of carbohydrate and polypeptide portions of immunoprecipitated fibronectin were compared. The oligosaccharides on fibronectin were analyzed by gel electrophoresis for alterations in sensitivity to the enzyme endo-beta-N-acetylgluosaminidase H, which specifically cleaves the 'high-mannose' class of asparagine-linked oligosaccharide. Incorporated mannose was removed only at early time points, suggesting that the structure of fibronectin oligosaccharides was altered due to processing. This possibility was confirmed by the analysis of glycopeptides generated by exhaustive pronase digestion. Two major glycopeptide structures were detected; their properties correspond to a 'high-mannose' oligosaccharide precursor and a 'complex' carbohydrate product. The precursor-product relationship of these two forms of oligosaccharide chains was demonstrated by pulse-chase labeling experiments. The precursor glycopeptide had an apparent size (Mr 2100) comparable to (Man)9GlcNAc (Mr 2080), and was sensitive to endo-beta-N-acetylglucosaminidase H; nearly all of the labeled mannose incorporated in a 10 min pulse was released from fibronectin glycopeptides by this enzyme. During a 90 min chase period, the glycopeptides became larger and increasingly resistant to endo-beta-N-acetylglucosaminidase H cleavage. The final 'complex' or processed oligosaccharide structure contained approximately two-thirds less [3H]mannose, was insensitive to endo-beta-N-acetylglucosaminidase H and had an apparent Mr of 2300 as estimated by gel filtration. We conclude that the carbohydrate portion of fibronectin is synthesized as a 'high-mannose' intermediate and is subsequently processed to give the characteristic 'complex' oligosaccharide chains of fibronectin.

Biological responses to PDGF-BB versus PDGF-DD in human mesangial cells.

PubMed

van Roeyen, C R C; Ostendorf, T; Denecke, B; Bokemeyer, D; Behrmann, I; Strutz, F; Lichenstein, H S; LaRochelle, W J; Pena, C E; Chaudhuri, A; Floege, J

2006-04-01

Platelet-derived growth factor (PDGF)-BB and PDGF-DD mediate mesangial cell proliferation in vitro and in vivo. While PDGF-BB is a ligand for the PDGF alpha- and beta-receptor chains, PDGF-DD binds more selectively to the beta-chain, suggesting potential differences in the biological activities. Signal transduction and regulation of gene expression induced by PDGF-BB and -DD were compared in primary human mesangial cells (HMCs), which expressed PDGF alpha- and beta-receptor subunits. The growth factor concentrations used were chosen based on their equipotency in inducing HMCs proliferation and binding to the betabeta-receptor. Both growth factors, albeit at different concentrations induced phosphorylation and activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2. In addition, PDGFs led to the phosphorylation and activation of signal transducers and activators of transcription 1 (STAT1) and STAT3. HMCs proliferation induced by either PDGF-BB or -DD could be blocked by signal transduction inhibitors of the mitogen-activated protein kinase-, Janus kinase (JAK)/STAT-, or phosphatidyl-inositol 3-kinase pathways. Using a gene chip array and subsequent verification by real-time reverse transcriptase (RT)-polymerase chain reaction, we found that in HMC genes for matrix metalloproteinase 13 (MMP-13) and MMP-14 and, to a low extent, cytochrome B5 and cathepsin L were exclusively regulated by PDGF-BB, whereas no exclusive gene regulation was detected by PDGF-DD. However, at the protein level, both MMP-13 and -14 were equally induced by PDGF-BB and -DD. PDGF-BB and -DD effect similar biological responses in HMCs albeit at different potencies. Rare apparently differential gene regulation did not result in different protein expression, suggesting that in HMCs both PDGFs exert their biological activity almost exclusively via the PDGF beta-receptor.

Assembly of the epithelial Na+ channel evaluated using sucrose gradient sedimentation analysis.

PubMed

Cheng, C; Prince, L S; Snyder, P M; Welsh, M J

1998-08-28

Three subunits, alpha, beta, and gamma, contribute to the formation of the epithelial Na+ channel. To investigate the oligomeric assembly of the channel complex, we used sucrose gradient sedimentation analysis to determine the sedimentation properties of individual subunits and heteromultimers comprised of multiple subunits. When the alpha subunit was expressed alone, it first formed an oligomeric complex with a sedimentation coefficient of 11 S, and then generated a higher order multimer of 25 S. In contrast, individual beta and gamma subunits predominately assembled into 11 S complexes. We obtained similar results with expression in cells and in vitro. When we co-expressed beta with alpha or with alpha plus gamma, the beta subunit assembled into a 25 S complex. Glycosylation of the alpha subunit was not required for assembly into a 25 S complex. We found that the alpha subunit formed intra-chain disulfide bonds. Although such bonds were not required to generate an oligomeric complex, under nonreducing conditions the alpha subunit formed a complex that migrated more homogeneously at 25 S. This suggests that intra-chain disulfide bonds may stabilize the complex. These data suggest that the epithelial Na+ channel subunits form high order oligomeric complexes and that the alpha subunit contains the information that facilitates such formation. Interestingly, the ability of the alpha, but not the beta or gamma, subunit to assemble into a 25 S homomeric complex correlates with the ability of these subunits to generate functional channels when expressed alone.

Peptide design using alpha,beta-dehydro amino acids: from beta-turns to helical hairpins.

PubMed

Mathur, Puniti; Ramakumar, S; Chauhan, V S

2004-01-01

Incorporation of alpha,beta-dehydrophenylalanine (DeltaPhe) residue in peptides induces folded conformations: beta-turns in short peptides and 3(10)-helices in larger ones. A few exceptions-namely, alpha-helix or flat beta-bend ribbon structures-have also been reported in a few cases. The most favorable conformation of DeltaPhe residues are (phi,psi) approximately (-60 degrees, -30 degrees ), (-60 degrees, 150 degrees ), (80 degrees, 0 degrees ) or their enantiomers. DeltaPhe is an achiral and planar residue. These features have been exploited in designing DeltaPhe zippers and helix-turn-helix motifs. DeltaPhe can be incorporated in both right and left-handed helices. In fact, consecutive occurrence of three or more DeltaPhe amino acids induce left-handed screw sense in peptides containing L-amino acids. Weak interactions involving the DeltaPhe residue play an important role in molecular association. The C--H.O==C hydrogen bond between the DeltaPhe side-chain and backbone carboxyl moiety, pi-pi stacking interactions between DeltaPhe side chains belonging to enantiomeric helices have shown to stabilize folding. The unusual capability of a DeltaPhe ring to form the hub of multicentered interactions namely, a donor in aromatic C--H.pi and C--H.O==C and an acceptor in a CH(3).pi interaction suggests its exploitation in introducing long-range interactions in the folding of supersecondary structures. Copyright 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004

Hemoglobin Abraham Lincoln, beta32 (B14) leucine leads to proline. An unstable variant producing severe hemolytic disease.

PubMed

Honig, G R; Green, D; Shamsuddin, M; Vida, L N; Mason, R G; Gnarra, D J; Maurer, H S

1973-07-01

An unstable hemoglobin variant was identified in a Negro woman with hemolytic anemia since infancy. A splenectomy had been performed when the patient was a child. The anemia was accompanied by erythrocyte inclusion bodies and excretion of darkly pigmented urine. Neither parent of the proposita demonstrated any hematologic abnormality, and it appeared that this hemoglobin variant arose as a new mutation. Erythrocyte survival in the patient was greatly reduced: the erythrocyte t(1/2) using radiochromium as a tag was 2.4 days, and a reticulocyte survival study performed after labeling the cells with L-[(14)C]leucine indicated a t(1/2) of 7.2 days. When stroma-free hemolysates were heated at 50 degrees C, 16-20% of the hemoglobin precipitated. The thermolability was prevented by the addition of hemin, carbon monoxide, or dithionite, suggesting an abnormality of heme binding. An increased rate of methemoglobin formation was also observed after incubation of erythrocytes at 37 degrees C. The abnormal hemoglobin could not be separated from hemoglobin A by electrophoresis or chromatography, but it was possible to isolate the variant beta-chain by precipitation with p-hydroxymercuribenzoate. Purification of the beta-chain by column chromatography followed by peptide mapping and amino acid analysis demonstrated a substitution of proline for beta32 leucine. It appears likely that a major effect of this substitution is a disruption of the normal orientation of the adjacent leucine residue at beta31 to impair heme stabilization.

Three outer arm dynein heavy chains of Chlamydomonas reinhardtii operate in a coordinated fashion both in vitro and in vivo.

PubMed

Takazaki, Hiroko; Liu, Zhongmei; Jin, Mingyue; Kamiya, Ritsu; Yasunaga, Takuo

2010-07-01

Outer arm dynein (OAD) in cilia and flagella contains two to three nonidentical heavy chains (HCs) that possess motor activity. In Chlamydomonas, flagellar OAD contains three HCs, alpha-, beta-, and gamma-HCs, each appearing to have a distinct role. To determine the precise molecular mechanism of their function, cross-sectional electron micrographs of wild-type and single HC-disruption mutants were compared and statistically analyzed. While the alpha-HC mutant displayed an OAD of lower density, which was attributed to a lack of alpha-HC, the OAD of beta- and gamma-HC mutants not only lacked the corresponding HC, but was also significantly affected in its structure, particularly with respect to the localization of alpha-HC. The lack of beta-HC induced mislocalization of alpha-HC, while a disruption of the gamma-HC gene resulted in the synchronized movement of alpha-HC and beta-HC in the manners for stacking. Interestingly, using cryo-electron microscopy, purified OADs were typically observed consisting of two stacked heads and an independent single head, which presumably corresponded to gamma-HC. This conformation is different from previous reports in which the three HCs displayed a stacked form in flagella observed by cryo-electron tomography and a bouquet structure on mica in deep-etch replica images. These results suggest that gamma-HC supports the tight stacking arrangement of inter or intra alpha-/beta-HC to facilitate the proper functioning of OAD. 2010 Wiley-Liss, Inc.

Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-beta-mannosidase from Aspergillus niger BK01.

PubMed

Do, Bien-Cuong; Dang, Thi-Thu; Berrin, Jean-Guy; Haltrich, Dietmar; To, Kim-Anh; Sigoillot, Jean-Claude; Yamabhai, Montarop

2009-11-13

Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-beta-mannosidases (1,4-beta-D-mannanases) catalyze the random hydrolysis of beta-1,4-mannosidic linkages in the main chain of beta-mannans. Biodegradation of beta-mannans by the action of thermostable mannan endo-1,4-beta-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS). A gene encoding mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannan mannanohydrolase (E.C. 3.2.1.78), commonly termed beta-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5), was cloned and successfully expressed heterologously (up to 243 microg of active recombinant protein per mL) in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant beta-mannanase is highly thermostable with a half-life time of approximately 56 h at 70 degrees C and pH 4.0. The optimal temperature (10-min assay) and pH value for activity are 80 degrees C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity), locust bean gum galactomannan, carob galactomannan (low viscosity), and 1,4-beta-D-mannan (from carob) are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these substrates are 215 s-1, 330 s-1, 292 s-1 and 148 s-1, respectively. Judged from the specificity constants kcat/Km, glucomannan is the preferred substrate of the A. niger beta -mannanase. Analysis by thin layer chromatography showed that the main product from enzymatic hydrolysis of locust bean gum is mannobiose, with only low amounts of mannotriose and higher manno-oligosaccharides formed. This study is the first report on the cloning and expression of a thermostable mannan endo-1,4-beta-mannosidase from A. niger in Pichia pastoris. The efficient expression and ease of purification will significantly decrease the production costs of this enzyme. Taking advantage of its acidic pH optimum and high thermostability, this recombinant beta-mannanase will be valuable in various biotechnological applications.

Relationship between subclinical rejection and genotype, renal messenger RNA, and plasma protein transforming growth factor-beta1 levels.

PubMed

Hueso, Miguel; Navarro, Estanis; Moreso, Francesc; Beltrán-Sastre, Violeta; Ventura, Francesc; Grinyó, Josep M; Serón, Daniel

2006-05-27

Transforming growth factor (TGF)-beta(1) is increased in allograft rejection and its production is associated with single nucleotide polymorphisms (SNPs). The contribution of SNPs at codons 10 and 25 of the TGF-beta(1) gene to renal allograft damage was assessed in 6-month protocol biopsies and their association with TGF-beta(1) production. TGF-beta(1) genotypes were evaluated by polymerase chain reaction (PCR)/restriction fragment length polymorphism. Intragraft TGF-beta(1) messenger RNA (mRNA) was measured by real-time PCR and TGF-beta(1) plasma levels were assessed by enzyme-linked immunosorbent assay. Eighty consecutive patients were included. Allele T at codon 10 (risk ratio, 6.7; P = 0.02) and an episode of acute rejection before protocol biopsy (risk ratio, 6.2; P = 0.01) were independent predictors of subclinical rejection (SCR). TGF-beta(1) plasma levels, but not those of TGF-beta(1) mRNA, were increased in patients with SCR (2.59 ng/mL +/- 0.91 [n = 22] vs. 2.05 ng/mL +/- 0.76 [n = 43]; P = 0.01). There was no association between allele T and TGF-beta(1) plasma or intragraft levels. Allele T at codon 10 of the TGF-beta(1) gene is associated with a higher incidence of SCR.

Difficulty in losing weight by behavioral intervention for women with Trp64Arg polymorphism of the beta3-adrenergic receptor gene.

PubMed

Shiwaku, K; Nogi, A; Anuurad, E; Kitajima, K; Enkhmaa, B; Shimono, K; Yamane, Y

2003-09-01

Trp64Arg mutation in the beta(3)-adrenergic receptor (beta(3)AR) gene is relatively common in Japanese people. However, it has not been clear whether persons with Trp64Arg mutation in the beta(3)AR gene tend to have obesity and difficulty in losing weight even with a restricted diet and exercise. We investigated the response of body weight and metabolic factors to behavioral intervention in Japanese women with Trp64Arg mutation in the beta(3)AR gene. A 3-month behavioral intervention study using a combination of diet and exercise programs. A total of 76 perimenopausal women with no clinical symptoms (age: 54.7+/-7.7 y, body mass index (BMI): 21.0-33.0 kg/m(2)). Anthropometric measurements (weight, height, body fat, waist circumference, hip circumference, skin fold, resting energy expenditure and blood pressure) and metabolic measurements (serum levels of cholesterol, triglyceride, phospholipid, nonesterified fatty acid, glucose, insulin and leptin) and determination of the beta(3)AR genotype by polymerase chain reaction followed by BstNI digestion. At the baseline of BMI, body weight, body fat, waist circumference, hip circumference, the arm skin fold, resting energy expenditure, or blood lipid and glucose profiles, there was no significant difference in participants with/without mutation of the beta(3)AR gene. The intervention yielded a body weight reduction in 69 and 48%, and induced a significant difference in weight loss (-0.74 and -0.01 kg) for women with wild-type and Trp64Arg mutation, respectively. Significant differences of anthropometric parameters were found in body weight, BMI, waist and hip circumferences and blood pressure of wild type by the intervention. However, women with Trp64Arg mutation did not show significant changes in these anthropometric parameters, except for hip circumference. A significant difference was found in high-density lipoprotein cholesterol (HDL-C) and in the low-density lipoprotein cholesterol/HDL-C ratio in both genotypes. The results of the present study suggest that the Trp64Arg mutation of the beta(3)AR gene is associated with difficulty in losing weight through behavioral intervention, although it is not related to obesity-related phenotypes and resting energy expenditure before the intervention.

Decreased enzyme activity and contents of hepatic branched-chain alpha-keto acid dehydrogenase complex subunits in a rat model for type 2 diabetes mellitus.

PubMed

Bajotto, Gustavo; Murakami, Taro; Nagasaki, Masaru; Sato, Yuzo; Shimomura, Yoshiharu

2009-10-01

The mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC) is responsible for the committed step in branched-chain amino acid catabolism. In the present study, we examined BCKDC regulation in Otsuka Long-Evans Tokushima Fatty (OLETF) rats both before (8 weeks of age) and after (25 weeks of age) the onset of type 2 diabetes mellitus. Long-Evans Tokushima Otsuka (LETO) rats were used as controls. Plasma branched-chain amino acid and branched-chain alpha-keto acid concentrations were significantly increased in young and middle-aged OLETF rats. Although the hepatic complex was nearly 100% active in all animals, total BCKDC activity and protein abundance of E1alpha, E1beta, and E2 subunits were markedly lower in OLETF than in LETO rats at 8 and 25 weeks of age. In addition, hepatic BCKDC activity and protein amounts were significantly decreased in LETO rats aged 25 weeks than in LETO rats aged 8 weeks. In skeletal muscle, E1beta and E2 proteins were significantly reduced, whereas E1alpha tended to increase in OLETF rats. Taken together, these results suggest that (1) whole-body branched-chain alpha-keto acid oxidation capacity is extremely reduced in OLETF rats independently of diabetes development, (2) the aging process decreases BCKDC activity and protein abundance in the liver of normal rats, and (3) differential posttranscriptional regulation for the subunits of BCKDC may exist in skeletal muscle.

TGF-beta3 is expressed in taste buds and inhibits proliferation of primary cultured taste epithelial cells.

PubMed

Nakamura, Shin-ichi; Kawai, Takayuki; Kamakura, Takashi; Ookura, Tetsuya

2010-01-01

Transforming growth factor-betas (TGF-betas), expressed in various tissues, play important roles in embryonic development and adult tissue homeostasis through their effects on cell proliferation, cell differentiation, cell death, and cell motility. However, expression of TGF-beta signaling components and their biological effect on taste epithelia has not been elucidated. We performed expression analysis of TGF-beta signaling components in taste epithelia and found that the TGF-beta3 mRNA was specifically expressed in taste buds. Type II TGF-betas receptor (TbetaR-II) mRNA was specifically expressed in the tongue epithelia including the taste epithelia. To elucidate the biological function of TGF-beta3 in taste epithelia, we performed proliferation assay with primary cultured taste epithelial cells. In the presence of TGF-beta3, percentage of BrdU-labeled cells decreased significantly, suggesting that the TGF-beta3 inhibited the proliferation of cultured taste epithelial cells through inhibiting cell-cycle entry into S phase. By quantitative reverse transcription-polymerase chain reaction assay, we found that the TGF-beta3 resulted in an increased level of expression of p15Ink4b and p21Cip1, suggesting that the TGF-beta3 inhibited the taste epithelial cell proliferation through inhibiting G1cyclin-Cdk complexes. Taken together, these results suggested that the TGF-beta3 may regulate taste epithelial cell homeostasis through controlling cell proliferation.

Extended 3{beta}-alkyl steranes and 3-alkyl triaromatic steroids in crude oils and rock extracts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dahl, J.; Moldowan, J.M.; Summons, R.E.

1995-09-01

In oils and Precambian- to Miocene-age source rocks from varying depositional environments, we have conclusively identified several novel 3-alkyl sterane and triaromatic steroid series, including (1) 3{beta}-n-pentyl steranes, (2) 3{beta}-isopentyl steranes, (3) 3{beta}-n-hexyl steranes, (4) 3{beta}-n-hepatyl steranes, (5) 3,4-dimethyl steranes, (6) 3{beta}-butyl,4-methyl steranes, (7) triaromatic 3-n-pentyl steroids, and (8) triaromatic 3-isopentyl steroids. We have also tentatively identified additional homologs with 3-alkyl substituents as large as C{sub 11}. The relative abundances of these compounds vary substantially between samples, as indicated by (1) the ratio of 3{beta}-n-pentyl steranes to 3{beta}-isopentyl steranes and (2) the ratio of 3-n-pentyl triaromatic steroids to 3-isopentyl triaromaticmore » steroids. These data suggest possible utility for these parameters as tools for oil-source rock correlations and reconstruction of depositional environments. Although no 3-alkyl steroid natural products are currently known, several lines of evidence suggest that 3{beta}-alkyl steroids result from bacterial side-chain additions to diagenetic {delta}{sup 2}-sterenes.« less

Alpha-beta T cells provide protection against lethal encephalitis in the murine model of VEEV infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paessler, Slobodan; Yun, Nadezhda E.; Judy, Barbara M.

2007-10-25

We evaluated the safety and immunogenicity of a chimeric alphavirus vaccine candidate in mice with selective immunodeficiencies. This vaccine candidate was highly attenuated in mice with deficiencies in the B and T cell compartments, as well as in mice with deficient gamma-interferon responsiveness. However, the level of protection varied among the strains tested. Wild type mice were protected against lethal VEEV challenge. In contrast, alpha/beta ({alpha}{beta}) TCR-deficient mice developed lethal encephalitis following VEEV challenge, while mice deficient in gamma/delta ({gamma}{delta}) T cells were protected. Surprisingly, the vaccine potency was diminished by 50% in animals lacking interferon-gamma receptor alpha chain (R1)-chainmore » and a minority of vaccinated immunoglobulin heavy chain-deficient ({mu}MT) mice survived challenge, which suggests that neutralizing antibody may not be absolutely required for protection. Prolonged replication of encephalitic VEEV in the brain of pre-immunized mice is not lethal and adoptive transfer experiments indicate that CD3{sup +} T cells are required for protection.« less

Expression of platelet-derived growth factor and its receptors in proliferative disorders of fibroblastic origin.

PubMed

Smits, A; Funa, K; Vassbotn, F S; Beausang-Linder, M; af Ekenstam, F; Heldin, C H; Westermark, B; Nistér, M

1992-03-01

Platelet-derived growth factor (PDGF) is known to stimulate the proliferation of connective tissue-derived cells in vitro. Less is known about its functions in vivo, and the role of PDGF in the development of human tumors has not been clarified. The authors have investigated the occurrence of PDGF and PDGF receptors in a series of proliferative disorders of fibroblastic origin using immunohistochemical and in situ hybridization techniques. High expression of PDGF beta-receptor mRNA and protein was found in the malignant tumors, and also in some benign lesions, such as dermatofibroma. In all these cases, benign as well as malignant, the PDGF B-chain mRNA, and less clearly, the PDGF A-chain mRNA, were coexpressed with the beta-receptor. In contrast, high expression of PDGF alpha-receptor mRNA was only found in fully malignant lesions, i.e., malignant fibrous histiocytoma. These data indicate that an autocrine growth stimulation via the PDGF beta-receptor could occur in an early phase of tumorigenesis, and may be a necessary but insufficient event for the progression into fully malignant human connective tissue lesions.

Metallo-Beta-Lactamase Producing Pseudomonas aeruginosa in a Healthcare Setting in Alexandria, Egypt.

PubMed

Abaza, Amani F; El Shazly, Soraya A; Selim, Heba S A; Aly, Gehan S A

2017-09-27

Pseudomonas aeruginosa has emerged as a major healthcare associated pathogen that creates a serious public health disaster in both developing and developed countries. In this work we aimed at studying the occurrence of metallo-beta-lactamase (MBL) producing P. aeruginosa in a healthcare setting in Alexandria, Egypt. This cross sectional study included 1583 clinical samples that were collected from patients admitted to Alexandria University Students' Hospital. P. aeruginosa isolates were identified using standard microbiological methods and were tested for their antimicrobial susceptibility patterns using single disc diffusion method according to the Clinical and Laboratory Standards Institute recommendations. Thirty P. aeruginosa isolates were randomly selected and tested for their MBL production by both phenotypic and genotypic methods. Diagnostic Epsilometer test was done to detect metallo-beta-lactamase enzyme producers and polymerase chain reaction test was done to detect imipenemase (IMP), Verona integron-encoded (VIM) and Sao Paulo metallo-beta-lactamase (IMP) encoding genes. Of the 1583 clinical samples, 175 (11.3%) P. aeruginosa isolates were identified. All the 30 (100%) selected P. aeruginosa isolates that were tested for MBL production by Epsilometer test were found to be positive; where 19 (63.3%) revealed blaSPM gene and 11 (36.7%) had blaIMP gene. blaVIM gene was not detected in any of the tested isolates. Isolates of MBL producing P. aeruginosa were highly susceptible to polymyxin B 26 (86.7%) and highly resistant to amikacin 26 (86.7%). MBL producers were detected phenotypically by Epsilometer test in both carbapenem susceptible and resistant P. aeruginosa isolates. blaSPM was the most commonly detected MBL gene in P. aeruginosa isolates.

A Single Mutation at the Sheet Switch Region Results in Conformational Changes Favoring 6 Light-Chain Fibrillogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hernández-Santoyo, A.; Del Pozo Yauner, L; Fuentes-Silva, D

Systemic amyloid light-chain (LC) amyloidosis is a disease process characterized by the pathological deposition of monoclonal LCs in tissue. All LC subtypes are capable of fibril formation although {lambda} chains, particularly those belonging to the {lambda}6 type, are overrepresented. Here, we report the thermodynamic and in vitro fibrillogenic properties of several mutants of the {lambda}6 protein 6aJL2 in which Pro7 and/or His8 was substituted by Ser or Pro. The H8P and H8S mutants were almost as stable as the wild-type protein and were poorly fibrillogenic. In contrast, the P7S mutation decreased the thermodynamic stability of 6aJL2 and greatly enhanced itsmore » capacity to form amyloid-like fibrils in vitro. The crystal structure of the P7S mutant showed that the substitution induced both local and long-distance effects, such as the rearrangement of the VL (variable region of the light chain)-VL interface. This mutant crystallized in two orthorhombic polymorphs, P2{sub 1}2{sub 1}2{sub 1} and C222{sub 1}. In the latter, a monomer that was not arranged in the typical Bence-Jones dimer was observed for the first time. Crystal-packing analysis of the C222{sub 1} lattice showed the establishment of intermolecular {beta}-{beta} interactions that involved the N-terminus and {beta}-strand B and that these could be relevant in the mechanism of LC fibril formation. Our results strongly suggest that Pro7 is a key residue in the conformation of the N-terminal sheet switch motif and, through long-distance interactions, is also critically involved in the contacts that stabilized the VL interface in {lambda}6 LCs.« less

Loop III region of platelet-derived growth factor (PDGF) B-chain mediates binding to PDGF receptors and heparin.

PubMed Central

Schilling, D; Reid IV, J D; Hujer, A; Morgan, D; Demoll, E; Bummer, P; Fenstermaker, R A; Kaetzel, D M

1998-01-01

Site-directed mutagenesis of the platelet-derived growth factor (PDGF) B-chain was conducted to determine the importance of cationic amino acid residues (Arg160-Lys161-Lys162; RKK) located within the loop III region in mediating the biological and cell-association properties of the molecule. Binding to both PDGF alpha-and beta-receptors was inhibited by the conversion of all three cationic residues into anionic glutamates (RKK-->EEE), whereas an RKK-->SSS mutant also exhibited a modest loss in affinity for beta-receptors. Replacements with serine at either Arg160 (RKK-->SKK) or at all three positions (RKK-->SSS) had little effect on binding to alpha-receptors. Replacements with either glutamic or serine residues at any of the three positions also resulted in significant inhibition of heparin-binding activity. Furthermore, the RKK-->EEE mutant exhibited decreased association with the cell surface and accumulated in the culture medium as 29-32 kDa forms. Stable transfection of U87 astrocytoma cells with RKK-->EEE mutants of either the A-chain or the B-chain inhibited malignant growth in athymic nude mice. Despite altered receptor-binding activities, each of the loop III mutants retained full mitogenic activity when applied to cultured Swiss 3T3 cells. CD spectrophotometric analysis of the RKK-->EEE mutant revealed a secondary structure indistinguishable from the wild type, with a high degree of beta-sheet structure and random coil content (50% and 43% respectively). These findings indicate an important role of the Arg160-Lys161-Lys162 sequence in mediating the biological and cell-associative activities of the PDGF-BB homodimer, and reveal that the mitogenic activity of PDGF-BB is insufficient to mediate its full oncogenic properties. PMID:9677323

Life-time expression of the proteins peroxiredoxin, beta-synuclein, PARK7/DJ-1, and stathmin in the primary visual and primary somatosensory cortices in rats

PubMed Central

Böhm, Michael R. R.; Melkonyan, Harutyun; Thanos, Solon

2015-01-01

Four distinct proteins are regulated in the aging neuroretina and may be regulated in the cerebral cortex, too: peroxiredoxin, beta-synuclein, PARK[Parkinson disease(autosomal recessive, early onset)]7/DJ-1, and Stathmin. Thus, we performed a comparative analysis of these proteins in the the primary somatosensory cortex (S1) and primary visual cortex (V1) in rats, in order to detect putative common development-, maturation- and age-related changes. The expressions of peroxiredoxin, beta-synuclein, PARK[Parkinson disease (autosomal recessive, early onset)]7/DJ-1, and Stathmin were compared in the newborn, juvenile, adult, and aged S1 and V1. Western blot (WB), quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and immunohistochemistry (IHC) analyses were employed to determine whether the changes identified by proteomics were verifiable at the cellular and molecular levels. All of the proteins were detected in both of the investigated cortical areas. Changes in the expressions of the four proteins were found throughout the life-time of the rats. Peroxiredoxin expression remained unchanged over life-time. Beta-Synuclein expression was massively increased up to the adult stage of life in both the S1 and V1. PARK[Parkinson disease (autosomal recessive, early onset)]7/DJ-1 exhibited a massive up-regulation in both the S1 and V1 at all ages. Stathmin expression was massively down regulated after the neonatal period in both the S1 and V1. The detected protein alterations were analogous to their retinal profiles. This study is the first to provide evidence that peroxiredoxin, beta-synuclein, PARK[Parkinson disease (autosomal recessive, early onset)]7/DJ-1, and Stathmin are associated with postnatal maturation and aging in both the S1 and V1 of rats. These changes may indicate their involvement in key functional pathways and may account for the onset or progression of age-related pathologies. PMID:25788877

Heterozygous Hb Hope [beta136(H14)Gly --> Asp] in association with heterozygous beta0-thalassemia with apparent homozygous expression, in a Spanish patient.

PubMed

Beneitez, David; Carrera, Alícia; Duran-Suárez, Joan Ramón; Paz, Victoria; León, Antonio; García Talavera, Juan

2006-01-01

Hb Hope [beta136(H14)Gly --> Asp (GGT --> GAT)] has been found alone or in combination with other globin gene mutations in several African-American families, as well as in Japanese, Thai, Laotian, Cuban and Mauritanian families. We report the hematological and molecular characteristics of a heterozygous association of Hb Hope with beta0-thalassemia (thal) in a Spanish patient, in whom the level of expression of abnormal hemoglobin (Hb) by cation exchange high performance liquid chromatography (HPLC) and electrophoresis suggested initially a homozygous expression of the abnormal Hb, although sequencing of the polymerase chain reaction (PCR)-amplified beta-globin gene demonstrated a heterozygous genotype for Hb Hope. To the best of our knowledge, this is the first description of a case of Hb Hope in a Spanish family.

Synthesis of 3 alpha,7 alpha,12 alpha,25-tetrahydroxy-5 beta-cholestan-24-one, an intermediate in the 25-hydroxylation pathway of cholic acid biosynthesis from cholesterol.

PubMed

Dayal, B; Tint, G S; Batta, A K; Shefer, S; Salen, G; Bose, A K; Pramanik, B N

1983-02-01

This paper describes the chemical synthesis of 3 alpha,7 alpha,12 alpha,25-tetrahydroxy-5 beta-cholestan-24-one via selective oxidation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha, 24 xi,25-pentol with silver carbonate on celite. The structure of this 24-keto bile alcohol was confirmed by gas-liquid chromatography and mass spectrometry. Synthesis of this compound via pyridinium chlorochromate oxidation of the triacetoxy derivative of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 xi,25-pentol followed by saponification further established its structure. 3 alpha,7 alpha,12 alpha,25-Tetrahydroxy-5 beta-cholestan-24-one was required for the in vivo and in vitro studies of side-chain oxidation and cleavage in the 25-hydroxylation pathway of cholic acid biosynthesis.

Beta-Cryptoxanthin as a source of Vitamin A.

USDA-ARS?s Scientific Manuscript database

Beta-cryptoxanthin is a common carotenoid that is found in fruit, and in human blood and tissues. Foods that are rich in beta-cryptoxanthin include tangerines, persimmons, and oranges. Beta-cryptoxanthin has several functions that are important for human health, including roles in antioxidant defens...

RHEB expression in fibroadenomas of the breast.

PubMed

Eom, Minseob; Han, Airi; Yi, Sang Yeop; Shin, John Junghun; Cui, Ying; Park, Kwang Hwa

2008-04-01

Although fibroadenoma is one of the most common types of benign breast tumor, genes specific to the tumor have not been identified. Microarrays were used to identify differentially expressed genes between fibroadenoma and infiltrating ductal carcinoma. The comparative expression of one of the identified genes, RAS homolog enriched in the brain (RHEB), was further explored using reverse transcriptase-polymerase chain reaction (RT-PCR). Microarray analysis was performed on tissue samples from five patients with fibroadenoma. In the fibroadenoma samples, the genes HDAC1, ROS1, TNFRSF10A, WASP2, TYRP1, WEE1, and RHEB were expressed at levels more than twofold higher than in the normal tissues. RT-PCR for RHEB indicated increased expression of RHEB in fibroadenoma compared to breast cancer. When studied with real-time PCR, the average RHEB/beta-actin ratio in fibroadenoma samples was 1.99, 2.46-fold greater than the average RHEB/beta-actin ratio in breast carcinoma of 0.81 (P < 0.01). Immunohistochemistry and PCR followed by microdissection shows increased expression of RHEB in epithelial cells compared to the stromal cells of fibroadenoma. Therefore, RHEB could be used cytopathologically to distinguish fibroadenoma from malignant breast carcinomas as a secondary diagnostic tool.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Cong; Sawaya, Michael R.; Eisenberg, David

{beta}{sub 2}-microglobulin ({beta}{sub 2}-m) is the light chain of the type I major histocompatibility complex. It deposits as amyloid fibrils within joints during long-term hemodialysis treatment. Despite the devastating effects of dialysis-related amyloidosis, full understanding of how fibrils form from soluble {beta}{sub 2}-m remains elusive. Here we show that {beta}{sub 2}-m can oligomerize and fibrillize via three-dimensional domain swapping. Isolating a covalently bound, domain-swapped dimer from {beta}{sub 2}-m oligomers on the pathway to fibrils, we were able to determine its crystal structure. The hinge loop that connects the swapped domain to the core domain includes the fibrillizing segment LSFSKD, whosemore » atomic structure we also determined. The LSFSKD structure reveals a class 5 steric zipper, akin to other amyloid spines. The structures of the dimer and the zipper spine fit well into an atomic model for this fibrillar form of {beta}{sub 2}-m, which assembles slowly under physiological conditions.« less

Beta-structures in fibrous proteins.

PubMed

Kajava, Andrey V; Squire, John M; Parry, David A D

2006-01-01

The beta-form of protein folding, one of the earliest protein structures to be defined, was originally observed in studies of silks. It was then seen in early studies of synthetic polypeptides and, of course, is now known to be present in a variety of guises as an essential component of globular protein structures. However, in the last decade or so it has become clear that the beta-conformation of chains is present not only in many of the amyloid structures associated with, for example, Alzheimer's Disease, but also in the prion structures associated with the spongiform encephalopathies. Furthermore, X-ray crystallography studies have revealed the high incidence of the beta-fibrous proteins among virulence factors of pathogenic bacteria and viruses. Here we describe the basic forms of the beta-fold, summarize the many different new forms of beta-structural fibrous arrangements that have been discovered, and review advances in structural studies of amyloid and prion fibrils. These and other issues are described in detail in later chapters.

Insulin-producing cells could not mimic the physiological regulation of insulin secretion performed by pancreatic beta cells

PubMed Central

2013-01-01

Objective The aim of this study was to compare the difference between insulin-producing cells (IPCs) and normal human pancreatic beta cells both in physiological function and morphological features in cellular level. Methods The levels of insulin secretion were measured by enzyme-linked immunosorbent assay. The insulin gene expression was determined by real-time quantitative polymerase chain reaction. The morphological features were detected by atomic force microscopy (AFM) and laser confocal scanning microscopy. Results IPCs and normal human pancreatic beta cells were similar to each other under the observation in AFM with the porous structure features in the cytoplasm. Both number of membrane particle size and average roughness of normal human beta cells were higher than those of IPCs. Conclusions Our results firstly revealed that the cellular ultrastructure of IPCs was closer to that of normal human pancreatic beta cells, but they still could not mimic the physiological regulation of insulin secretion performed by pancreatic beta cells. PMID:23421382

Synthesis of anatase TiO2 nanoparticles with beta-cyclodextrin as a supramolecular shell.

PubMed

Li, Landong; Sun, Xiaohong; Yang, Yali; Guan, Naijia; Zhang, Fuxiang

2006-11-20

We report a novel, green hydrothermal-synthesis route to well-dispersed anatase TiO2 nanoparticles with particle sizes of 9-16 nm in the presence of beta-CD (beta-cyclodextrin). During the synthesis process, the CD-containing synthesis mixture assembled in both longitudinal and latitudinal directions. Driven by the interaction between molecules, the beta-CDs assembled in the longitudinal direction to form long-chain compounds, whereas in the latitudinal direction, they tended to form regular aggregates through coordination with the Ti species from the hydrolysis of tetrabutyl titanate. In view of the effect of the coordination and the steric hindrance of beta-CDs as a supramolecular shell, homogeneous nuclei and slow growth of TiO2 crystals during the synthesis process was observed, which was responsible for the formation of uniform TiO2 nanoparticles. The low beta-CD dosage and the high product yield (>90%) demonstrated well the potential of this synthesis route in the large-scale industrial production of anatase nanoparticles.

Polymer support oligonucleotide synthesis XVIII: use of beta-cyanoethyl-N,N-dialkylamino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product.

PubMed Central

Sinha, N D; Biernat, J; McManus, J; Köster, H

1984-01-01

Various 5'O-N-protected deoxynucleoside-3'-O-beta-cyanoethyl-N,N-dialkylamino-/N- morpholinophosphoramidites were prepared from beta-cyanoethyl monochlorophosphoramidites of N,N-dimethylamine, N,N-diisopropylamine and N-morpholine. These active deoxynucleoside phosphates have successfully been used for oligodeoxynucleotide synthesis on controlled pore glass as polymer support and are very suitable for automated DNA-synthesis due to their stability in solution. The intermediate dichloro-beta- cyanoethoxyphosphine can easily be prepared free from any PC1(3) contamination. The active monomers obtained from beta-cyanoethyl monochloro N,N- diisopropylaminophosphoramidites are favoured. Cleavage of the oligonucleotide chain from the polymer support, N-deacylation and deprotection of beta-cyanoethyl group from the phosphate triester moiety can be performed in one step with concentrated aqueous ammonia. Mixed oligodeoxynucleotides are characterized by the sequencing method of Maxam and Gilbert. Images PMID:6547529

Structural and sequence features of two residue turns in beta-hairpins.

PubMed

Madan, Bharat; Seo, Sung Yong; Lee, Sun-Gu

2014-09-01

Beta-turns in beta-hairpins have been implicated as important sites in protein folding. In particular, two residue β-turns, the most abundant connecting elements in beta-hairpins, have been a major target for engineering protein stability and folding. In this study, we attempted to investigate and update the structural and sequence properties of two residue turns in beta-hairpins with a large data set. For this, 3977 beta-turns were extracted from 2394 nonhomologous protein chains and analyzed. First, the distribution, dihedral angles and twists of two residue turn types were determined, and compared with previous data. The trend of turn type occurrence and most structural features of the turn types were similar to previous results, but for the first time Type II turns in beta-hairpins were identified. Second, sequence motifs for the turn types were devised based on amino acid positional potentials of two-residue turns, and their distributions were examined. From this study, we could identify code-like sequence motifs for the two residue beta-turn types. Finally, structural and sequence properties of beta-strands in the beta-hairpins were analyzed, which revealed that the beta-strands showed no specific sequence and structural patterns for turn types. The analytical results in this study are expected to be a reference in the engineering or design of beta-hairpin turn structures and sequences. © 2014 Wiley Periodicals, Inc.

Biochemical basis of type IB (E1beta ) mutations in maple syrup urine disease. A prevalent allele in patients from the Druze kindred in Israel.

PubMed

Wynn, R M; Chuang, J L; Sansaricq, C; Mandel, H; Chuang, D T

2001-09-28

Maple syrup urine disease (MSUD) is a metabolic disorder associated with often-fatal ketoacidosis, neurological derangement, and mental retardation. In this study, we identify and characterize two novel type IB MSUD mutations in Israeli patients, which affect the E1beta subunit in the decarboxylase (E1) component of the branched-chain alpha-ketoacid dehydrogenase complex. The recombinant mutant E1 carrying the prevalent S289L-beta (TCG --> TTG) mutation in the Druze kindred exists as a stable inactive alphabeta heterodimer. Based on the human E1 structure, the S289L-beta mutation disrupts the interactions between Ser-289-beta and Glu-290-beta', and between Arg-309-beta and Glu-290-beta', which are essential for native alpha(2)beta(2) heterotetrameric assembly. The R133P-beta (CGG --> CCG) mutation, on the other hand, is inefficiently expressed in Escherichia coli as heterotetramers in a temperature-dependent manner. The R133P-beta mutant E1 exhibits significant residual activity but is markedly less stable than the wild-type, as measured by thermal inactivation and free energy change of denaturation. The R133P-beta substitution abrogates the coordination of Arg-133-beta to Ala-95-beta, Glu-96-beta, and Ile-97-beta, which is important for strand-strand interactions and K(+) ion binding in the beta subunit. These findings provide new insights into folding and assembly of human E1 and will facilitate DNA-based diagnosis for MSUD in the Israeli population.

Beta-papillomaviruses in anogenital hairs plucked from healthy individuals.

PubMed

Potocnik, Marko; Kocjan, Bostjan J; Seme, Katja; Luzar, Bostjan; Babic, Dunja Z; Poljak, Mario

2006-12-01

A total of 150 specimens of anogenital hairs plucked from the scrotal, pubic, and perianal region of 51 immunocompetent healthy male individuals were tested for the presence of beta-papillomaviruses (beta-HPV) using the nested M(a)/H(a) polymerase chain reaction. Beta-HPV were found in a total of 38 (25.3%) of 150 hair samples. According to the sampling sites, beta-HPV were detected in 18/51 (35.3%), 13/50 (26.0%), and 7/49 (14.3%) plucked hair samples obtained from the pubic, scrotal, and perianal region, respectively. The prevalence of beta-HPV in the plucked pubic hairs was significantly higher than in the perianal hairs (P = 0.013). In contrast, the difference in the prevalence of beta-HPV in the pubic and scrotal hairs as well as in scrotal and perianal hairs did not reach statistical significance (P = 0.302 and P = 0.227, respectively). The difference in the lifetime-cumulative sun exposure is the most likely explanation for the differences obtained on beta-HPV prevalence. Beta-HPV genotype HPV-38 was detected most frequently, followed by HPV-36, HPV-15, and HPV-14D. In addition to the beta-HPV recognized officially five partial DNA sequences suggesting putative new HPV genotypes were identified. (c) 2006 Wiley-Liss, Inc.

Structural characterisation of galactoglucomannan secreted by suspension-cultured cells of Nicotiana plumbaginifolia.

PubMed

Sims, I M; Craik, D J; Bacic, A

1997-08-25

Galactoglucomannan (GGM) from cultures of Nicotiana plumbaginifolia has Man:Glc:Gal:Ara:Xyl in 1.0:1.1:1.0:0.1:0.04 ratio. Linkage analysis contained 4- and 4,6-Manp, 4-Glcp, terminal Galp and 2-Galp, small amounts and terminal Arap and terminal Xylp, and approximately 0.03 mol acetyl per mol of glucosyl residue. Treatment with alpha- and beta-D-galactosidases showed that the majority of the side-chains were either single Galp-alpha-(1-->residues or the disaccharide Galp-beta-(1-->2)-Galp-alpha-(1-->linked to O-6 of the 4-Manp residues of the glucomannan backbone. Analysis of the oligosaccharides generated by endo-(1-->4)-beta-mannanase digestion confirmed that the GGM comprises a backbone of predominantly alternating-->4)-D-Manp-beta-(1-->and-->4)-D-Glcp-beta-(1-->branch ed at O-6 of 65% of the 4-Manp residues. The major oligosaccharide identified was D-Glcp-beta-(1-->4)-[D-Galp-beta-(1-->2)-D-Galp-alpha-(1-->6)]-D-Man p-beta-(1-->4)-D-Glcp-beta-(1-->4)-[D-Galp-alpha-(1-->6)]-D-Manp -beta-(1-->(27%), and most of the other oligosaccharides produced in significant quantities were based on this structure.

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

PubMed

Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-05-15

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes.

Discovery of nonsteroidal 17beta-hydroxysteroid dehydrogenase 1 inhibitors by pharmacophore-based screening of virtual compound libraries.

PubMed

Schuster, Daniela; Nashev, Lyubomir G; Kirchmair, Johannes; Laggner, Christian; Wolber, Gerhard; Langer, Thierry; Odermatt, Alex

2008-07-24

17Beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) plays a pivotal role in the local synthesis of the most potent estrogen estradiol. Its expression is a prognostic marker for the outcome of patients with breast cancer and inhibition of 17beta-HSD1 is currently under consideration for breast cancer prevention and treatment. We aimed to identify nonsteroidal 17beta-HSD1 inhibitor scaffolds by virtual screening with pharmacophore models built from crystal structures containing steroidal compounds. The most promising model was validated by comparing predicted and experimentally determined inhibitory activities of several flavonoids. Subsequently, a virtual library of nonsteroidal compounds was screened against the 3D pharmacophore. Analysis of 14 selected compounds yielded four that inhibited the activity of human 17beta-HSD1 (IC 50 below 50 microM). Specificity assessment of identified 17beta-HSD1 inhibitors emphasized the importance of including related short-chain dehydrogenase/reductase (SDR) members to analyze off-target effects. Compound 29 displayed at least 10-fold selectivity over the related SDR enzymes tested.

9-Methyl-beta-carboline has restorative effects in an animal model of Parkinson's disease.

PubMed

Wernicke, Catrin; Hellmann, Julian; Zieba, Barbara; Kuter, Katarzyna; Ossowska, Krystyna; Frenzel, Monika; Dencher, Norbert A; Rommelspacher, Hans

2010-01-01

In a previous study, a primary culture of midbrain cells was exposed to 9-methyl-beta-carboline for 48 h, which caused an increase in the number of tyrosine hydroxylase-positive cells. Quantitative RT-PCR revealed increased transcription of genes participating in the maturation of dopaminergic neurons. These in vitro findings prompted us to investigate the restorative actions of 9-methyl-beta-carboline in vivo. The compound was delivered for 14 days into the left cerebral ventricle of rats pretreated with the neurotoxin 1-methyl-4-phenyl-pyridinium ion (MPP+) for 28 days applying a dose which lowered dopamine by approximately 50%. Interestingly, 9-methyl-beta-carboline reversed the dopamine-lowering effect of the neurotoxin in the left striatum. Stereological counts of tyrosine hydroxylase-immunoreactive cells in the substantia nigra revealed that the neurotoxin caused a decrease in the number of those cells. However, when treated subsequently with 9-methyl-beta-carboline, the number reached normal values. In search of an explanation for the restorative activity, we analyzed the complexes that compose the respiratory chain in striatal mitochondria by 2-dimension gel electrophoresis followed by MALDI-TOF peptide mass fingerprinting.We found no changes in the overall composition of the complexes. However, the activity of complex I was increased by approximately 80% in mitochondria from rats treated with MPP+ and 9-methyl-beta-carboline compared to MPP+ and saline and to sham-operated rats, as determined by measurements of nicotinamide adenine dinucleotide dehydrogenase activity. Microarray technology and single RT-PCR revealed the induction of neurotrophins: brain-derived neurotrophic factor, conserved dopamine neurotrophic factor, cerebellin 1 precursor protein, and ciliary neurotrophic factor. Selected western blots yielded consistent results. The findings demonstrate restorative effects of 9-methyl-beta-carboline in an animal model of Parkinson's disease that improve the effectiveness of the respiratory chain and promote the transcription and expression of neurotrophin-related genes.

Determination of harmane and harmine in human blood using reversed-phased high-performance liquid chromatography and fluorescence detection.

PubMed

Zheng, W; Wang, S; Barnes, L F; Guan, Y; Louis, E D

2000-03-15

A number of tremorogenic beta-carboline alkaloids have been found in common plant-derived foodstuffs, beverages, and inhaled substances. Because of their natural presence in the food chain, there is a growing concern regarding the potential risks of certain essential tremors associated with the long-term, low-level dietary exposure to these alkaloids. The purpose of this study was to develop an effective analytical method to determine blood levels of two major beta-carboline derivatives, harmane and harmine. Human blood was extracted with ethyl acetate and methyl-t-butyl ether (2:98) under an alkaline condition. After evaporation of organic solvent, the samples were reconstructed in methanol. The samples were fractionated on a 250 x 4.6-mm C18 reversed-phase column with an isocratic mobile system consisting of 17.5 mM potassium phosphate buffer (ph 6.5) and methanol (30:70), followed by an on-line fluorescence detection. The method had the detection limit to determine 206 and 81 pg/ml of harmane and harmine, respectively, in 10 ml of human blood. The intraday precision (C.V.) at 25 ng/ml was less than 6.7 and 3.4% for harmane and harmine, respectively. The interday precision was 7.3% for harmane and 5.4% for harmine. The method has proven sensitive, reproducible, and thus useful for both laboratory and clinical studies of beta-carboline toxicities. Copyright 2000 Academic Press.

Variability and repertoire size of T-cell receptor V alpha gene segments.

PubMed

Becker, D M; Pattern, P; Chien, Y; Yokota, T; Eshhar, Z; Giedlin, M; Gascoigne, N R; Goodnow, C; Wolf, R; Arai, K

The immune system of higher organisms is composed largely of two distinct cell types, B lymphocytes and T lymphocytes, each of which is independently capable of recognizing an enormous number of distinct entities through their antigen receptors; surface immunoglobulin in the case of the former, and the T-cell receptor (TCR) in the case of the latter. In both cell types, the genes encoding the antigen receptors consist of multiple gene segments which recombine during maturation to produce many possible peptides. One striking difference between B- and T-cell recognition that has not yet been resolved by the structural data is the fact that T cells generally require a major histocompatibility determinant together with an antigen whereas, in most cases, antibodies recognize antigen alone. Recently, we and others have found that a series of TCR V beta gene sequences show conservation of many of the same residues that are conserved between heavy- and light-chain immunoglobulin V regions, and these V beta sequences are predicted to have an immunoglobulin-like secondary structure. To extend these studies, we have isolated and sequenced eight additional alpha-chain complementary cDNA clones and compared them with published sequences. Analyses of these sequences, reported here, indicate that V alpha regions have many of the characteristics of V beta gene segments but differ in that they almost always occur as cross-hybridizing gene families. We conclude that there may be very different selective pressures operating on V alpha and V beta sequences and that the V alpha repertoire may be considerably larger than that of V beta.

Synthesis of 4-O-glycosylated 1,5-anhydro-D-fructose and of 1,5-anhydro-D-tagatose from a common intermediate 2,3-O-isopropylidene-D-fructose.

PubMed

Agoston, Károly; Dékány, Gyula; Lundt, Inge

2009-05-26

Four novel disaccharides of glycosylated 1,5-anhydro-D-ketoses have been prepared: 1,5-anhydro-4-O-beta-D-glucopyranosyl-D-fructose, 1,5-anhydro-4-O-beta-D-galactopyranosyl-D-fructose, 1,5-anhydro-4-O-beta-D-glucopyranosyl-D-tagatose, and 1,5-anhydro-4-O-beta-D-galactopyranosyl-D-tagatose. The common intermediate, 1,5-anhydro-2,3-O-isopropylidene-beta-D-fructopyranose, was prepared from D-fructose and was converted into the D-tagatose derivative by oxidation followed by stereoselective reduction to the 4-epimer. The anhydroketoses thus prepared were glycosylated and deprotected to give the disaccharides.

Mesalamine inhibits epithelial beta-catenin activation in chronic ulcerative colitis.

PubMed

Brown, Jeffrey B; Lee, Goo; Managlia, Elizabeth; Grimm, Gery R; Dirisina, Ramanarao; Goretsky, Tatiana; Cheresh, Paul; Blatner, Nichole R; Khazaie, Khashayarsha; Yang, Guang-Yu; Li, Linheng; Barrett, Terrence A

2010-02-01

Mesalamine is a mainstay therapeutic agent in chronic ulcerative colitis (CUC) in which condition it reverses crypt architectural changes and reduces colitis-associated cancer (CAC). The present study addressed the possibility that mesalamine reduces beta-catenin-associated progenitor cell activation, Akt-phosphorylated beta-catenin(Ser552) (P-beta-catenin), and colitis-induced dysplasia (CID). Effects of mesalamine on P-beta-catenin staining and function were assessed by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) in biopsy specimens of CUC in mild or "refractory" severe mucosal inflammation. Effects of mesalamine on epithelial proliferation and activation of Akt and beta-catenin were assessed in interleukin (IL)-10(-/-) colitis and CID by immunohistochemistry and Western blotting. Dysplasia was assessed by counting the number and lengths of lesions per colon. Data from IL-10(-/-) and human colitis samples show that mesalamine reduced Akt activation and P-beta-catenin levels in the middle and upper crypt. Reductions in P-beta-catenin in CUC biopsy specimens with severe inflammation suggested that mesalamine reduced P-beta-catenin levels in tissue refractory to mesalamine's anti-inflammatory effects. In IL-10(-/-) mice, mesalamine reduced CID concordant with inhibition of crypt Akt and beta-catenin signaling. The results are consistent with the model that mesalamine contributes to chemoprevention in CAC by reducing beta-catenin signaling within intestinal progenitors.

Short-chain fatty acids and poly-beta-hydroxyalkanoates: (New) Biocontrol agents for a sustainable animal production.

PubMed

Defoirdt, Tom; Boon, Nico; Sorgeloos, Patrick; Verstraete, Willy; Bossier, Peter

2009-01-01

Because of the risk of antibiotic resistance development, there is a growing awareness that antibiotics should be used more carefully in animal production. However, a decreased use of antibiotics could result in a higher frequency of pathogenic bacteria, which in its turn could lead to a higher incidence of infections. Short-chain fatty acids (SCFAs) have long been known to exhibit bacteriostatic activity. These compounds also specifically downregulate virulence factor expression and positively influence the gastrointestinal health of the host. As a consequence, there is currently considerable interest in SCFAs as biocontrol agents in animal production. Polyhydroxyalkanoates (PHAs) are polymers of beta-hydroxy short-chain fatty acids. Currently, PHAs are applied as replacements for synthetic polymers. These biopolymers can be depolymerised by many different microorganisms that produce extracellular PHA depolymerases. Interestingly, different studies provided some evidence that PHAs can also be degraded upon passage through the gastrointestinal tract of animals and consequently, adding these compounds to the feed might result in biocontrol effects similar to those described for SCFAs.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon, Hyock Joo; Abi-Mosleh, Lina; Wang, Michael L.

LDL delivers cholesterol to lysosomes by receptor-mediated endocytosis. Exit of cholesterol from lysosomes requires two proteins, membrane-bound Niemann-Pick C1 (NPC1) and soluble NPC2. NPC2 binds cholesterol with its isooctyl side chain buried and its 3{beta}-hydroxyl exposed. Here, we describe high-resolution structures of the N-terminal domain (NTD) of NPC1 and complexes with cholesterol and 25-hydroxycholesterol. NPC1(NTD) binds cholesterol in an orientation opposite to NPC2: 3{beta}-hydroxyl buried and isooctyl side chain exposed. Cholesterol transfer from NPC2 to NPC1(NTD) requires reorientation of a helical subdomain in NPC1(NTD), enlarging the opening for cholesterol entry. NPC1 with point mutations in this subdomain (distinct from themore » binding subdomain) cannot accept cholesterol from NPC2 and cannot restore cholesterol exit from lysosomes in NPC1-deficient cells. We propose a working model wherein after lysosomal hydrolysis of LDL-cholesteryl esters, cholesterol binds NPC2, which transfers it to NPC1(NTD), reversing its orientation and allowing insertion of its isooctyl side chain into the outer lysosomal membranes.« less

Characterization of human DHRS4: an inducible short-chain dehydrogenase/reductase enzyme with 3beta-hydroxysteroid dehydrogenase activity.

PubMed

Matsunaga, Toshiyuki; Endo, Satoshi; Maeda, Satoshi; Ishikura, Shuhei; Tajima, Kazuo; Tanaka, Nobutada; Nakamura, Kazuo T; Imamura, Yorishige; Hara, Akira

2008-09-15

Human DHRS4 is a peroxisomal member of the short-chain dehydrogenase/reductase superfamily, but its enzymatic properties, except for displaying NADP(H)-dependent retinol dehydrogenase/reductase activity, are unknown. We show that the human enzyme, a tetramer composed of 27kDa subunits, is inactivated at low temperature without dissociation into subunits. The cold inactivation was prevented by a mutation of Thr177 with the corresponding residue, Asn, in cold-stable pig DHRS4, where this residue is hydrogen-bonded to Asn165 in a substrate-binding loop of other subunit. Human DHRS4 reduced various aromatic ketones and alpha-dicarbonyl compounds including cytotoxic 9,10-phenanthrenequinone. The overexpression of the peroxisomal enzyme in cultured cells did not increase the cytotoxicity of 9,10-phenanthrenequinone. While its activity towards all-trans-retinal was low, human DHRS4 efficiently reduced 3-keto-C(19)/C(21)-steroids into 3beta-hydroxysteroids. The stereospecific conversion to 3beta-hydroxysteroids was observed in endothelial cells transfected with vectors expressing the enzyme. The mRNA for the enzyme was ubiquitously expressed in human tissues and several cancer cells, and the enzyme in HepG2 cells was induced by peroxisome-proliferator-activated receptor alpha ligands. The results suggest a novel mechanism of cold inactivation and role of the inducible human DHRS4 in 3beta-hydroxysteroid synthesis and xenobiotic carbonyl metabolism.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Ying; Li, Jianguo, E-mail: 2010lijianguo@sina.cn

Highlights: Black-Right-Pointing-Pointer Carbachol reduced the lipopolysaccharide-induced intestinal barrier breakdown. Black-Right-Pointing-Pointer Carbachol ameliorated the lipopolysaccharide-induced ileal tight junction damage. Black-Right-Pointing-Pointer Carbachol prevented the LPS-induced NF-{kappa}{beta} and myosin light-chain kinase activation. Black-Right-Pointing-Pointer Carbachol exerted its beneficial effects in an {alpha}7 nicotinic receptor-dependent manner. -- Abstract: Carbachol is a cholinergic agonist that protects the intestines after trauma or burn injury. The present study determines the beneficial effects of carbachol and the mechanisms by which it ameliorates the lipopolysaccharide (LPS)-induced intestinal barrier breakdown. Rats were injected intraperitoneally with 10 mg/kg LPS. Results showed that the gut barrier permeability was reduced, the ultrastructural disruption ofmore » tight junctions (TJs) was prevented, the redistribution of zonula occludens-1 and claudin-2 proteins was partially reversed, and the nuclear factor-kappa beta (NF-{kappa}{beta}) and myosin light-chain kinase (MLCK) activation in the intestinal epithelium were suppressed after carbachol administration in LPS-exposed rats. Pretreatment with the {alpha}7 nicotinic acetylcholine receptor ({alpha}7nAchR) antagonist {alpha}-bungarotoxin blocked the protective action of carbachol. These results suggested that carbachol treatment can protect LPS-induced intestinal barrier dysfunction. Carbachol exerts its beneficial effect on the amelioration of the TJ damage by inhibiting the NF-{kappa}{beta} and MLCK pathways in an {alpha}7nAchR-dependent manner.« less

Trafficking of cell surface beta-amyloid precursor protein: retrograde and transcytotic transport in cultured neurons

PubMed Central

1995-01-01

Amyloid beta-protein (A beta), the principal constituent of senile plaques seen in Alzheimer's disease (AD), is derived by proteolysis from the beta-amyloid precursor protein (beta PP). The mechanism of A beta production in neurons, which are hypothesized to be a rich source of A beta in brain, remains to be defined. In this study, we describe a detailed localization of cell surface beta PP and its subsequent trafficking in primary cultured neurons. Full-length cell surface beta PP was present primarily on perikarya and axons, the latter with a characteristic discontinuous pattern. At growth cones, cell surface beta PP was inconsistently detected. By visualizing the distribution of beta PP monoclonal antibodies added to intact cultures, beta PP was shown to be internalized from distal axons or terminals and retrogradely transported back to perikarya in organelles which colocalized with fluid-phase endocytic markers. Retrograde transport of beta PP was shown in both hippocampal and peripheral sympathetic neurons, the latter using a compartment culture system that isolated cell bodies from distal axons and terminals. In addition, we demonstrated that beta PP from distal axons was transcytotically transported to the surface of perikarya from distal axons in sympathetic neurons. Indirect evidence of this transcytotic pathway was obtained in hippocampal neurons using antisense oligonucleotide to the kinesin heavy chain to inhibit anterograde beta PP transport. Taken together, these results demonstrate novel aspects of beta PP trafficking in neurons, including retrograde axonal transport and transcytosis. Moreover, the axonal predominance of cell surface beta PP is unexpected in view of the recent report of polarized sorting of beta PP to the basolateral domain of MDCK cells. PMID:7721945

Structural investigation of C4b-binding protein by molecular modeling: localization of putative binding sites.

PubMed

Villoutreix, B O; Härdig, Y; Wallqvist, A; Covell, D G; García de Frutos, P; Dahlbäck, B

1998-06-01

C4b-binding protein (C4BP) contributes to the regulation of the classical pathway of the complement system and plays an important role in blood coagulation. The main human C4BP isoform is composed of one beta-chain and seven alpha-chains essentially built from three and eight complement control protein (CCP) modules, respectively, followed by a nonrepeat carboxy-terminal region involved in polymerization of the chains. C4BP is known to interact with heparin, C4b, complement factor I, serum amyloid P component, streptococcal Arp and Sir proteins, and factor VIII/VIIIa via its alpha-chains and with protein S through its beta-chain. The principal aim of the present study was to localize regions of C4BP involved in the interaction with C4b, Arp, and heparin. For this purpose, a computer model of the 8 CCP modules of C4BP alpha-chain was constructed, taking into account data from previous electron microscopy (EM) studies. This structure was investigated in the context of known and/or new experimental data. Analysis of the alpha-chain model, together with monoclonal antibody studies and heparin binding experiments, suggests that a patch of positively charged residues, at the interface between the first and second CCP modules, plays an important role in the interaction between C4BP and C4b/Arp/Sir/heparin. Putative binding sites, secondary-structure prediction for the central core, and an overall reevaluation of the size of the C4BP molecule are also presented. An understanding of these intermolecular interactions should contribute to the rational design of potential therapeutic agents aiming at interfering specifically some of these protein-protein interactions.

Two-dimensional sup 1 H nuclear magnetic resonance study of AaH IT, an anti-insect toxin from the scorpion Androctonus australis Hector. Sequential resonance assignments and folding of the polypeptide chain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Darbon, H.; Weber, C.; Braun, W.

1991-02-19

Sequence-specific nuclear magnetic resonance assignments for the polypeptide backbone and for most of the amino acid side-chain protons, as well as the general folding of AaH IT, are described. AaH IT is a neurotoxin purified from the venom of the scorpion Androctonus australis Hector and is specifically active on the insect nervous system. The secondary structure and the hydrogen-bonding patterns in the regular secondary structure elements are deduced from nuclear Overhauser effects and the sequence locations of the slowly exchanging amide protons. The backbone folding is determined by distance geometry calculations with the DISMAN program. The regular secondary structure includesmore » two and a half turns of {alpha}-helix running from residues 21 to 30 and a three-stranded antiparallel {beta}-sheet including peptides 3-5, 34-38, and 41-46. Two tight turns are present, one connecting the end of the {alpha}-helix to an external strand of the {beta}-sheet, i.e., turn 31-34, and another connecting this same strand to the central one, i.e., turn 38-41. The differences in the specificity of these related proteins, which are able to discriminate between mammalian and insect voltage-dependent sodium channels of excitable tissues, are most probably brought about by the position of the C-terminal peptide with regard to a hydrophobic surface common to all scorpion toxins examined thus far. Thus, the interaction of a given scorpion toxin with its receptor might well be governed by the presence of this solvent-exposed hydrophobic surface, whereas adjacent areas modulate the specificity of the interaction.« less

Nutrition modulation of cachexia/proteolysis.

PubMed

Siddiqui, Rafat; Pandya, Darshak; Harvey, Kevin; Zaloga, Gary P

2006-04-01

Cachexia represents progressive wasting of muscle and adipose tissue and is associated with increased morbidity and mortality. Although anorexia usually accompanies cachexia, cachexia rarely responds to increased food intake alone. Our knowledge of the underlying mechanisms responsible for cachexia remains incomplete. However, most states of cachexia are associated with underlying inflammatory processes and/or cancer. These processes activate protein degradation and lipolytic pathways, resulting in tissue loss. In this article, we briefly review the pathophysiology of cachexia and discuss the role of specific nutrient supplements for the treatment of cachexia. The branched chain amino acid leucine, the leucine metabolite beta-hydroxy-beta-methylbutyrate, arginine, glutamine, omega-3 long chain fatty acids, conjugated linoleic acid, and polyphenols have demonstrated some efficacy in animal and/or human studies. Optimal treatment for cachexia is likely aimed at maximizing muscle and adipose synthesis while minimizing degradation.

Immunoglobulin heavy and light chains and T-cell receptor beta and gamma chains PCR assessment on cytological samples. A study comparing FTA cards and cryopreserved lymph node fine-needle cytology.

PubMed

Peluso, A L; Cozzolino, I; Bottiglieri, A; Lucchese, L; Di Crescenzo, R M; Langella, M; Selleri, C; Zeppa, P

2017-06-01

To evaluate and compare the DNA yield and quality extracted from lymph node fine needle cytology (FNC) samples stored on FTA cards to those cryopreserved, and to assess the immunoglobulin heavy and light chains (IGHK) and T-Cell receptor beta and gamma chains (TCRBG) PCR tests. DNA extractions were performed on FNC of 80 non-Hodgkin lymphomas (NHL), four myelomas and 56 benign reactive hyperplasias (BRH) cryopreserved and stored on FTA cards. The JAK2 gene was amplified to assess the DNA integrity and the IGHK/TCRBG clonality status was tested. IGHK monoclonality was found in 99% of B-cell NHL and 100% of myeloma. TCRBG monoclonality was found in 100% of T-cell NHL. TCRBG polyclonality was detected in 97% of B-cell NHL, 100% of myeloma and 96% of BRH. IGHK/TCRBG PCR data were confirmed by histological and/or follow-up controls. No differences were found in the DNA quality between cryopreservation and FTA cards storage methods. IGHK/TCRBG PCR of the lymphoproliferative process on FTA cards is comparable to those cryopreserved. FTA cards can be used to store lymph node FNC for further molecular investigations. © 2016 John Wiley & Sons Ltd.

Mechanism of insulin fibrillation: the structure of insulin under amyloidogenic conditions resembles a protein-folding intermediate.

PubMed

Hua, Qing-xin; Weiss, Michael A

2004-05-14

Insulin undergoes aggregation-coupled misfolding to form a cross-beta assembly. Such fibrillation has long complicated its manufacture and use in the therapy of diabetes mellitus. Of interest as a model for disease-associated amyloids, insulin fibrillation is proposed to occur via partial unfolding of a monomeric intermediate. Here, we describe the solution structure of human insulin under amyloidogenic conditions (pH 2.4 and 60 degrees C). Use of an enhanced sensitivity cryogenic probe at high magnetic field avoids onset of fibrillation during spectral acquisition. A novel partial fold is observed in which the N-terminal segments of the A- and B-chains detach from the core. Unfolding of the N-terminal alpha-helix of the A-chain exposes a hydrophobic surface formed by native-like packing of the remaining alpha-helices. The C-terminal segment of the B-chain, although not well ordered, remains tethered to this partial helical core. We propose that detachment of N-terminal segments makes possible aberrant protein-protein interactions in an amyloidogenic nucleus. Non-cooperative unfolding of the N-terminal A-chain alpha-helix resembles that observed in models of proinsulin folding intermediates and foreshadows the extensive alpha --> beta transition characteristic of mature fibrils.

The structure and dynamics of rat apo-cellular retinol-binding protein II in solution: comparison with the X-ray structure.

PubMed

Lu, J; Lin, C L; Tang, C; Ponder, J W; Kao, J L; Cistola, D P; Li, E

1999-03-05

The structure and dynamics of rat apo-cellular retinol binding protein II (apo-CRBP II) in solution has been determined by multidimensional NMR analysis of uniformly enriched recombinant rat 13C, 15N-apo-CRBP II and 15N-apo-CRBP II. The final ensemble of 24 NMR structures has been calculated from 3274 conformational restraints or 24.4 restraints/residue. The average root-mean-square deviation of the backbone atoms for the final 24 structures relative to their mean structure is 1.06 A. Although the average solution structure is very similar to the crystal structure, it differs at the putative entrance to the binding cavity, which is formed by the helix-turn-helix motif, the betaC-betaD turn and the betaE-betaF turn. The mean coordinates of the main-chain atoms of amino acid residues 28-38 are displaced in the solution structure relative to the crystal structure. The side-chain of F58, located on the betaC-betaD turn, is reoriented such that it interacts with L37 and no longer blocks entry into the ligand-binding pocket. Residues 28-35, which form the second helix of the helix-turn-helix motif in the crystal structure, do not exhibit a helical conformation in the solution structure. The solution structure of apo-CRBP II exhibits discrete regions of backbone disorder which are most pronounced at residues 28-32, 37-38 and 73-76 in the betaE-betaF turn as evaluated by the consensus chemical shift index, the root-mean-square deviation, amide 1H exchange rates and 15N relaxation studies. These studies indicate that fluctuations in protein conformation occur on the microseconds to ms time-scale in these regions of the protein. Some of these exchange processes can be directly observed in the three-dimensional 15N-resolved NOESY spectrum. These results suggest that in solution, apo-CRBP II undergoes conformational changes on the microseconds to ms time-scale which result in increased access to the binding cavity. Copyright 1999 Academic Press.

Proteopedia: Rossmann Fold: A Beta-Alpha-Beta Fold at Dinucleotide Binding Sites

ERIC Educational Resources Information Center

Hanukoglu, Israel

2015-01-01

The Rossmann fold is one of the most common and widely distributed super-secondary structures. It is composed of a series of alternating beta strand (ß) and alpha helical (a) segments wherein the ß-strands are hydrogen bonded forming a ß-sheet. The initial beta-alpha-beta (ßaß) fold is the most conserved segment of Rossmann folds. As this segment…

Prevalence of major depressive disorder in patients receiving beta-blocker therapy versus other medications.

PubMed

Carney, R M; Rich, M W; teVelde, A; Saini, J; Clark, K; Freedland, K E

1987-08-01

Depression is believed to be a common side effect in patients receiving beta-blocker therapy. However, diagnoses of depression defined by current diagnostic criteria may not be more common in patients receiving beta-blockers than in patients with the same medical disorder receiving other medications. Seventy-seven patients undergoing elective cardiac catheterization for evaluation of chest pain received a semi-structured diagnostic psychiatric interview. Twenty-one percent of the patients receiving beta-blockers and 33 percent of the patients receiving medications other than beta-blockers met the current American Psychiatric Association criteria for major depressive disorder (DSM-III) (p = NS). The mean heart rate and state anxiety scores for patients taking beta-blockers were significantly lower than those measured in patients taking medications other than beta-blockers. No other medical or demographic differences were observed between the two groups. Despite the methodologic limitations of the study, there does not appear to be a difference in the point prevalence of depression between patients receiving beta-blockers and those receiving other medications.

beta-Thalassemia present in cis to a new beta-chain structural variant, Hb Vicksburg [beta 75 (E19)Leu leads to 0].

PubMed Central

Adams, J G; Steinberg, M H; Newman, M V; Morrison, W T; Benz, E J; Iyer, R

1981-01-01

Hemoglobin Vicksburg was discovered in a 6-year-old Black boy who had been anemic since infancy. Examination of his hemolysate revealed 87.5% Hb F, 2.4% Hb A2, and 7.6% Hb Vicksburg, which had the electrophoretic and chromatographic properties of Hb A. Structural analysis of Hb Vicksburg demonstrated a deletion of leucine at beta 75(E19), a new variant. Hb Vicksburg was neither unstable nor subject to posttranslational degradation. The alpha/non-alpha biosynthetic ratio was 2.6. Because the proband appeared to be a mixed heterozygote for Hb Vicksburg and beta 0-thalassemia, Hb Vicksburg should have comprised the major portion of the hemolysate. Thus, Hb Vicksburg was synthesized at a rate considerably lower than would be expected on the basis of gene dosage. There was no reason to suspect abnormal translation of beta Vicksburg mRNA; in individuals with Hb St. Antoine (beta 74 and beta 75 deleted), the abnormal hemoglobin comprised 25% of the hemolysate in the simple heterozygote yet was unstable. Deletion of beta 75, therefore, would not in itself appear to lead to diminished synthesis. There was a profound deficit of beta Vicksburg mRNA when measured by liquid hybridization analysis with beta cDNA. The most plausible explanation for the low output of Hb Vicksburg is that a mutation for beta +-thalassemia is present in cis to the structural mutation. PMID:6165992

beta-Thalassemia present in cis to a new beta-chain structural variant, Hb Vicksburg [beta 75 (E19)Leu leads to 0].

PubMed

Adams, J G; Steinberg, M H; Newman, M V; Morrison, W T; Benz, E J; Iyer, R

1981-01-01

Hemoglobin Vicksburg was discovered in a 6-year-old Black boy who had been anemic since infancy. Examination of his hemolysate revealed 87.5% Hb F, 2.4% Hb A2, and 7.6% Hb Vicksburg, which had the electrophoretic and chromatographic properties of Hb A. Structural analysis of Hb Vicksburg demonstrated a deletion of leucine at beta 75(E19), a new variant. Hb Vicksburg was neither unstable nor subject to posttranslational degradation. The alpha/non-alpha biosynthetic ratio was 2.6. Because the proband appeared to be a mixed heterozygote for Hb Vicksburg and beta 0-thalassemia, Hb Vicksburg should have comprised the major portion of the hemolysate. Thus, Hb Vicksburg was synthesized at a rate considerably lower than would be expected on the basis of gene dosage. There was no reason to suspect abnormal translation of beta Vicksburg mRNA; in individuals with Hb St. Antoine (beta 74 and beta 75 deleted), the abnormal hemoglobin comprised 25% of the hemolysate in the simple heterozygote yet was unstable. Deletion of beta 75, therefore, would not in itself appear to lead to diminished synthesis. There was a profound deficit of beta Vicksburg mRNA when measured by liquid hybridization analysis with beta cDNA. The most plausible explanation for the low output of Hb Vicksburg is that a mutation for beta +-thalassemia is present in cis to the structural mutation.

Beta thalassemia in 31,734 cases with HBB gene mutations: Pathogenic and structural analysis of the common mutations; Iran as the crossroads of the Middle East.

PubMed

Mahdieh, Nejat; Rabbani, Bahareh

2016-11-01

Thalassemia is one of the most common single gene disorders worldwide. Nearly 80 to 90 million with minor beta thalassemia and 60-70 thousand affected infants are born annually worldwide. A comprehensive search on several databases including PubMed, InterScience, British Library Direct, and Science Direct was performed extracting papers about mutation detection and frequency of beta thalassemia. All papers reporting on the mutation frequency of beta thalassemia patients were selected to analyze the frequency of mutations in different regions and various ethnicities. Mutations of 31,734 individuals were identified. Twenty common mutations were selected for further analysis. Genotype-phenotype correlation, interactome, and in silico analyses of the mutations were performed using available bioinformatics tools. Secondary structure prediction was achieved for two common mutations with online tools. The mutations were also common among the countries neighboring Iran, which are responsible for 71% to 98% of mutations. Computational analyses could be used in addition to segregation and expression analysis to assess the extent of pathogenicity of the variant. The genetics of beta thalassemia in Iran is more extensively heterogeneous than in neighboring countries. Some common mutations have arisen historically from Iran and moved to other populations due to population migrations. Also, due to genetic drift, the frequencies of some mutations have increased in small populations. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structural and molecular characterization of the prefoldin beta subunit from Thermococcus strain KS-1.

PubMed

Kida, Hiroshi; Sugano, Yuri; Iizuka, Ryo; Fujihashi, Masahiro; Yohda, Masafumi; Miki, Kunio

2008-11-14

Prefoldin (PFD) is a heterohexameric molecular chaperone that is found in eukaryotic cytosol and archaea. PFD is composed of alpha and beta subunits and forms a "jellyfish-like" structure. PFD binds and stabilizes nascent polypeptide chains and transfers them to group II chaperonins for completion of their folding. Recently, the whole genome of Thermococcus kodakaraensis KOD1 was reported and shown to contain the genes of two alpha and two beta subunits of PFD. The genome of Thermococcus strain KS-1 also possesses two sets of alpha (alpha1 and alpha2) and beta subunits (beta1 and beta2) of PFD (TsPFD). However, the functions and roles of each of these PFD subunits have not been investigated in detail. Here, we report the crystal structure of the TsPFD beta1 subunit at 1.9 A resolution and its functional analysis. TsPFD beta1 subunits form a tetramer with four coiled-coil tentacles resembling the jellyfish-like structure of heterohexameric PFD. The beta hairpin linkers of beta1 subunits assemble to form a beta barrel "body" around a central fourfold axis. Size-exclusion chromatography and multi-angle light-scattering analyses show that the beta1 subunits form a tetramer at pH 8.0 and a dimer of tetramers at pH 6.8. The tetrameric beta1 subunits can protect against aggregation of relatively small proteins, insulin or lysozyme. The structural and biochemical analyses imply that PFD beta1 subunits act as molecular chaperones in living cells of some archaea.

The mechanism of chromosome 7 inversion in human lymphocytes expressing chimeric gamma beta TCR.

PubMed

Retière, C; Halary, F; Peyrat, M A; Le Deist, F; Bonneville, M; Hallet, M M

1999-01-15

Functional chimeric TCR chains, encoded by V gamma J gamma C beta or V gamma J beta C beta hybrid gene TCR, are expressed at the surface of a small fraction of alpha beta T lymphocytes in healthy individuals. Their frequency is dramatically increased in patients with ataxia-telangiectasia, a syndrome associated with inherited genomic instability. As the TCR gamma and beta loci are in an inverted orientation on chromosome 7, the generation of such hybrid genes requires at least an inversion event. Until now, neither the sequences involved in this genetic mechanism nor the number of recombinations leading to the formation of functional transcriptional units have been characterized. In this manuscript, we demonstrate that at least two rearrangements, involving classical recombination signal sequence and the V(D)J recombinase complex, lead to the formation of productive hybrid genes. A primary inversion 7 event between D beta and J gamma genic segments generates C gamma V beta and C beta V gamma hybrid loci. Within the C gamma V beta locus, secondary rearrangements between V gamma and J gamma or V gamma and J beta elements generate functional genes. Besides, our results suggest that secondary rearrangements were blocked in the C beta V gamma locus of normal but not ataxia-telangiectasia T lymphocytes. We also provide formal evidence that the same D beta-3' recombination signal sequence can be used in successive rearrangements with J gamma and J beta genic segments, thus showing that a signal joint has been involved in a secondary recombination event.

Salt bridge residues between I-Ak dimer of dimers alpha-chains modulate antigen presentation.

PubMed

Yadati, S; Nydam, T; Demian, D; Wade, T K; Gabriel, J L; Barisas, B G; Wade, W F

1999-03-15

Class II dimers of dimers are predicted to have functional significance in antigen presentation. The putative contact amino acids of the I-Ak class II dimer of dimers have been identified by molecular modeling based on the DR1 crystal structure (Nydam et al., Int. Immunol. 10, 1237,1998). We have previously reported the role in antigen presentation of dimer of dimers contact amino acids located in the C-terminal domains of the alpha- and beta-chains of class II. Our calculations show that residues Ealpha89 and Ralpha145 in the alpha2-domain form an inter alpha-chain salt bridge between pairs of alphabeta-heterodimers. Other residues, Qalpha92 and Nalpha115, may be involved in close association in that part of the alpha-chain. We investigated the role of these amino acids on class II expression and antigen presentation. Class II composed of an Ealpha89K substituted alpha-chain paired with a wt beta-chain exhibited inhibited antigen presentation and expression of alpha-chain serologic epitopes. In contrast, mutation of Ralpha145E had less affect on antigen presentation and did not affect I-Ak serologic epitopes. Interchanging charges of the salt bridge residues by expressing both Ralpha145E and Ealpha89K on the same chain obviated the large negative effect of the Ealpha89K mutation on antigen presentation but not on the serologic epitopes. Our results are similar for those reported for mutation of DR3's inter-chain salt bridge with the exception that double mutants did not moderate the DR3 defect. Interestingly, the amino acids differences between I-A and DR change the location of the inter-chain salt bridges. In DR1 these residues are located at positions Ealpha88 and Kalpha111; in I-Ak these residues are located at position Ealpha89 and Ralpha145. Inter alpha-chain salt bridges are thus maintained in various class II molecules by amino acids located in different parts of the alpha2-domain. This conservation of structure suggests that considerable functional importance may attach to the ionic interactions.

Recombinant follicle-stimulating hormone: new biotechnology for infertility.

PubMed

Prevost, R R

1998-01-01

The frequency of infertility in developed countries is approximately 8-10%. New drugs are available for assisted reproduction techniques. Two recombinant follicle-stimulating hormone (FSH) products, follitropin-beta (Follistim in the United States, Puregon in Europe) and follitropin-alpha (Gonal-F), join compounds derived through transfecting nonhuman cell lines with genetic material capable of replicating identical amino acid sequences to human compounds. The cell line used for recombinant (r)-FSH production is the Chinese hamster ovary (CHO). Previously, the only agents that showed benefit in controlled ovulatory stimulation were derived from the urine of menopausal women. Those compounds contain additional substances, such as urinary proteins and various amounts of luteininzing hormone. The amino acid sequence of r-FSH is identical to that of human FSH, but the two recombinant products exist in many different isoforms and differ from each other and from human FSH due to varied carbohydrate side chains. Due to variation in the carbohydrate side chains, follitropin-beta in solution has a higher pH than urine-derived FSH, which enhances receptor affinity and therefore is a greater inducer of folliculogenesis. Follitropin-beta does not cause endogenous production of anti-CHO or anti-FSH antibodies, and is well tolerated.

Expression of platelet-derived growth factor and its receptors in proliferative disorders of fibroblastic origin.

PubMed Central

Smits, A.; Funa, K.; Vassbotn, F. S.; Beausang-Linder, M.; af Ekenstam, F.; Heldin, C. H.; Westermark, B.; Nistér, M.

1992-01-01

Platelet-derived growth factor (PDGF) is known to stimulate the proliferation of connective tissue-derived cells in vitro. Less is known about its functions in vivo, and the role of PDGF in the development of human tumors has not been clarified. The authors have investigated the occurrence of PDGF and PDGF receptors in a series of proliferative disorders of fibroblastic origin using immunohistochemical and in situ hybridization techniques. High expression of PDGF beta-receptor mRNA and protein was found in the malignant tumors, and also in some benign lesions, such as dermatofibroma. In all these cases, benign as well as malignant, the PDGF B-chain mRNA, and less clearly, the PDGF A-chain mRNA, were coexpressed with the beta-receptor. In contrast, high expression of PDGF alpha-receptor mRNA was only found in fully malignant lesions, i.e., malignant fibrous histiocytoma. These data indicate that an autocrine growth stimulation via the PDGF beta-receptor could occur in an early phase of tumorigenesis, and may be a necessary but insufficient event for the progression into fully malignant human connective tissue lesions. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:1372158

Sulfatide promotes the folding of proinsulin, preserves insulin crystals, and mediates its monomerization.

PubMed

Osterbye, T; Jørgensen, K H; Fredman, P; Tranum-Jensen, J; Kaas, A; Brange, J; Whittingham, J L; Buschard, K

2001-06-01

Sulfatide is a glycolipid that has been associated with insulin-dependent diabetes mellitus. It is present in the islets of Langerhans and follows the same intracellular route as insulin. However, the role of sulfatide in the beta cell has been unclear. Here we present evidence suggesting that sulfatide promotes the folding of reduced proinsulin, indicating that sulfatide possesses molecular chaperone activity. Sulfatide associates with insulin by binding to the insulin domain A8--A10 and most likely by interacting with the hydrophobic side chains of the dimer-forming part of the insulin B-chain. Sulfatide has a dual effect on insulin. It substantially reduces deterioration of insulin hexamer crystals at pH 5.5, conferring stability comparable to those in beta cell granules. Sulfatide also mediates the conversion of insulin hexamers to the biological active monomers at neutral pH, the pH at the beta-cell surface. Finally, we report that inhibition of sulfatide synthesis with chloroquine and fumonisine B1 leads to inhibition of insulin granule formation in vivo. Our observations suggest that sulfatide plays a key role in the folding of proinsulin, in the maintenance of insulin structure, and in the monomerization process.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Antonacci, R.; Colombo, I.; Volta, M.

The electron-transfer flavoprotein (ETF), located in the mitochondrial matrix, is a nuclear-encoded enzyme delivering to the respiratory chain electrons by straight-chain acyl-CoA dehydrogenases and other dehydrogenases. ETF is composed of a 35-kDa [alpha]-subunit that is cleaved to a 32-kDa protein during mitochondrial import (ETFA) and a [beta]-subunit that reaches the mitochondrion unmodified (ETFB). The cDNA encoding both these subunits has been cloned and sequenced. 14 refs., 1 fig.

Studies on long chain cis- and trans-acyl-CoA esters and Acyl-CoA dehydrogenase from rat heart mitochondria.

PubMed

Korsrud, G O; Conacher, H B; Jarvis, G A; Beare-Rogers, J L

1977-02-01

The beta-oxidation of long chain fatty acids was investigated in a preparation of rat heart mitochondria. The acyl-CoA esters of the cis and trans isomers of delta9-hexadecenoic, delta9-octadecenoic, delta11-eicosenoic, and delta13-docosenoic acids were prepared. Rates of the acyl-CoA reaction were determined with an extract from rat heart mitochondria. The apparent Michaelis constant (Km) and maximum velocity (Vmax) were calculated for each substrate. In general, apparent Vmax values decreased with increasing chain length of the monoenoic substrates. Reduced activity of acyl-CoA dehydrogenase with long chain acyl-CoA esters could have contributed to accumulation of lipids in hearts of rats fed diets containing long chain fatty acids.

Enzymatic synthesis of dimaltosyl-{beta}-cyclodextrin via a transglycosylation reaction using TreX, a Sulfolobus solfataricus P2 debranching enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, Hee-Kwon; Cha, Hyunju; Yang, Tae-Joo

2008-02-01

Di-O-{alpha}-maltosyl-{beta}-cyclodextrin ((G2){sub 2}-{beta}-CD) was synthesized from 6-O-{alpha}-maltosyl-{beta}-cyclodextrin (G2-{beta}-CD) via a transglycosylation reaction catalyzed by TreX, a debranching enzyme from Sulfolobus solfataricus P2. TreX showed no activity toward glucosyl-{beta}-CD, but a transfer product (1) was detected when the enzyme was incubated with maltosyl-{beta}-CD, indicating specificity for a branched glucosyl chain bigger than DP2. Analysis of the structure of the transfer product (1) using MALDI-TOF/MS and isoamylase or glucoamylase treatment revealed it to be dimaltosyl-{beta}-CD, suggesting that TreX transferred the maltosyl residue of a G2-{beta}-CD to another molecule of G2-{beta}-CD by forming an {alpha}-1,6-glucosidic linkage. When [{sup 14}C]-maltose and maltosyl-{beta}-CD were reactedmore » with the enzyme, the radiogram showed no labeled dimaltosyl-{beta}-CD; no condensation product between the two substrates was detected, indicating that the synthesis of dimaltosyl-{beta}-CD occurred exclusively via transglycosylation of an {alpha}-1,6-glucosidic linkage. Based on the HPLC elution profile, the transfer product (1) was identified to be isomers of 6{sup 1},6{sup 3}- and 6{sup 1},6{sup 4}-dimaltosyl-{beta}-CD. Inhibition studies with {beta}-CD on the transglycosylation activity revealed that {beta}-CD was a mixed-type inhibitor, with a K{sub i} value of 55.6 {mu}mol/mL. Thus, dimaltosyl-{beta}-CD can be more efficiently synthesized by a transglycosylation reaction with TreX in the absence of {beta}-CD. Our findings suggest that the high yield of (G2){sub 2}-{beta}-CD from G2-{beta}-CD was based on both the transglycosylation action mode and elimination of the inhibitory effect of {beta}-CD.« less

A new yeast producing beta-glucosidase and tolerant to lignocellulose hydrolysate inhibitors for cellulosic ethanol production using SSF

USDA-ARS?s Scientific Manuscript database

Conventional cellulose-to-ethanol conversion requires cellulose degradation in order to be utilized for growth and fermentation by common ethanologenic yeast. Cellulose is commonly enzymatically degraded into cellobiose by cellulase and subsequently cellobiose broken down into glucose by beta-glucos...

Isolation and characterization of a cDNA from Cuphea lanceolata encoding a beta-ketoacyl-ACP reductase.

PubMed

Klein, B; Pawlowski, K; Höricke-Grandpierre, C; Schell, J; Töpfer, R

1992-05-01

A cDNA encoding beta-ketoacyl-ACP reductase (EC 1.1.1.100), an integral part of the fatty acid synthase type II, was cloned from Cuphea lanceolata. This cDNA of 1276 bp codes for a polypeptide of 320 amino acids with 63 N-terminal residues presumably representing a transit peptide and 257 residues corresponding to the mature protein of 27 kDa. The encoded protein shows strong homology with the amino-terminal sequence and two tryptic peptides from avocado mesocarp beta-ketoacyl-ACP reductase, and its total amino acid composition is highly similar to those of the beta-ketoacyl-ACP reductases of avocado and spinach. Amino acid sequence homologies to polyketide synthase, beta-ketoreductases and short-chain alcohol dehydrogenases are discussed. An engineered fusion protein lacking most of the transit peptide, which was produced in Escherichia coli, was isolated and proved to possess beta-ketoacyl-ACP reductase activity. Hybridization studies revealed that in C. lanceolata beta-ketoacyl-ACP reductase is encoded by a small family of at least two genes and that members of this family are expressed in roots, leaves, flowers and seeds.

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

PubMed Central

Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-01-01

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes. Images PMID:1533935

Natural derivatives of curcumin attenuate the Wnt/{beta}-catenin pathway through down-regulation of the transcriptional coactivator p300

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ryu, Min-Jung; Cho, Munju; Song, Jie-Young

2008-12-26

Curcumin, a component of turmeric (Curcuma longa), has been reported to suppress {beta}-catenin response transcription (CRT), which is aberrantly activated in colorectal cancer. However, the effects of its natural analogs (demethoxycurcumin [DMC] and bisdemethoxycurcumin [BDMC]) and metabolite (tetrahydrocurcumin [THC]) on the Wnt/{beta}-catenin pathway have not been investigated. Here, we show that DMC and BDMC suppressed CRT that was activated by Wnt3a conditioned-medium (Wnt3a-CM) without altering the level of intracellular {beta}-catenin, and inhibited the growth of various colon cancer cells, with comparable potency to curcumin. Additionally, DMC and BDMC down-regulated p300, which is a positive regulator of the Wnt/{beta}-catenin pathway. Notably,more » THC also inhibited CRT and cell proliferation, but to a much lesser degree than curcumin, DMC, or BDMC, indicating that the conjugated bonds in the central seven-carbon chain of curcuminoids are essential for the inhibition of Wnt/{beta}-catenin pathway and the anti-proliferative activity of curcuminoids. Thus, our findings suggest that curcumin derivatives inhibit the Wnt/{beta}-catenin pathway by decreasing the amount of the transcriptional coactivator p300.« less

Molecular cloning of a novel widely expressed human 80 kDa 17 beta-hydroxysteroid dehydrogenase IV.

PubMed Central

Adamski, J; Normand, T; Leenders, F; Monté, D; Begue, A; Stéhelin, D; Jungblut, P W; de Launoit, Y

1995-01-01

Reactions of oestrogens and androgens at position C-17 are catalysed by 17 beta-hydroxysteroid dehydrogenases (17 beta-HSDs). Cloning of the cDNA of a novel human 17 beta-HSD IV and expression of its mRNA are described. A probe derived from the recently discovered porcine 17 beta-oestradiol dehydrogenase (17 beta-EDH) was used to isolate a 2.6 kb human cDNA encoding a continuous protein of 736 amino acids of high (84%) similarity to the porcine 17 beta-EDH. The calculated molecular mass of the human enzyme is 79,595 Da. Other sequence similarities shared by the two enzymes are: an N-terminal sequence which is similar to that of members of the short-chain alcohol dehydrogenase family; amino acids 343-607 which are similar to the C-terminal domains of a trifunctional Candida tropicalis enzyme and the FOX2 gene product of Saccharomyces cerevisiae; amino acids 596-736 which are similar to human sterol carrier protein 2. The previously cloned human 17 beta-HSD I, II and III are less than 25% identical with 17 beta-HSD IV. mRNA for HSD IV is a single species of 3.0 kb, present in many tissues with highest concentrations in liver, heart, prostate and testes. When over-expressed in mammalian cells, the human 17 beta-HSD IV enzyme displays a specific unidirectional oxidative 17 beta-HSD activity. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7487879

Genetics Home Reference: peroxisomal acyl-CoA oxidase deficiency

MedlinePlus

... of certain fat molecules called very long-chain fatty acids (VLCFAs). Specifically, it is involved in the first step of a process called the peroxisomal fatty acid beta-oxidation pathway. This process shortens the VLCFA ...

Sex-related difference in the inductions by perfluoro-octanoic acid of peroxisomal beta-oxidation, microsomal 1-acylglycerophosphocholine acyltransferase and cytosolic long-chain acyl-CoA hydrolase in rat liver.

PubMed Central

Kawashima, Y; Uy-Yu, N; Kozuka, H

1989-01-01

Inductions by perfluoro-octanoic acid (PFOA) of hepatomegaly, peroxisomal beta-oxidation, microsomal 1-acylglycerophosphocholine acyltransferase and cytosolic long-chain acyl-CoA hydrolase were compared in liver between male and female rats. Marked inductions of these four parameters were seen concurrently in liver of male rats, whereas the inductions in liver of female rats were far less pronounced. The sex-related difference in the response of rat liver to PFOA was much more marked than that seen with p-chlorophenoxyisobutyric acid (clofibric acid) or 2,2'-(decamethylenedithio)diethanol (tiadenol). Hormonal manipulations revealed that this sex-related difference in the inductions is strongly dependent on sex hormones, namely that testosterone is necessary for the inductions, whereas oestradiol prevented the inductions by PFOA. PMID:2570571

Pathophysiologically based drug treatment of sickle cell disease.

PubMed

Steinberg, Martin H

2006-04-01

Sickle cell disease is a systemic disorder that is caused by a mutation (Glu6Val) in the gene that encodes beta globin. The sickle hemoglobin molecule (HbS) is a tetramer of two alpha-globin chains and two sickle beta-globin chains, and has the tendency to polymerize when deoxygenated. HbS facilitates abnormal interactions between the sickle erythrocyte and leukocytes and endothelial cells, which trigger a complex pathobiology. This multifaceted pathophysiology provides the opportunity to interrupt the disease at multiple sites, including polymerization of HbS, erythrocyte density and cell-cell interactions. For example, it is possible to induce higher concentrations of fetal hemoglobin, which disrupts the pathology-initiating step of HbS polymerization. Furthermore, it is possible to improve the hydration of sickle erythrocytes and it might be feasible to counteract the endothelial, inflammatory and oxidative abnormalities of sickle cell disease. A therapeutic approach that targets several sites of pathobiology might be most promising.

How main-chains of proteins explore the free-energy landscape in native states.

PubMed

Senet, Patrick; Maisuradze, Gia G; Foulie, Colette; Delarue, Patrice; Scheraga, Harold A

2008-12-16

Understanding how a single native protein diffuses on its free-energy landscape is essential to understand protein kinetics and function. The dynamics of a protein is complex, with multiple relaxation times reflecting a hierarchical free-energy landscape. Using all-atom molecular dynamics simulations of an alpha/beta protein (crambin) and a beta-sheet polypeptide (BS2) in their "native" states, we demonstrate that the mean-square displacement of dihedral angles, defined by 4 successive C(alpha) atoms, increases as a power law of time, t(alpha), with an exponent alpha between 0.08 and 0.39 (alpha = 1 corresponds to Brownian diffusion), at 300 K. Residues with low exponents are located mainly in well-defined secondary elements and adopt 1 conformational substate. Residues with high exponents are found in loops/turns and chain ends and exist in multiple conformational substates, i.e., they move on multiple-minima free-energy profiles.

[Mutation analysis of beta myosin heavy chain gene in hypertrophic cardiomyopathy families].

PubMed

Fan, Xin-ping; Yang, Zhong-wei; Feng, Xiu-li; Yang, Fu-hui; Xiao, Bai; Liang, Yan

2011-08-01

To detect the gene mutations of beta-myosin heavy chain gene (MYH7) in Chinese pedigrees with hypertrophic cardiomyopathy (HCM), and to analyze the correlation between the genotype and phenotype. Exons 3, 5, 7-9, 11-16 and 18-23 of the MYH7 gene were amplified with PCR in three Chinese pedigrees with HCM. The products were sequenced. Sequence alignment between the detected and the standard sequences was performed. A missense mutation of Thr441Met in exon 14 was identified in a pedigree, which was not detected in the controls. Several synonymous mutations of MYH7 gene were detected in the three pedigrees. The mutation of Thr441Met, located in the actin binding domain of the globular head, was first identified in Chinese. It probably caused HCM. HCM is a heterogeneous disease. Many factors are involved in the process of its occurrence and development.

Inhibition of glycogen synthase kinase 3beta during heart failure is protective.

PubMed

Hirotani, Shinichi; Zhai, Peiyong; Tomita, Hideharu; Galeotti, Jonathan; Marquez, Juan Pablo; Gao, Shumin; Hong, Chull; Yatani, Atsuko; Avila, Jesús; Sadoshima, Junichi

2007-11-26

Glycogen synthase kinase (GSK)-3, a negative regulator of cardiac hypertrophy, is inactivated in failing hearts. To examine the histopathological and functional consequence of the persistent inhibition of GSK-3beta in the heart in vivo, we generated transgenic mice with cardiac-specific overexpression of dominant negative GSK-3beta (Tg-GSK-3beta-DN) and tetracycline-regulatable wild-type GSK-3beta. GSK-3beta-DN significantly reduced the kinase activity of endogenous GSK-3beta, inhibited phosphorylation of eukaryotic translation initiation factor 2B epsilon, and induced accumulation of beta-catenin and myeloid cell leukemia-1, confirming that GSK-3beta-DN acts as a dominant negative in vivo. Tg-GSK-3beta-DN exhibited concentric hypertrophy at baseline, accompanied by upregulation of the alpha-myosin heavy chain gene and increases in cardiac function, as evidenced by a significantly greater Emax after dobutamine infusion and percentage of contraction in isolated cardiac myocytes, indicating that inhibition of GSK-3beta induces well-compensated hypertrophy. Although transverse aortic constriction induced a similar increase in hypertrophy in both Tg-GSK-3beta-DN and nontransgenic mice, Tg-GSK-3beta-DN exhibited better left ventricular function and less fibrosis and apoptosis than nontransgenic mice. Induction of the GSK-3beta transgene in tetracycline-regulatable wild-type GSK-3beta mice induced left ventricular dysfunction and premature death, accompanied by increases in apoptosis and fibrosis. Overexpression of GSK-3beta-DN in cardiac myocytes inhibited tumor necrosis factor-alpha-induced apoptosis, and the antiapoptotic effect of GSK-3beta-DN was abrogated in the absence of myeloid cell leukemia-1. These results suggest that persistent inhibition of GSK-3beta induces compensatory hypertrophy, inhibits apoptosis and fibrosis, and increases cardiac contractility and that the antiapoptotic effect of GSK-3beta inhibition is mediated by myeloid cell leukemia-1. Thus, downregulation of GSK-3beta during heart failure could be compensatory.

Prediction and analysis of beta-turns in proteins by support vector machine.

PubMed

Pham, Tho Hoan; Satou, Kenji; Ho, Tu Bao

2003-01-01

Tight turn has long been recognized as one of the three important features of proteins after the alpha-helix and beta-sheet. Tight turns play an important role in globular proteins from both the structural and functional points of view. More than 90% tight turns are beta-turns. Analysis and prediction of beta-turns in particular and tight turns in general are very useful for the design of new molecules such as drugs, pesticides, and antigens. In this paper, we introduce a support vector machine (SVM) approach to prediction and analysis of beta-turns. We have investigated two aspects of applying SVM to the prediction and analysis of beta-turns. First, we developed a new SVM method, called BTSVM, which predicts beta-turns of a protein from its sequence. The prediction results on the dataset of 426 non-homologous protein chains by sevenfold cross-validation technique showed that our method is superior to the other previous methods. Second, we analyzed how amino acid positions support (or prevent) the formation of beta-turns based on the "multivariable" classification model of a linear SVM. This model is more general than the other ones of previous statistical methods. Our analysis results are more comprehensive and easier to use than previously published analysis results.

Are genetic variants in the platelet-derived growth factor [beta] gene associated with chronic pancreatitis?

PubMed

Muddana, Venkata; Park, James; Lamb, Janette; Yadav, Dhiraj; Papachristou, Georgios I; Hawes, Robert H; Brand, Randall; Slivka, Adam; Whitcomb, David C

2010-11-01

Platelet-derived growth factor [beta] (PDGF-[beta]) is a major signal in proliferation and matrix synthesis through activated pancreatic stellate cells, leading to fibrosis of the pancreas. Recurrent acute pancreatitis (RAP) seems to predispose to chronic pancreatitis (CP) in some patients but not others. We tested the hypothesis that 2 known PDGF-[beta] polymorphisms are associated with progression from RAP to CP. We also tested the hypothesis that PDGF-[beta] polymorphisms in combination with environmental risk factors such as alcohol and smoking are associated with CP. Three hundred eighty-two patients with CP (n = 176) and RAP (n = 206) and 251 controls were evaluated. Platelet-derived growth factor [beta] polymorphisms +286 A/G (rs#1800818) seen in 5'-UTR and +1135 A/C (rs#1800817) in first intron were genotyped using single-nucleotide polymorphism polymerase chain reaction approach and confirmed by DNA sequencing. The genotypic frequencies for PDGF-[beta] polymorphisms in positions +286 and +1135 were found to be similar in controls and patients with RAP and CP. There was no difference in genotypic frequencies among RAP, CP, and controls in subjects in the alcohol and smoking subgroups. Known variations in the PDGF-[beta] gene do not have a significant effect on promoting or preventing fibrogenesis in pancreatitis. Further evaluation of this important pathway is warranted.

Molecular investigations of β-thalassemic children in Erbil governorate

NASA Astrophysics Data System (ADS)

Hasan, Ahmad N.; Al-Attar, Mustafa S.

2017-09-01

The present work studies the molecular investigation of 40 thalassemic carriers using polymerase chain reaction. Forty thalassemic carriers who were registered and treated at Erbil thalassemic center and twenty apparently healthy children have been included in the present study. Ages of both groups ranged between 1-18 years. Four primers used to detect four different beta thalassemia mutations they were codon 8/9, codon 8, codon 41/42 and IVS-1-5. The two most common mutations detected among thalassemia group were Cd8/9 with 8 cases (20%) and Cd-8 with 6 cases (15%) followed by codon 41/42 with 4 cases (10%) which investigated and detected for the first time in Erbil governorate through the present study and finally IVS-1-5 with 3 cases (7.5%), while no any cases detected among control group.

A theoretical case study of type I and type II beta-turns.

PubMed

Czinki, Eszter; Császár, Attila G; Perczel, András

2003-03-03

NMR chemical shielding anisotropy tensors have been computed by employing a medium size basis set and the GIAO-DFT(B3LYP) formalism of electronic structure theory for all of the atoms of type I and type II beta-turn models. The models contain all possible combinations of the amino acid residues Gly, Ala, Val, and Ser, with all possible side-chain orientations where applicable in a dipeptide. The several hundred structures investigated contain either constrained or optimized phi, psi, and chi dihedral angles. A statistical analysis of the resulting large database was performed and multidimensional (2D and 3D) chemical-shift/chemical-shift plots were generated. The (1)H(alpha-13)C(alpha), (13)C(alpha-1)H(alpha-13)C(beta), and (13)C(alpha-1)H(alpha-13)C' 2D and 3D plots have the notable feature that the conformers clearly cluster in distinct regions. This allows straightforward identification of the backbone and side-chain conformations of the residues forming beta-turns. Chemical shift calculations on larger For-(L-Ala)(n)-NH(2) (n=4, 6, 8) models, containing a single type I or type II beta-turn, prove that the simple models employed are adequate. A limited number of chemical shift calculations performed at the highly correlated CCSD(T) level prove the adequacy of the computational method chosen. For all nuclei, statistically averaged theoretical and experimental shifts taken from the BioMagnetic Resonance Bank (BMRB) exhibit good correlation. These results confirm and extend our previous findings that chemical shift information from selected multiple-pulse NMR experiments could be employed directly to extract folding information for polypeptides and proteins.

The effects of forest conversion to oil palm on ground-foraging ant communities depend on beta diversity and sampling grain.

PubMed

Wang, Wendy Y; Foster, William A

2015-08-01

Beta diversity - the variation in species composition among spatially discrete communities - and sampling grain - the size of samples being compared - may alter our perspectives of diversity within and between landscapes before and after agricultural conversion. Such assumptions are usually based on point comparisons, which do not accurately capture actual differences in total diversity. Beta diversity is often not rigorously examined. We investigated the beta diversity of ground-foraging ant communities in fragmented oil palm and forest landscapes in Sabah, Malaysia, using diversity metrics transformed from Hill number equivalents to remove dependences on alpha diversity. We compared the beta diversities of oil palm and forest, across three hierarchically nested sampling grains. We found that oil palm and forest communities had a greater percentage of total shared species when larger samples were compared. Across all grains and disregarding relative abundances, there was higher beta diversity of all species among forest communities. However, there were higher beta diversities of common and very abundant (dominant) species in oil palm as compared to forests. Differences in beta diversities between oil palm and forest were greatest at the largest sampling grain. Larger sampling grains in oil palm may generate bigger species pools, increasing the probability of shared species with forest samples. Greater beta diversity of all species in forest may be attributed to rare species. Oil palm communities may be more heterogeneous in common and dominant species because of variable community assembly events. Rare and also common species are better captured at larger grains, boosting differences in beta diversity between larger samples of forest and oil palm communities. Although agricultural landscapes support a lower total diversity than natural forests, diversity especially of abundant species is still important for maintaining ecosystem stability. Diversity in agricultural landscapes may be greater than expected when beta diversity is accounted for at large spatial scales.

Crystal Structures of Xanthomonas campestris OleA Reveal Features That Promote Head-to-Head Condensation of Two Long-Chain Fatty Acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goblirsch, BR; Frias, JA; Wackett, LP

2012-05-22

OleA is a thiolase superfamily enzyme that has been shown to catalyze the condensation of two long-chain fatty acylcoenzyme A (CoA) substrates. The enzyme is part of a larger gene cluster responsible for generating long-chain olefin products, a potential biofuel precursor. In thiolase superfamily enzymes, catalysis is achieved via a ping-pong mechanism. The first substrate forms a covalent intermediate with an active site cysteine that is followed by reaction with the second substrate. For OleA, this conjugation proceeds by a nondecarboxylative Claisen condensation. The OleA from Xanthomonas campestris has been crystallized and its structure determined, along with inhibitor-bound and xenon-derivatizedmore » structures, to improve our understanding of substrate positioning in the context of enzyme turnover. OleA is the first characterized thiolase superfamily member that has two long-chain alkyl substrates that need to be bound simultaneously and therefore uniquely requires an additional alkyl binding channel. The location of the fatty acid biosynthesis inhibitor, cerulenin, that possesses an alkyl chain length in the range of known OleA substrates, in conjunction with a single xenon binding site, leads to the putative assignment of this novel alkyl binding channel. Structural overlays between the OleA homologues, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase and the fatty acid biosynthesis enzyme FabH, allow assignment of the two remaining channels: one for the thioester-containing pantetheinate arm and the second for the alkyl group of one substrate. A short beta-hairpin region is ordered in only one of the crystal forms, and that may suggest open and closed states relevant for substrate binding. Cys143 is the conserved catalytic cysteine within the superfamily, and the site of alkylation by cerulenin. The alkylated structure suggests that a glutamic acid residue (Glu117 beta) likely promotes Claisen condensation by acting as the catalytic base. Unexpectedly, Glu117 beta comes from the other monomer of the physiological dimer.« less

Crystal Structures of Xanthomonas campestris OleA Reveal Features That Promote Head-to-Head Condensation of Two Long-Chain Fatty Acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goblirsch, Brandon R.; Frias, Janice A.; Wackett, Lawrence P.

2012-10-25

OleA is a thiolase superfamily enzyme that has been shown to catalyze the condensation of two long-chain fatty acyl-coenzyme A (CoA) substrates. The enzyme is part of a larger gene cluster responsible for generating long-chain olefin products, a potential biofuel precursor. In thiolase superfamily enzymes, catalysis is achieved via a ping-pong mechanism. The first substrate forms a covalent intermediate with an active site cysteine that is followed by reaction with the second substrate. For OleA, this conjugation proceeds by a nondecarboxylative Claisen condensation. The OleA from Xanthomonas campestris has been crystallized and its structure determined, along with inhibitor-bound and xenon-derivatizedmore » structures, to improve our understanding of substrate positioning in the context of enzyme turnover. OleA is the first characterized thiolase superfamily member that has two long-chain alkyl substrates that need to be bound simultaneously and therefore uniquely requires an additional alkyl binding channel. The location of the fatty acid biosynthesis inhibitor, cerulenin, that possesses an alkyl chain length in the range of known OleA substrates, in conjunction with a single xenon binding site, leads to the putative assignment of this novel alkyl binding channel. Structural overlays between the OleA homologues, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase and the fatty acid biosynthesis enzyme FabH, allow assignment of the two remaining channels: one for the thioester-containing pantetheinate arm and the second for the alkyl group of one substrate. A short {beta}-hairpin region is ordered in only one of the crystal forms, and that may suggest open and closed states relevant for substrate binding. Cys143 is the conserved catalytic cysteine within the superfamily, and the site of alkylation by cerulenin. The alkylated structure suggests that a glutamic acid residue (Glu117{beta}) likely promotes Claisen condensation by acting as the catalytic base. Unexpectedly, Glu117{beta} comes from the other monomer of the physiological dimer.« less

Beta-cryptoxanthin: A vitamin A-forming carotenoid

USDA-ARS?s Scientific Manuscript database

Beta-cryptoxanthin is a common carotenoid. It is generally the fourth most abundant in human blood but can achieve high concentrations especially in Japanese and Spanish populations. Its richest food sources include mandarin oranges, persimmons, oranges, papayas, pumpkin, and red sweet peppers. Beta...

Chromosome localization of human genes for clathrin adaptor polypeptides AP2{beta} and AP50 and the clathrin-binding protein, VCP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Druck, T.; Gu, Y.; Prabhala, G.

1995-11-01

Clathrin-coated vesicles, involved in endocytosis and Golgi processing, have a surface lattice containing clathrin triskelia and stoichiometric amounts of additional components termed {open_quotes}assembly proteins,{close_quotes} or APs. The AP form at the plasma membrane, AP2, is composed of two large subunits of 100-115 kDa, denoted AP2{alpha} and AP2{beta}, a medium chain of 50 kDa, designated AP50, and a small chain. We have determined human chromosomal locations of genes for a large AP2{beta} (CLAPB1) and a medium (CLAPM1) AP subunit and of a novel clathrin-binding protein, VCP, that binds clathrin simultaneously with A1`s. Chromosomal in situ hybridization of a human genomic clonemore » demonstrated that the CLAPM1 gene mapped to chromosome region 3q28. The gene for the CLAPB1 large subunit was mapped to 17q11.2-q12 by PCR amplification of an AP2{beta} fragment from a panel of rodent-human hybrid DNAs. To map the human VCP sequence, a human-specific probe was made by RT-PCR of human mRNA using oligonucleotide primers from conserved regions of the porcine sequence. The amplified human fragment served as probe on Southern blots of hybrid DNAs to determine that the human VCP locus maps to chromosome region 9pter-q34. 13 refs., 2 figs.« less

Biosynthesis and processing of platelet GPIIb-IIIa in human megakaryocytes.

PubMed

Duperray, A; Berthier, R; Chagnon, E; Ryckewaert, J J; Ginsberg, M; Plow, E; Marguerie, G

1987-06-01

Platelet membrane glycoprotein IIb-IIIa forms a calcium-dependent heterodimer and constitutes the fibrinogen receptor on stimulated platelets. GPIIb is a two-chain protein containing disulfide-linked alpha and beta subunits. GPIIIa is a single chain protein. These proteins are synthesized in the bone marrow by megakaryocytes, but the study of their synthesis has been hampered by the difficulty in obtaining enriched population of megakaryocytes in large numbers. To examine the biosynthesis and processing of GPIIb-IIIa, purified human megakaryocytes were isolated from liquid cultures of cryopreserved leukocytes stem cell concentrates from patients with chronic myelogenous leukemia. Immunoprecipitation of [35S]methionine pulse-chase-labeled cell extracts by antibodies specific for the alpha or beta subunits of GPIIb indicated that GPIIb was derived from a precursor of Mr 130,000 that contains the alpha and beta subunits. This precursor was converted to GPIIb with a half-life of 4-5 h. No precursor form of GPIIIa was detected. The glycosylation of GPIIb-IIIa was examined in megakaryocytes by metabolic labeling in the presence of tunicamycin, monensin, or treatment with endoglycosidase H. The polypeptide backbones of the GPIIb and the GPIIIa have molecular masses of 120 and 90 kD, respectively. High-mannose oligosaccharides are added to these polypeptide backbones co-translationally. The GPIIb precursor is then processed with conversion of high-mannose to complex type carbohydrates yielding the mature subunits GPIIb alpha (Mr 116,000) and GPIIb beta (Mr 25,000). No posttranslational processing of GPIIIa was detected.

RNA interference of Arabidopsis beta-amylase8 prevents maltose accumulation upon cold shock and increases sensitivity of PSII photochemical efficiency to freezing stress.

PubMed

Kaplan, Fatma; Guy, Charles L

2005-12-01

It has been suggested that beta-amylase (BMY) induction during temperature stress in Arabidopsis could lead to starch-dependent maltose accumulation, and that maltose may contribute to protection of the electron transport chain and proteins in the chloroplast stroma during acute stress. A time-course transcript profiling analysis for cold shock at 4 degrees C revealed that BMY8 (At4g17090) was induced specifically in response to cold shock, while major induction was not observed for any of the other eight beta-amylases. A parallel metabolite-profiling analysis revealed a robust transient maltose accumulation during cold shock. BMY8 RNAi lines with lower BMY8 expression exhibited a starch-excess phenotype, and a dramatic decrease in maltose accumulation during a 6-h cold shock at 4 degrees C. The decreased maltose content was also accompanied by decreased glucose, fructose and sucrose content in the BMY8 RNAi plants, consistent with the roles of beta-amylase and maltose in transitory starch metabolism. BMY8 RNAi lines with reduced soluble sugar content exhibited diminished chlorophyll fluorescence as F(v)/F(m) ratio compared with wild type, suggesting that PSII photochemical efficiency was more sensitive to freezing stress. Together, carbohydrate analysis and freezing stress results of BMY8 RNAi lines indicate that increased maltose content, by itself or together through a maltose-dependent increase in other soluble sugars, contributes to the protection of the photosynthetic electron transport chain during freezing stress.

Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet "Beta vulgaris L.": GMO application.

PubMed

Chaouachi, Maher; Alaya, Akram; Ali, Imen Ben Haj; Hafsa, Ahmed Ben; Nabi, Nesrine; Bérard, Aurélie; Romaniuk, Marcel; Skhiri, Fethia; Saïd, Khaled

2013-01-01

KEY MESSAGE : Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis. The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R (2) > 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.

Hydrogen-bonded turns in proteins: the case for a recount.

PubMed

Panasik, Nick; Fleming, Patrick J; Rose, George D

2005-11-01

Beta-turns are sites at which proteins change their overall chain direction, and they occur with high frequency in globular proteins. The Protein Data Bank has many instances of conformations that resemble beta-turns but lack the characteristic N-H(i) --> O=C(i - 3) hydrogen bond of an authentic beta-turn. Here, we identify potential hydrogen-bonded beta-turns in the coil library, a Web-accessible database utility comprised of all residues not in repetitive secondary structure, neither alpha-helix nor beta-sheet (http://www.roselab.jhu.edu/coil). In particular, candidate turns were identified as four-residue segments satisfying highly relaxed geometric criteria but lacking a strictly defined hydrogen bond. Such candidates were then subjected to a minimization protocol to determine whether slight changes in torsion angles are sufficient to shift the conformation into reference-quality geometry without deviating significantly from the original structure. This approach of applying constrained minimization to known structures reveals a substantial population of previously unidentified, stringently defined, hydrogen-bonded beta-turns. In particular, 33% of coil library residues were classified as beta-turns prior to minimization. After minimization, 45% of such residues could be classified as beta-turns, with another 8% in 3(10) helixes (which closely resemble type III beta-turns). Of the remaining coil library residues, 37% have backbone dihedral angles in left-handed polyproline II structure.

A novel role of HLA class I in the pathology of medulloblastoma.

PubMed

Smith, Courtney; Santi, Mariarita; Rajan, Bhargavi; Rushing, Elisabeth J; Choi, Mi Rim; Rood, Brian R; Cornelison, Robert; MacDonald, Tobey J; Vukmanovic, Stanislav

2009-07-12

MHC class I expression by cancer cells enables specific antigen recognition by the immune system and protection of the host. However, in some cancer types MHC class I expression is associated with an unfavorable outcome. We explored the basis of MHC class I association with unfavorable prognostic marker expression in the case of medulloblastoma. We investigated expression of four essential components of MHC class I (heavy chain, beta2m, TAP1 and TAP2) in 10 medulloblastoma mRNA samples, a tissue microarray containing 139 medulloblastoma tissues and 3 medulloblastoma cell lines. Further, in medulloblastoma cell lines we evaluated the effects of HLA class I engagement on activation of ERK1/2 and migration in vitro. The majority of specimens displayed undetectable or low levels of the heavy chains. Medulloblastomas expressing high levels of HLA class I displayed significantly higher levels of anaplasia and c-myc expression, markers of poor prognosis. Binding of beta2m or a specific antibody to open forms of HLA class I promoted phosphorylation of ERK1/2 in medulloblastoma cell line with high levels, but not in the cell line with low levels of HLA heavy chain. This treatment also promoted ERK1/2 activation dependent migration of medulloblastoma cells. MHC class I expression in medulloblastoma is associated with anaplasia and c-myc expression, markers of poor prognosis. Peptide- and/or beta2m-free forms of MHC class I may contribute to a more malignant phenotype of medulloblastoma by modulating activation of signaling molecules such as ERK1/2 that stimulates cell mobility.

Beta-Cryptoxanthin- and alpha-carotene-rich foods have greater apparent bioavailability than beta-carotene-rich foods in Western diets

USDA-ARS?s Scientific Manuscript database

Background: Beta-carotene (BC), beta-cryptoxanthin (CX) and alpha-carotene (AC) are common carotenoids that form vitamin A (VA). Conversion ratios for VA formation have been set at 12:1 for BC- and 24:1 for CX- and AC-rich foods, respectively. These conversion ratios are based on chemical structure...

A jojoba beta-Ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants.

PubMed Central

Lassner, M W; Lardizabal, K; Metz, J G

1996-01-01

beta-Ketoacyl-coenzyme A (CoA) synthase (KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoA. This reaction is the initial step of the microsomal fatty acyl-CoA elongation pathway responsible for formation of very long chain fatty acids (VLCFAs, or fatty acids with chain lengths > 18 carbons). Manipulation of this pathway is significant for agriculture, because it is the basis of conversion of high erucic acid rapeseed into canola. High erucic acid rapeseed oil, used as an industrial feedstock, is rich in VLCFAs, whereas the edible oil extracted from canola is essentially devoid of VLCFAs. Here, we report the cloning of a cDNA from developing jojoba embryos involved in microsomal fatty acid elongation. The jojoba cDNA is homologous to the recently cloned Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene that has been suggested to encode KCS. We characterize the jojoba enzyme and present biochemical data indicating that the jojoba cDNA does indeed encode KCS. Transformation of low erucic acid rapeseed with the jojoba cDNA restored KCS activity to developing embryos and altered the transgenic seed oil composition to contain high levels of VLCFAs. The data reveal the key role KCS plays in determining the chain lengths of fatty acids found in seed oils. PMID:8742713

A jojoba beta-Ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants.

PubMed

Lassner, M W; Lardizabal, K; Metz, J G

1996-02-01

beta-Ketoacyl-coenzyme A (CoA) synthase (KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoA. This reaction is the initial step of the microsomal fatty acyl-CoA elongation pathway responsible for formation of very long chain fatty acids (VLCFAs, or fatty acids with chain lengths > 18 carbons). Manipulation of this pathway is significant for agriculture, because it is the basis of conversion of high erucic acid rapeseed into canola. High erucic acid rapeseed oil, used as an industrial feedstock, is rich in VLCFAs, whereas the edible oil extracted from canola is essentially devoid of VLCFAs. Here, we report the cloning of a cDNA from developing jojoba embryos involved in microsomal fatty acid elongation. The jojoba cDNA is homologous to the recently cloned Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene that has been suggested to encode KCS. We characterize the jojoba enzyme and present biochemical data indicating that the jojoba cDNA does indeed encode KCS. Transformation of low erucic acid rapeseed with the jojoba cDNA restored KCS activity to developing embryos and altered the transgenic seed oil composition to contain high levels of VLCFAs. The data reveal the key role KCS plays in determining the chain lengths of fatty acids found in seed oils.

Thermodynamic study on competitive solubilization of cholesterol and beta-sitosterol in bile salt micelles.

PubMed

Matsuoka, Keisuke; Hirosawa, Takashi; Honda, Chikako; Endo, Kazutoyo; Moroi, Yoshikiyo; Shibata, Osamu

2007-07-01

Differences in the preferential solubilization of cholesterol and competitive solubilizates (beta-sitosterol and aromatic compounds) in bile salt micelles was systematically studied by changing the molar ratio of cholesterol to competitive solubilizates. The cholesterol solubility in a mixed binary system (cholesterol and beta-sitosterol) was almost half that of the cholesterol alone system, regardless of the excess beta-sitosterol quantity added. On the other hand, the mutual solubilities of cholesterol and pyrene were not inhibited by their presence in binary mixed crystals. Finally, the cholesterol solubility was measured by changing the alkyl chain length of n-alkylbenzenes. When tetradecylbenzene was added to the bile solution, the cholesterol solubility decreased slightly and was below the original cholesterol solubility. Based on Gibbs energy change (DeltaG degrees ) for solubilization, chemicals that inhibit cholesterol solubility in their combined crystal systems showed a larger negative DeltaG degrees value than cholesterol alone.

Oxygen binding properties of hemoglobin from the white rhinoceros (beta 2-GLU) and the tapir.

PubMed

Baumann, R; Mazur, G; Braunitzer, G

1984-04-01

The beta-chain of rhinoceros hemoglobin contains glutamic acid at position beta 2, and important site for the binding of organic phosphates. We have investigated the oxygen binding properties of this hemoglobin and its interaction with ATP, 2,3-diphosphoglycerate, CO2 and chloride. The results show that the presence of GLU at position beta 2 nearly abolishes the effect of organic phosphates and CO2, whereas the oxygen-linked binding of chloride is not affected. Thus rhinoceros hemoglobin has only protons and chloride anions as major allosteric effectors for the control of its oxygen affinity. From the results obtained with hemoglobin solutions it can be calculated that the blood oxygen affinity of the rhinoceros must be rather high with a P50 of about 20 torr at pH 7.4 and 37 degrees C, which conforms with observations obtained for other large mammals.

Rigidity and pH dependent Morphology of Beta-Lactoglobulin Spherulites

NASA Astrophysics Data System (ADS)

Gayetsky, Lisa; Armstead, Douglas

2008-03-01

Beta-Lactoglobulin is a milk protein that will denature in acidic solution (less than 2.0 pH) and if heated for extended periods (greater than 18 hours) it will form radial structures called Spherulites. Spherulites, along with the amyloid fibrils that compose them, are of practical importance because they form in the human body and cause the amyloidosis diseases. Different amyloidosis are caused by different types of denatured proteins occurring in different parts of the body. Since it is believed that Spherulite formation is a generic protein characteristic, Beta-Lactoglobulin is a legitimate and easy to use protein to study these structures. In this study we are quantifying the shape of Beta-Lactoglobulin Spherulites to determine if the pH of the protein solution has an impact on the morphology due to side chain interactions or other causes. We are also testing the rigidity of these structures to determine the relevance of small shape changes.

N-substituted imidazolines and ethylenediamines and their action on alpha- and beta-adrenergic receptors.

PubMed

Hamada, A; Yaden, E L; Horng, J S; Ruffolo, R R; Patil, P N; Miller, D D

1985-09-01

A series of N-substituted imidazolines and ethylenediamines were synthesized and examined for their activity in alpha- and beta-adrenergic systems. The length of the intermediate side chain between the catechol and imidazoline ring or the amine of the ethylenediamine segment was shown to affect the adrenergic activity. N-[2-(3,4-Dihydroxyphenyl)ethyl]imidazoline hydrochloride (2) and N-[2-(3,4-dihydroxyphenyl)ethyl]ethylenediamine dihydrochloride (4), both with two methylene groups between the catechol and amine segment, were found to be somewhat selective for alpha 2-adrenergic receptors while 1-(3,4-dihydroxybenzyl)imidazoline hydrochloride (1) and N-2-(3,4-dihydroxybenzyl)ethylenediamine dihydrochloride (3), both with one methylene group between the catechol and amine segment, were more selective for alpha1-adrenergic receptors in a pithed rat model. Of the four compounds examined, only compound 2 showed significant direct activity on beta1- and beta2-adrenergic receptors.

[Beta thalassemia major and pregnancy during adolescence: report of two cases].

PubMed

Trigo, Lucas Augusto Monteiro Castro; Surita, Fernanda Garanhani; Parpinelli, Mary Angela; Pereira, Belmiro Gonçalves; Fertrin, Kleber Yotsumoto; Costa, Maria Laura

2015-06-01

Beta thalassemia major is a rare hereditary blood disease in which impaired synthesis of beta globin chains causes severe anemia. Medical treatment consists of chronic blood transfusions and iron chelation. We describe two cases of adolescents with beta thalassemia major with unplanned pregnancies and late onset of prenatal care. One had worsening of anemia with increased transfusional requirement, fetal growth restriction, and placental senescence. The other was also diagnosed with hypothyroidism and low maternal weight, and was admitted twice during pregnancy due to dengue shock syndrome and influenza H1N1-associated respiratory infection. She also developed fetal growth restriction and underwent vaginal delivery at term complicated by uterine hypotonia. Both patients required blood transfusions after birth and chose medroxyprogesterone as a contraceptive method afterwards. This report highlights the importance of medical advice on contraceptive methods for these women and the role of a specialized prenatal follow-up in association with a hematologist.

Maple syrup urine disease: identification and carrier-frequency determination of a novel founder mutation in the Ashkenazi Jewish population.

PubMed

Edelmann, L; Wasserstein, M P; Kornreich, R; Sansaricq, C; Snyderman, S E; Diaz, G A

2001-10-01

Maple syrup urine disease (MSUD) is a rare, autosomal recessive disorder of branched-chain amino acid metabolism. We noted that a large proportion (10 of 34) of families with MSUD that were followed in our clinic were of Ashkenazi Jewish (AJ) descent, leading us to search for a common mutation within this group. On the basis of genotyping data suggestive of a conserved haplotype at tightly linked markers on chromosome 6q14, the BCKDHB gene encoding the E1beta subunit was sequenced. Three novel mutations were identified in seven unrelated AJ patients with MSUD. The locations of the affected residues in the crystal structure of the E1beta subunit suggested possible mechanisms for the deleterious effects of these mutations. Large-scale population screening of AJ individuals for R183P, the mutation present in six of seven patients, revealed that the carrier frequency of the mutant allele was approximately 1/113; the patient not carrying R183P had a previously described homozygous mutation in the gene encoding the E2 subunit. These findings suggested that a limited number of mutations might underlie MSUD in the AJ population, potentially facilitating prenatal diagnosis and carrier detection of MSUD in this group.

Characterization of interleukin-4-stimulated nasal polyp fibroblasts.

PubMed

Steinke, John W; Crouse, Charles D; Bradley, Dewayne; Hise, Kathleen; Lynch, Kevin; Kountakis, Stilianos E; Borish, Larry

2004-02-01

Chronic hyperplastic eosinophilic sinusitis is an inflammatory disease that results in the accumulation of eosinophils, fibroblasts, mast cells, and goblet cells at the site of injury. A common feature of this disease is the presence of nasal polyposis (NP). The current studies were designed to assess the contribution of interleukin (IL)-4 to fibroblast-mediated inflammation in chronic hyperplastic eosinophilic sinusitis/NP. In addition, we hypothesized that cysteinyl leukotrienes (CysLT) may directly influence fibroblast-mediated fibrotic and remodeling pathways in this disorder. Fibroblasts were isolated from NP tissue. All fibroblast lines expressed the IL-4 receptor. IL-4 induced changes in mRNA and protein expression of fibrotic (transforming growth factor-beta1 and -beta2) and inflammatory cytokines and chemokines (IL-6 and CCL11) by fibroblasts as measured by semiquantitative and quantitative polymerase chain reaction, RNase protection assay, and enzyme-linked immunosorbent assay. The expression of CysLT and other proinflammatory lipid receptors on fibroblasts was evaluated. CysLT1 and CysLT2 receptors were not expressed on fibroblasts; however, LPA(1) receptor was constitutively expressed and LPA(2) receptor expression was upregulated by IL-4. The metabolic cascade involved in CysLT synthesis was not expressed in fibroblasts and could not be induced by IL-4 treatment.

TGF-beta inhibits IL-1beta-activated PAR-2 expression through multiple pathways in human primary synovial cells.

PubMed

Tsai, Shin-Han; Sheu, Ming-Thau; Liang, Yu-Chih; Cheng, Hsiu-Tan; Fang, Sheng-Shiung; Chen, Chien-Ho

2009-10-23

To investigate the mechanism how Transforming growth factor-beta(TGF-beta) represses Interleukin-1beta (IL-1beta)-induced Proteinase-Activated Receptor-2 (PAR-2) expression in human primary synovial cells (hPSCs). Human chondrocytes and hPSCs isolated from cartilages and synovium of Osteoarthritis (OA) patients were cultured with 10% fetal bovine serum media or serum free media before treatment with IL-1beta, TGF-beta1, or Connective tissue growth factor (CTGF). The expression of PAR-2 was detected using reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting. Collagen zymography was performed to assess the activity of Matrix metalloproteinases-13 (MMP-13). It was demonstrated that IL-1beta induces PAR-2 expression via p38 pathway in hPSCs. This induction can be repressed by TGF-beta and was observed to persist for at least 48 hrs, suggesting that TGF-beta inhibits PAR-2 expression through multiple pathways. First of all, TGF-beta was able to inhibit PAR-2 activity by inhibiting IL-1beta-induced p38 signal transduction and secondly the inhibition was also indirectly due to MMP-13 inactivation. Finally, TGF-beta was able to induce CTGF, and in turn CTGF represses PAR-2 expression by inhibiting IL-1beta-induced phospho-p38 level. TGF-beta could prevent OA from progression with the anabolic ability to induce CTGF production to maintain extracellular matrix (ECM) integrity and to down regulate PAR-2 expression, and the anti-catabolic ability to induce Tissue inhibitors of metalloproteinase-3 (TIMP-3) production to inhibit MMPs leading to avoid PAR-2 over-expression. Because IL-1beta-induced PAR-2 expressed in hPSCs might play a significantly important role in early phase of OA, PAR-2 repression by exogenous TGF-beta or other agents might be an ideal therapeutic target to prevent OA from progression.

Fc gamma RII/III and CD2 expression mark distinct subpopulations of immature CD4-CD8- murine thymocytes: in vivo developmental kinetics and T cell receptor beta chain rearrangement status.

PubMed

Rodewald, H R; Awad, K; Moingeon, P; D'Adamio, L; Rabinowitz, D; Shinkai, Y; Alt, F W; Reinherz, E L

1993-04-01

We have recently identified a dominant wave of CD4-CD8- (double-negative [DN]) thymocytes in early murine fetal development that express low affinity Fc gamma receptors (Fc gamma RII/III) and contain precursors for Ti alpha/beta lineage T cells. Here we show that Fc gamma RII/III is expressed in very immature CD4low single-positive (SP) thymocytes and that Fc gamma RII/III expression is downregulated within the DN subpopulation and before the CD3-CD8low SP stage in T cell receptor (TCR)-alpha/beta lineage-committed thymocytes. DN Fc gamma RII/III+ thymocytes also contain a small fraction of TCR-gamma/delta lineage cells in addition to TCR-alpha/beta progenitors. Fetal day 15.5 DN TCR-alpha/beta lineage progenitors can be subdivided into three major subpopulations as characterized by cell surface expression of Fc gamma RII/III vs. CD2 (Fc gamma RII/III+CD2-, Fc gamma RII/III+CD2+, Fc gamma RII/III-CD2+). Phenotypic analysis during fetal development as well as adoptive transfer of isolated fetal thymocyte subpopulations derived from C57B1/6 (Ly5.1) mice into normal, nonirradiated Ly5.2 congenic recipient mice identifies one early differentiation sequence (Fc gamma RII/III+CD2(-)-->Fc gamma RII/III+CD2(+)-->Fc gamma RII/III-CD2+) that precedes the entry of DN thymocytes into the CD4+CD8+ double-positive (DP) TCRlow/- stage. Unseparated day 15.5 fetal thymocytes develop into DP thymocytes within 2.5 d and remain at the DP stage for > 48 h before being selected into either CD4+ or CD8+ SP thymocytes. In contrast, Fc gamma RII/III+CD2- DN thymocytes follow this same developmental pathway but are delayed by approximately 24 h before entering the DP compartment, while Fc gamma RII/III-CD2+ display accelerated development by approximately 24 h compared with total day 15.5 thymocytes. Fc gamma RII/III-CD2+ are also more developmentally advanced than Fc gamma RII/III+CD2- fetal thymocytes with respect to their TCR beta chain V(D)J rearrangement. At day 15.5 in gestation, beta chain V(D)J rearrangement is mostly, if not entirely, restricted to the Fc gamma RII/III-CD2+ subset of DN fetal thymocytes. Consistent with this analysis in fetal thymocytes, > 90% of adult thymocytes derived from mice carrying a disrupting mutation at the recombination-activating gene 2 locus (RAG-2-/-) on both alleles are developmentally arrested at the DN CD2- stage. In addition, there is a fivefold increase in the relative percentage of thymocytes expressing Fc gamma RII/III in TCR and immunoglobulin gene rearrangement-incompetent homozygous RAG-2-/- mice (15% Fc gamma RII/III+) versus rearrangement-competent heterozygous RAG-2+/- mice (< 3% Fc gamma RII/III+). Thus, Fc gamma RII/III expression defines an early DN stage preceding V beta(D beta)I beta rearrangement, which in turn is followed by surface expression of CD2. Loss of Fc gamma RII/III and acquisition of CD2 expression characterize a late DN stage immediately before the conversion into DP thymocytes.

Jacalin: a jackfruit (Artocarpus heterophyllus) seed-derived lectin of versatile applications in immunobiological research.

PubMed

Kabir, S

1998-03-15

Jacalin, the major protein from the jackfruit (Artocarpus heterophyllus) seeds, is a tetrameric two-chain lectin (molecular mass 65 kDa) combining a heavy alpha chain of 133 amino acid residues with a light beta chain of 20-21 amino acid residues. It is highly specific for the alpha-O-glycoside of the disaccharide Thomsen-Friedenreich antigen (Gal beta1-3GalNAc), even in its sialylated form. This property has made jacalin suitable for studying various O-linked glycoproteins, particularly human IgA1. Jacalin's uniqueness in being strongly mitogenic for human CD4+ T lymphocytes has made it a useful tool for the evaluation of the immune status of patients infected with human immunodeficiency virus (HIV)-1. The abundance of source material for the production of jacalin, its ease of purification, yield and stability have made it an attractive cost-effective lectin. It has found applications in diverse areas such as the isolation of human plasma glycoproteins (IgA1, C1-inhibitor, hemopexin, alpha2-HSG), the investigation of IgA-nephropathy, the analysis of O-linked glycoproteins and the detection of tumours.

Acute high-altitude hypoxic brain injury: Identification of ten differential proteins

PubMed Central

Li, Jianyu; Qi, Yuting; Liu, Hui; Cui, Ying; Zhang, Li; Gong, Haiying; Li, Yaxiao; Li, Lingzhi; Zhang, Yongliang

2013-01-01

Hypobaric hypoxia can cause severe brain damage and mitochondrial dysfunction, and is involved in hypoxic brain injury. However, little is currently known about the mechanisms responsible for mitochondrial dysfunction in hypobaric hypoxic brain damage. In this study, a rat model of hypobaric hypoxic brain injury was established to investigate the molecular mechanisms associated with mitochondrial dysfunction. As revealed by two-dimensional electrophoresis analysis, 16, 21, and 36 differential protein spots in cerebral mitochondria were observed at 6, 12, and 24 hours post-hypobaric hypoxia, respectively. Furthermore, ten protein spots selected from each hypobaric hypoxia subgroup were similarly regulated and were identified by mass spectrometry. These detected proteins included dihydropyrimidinase-related protein 2, creatine kinase B-type, isovaleryl-CoA dehydrogenase, elongation factor Ts, ATP synthase beta-subunit, 3-mercaptopyruvate sulfurtransferase, electron transfer flavoprotein alpha-subunit, Chain A of 2-enoyl-CoA hydratase, NADH dehydrogenase iron-sulfur protein 8 and tropomyosin beta chain. These ten proteins are all involved in the electron transport chain and the function of ATP synthase. Our findings indicate that hypobaric hypoxia can induce the differential expression of several cerebral mitochondrial proteins, which are involved in the regulation of mitochondrial energy production. PMID:25206614

Expression of beta-dystroglycan is reduced or absent in many human carcinomas.

PubMed

Cross, S S; Lippitt, J; Mitchell, A; Hollingsbury, F; Balasubramanian, S P; Reed, M W R; Eaton, C; Catto, J W; Hamdy, F; Winder, S J

2008-11-01

Dystroglycan is an important structural and signalling protein that is expressed in most human cells. alpha-Dystroglycan has been investigated and found to be reduced in human cancers, but there is only one published study on the expression of beta-dystroglycan in human cancer and that was only on small numbers of breast and prostatic cancers. The aim was to conduct a comprehensive immunohistochemical survey of the expression of beta-dystroglycan in normal human tissues and common cancers. Triplicate tissue microarrays of 681 samples of normal human tissues and common cancers were stained using an antibody directed against the cytoplasmic component of beta-dystroglycan. beta-Dystroglycan was strongly expressed at the intercellular junctions and basement membranes of all normal human epithelia. Expression of beta-dystroglycan was absent or markedly reduced in 100% of oesophageal adenocarcinomas, 97% of colonic cancers, 100% of transitional cell carcinomas of the urothelium and 94% of breast cancers. In the breast cancers, the only tumours that showed any retention of beta-dystroglycan expression were small low-grade oestrogen receptor-positive tumours. The only cancers that showed retention of beta-dystroglycan expression were cutaneous basal cell carcinomas. There is loss or marked reduction of beta-dystroglycan expression (by immunohistochemistry) in the vast majority of human cancers surveyed. Since beta-dystroglycan is postulated to have a tumour suppressor effect, this loss may have important functional significance.

Specific and straightforward molecular investigation of β-thalassemia mutations in the Malaysian Malays and Chinese using direct TaqMan genotyping assays.

PubMed

Kho, S L; Chua, K H; George, E; Tan, J A M A

2013-07-15

Beta-thalassemia is a life-threatening inherited blood disorder. Rapid characterization of β-globin gene mutations is necessary because of the high frequency of Malaysian β-thalassemia carriers. A combination real-time polymerase chain reaction genotyping assay using TaqMan probes was developed to confirm β-globin gene mutations. In this study, primers and probes were designed to specifically identify 8 common β-thalassemia mutations in the Malaysian Malay and Chinese ethnic groups using the Primer Express software. "Blind tests" using DNA samples from healthy individuals and β-thalassemia patients with different genotypes were performed to determine the specificity and sensitivity of this newly designed assay. Our results showed 100% sensitivity and specificity for this novel assay. In conclusion, the TaqMan genotyping assay is a straightforward assay that allows detection of β-globin gene mutations in less than 40 min. The simplicity and reproducibility of the TaqMan genotyping assay permit its use in laboratories as a rapid and cost-effective diagnostic tool for confirmation of common β-thalassemia mutations in Malaysia.

A beta1-adrenergic receptor CaM kinase II-dependent pathway mediates cardiac myocyte fetal gene induction.

PubMed

Sucharov, Carmen C; Mariner, Peter D; Nunley, Karin R; Long, Carlin; Leinwand, Leslie; Bristow, Michael R

2006-09-01

Beta-adrenergic signaling plays an important role in the natural history of dilated cardiomyopathies. Chronic activation of beta-adrenergic receptors (beta1-AR and beta2-AR) during periods of cardiac stress ultimately harms the failing heart by mechanisms that include alterations in gene expression. Here, we show that stimulation of beta-ARs with isoproterenol in neonate rat ventricular myocytes causes a "fetal" response in the relative activities of the human cardiac fetal and/or adult gene promoters that includes repression of the human and rat alpha-myosin heavy chain (alpha-MyHC) promoters with simultaneous activation of the human atrial natriuretic peptide (ANP) and rat beta-MyHC promoters. We also show that the promoter changes correlate with changes in endogenous gene expression as measured by mRNA expression. Furthermore, we show that these changes are specifically mediated by the beta1-AR, but not the beta2-AR, and are independent of alpha1-AR stimulation. We also demonstrate that the fetal gene response is independent of cAMP and protein kinase A, whereas inhibition of Ca2+/calmodulin-dependent protein kinase (CaMK) pathway blocks isoproterenol-mediated fetal gene program induction. Finally, we show that induction of the fetal program is dependent on activation of the L-type Ca2+ channel. We conclude that in neonatal rat cardiac myocytes, agonist-occupied beta1-AR mobilizes Ca2+ stores to activate fetal gene induction through cAMP independent pathways that involve CaMK.

Isolation and characterization of a novel endo-beta-galactofuranosidase from Bacillus sp.

PubMed

Ramli, N; Fujinaga, M; Tabuchi, M; Takegawa, K; Iwahara, S

1995-10-01

A soil bacterium capable of growing on a polysaccharide-containing beta(1-->6)galactofuranoside residues derived from the acidic polysaccharide of Fusarium sp. as a carbon source has been isolated. From various bacteriological characteristics, the organism was identified as a Bacillus sp. The bacterium produced beta-galactofuranosidase inductively in the culture media. The most effective inducer for the beta-galactofuranosidase production was a polysaccharide containing beta(1-->5) or beta(1-->6)-linked galactofuranoside residues, but gum arabic, gum guar, gum ghati, arabinogalactam, araban, and pectic acid did not induce the enzyme. The enzyme had three different molecular weight forms. The low molecular-weight form was purified by a combination of Toyopearl HW-55 and DEAE-Toyopearl 650S column chromatographies, and preparative polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 67,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 6 and 37 degrees C, and was stable between pH 4 to 8 at 5 degrees C. The action of the enzyme was inhibited by the addition of Cd2+, Co2+, Hg2+, Zn2+, iodoacetic acid, and EDTA. The purified enzyme cleaved beta(1-->5) and beta(1-->6)-linked galactofuranosyl chains. Based upon the mode of liberation of galactofuranosyl residues from pyridylamino-beta(1-->6)-linked galactofuranoside oligomers, the enzyme can be classified as an endo-beta-galactofuranosidase that randomly hydrolyzes the linkage.

Application of a novel design paradigm to generate general nonpeptide combinatorial templates mimicking beta-turns: synthesis of ligands for melanocortin receptors.

PubMed

Webb, Thomas R; Jiang, Luyong; Sviridov, Sergey; Venegas, Ruben E; Vlaskina, Anna V; McGrath, Douglas; Tucker, John; Wang, Jian; Deschenes, Alain; Li, Rongshi

2007-01-01

We report the further application of a novel approach to template and ligand design by the synthesis of agonists of the melanocortin receptor. This design method uses the conserved structural data from the three-dimensional conformations of beta-turn peptides to design rigid nonpeptide templates that mimic the orientation of the main chain C-alpha atoms in a peptide beta-turn. We report details on a new synthesis of derivatives of template 1 that are useful for the synthesis of exploratory libraries. The utility of this technique is further exemplified by several iterative rounds of high-throughput synthesis and screening, which result in new partially optimized nonpeptide agonists for several melanocortin receptors.

Synthesis of polyhydroxysterols (III): synthesis and structural elucidation of 24-methylenecholest-4-en-3beta,6 alpha-diol.

PubMed

Cui, Jian Guo; Lin, Cui Wu; Zeng, Long Mei; Su, Jing Yu

2002-12-01

Using stigmasterol as the starting material, 24-methylenecholest-4-en-3beta,6 alpha-diol (2) was synthesized in eight steps in 13% overall yield. The introduction of the sterol side-chain was carried out using (3-methyl-2-oxobutyl)-triphenylarsonium bromide (11) and K(2)CO(3) in a solid-liquid phase-transfer Wittig reaction. Construction of the steroidal nucleus was finished by oxidation of 24-methylenecholest-5-en-3beta-ol (9) with pyridinium chlorochromate (PCC) in dichloromethane at ambient temperature and by reduction of 24-methylenecholest-4-en-3,6-dione (10) with NaBH(4) in the presence of CeCl(3).7H(2)O.

Secondary structure inducing potential of beta-amino acids: torsion angle clustering facilitates comparison and analysis of the conformation during MD trajectories.

PubMed

Guthöhrlein, E W; Malesević, M; Majer, Z; Sewald, N

2007-01-01

While numerous examples of beta-peptides--exclusively composed of beta-amino acids--have been investigated during the past decade, there are only few reports on the conformational preference of a single beta-amino acid when incorporated into a cyclopeptide. The conformational bias of beta-amino acids on the secondary structure of cyclopeptides has been investigated by NMR spectroscopy in combination with distance geometry (DG) and molecular dynamics (MD) calculations using experimental constraints. The atomic coordinate RMSD criterion usually employed for clustering of conformations after DG and MD calculations does not necessarily group similar peptide conformations, as there is an insufficient correlation between atomic coordinates and torsion angles. To improve on this shortcoming and to eliminate any arbitrary decisions during this process, a torsion angle clustering procedure has been implemented. For the cyclic pentapeptides cyclo-(-Val-beta-Hala-Phe-Leu-Ile-) 1 and cyclo-(-Ser-Pro-Leu-beta-Hasn-Asp-) 3, the beta-amino acid is found in the central position of an extended gamma-turn (pseudo gamma-turn, Psigamma-turn), while the beta-Hpro residue in the cyclic hexapeptide cyclo-(-Ser-beta-Hpro-Leu-Asn-Ile-Asp-) 5 preferentially occupies position i+1 of a pseudo beta-turn (Psibeta-turn). These results further corroborate the hypothesis of beta-amino acids being reliable inducers of secondary structure in cyclic penta- and hexapeptides. They can be employed in the de novo design of biologically active cyclopeptides in pharmaceutical research, since the three-dimensional presentation of pharmacophoric groups in the side chains can be tailored by incorporation of beta-amino acids in strategic sequential positions. (c) 2007 Wiley Periodicals, Inc.

How to polymerize ethylene in a highly controlled fashion?

PubMed

Kempe, Rhett

2007-01-01

Very fast, reversible, polyethylene (PE) chain transfer or complex-catalysed "Aufbaureaktion" describes a "living" chain-growing process on a main-group metal or zinc atom; this process is catalysed by an organo-transition-metal or lanthanide complex. PE chains are transferred very fast between the two metal sites and chain growth takes place through ethylene insertion into the transition-metal- or lanthanide-carbon bond-coordinative chain-transfer polymerisation (CCTP). The transferred chains "rest" at the main-group or zinc centre, at which chain-termination processes like beta-H transfer/elimination are of low significance. Such protocols can be used to synthesise very narrowly distributed PE materials (M(w)/M(n)<1.1 up to a molecular weight of about 4000 g mol(-1)) with differently functionalised end groups. Higher molecular-weight polymers can be obtained with a slightly increased M(w)/M(n), since diffusion control and precipitation of the polymers influences the chain-transfer process. Recently, a few transition-metal- or lanthanide-based catalyst systems that catalyse such a highly reversible chain-growing process have been described. They are summarised and compared within this contribution.

Solution structure for Pandinus toxin K-alpha (PiTX-K alpha), a selective blocker of A-type potassium channels.

PubMed

Tenenholz, T C; Rogowski, R S; Collins, J H; Blaustein, M P; Weber, D J

1997-03-11

PiTX-K alpha, a 35-residue peptide recently isolated from the venom of Pandinus imperator, blocks the rapidly inactivating (A-type) K+ channel(s) in rat brain synaptosomes and the cloned Kv 1.2 potassium channel at very low toxin concentrations (6 nM and 32 pM, respectively) [Rogowski, R. S., Collins, J. H., O'Neil, T. J., Gustafson, T. A., Werkman, T. A., Rogawski, M. A., Tenenholz, T. C., Weber, D. J., & Blaustein, M. P. (1996) Mol. Pharmacol. 50, 1167-1177]. The three-dimensional structure of PiTX-K alpha was determined using NMR spectroscopy in order to understand its selectivity and affinity toward K+ channels. PiTX-K alpha was found to have an alpha-helix from residues 10 to 21 and two beta-strands (betaI, 26-28; betaII, 33-35) connected by a type II beta-turn to form a small antiparallel beta-sheet. Three disulfide bonds, which are conserved in all members of the charybdotoxin family (alpha-K toxins), anchor one face of the alpha-helix to the beta-sheet. The N-terminal portion of PiTX-K alpha has three fewer residues than other alpha-K toxins such as charybdotoxin. Rather than forming a third beta-strand as found for other alpha-K toxins, the N-terminal region of PiTX-K alpha adopts an extended conformation. This structural difference in PiTX-K alpha together with differences in sequence at Pro-10, Tyr-14, and Asn-25 (versus Ser-10, Trp-14, and Arg-25 in CTX) may explain why PiTX-K alpha does not block maxi-K+ channels. Differences in three-dimensional structure between PiTX-K alpha and charybdotoxin are also observed in both the tight turn and the loop that connects the first beta-strand to the alpha-helix. As a result, side chains of two residues (Tyr-23 and Arg-31) are in regions of PiTX-K alpha that probably interact with rapidly inactivating A-type K+ channels. The analogous residues in charybdotoxin are positioned differently on the toxin surface. Thus, the locations of Tyr-23 and Arg-31 side chains in PiTX-K alpha could explain why this toxin blocks A-type channels at much lower concentrations than does charybdotoxin.

Biochemistry of Ammonia Monoxygenase from Nitrosomonas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alan Hooper

2009-07-15

Major results. 1. CytochromecM552, a protein in the electron transfer chain to ammonia monooxygenase. Purification, modeling of protein structure based on primary structure, characterization of 4 hemes by magnetic spectroscopy, potentiometry, ligand binding and turnover. Kim, H. J., ,Zatsman, et al. 2008). 2. Characterization of proteins which thought to be involved in the AMO reaction or to protect AMO from toxic nitrogenous intermediates such as NO. Nitrosocyanin is a protein present only in bacteria which catalyze the ammonia monoxygenase reaction (1). Cytochrome c P460 beta and cytochrome c’ beta.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chasteler, R.M.; Nitschke, J.M.; Firestone, R.B.

Neutron-rich rare-earth isotopes were produced in multinucleon transfer reactions between {sup 170}Er ions and {sup nat}W targets. On-line mass separation was used together with {beta}- and {gamma}-ray spectroscopy in these studies. At mass {ital A}=169, the heaviest known dysprosium isotope, 39(8) s,{sup 169}Dy, was identified. It was observed to {beta}{sup {minus}} decay to the ground state of {sup 169}Ho or through a level at 1578 keV. In the {ital A}=171 mass chain, a partial decay scheme for 55(3)-s {sup 171}Ho was determined.

Chain of Custody Item Monitor Message Viewer v.1.0 Beta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schwartz, Steven Robert; Fielder, Laura; Hymel, Ross W.

The CoCIM Message Viewer software allows users to connect to and download messages from a Chain of Custody Item Monitor (CoCIM) connected to a serial port on the user’s computer. The downloaded messages are authenticated and displayed in a Graphical User Interface that allows the user a limited degree of sorting and filtering of the downloaded messages as well as the ability to save downloaded files or to open previously downloaded message history files.

Diagnostic difficulty of beta-thalassemia syndrome in a multi-transfused patient: contribution of myelogram and studying parents.

PubMed

Trawinski, Élisabeth; Fenneteau, Odile; Le Mouel, Lou; Ithier, Ghislaine; Couque, Nathalie

2017-10-01

We report the case of a 5 year old, initially followed for congenital sideroblastic anemia, whose explorations reveal a complex family hemoglobinopathy. Myelogram performed in children, reveals dystrophic mature erythroblasts with hemoglobinization defect and basophil punctuations. These abnormalities point towards an abnormal synthesis of heme or globin chains. Iterative transfusions in child do not allow interpreting a search for abnormal hemoglobin. However, the analysis carried out in his parents, with increased HBA2 rate and microcytosis concluded in beta-thalassemia trait for father and mother. Knowing that beta-thalassemia syndrome is a genetic condition, usually recessive, the presence of beta-thalassemia trait in parents is in favor of a beta-thalassemia syndrome in child. This diagnostic hypothesis is confirmed by molecular study of globin genes that will reveal a complex hemoglobinopathie for all family's members. The parents are carriers for heterozygous mutation of β + thalassemia that the sick child presents in homozygous state supporting the diagnosis of beta-thalassemia syndrome. Moreover, a triple α globin gene is present respectively at heterozygous state for mother and at homozygous state for father and child. The triple α globin gene is a known factor of aggravation of beta-thalassemia and this clinical case with continuum observed, perfectly illustrates the intricacies between α and β globin genes.

[Frequency of enzymes associated with reduced sensitivity to beta-lactam antibiotics in enterobacteria isolates, Caracas, Venezuela].

PubMed

Marcano, Daniel; De Jesús, Andreína; Hernández, Luis; Torres, Luis

2011-12-01

To determine the frequency of enzymatic mechanisms associated with reduced sensitivity to broad-spectrum beta-lactam antibiotics in enterobacteria isolates obtained at hospital centers in Caracas, Venezuela. A cross-sectional study was conducted on enterobacteria isolated from patients at eight hospital centers in Caracas, Venezuela, from 15 October 2009 to 15 January 2010. The species were identified using conventional biochemical tests, and their susceptibility to antimicrobial drugs was assessed by antibiogram (Kirby-Bauer method), using the 2010 performance standards published by the Clinical and Laboratory Standards Institute. Beta-lactam-resistant genes were detected using an enhanced polymerase chain reaction assay. Of 1 235 isolates, 207 (16.8%) exhibited resistance to third- and fourth-generation cephalosporins, carbapenems, or both. They presented the following phenotypes: extended-spectrum beta-lactamase (ESBL), 93.8%; depressed AmpC, 4.3%; and carbapenemase, 1.9%. Further characterization of the first two phenotypes yielded the following breakdown of types: SHV, 36.7%; CTX-M-1 group, 22.3%; TEM, 21.7%; CTX-M-1 group with impermeability, 5.2%; two-enzyme combinations, 4.5%; CTX-M-2 group, 4.3%; PER, 3.4%; and KPC, 1.9%. The SHV type was predominant in the public hospital strains, whereas the CTX-M-1 group was most common in the strains from the private hospitals. Of the enzymatic mechanisms investigated, the SHV type was the most frequent, followed by the CTX-M-1 group and the TEM type. Also, a high percentage of type KPC was found. The research reported here is one of only a few multicenter studies that have been conducted in Venezuela to evaluate the frequency of this type of antimicrobial resistance mechanism, including phenotypical and molecular characterization. It was shown that the detection methods require proper interpretation of sensitivity profiles and molecular confirmation of the mechanism present.

Modulation of cardiac myocyte phenotype in vitro by the composition and orientation of the extracellular matrix.

PubMed

Simpson, D G; Terracio, L; Terracio, M; Price, R L; Turner, D C; Borg, T K

1994-10-01

Cellular phenotype is the result of a dynamic interaction between a cell's intrinsic genetic program and the morphogenetic signals that serve to modulate the extent to which that program is expressed. In the present study we have examined how morphogenetic information might be stored in the extracellular matrix (ECM) and communicated to the neonatal heart cell (NHC) by the cardiac alpha 1 beta 1 integrin molecule. A thin film of type I collagen (T1C) was prepared with a defined orientation. This was achieved by applying T1C to the peripheral edge of a 100 mm culture dish. The T1C was then drawn across the surface of the dish in a continuous stroke with a sterile cell scraper and allowed to polymerize. When NHCs were cultured on this substrate, they spread, as a population, along a common axis in parallel with the gel lattice and expressed an in vivo-like phenotype. Individual NHCs displayed an elongated, rod-like shape and disclosed parallel arrays of myofibrils. These phenotypic characteristics were maintained for at least 4 weeks in primary culture. The evolution of this tissue-like organizational pattern was dependent upon specific interactions between the NHCs and the collagen-based matrix that were mediated by the cardiac alpha 1 beta 1 integrin complex. This conclusion was supported by a variety of experimental results. Altering the tertiary structure of the matrix or blocking the extracellular domains of either the cardiac alpha 1 or beta 1 integrin chain inhibited the expression of the tissue-like pattern of organization. Neither cell-to-cell contact or contractile function were necessary to induce the formation of the rod-like cell shape. However, beating activity was necessary for the assembly of a well-differentiated myofibrillar apparatus. These data suggest that the cardiac alpha 1 beta 1 integrin complex serves to detect and transduce phenotypic information stored within the tertiary structure of the surrounding matrix.

Internal electron transfer between hemes and Cu(II) bound at cysteine beta93 promotes methemoglobin reduction by carbon monoxide.

PubMed

Bonaventura, C; Godette, G; Tesh, S; Holm, D E; Bonaventura, J; Crumbliss, A L; Pearce, L L; Peterson, J

1999-02-26

Previous studies showed that CO/H2O oxidation provides electrons to drive the reduction of oxidized hemoglobin (metHb). We report here that Cu(II) addition accelerates the rate of metHb beta chain reduction by CO by a factor of about 1000. A mechanism whereby electron transfer occurs via an internal pathway coupling CO/H2O oxidation to Fe(III) and Cu(II) reduction is suggested by the observation that the copper-induced rate enhancement is inhibited by blocking Cys-beta93 with N-ethylmaleimide. Furthermore, this internal electron-transfer pathway is more readily established at low Cu(II) concentrations in Hb Deer Lodge (beta2His --> Arg) and other species lacking His-beta2 than in Hb A0. This difference is consistent with preferential binding of Cu(II) in Hb A0 to a high affinity site involving His-beta2, which is ineffective in promoting electron exchange between Cu(II) and the beta heme iron. Effective electron transfer is thus affected by Hb type but is not governed by the R left arrow over right arrow T conformational equilibrium. The beta hemes in Cu(II)-metHb are reduced under CO at rates close to those observed for cytochrome c oxidase, where heme and copper are present together in the oxygen-binding site and where internal electron transfer also occurs.

Polymorphisms of the IL-1beta and IL-1beta-inducible genes in ulcerative colitis.

PubMed

Nohara, Hiroaki; Saito, Yuki; Higaki, Singo; Okayama, Naoko; Hamanaka, Yuichiro; Okita, Kiwamu; Hinoda, Yuji

2002-11-01

Ulcerative colitis (UC) is a chronic disorder of undetermined etiology, but a genetic predisposition to UC is well recognized. Among cytokines induced in UC, interleukin 1 (IL-1) appears to have a central role because of its immunological upregulatory and proinflammatory activities. The aim of this study was to assess whether UC is associated with polymorphisms of the IL-1beta gene and three additional genes inducible with IL-1beta in Japanese subjects. A total of 96 patients with UC and 106 ethnically matched controls were genotyped at polymorphic sites in IL-1beta, matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 3 (MMP-3), and inducible nitric oxide synthase (iNOS) genes, using polymerase chain reaction (PCR)-based methods. There was no significant difference in genotype distributions of IL-1beta, MMP-1, MMP-3, and iNOS genes between controls and UC patients in a Japanese population. Also, no significant association of those polymorphisms with various clinical parameters of the patients was found. However, concerning association of age at onset with clinical factors in UC, the frequency of pancolitis was significantly higher in UC patients with age at onset being less than 30 years than in those more than 30 years of age (P = 0.049). No association of the IL-1beta and three IL-1beta-inducible gene polymorphisms with UC was observed in a Japanese population.

The mechanism of cell death in human cultured colon adenocarcinoma cell line COLO 201 induced by beta-D-N-acetylglucosaminyl-p-nitrophenol.

PubMed

Kukidome, J; Kakizaki, I; Takagaki, K; Matsuki, A; Munakata, A; Endo, M

2001-05-01

COLO 201, human colon adenocarcinoma cells were incubated with artificial primers, p-nitrophenyl-glycoside derivatives at 1.0 mmol (mM) in the medium containing 10% fetal bovine serum to detect sugar chain elongation. However, when p-nitrophenyl-beta-N-acetylglucosamine (beta-GlcNAc-PNP) was added, the medium changed color to yellow and the cells were dead. To explain this finding, the cells were incubated with 1.0 mM each of beta-GlcNAc-PNP and 4-methylumbelliferyl-beta-N-acetylglucosamine, then the number of living cells was measured in a time course. In beta-GlcNAc-PNP, the living cells were decreased at 24 hours. The cells were survived with N-acetylglucosamine, whereas in the presence of p-nitrophenol (PNP) the living cells were decreased. It was suggested that PNP released from beta-GlcNAc-PNP induced the cell death. Activity of beta-D-N-acetylglucosaminidase was detected in fetal bovine serum. It was shown that PNP induced the cell death in time-and-dose dependent manner. Genomic DNA from COLO 201 analyzed by agarose gel electrophoresis was fragmentated. PNP analogues were tested for toxicity, and the results suggested that the phenolic OH-group linked to benzene ring and nitro-group linked to the structure in para-form (PNP) was the most effective.

Polymorphic one-dimensional (N2H4)2ZnTe: soluble precursors for the formation of hexagonal or cubic zinc telluride.

PubMed

Mitzi, David B

2005-10-03

Two hydrazine zinc(II) telluride polymorphs, (N2H4)2ZnTe, have been isolated, using ambient-temperature solution-based techniques, and the crystal structures determined: alpha-(N2H4)2ZnTe (1) [P21, a = 7.2157(4) Angstroms, b = 11.5439(6) Angstroms, c = 7.3909(4) Angstroms, beta = 101.296(1) degrees, Z = 4] and beta-(N2H4)2ZnTe (2) [Pn, a = 8.1301(5) Angstroms, b = 6.9580(5) Angstroms, c = 10.7380(7) Angstroms, beta = 91.703(1) degrees, Z = 4]. The zinc atoms in 1 and 2 are tetrahedrally bonded to two terminal hydrazine molecules and two bridging tellurium atoms, leading to the formation of extended one-dimensional (1-D) zinc telluride chains, with different chain conformations and packings distinguishing the two polymorphs. Thermal decomposition of (N2H4)2ZnTe first yields crystalline wurtzite (hexagonal) ZnTe at temperatures as low as 200 degrees C, followed by the more stable zinc blende (cubic) form at temperatures above 350 degrees C. The 1-D polymorphs are soluble in hydrazine and can be used as convenient precursors for the low-temperature solution processing of p-type ZnTe semiconducting films.

Beta-lactam hypersensitivity and cross-reactivity.

PubMed

Terico, Adrienne T; Gallagher, Jason C

2014-12-01

Penicillin is the most frequently reported cause of drug allergy, and cross-reactivity of penicillins with other beta-lactam antibiotics is an area of debate. This review evaluates the available data on immunoglobulin E-mediated penicillin hypersensitivity and cross-reactivity with cephalosporin, carbapenem, and monobactam antibiotics. A MEDLINE search was conducted from 1950 to October 2013, and selected references from review articles were also evaluated. There is a wide variety in reported incidences of cross-reactivity between penicillins and cephalosporins or carbapenems, with early retrospective studies suggesting up to 41.7% and 47.4% cross-reactivity, respectively. Conversely, the use of monobactam antibiotics is frequently employed in the case of a penicillin allergy, as prescribers believe that there is no cross-reactivity between the 2 drug classes. More recent prospective studies suggest that the rates of cross-reactivity with cephalosporins and carbapenems are <5% and <1%, respectively. Similarities in penicillin and cephalosporin side chains may play a role in cross-reactivity between these classes. Cross-reactivity with monobactams is essentially negligible; however, there are some clinical data to support an interaction between ceftazidime and aztreonam, due to the similarity of their side chains. The data reviewed suggest that avoidance of other beta-lactams in patients with type 1 hypersensitivity to penicillins should be reconsidered. © The Author(s) 2014.

Amyloid-beta aggregation: selective inhibition of aggregation in mixtures of amyloid with different chain lengths.

PubMed Central

Snyder, S W; Ladror, U S; Wade, W S; Wang, G T; Barrett, L W; Matayoshi, E D; Huffaker, H J; Krafft, G A; Holzman, T F

1994-01-01

One of the clinical manifestations of Alzheimer's disease is the deposition of the 39-43 residue amyloid-beta (A beta) peptide in aggregated fibrils in senile plaques. Characterization of the aggregation behavior of A beta is one of the critical issues in understanding the role of A beta in the disease process. Using solution hydrodynamics, A beta was observed to form three types of species in phosphate-buffered saline: insoluble aggregates with sedimentation coefficients of approximately 50,000 S and molecular masses of approximately 10(9) Da, "soluble aggregates" with sedimentation coefficients of approximately 30 S and masses of approximately 10(6) Da, and monomer. When starting from monomer, the aggregation kinetics of A beta 1-40 (A beta 40) and A beta 1-42 (A beta 42), alone and in combination, reveal large differences in the tendency of these peptides to aggregate as a function of pH and other solution conditions. At pH 4.1 and 7.0-7.4, aggregation is significantly slower than at pH 5 and 6. Under all conditions, aggregation of the longer A beta 42 was more rapid than A beta 40. Oxidation of Met-35 to the sulfoxide in A beta 40 enhances the aggregation rate over that of the nonoxidized peptide. Aggregation was found to be dependent upon temperature and to be strongly dependent on peptide concentration and ionic strength, indicating that aggregation is driven by a hydrophobic effect. When A beta 40 and A beta 42 are mixed together, A beta 40 retards the aggregation of A beta 42 in a concentration-dependent manner. Shorter fragments have a decreasing ability to interfere with A beta 42 aggregation. Conversely, the rate of aggregation of A beta 40 can be significantly enhanced by seeding slow aggregating solutions with preformed aggregates of A beta 42. Taken together, the inhibition of A beta 42 aggregation by A beta 40, the seeding of A beta 40 aggregation by A beta 42 aggregates, and the chemical oxidation of A beta 40 suggest that the relative abundance and rates of production of different-length A beta and its exposure to radical damage may be factors in the accumulation of A beta in plaques in vivo. Images FIGURE 6 PMID:7811936

Thalassaemia screening among students in a secondary school in Ampang, Malaysia.

PubMed

Jameela, S; Sabirah, S O Sharifah; Babam, J; Phan, C L; Visalachy, P; Chang, K M; Salwana, M A; Zuraidah, A; Subramanian, Y; Rahimah, A

2011-12-01

Thalassaemia is a common disorder in Malaysia. It is estimated that 4.5% of the population are carriers for beta- or alpha- thalassaemias. We set out to screen Form 4 students aged between 15 and 16 years old in a national school, for thalassaemia in March 2008. Written consent was obtained from 310 students. The carrier rate for the common thalassaemia syndromes was 6.8% (2.9% for beta-thalassaemia, 2.6% for HbE and 1.3% for two-gene deletion for alpha-thalassaemia). Carriers for beta-thalassaemia and two-gene deletion for alpha-thalassaemia were more common in the Chinese (4.3% and 1.4% respectively) while heterozygous HbE was more common in the Malays (3.8%). The laboratory cost of screening one student was RM 45 and the total number of man-hours spent in this screening activity was 600. This screening exercise showed that thalassaemia carriers are common among the Chinese and Malays and it is feasible to carry out a screening programme for secondary school students.

Mycophenolate mofetil modulates adhesion receptors of the beta1 integrin family on tumor cells: impact on tumor recurrence and malignancy

PubMed Central

Engl, Tobias; Makarević, Jasmina; Relja, Borna; Natsheh, Iyad; Müller, Iris; Beecken, Wolf-Dietrich; Jonas, Dietger; Blaheta, Roman A

2005-01-01

Background Tumor development remains one of the major obstacles following organ transplantation. Immunosuppressive drugs such as cyclosporine and tacrolimus directly contribute to enhanced malignancy, whereas the influence of the novel compound mycophenolate mofetil (MMF) on tumor cell dissemination has not been explored. We therefore investigated the adhesion capacity of colon, pancreas, prostate and kidney carcinoma cell lines to endothelium, as well as their beta1 integrin expression profile before and after MMF treatment. Methods Tumor cell adhesion to endothelial cell monolayers was evaluated in the presence of 0.1 and 1 μM MMF and compared to unstimulated controls. beta1 integrin analysis included alpha1beta1 (CD49a), alpha2beta1 (CD49b), alpha3beta1 (CD49c), alpha4beta1 (CD49d), alpha5beta1 (CD49e), and alpha6beta1 (CD49f) receptors, and was carried out by reverse transcriptase-polymerase chain reaction, confocal microscopy and flow cytometry. Results Adhesion of the colon carcinoma cell line HT-29 was strongly reduced in the presence of 0.1 μM MMF. This effect was accompanied by down-regulation of alpha3beta1 and alpha6beta1 surface expression and of alpha3beta1 and alpha6beta1 coding mRNA. Adhesion of the prostate tumor cell line DU-145 was blocked dose-dependently by MMF. In contrast to MMF's effects on HT-29 cells, MMF dose-dependently up-regulated alpha1beta1, alpha2beta1, alpha3beta1, and alpha5beta1 on DU-145 tumor cell membranes. Conclusion We conclude that MMF possesses distinct anti-tumoral properties, particularly in colon and prostate carcinoma cells. Adhesion blockage of HT-29 cells was due to the loss of alpha3beta1 and alpha6beta1 surface expression, which might contribute to a reduced invasive behaviour of this tumor entity. The enhancement of integrin beta1 subtypes observed in DU-145 cells possibly causes re-differentiation towards a low-invasive phenotype. PMID:15644133

A quantitative real-time RT-PCR assay to measure TGF-beta mRNA and its correlation with hematologic, plasma chemistry and organo-somatic indices responses in triamcinolone-treated Atlantic menhaden, Brevoortia tyrannus.

PubMed

Johnson, A K; Harms, C A; Levine, J F; Law, J McHugh

2006-01-01

A quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) assay was developed to measure transforming growth factor-beta (TGF-beta) in Atlantic menhaden (Brevoortia tyrannus), an estuarine-dependent species plagued by ulcerative skin lesions in the estuaries along the eastern United States. Atlantic menhaden were acclimated in a closed system for two weeks prior to initiation of the study. The synthetic glucocorticoid, triamcinolone acetonide (10mg/kg body weight) was administered by intracoelomic injection and its effect on the splenic mononuclear cell TGF-beta mRNA transcription, liver-somatic index, spleno-somatic index, hematology, and plasma chemistry were compared to untreated fish at 48 and 96h post-treatment. Triamcinolone-treated Atlantic menhaden showed suppression of TGF-beta mRNA production, neutrophilia, monocytosis, lymphopenia, and an increase in blood glucose concentrations. The health indices used in this study may help us interpret some of the changes observed during the development of ulcerative skin lesions in wild-caught menhaden.

Myosin storage myopathy associated with a heterozygous missense mutation in MYH7.

PubMed

Tajsharghi, Homa; Thornell, Lars-Eric; Lindberg, Christopher; Lindvall, Björn; Henriksson, Karl-Gösta; Oldfors, Anders

2003-10-01

Myosin constitutes the major part of the thick filaments in the contractile apparatus of striated muscle. MYH7 encodes the slow/beta-cardiac myosin heavy chain (MyHC), which is the main MyHC isoform in slow, oxidative, type 1 muscle fibers of skeletal muscle. It is also the major MyHC isoform of cardiac ventricles. Numerous missense mutations in the globular head of slow/beta-cardiac MyHC are associated with familial hypertrophic cardiomyopathy. We identified a missense mutation, Arg1845Trp, in the rod region of slow/beta-cardiac MyHC in patients with a skeletal myopathy from two different families. The myopathy was characterized by muscle weakness and wasting with onset in childhood and slow progression, but no overt cardiomyopathy. Slow, oxidative, type 1 muscle fibers showed large inclusions consisting of slow/beta-cardiac MyHC. The features were similar to a previously described entity: hyaline body myopathy. Our findings indicate that the mutated residue of slow/beta-cardiac MyHC is essential for the assembly of thick filaments in skeletal muscle. We propose the term myosin storage myopathy for this disease.

Differentiation of human-induced pluripotent stem cells into insulin-producing clusters.

PubMed

Shaer, Anahita; Azarpira, Negar; Vahdati, Akbar; Karimi, Mohammad Hosein; Shariati, Mehrdad

2015-02-01

In diabetes mellitus type 1, beta cells are mostly destroyed; while in diabetes mellitus type 2, beta cells are reduced by 40% to 60%. We hope that soon, stem cells can be used in diabetes therapy via pancreatic beta cell replacement. Induced pluripotent stem cells are a kind of stem cell taken from an adult somatic cell by "stimulating" certain genes. These induced pluripotent stem cells may be a promising source of cell therapy. This study sought to produce isletlike clusters of insulin-producing cells taken from induced pluripotent stem cells. A human-induced pluripotent stem cell line was induced into isletlike clusters via a 4-step protocol, by adding insulin, transferrin, and selenium (ITS), N2, B27, fibroblast growth factor, and nicotinamide. During differentiation, expression of pancreatic β-cell genes was evaluated by reverse transcriptase-polymerase chain reaction; the morphologic changes of induced pluripotent stem cells toward isletlike clusters were observed by a light microscope. Dithizone staining was used to stain these isletlike clusters. Insulin produced by these clusters was evaluated by radio immunosorbent assay, and the secretion capacity was analyzed with a glucose challenge test. Differentiation was evaluated by analyzing the morphology, dithizone staining, real-time quantitative polymerase chain reaction, and immunocytochemistry. Gene expression of insulin, glucagon, PDX1, NGN3, PAX4, PAX6, NKX6.1, KIR6.2, and GLUT2 were documented by analyzing real-time quantitative polymerase chain reaction. Dithizone-stained cellular clusters were observed after 23 days. The isletlike clusters significantly produced insulin. The isletlike clusters could increase insulin secretion after a glucose challenge test. This work provides a model for studying the differentiation of human-induced pluripotent stem cells to insulin-producing cells.

Cecal parameters of rats fed diets containing grapefruit polyphenols and inulin as single supplements or in a combination.

PubMed

Zduńczyk, Zenon; Juśkiewicz, Jerzy; Estrella, Isabel

2006-09-01

We compared the effects of grapefruit flavonoids and inulin, as single dietary components or in a combination, on cecal fermentation in rats adapted to a semipurified diet. The experimental diets contained 0.3% flavonoid extract and 5% or 10% inulin and a combination of both supplements. The large bowel metabolism assessment was based on cecal parameters: bulk effect, pH, microbial enzymes activity, and short-chain fatty acid production. Both supplements induced significant enlargement of the cecal digesta weight. Acidification of cecal digesta was more pronounced, with a higher inulin addition to the diet. Cecal pH was the highest with the flavonoid-rich diets and lowest in the case of a simultaneous addition of flavonoids and a high content of inulin. The flavonoid extract applied as a single dietary supplement was observed to decrease the activity of bacterial beta-glucosidase and beta- and alpha-galactosidases in the cecal digesta. In contrast, addition of the grapefruit extract to inulin-containing diets increased the activity of alpha-glucosidase, alpha-galactosidase, and beta-galactosidase. Great accumulation of cecal digesta in rats consuming the flavonoid-diet caused a considerable increase in the short-chain fatty acid pool, mainly acetic acid. Inulin added to the diet decreased the excessive enlargement of digesta caused by dietary flavonoids. Dietary addition of inulin to the flavonoid-diet also normalized hydration of cecal digesta and significantly decreased the pH of digesta. The presence of polyphenols in the inulin-containing diets did not change total short-chain fatty acid production in the cecum of rats. Our results suggested that simultaneous intake of inulin and polyphenols can decrease the detrimental effects of the latter on cecal fermentation.

Prenatal diagnosis of hemoglobinopathies: evaluation of techniques for analysing globin-chain synthesis in blood samples obtained by fetoscopy.

PubMed Central

Congote, L. F.; Hamilton, E. F.; Chow, J. C.; Perry, T. B.

1982-01-01

Three techniques for analysing hemoglobin synthesis in blood samples obtained by fetoscopy were evaluated. Of the fetuses studied, 12 were not at risk of genetic disorders, 10 were at risk of beta-thalassemia, 2 were at risk of sickle cell anemia and 1 was at risk of both diseases. The conventional method of prenatal diagnosis of hemoglobinopathies, involving the separation of globin chains labelled with a radioactive isotope on carboxymethyl cellulose (CMC) columns, was compared with a method involving globin-chain separation by high-pressure liquid chromatography (HPLC) and with direct analysis of labelled hemoglobin tetramers obtained from cell lysates by chromatography on ion-exchange columns. The last method is technically the simplest and can be used for diagnosing beta-thalassemia and sickle cell anemia. However, it gives spuriously high levels of adult hemoglobin in samples containing nonlabelled adult hemoglobin. HPLC is the fastest method for prenatal diagnosis of beta-thalassemia and may prove as reliable as the CMC method. Of the 13 fetuses at risk for hemoglobinopathies, 1 was predicted to be affected, and the diagnosis was confirmed in the abortus. Of 12 predicted to be unaffected, 1 was aborted spontaneously and was unavailable for confirmatory studies, as were 3 of the infants; however, the diagnosis was confirmed in seven cases and is awaiting confirmation when the infant in 6 months old in one case. Couples at risk of bearing a child with a hemoglobinopathy should be referred for genetic counselling before pregnancy or, at the latest, by the 12th week of gestation so that prenatal diagnosis can be attempted by amniocentesis, safer procedure, with restriction endonuclease analysis of the amniotic fluid cells. PMID:7139502

Characterization of a beta 1----3-N-acetylglucosaminyltransferase associated with synthesis of type 1 and type 2 lacto-series tumor-associated antigens from the human colonic adenocarcinoma cell line SW403.

PubMed

Holmes, E H

1988-01-01

Previous studies have indicated that activation of a normally unexpressed beta 1----3-N-acetylglucosaminyltransferase is responsible for the accumulation of a wide diversity of both type 1 and 2 lacto-series antigens in human colonic adenocarcinomas. A beta 1----3-N-acetylglucosaminyltransferase has been solubilized from the human colonic adenocarcinoma cell line SW403 by 0.2% Triton X-100 and some of its properties have been studied. The enzyme was active over a broad pH range from 5.8 to 7.5 and had a strict requirement for Mn2+ as a divalent metal ion. Transfer of N-acetylglucosamine (GlcNAc) to lactosylceramide was optimal when assayed in the presence of a final concentration of Triton CF-54 of 0.3%. Inclusion of CDPcholine in the reaction mixture stimulated the activity by protecting the UDP[14C]GlcNAc from hydrolysis by endogenous enzymes. The kinetic parameters of the enzyme were studied. Km values for acceptors nLc4 and nLc6 were determined to be 0.19 mM for each. However, the Vmax values calculated for these acceptors were 150 and 110 pmol/h/mg protein for nLc4 and nLc6, respectively, suggesting reduced potential for further elongation as the chain length increases. The Km for UDPGlcNAc was determined to be 0.17 mM. Studies of the acceptor specificity have indicated transfer of GlcNAc occurs mainly to type 2 chain nonfucosylated structures. However, elongation of the type 1 chain structure Lc4 was also detected.

A novel missense mutation, Leu390Val, in the cardiac beta-myosin heavy chain associated with pronounced septal hypertrophy in two families with hypertrophic cardiomyopathy.

PubMed

Havndrup, O; Bundgaard, H; Andersen, P S; Larsen, L A; Vuust, J; Kjeldsen, K; Christiansen, M

2000-12-01

An examination of the genetic background and phenotypic presentation of familial hypertrophic cardiomyopathy (FHC) with respect to specific mutations in the MYH7-gene encoding the cardiac beta-myosin heavy chain. Two families (n = 22) from a cohort of 67 families with FHC were studied at the National University Hospital, Rigshospitalet, Copenhagen. Clinical, non-invasive examinations of all included family members followed by molecular genetic analysis including PCR-single strand conformation polymorphism/heteroduplex (SSCP/HD) analysis and sequencing of exon 3-23 of the MYH7-gene. We found FHC associated with a missense mutation in two families, i.e. a C > G transversion at position g10124 and a G > T transversion at position g10126 causing the change of a leucine residue at codon 390 to a valine residue. The mutation is located in the actin-binding region of the beta-myosin heavy chain. The leucine residue is evolutionarily conserved in vertebrate myosins. In the two families, the phenotypic presentations in the clinically affected were characterized by asymmetric septal hypertrophy (septum diameter 18.8 (5.0) mm (mean (SD)) with only minor involvement of the left ventricular free wall (posterior wall diameter 11.0 (2.2) mm). Furthermore, the left ventricular systolic and diastolic functions were well preserved, even at a high age. The symptomatic status of the clinically affected patients depended on the presence or absence of a concomitant left ventricular outflow tract gradient. We report a novel missense mutation associated with FHC caused by a double nucleotide transversion. The penetrance of the mutation was not complete, but in clinically affected patients the mutation gives rise to an echocardiographic phenotype, predominantly characterized by pronounced septal hypertrophy.

Extension arm facilitated pegylation of alphaalpha-hemoglobin with modifications targeted exclusively to amino groups: functional and structural advantages of free Cys-93(beta) in the PEG-Hb adduct.

PubMed

Li, Dongxia; Hu, Tao; Manjula, Belur N; Acharya, Seetharama A

2009-11-01

Cys-93(beta) of hemoglobin (Hb) was reversibly protected as a mixed disulfide with thiopyridine during extension arm facilitated (EAF) PEGylation and its influence on the structural and functional properties of the EAF-PEG-Hb has been investigated. Avoiding PEGylation of Cys-93(beta) in the EAF-PEG-Hb lowers the level of perturbation of heme pocket, alpha1beta2 interface, autoxidation, heme loss, and the O(2) affinity, as compared to the EAF-PEG-Hb with PEGylation of Cys-93(beta).The structural and functional advantages of reversible protection of Cys-93(beta) during EAF PEGylation of oxy-Hb has been compared with Euro PEG-Hb generated by EAF PEGylation of deoxy Hb where Cys-93(beta) is free in the final product. The alphaalpha-fumaryl cross-linking and EAF PEGylation targeted exclusively to Lys residues has been combined together for generation of second-generation EAF-PEG-Hb with lower oxygen affinity. The PEG chains engineered on Lys as well as PEGylation of Cys-93(beta) independently contribute to the stabilization of oxy conformation of Hb and hence increase the oxygen affinity of Hb. However, oxygen affinity of the EAF-PEG-alphaalpha-Hb is more sensitive to the presence of PEGylation on Cys-93(beta) than that of the EAF-PEG-Hb. The present modified EAF PEGylation platform is expected to facilitate the design of novel versions of the EAF-PEG-Hbs that can now integrate the advantages of avoiding PEGylation of Cys-93(beta).

Construction and measurements of a vacuum-swing-adsorption radon-mitigation system

NASA Astrophysics Data System (ADS)

Schnee, R. W.; Bunker, R.; Ghulam, G.; Jardin, D.; Kos, M.; Tenney, A. S.

2013-08-01

Long-lived alpha and beta emitters in the 222Rn decay chain on (and near) detector surfaces may be the limiting background in many experiments attempting to detect dark matter or neutrinoless double beta decay, and in screening detectors. In order to reduce backgrounds from radon-daughter plate-out onto the wires of the Beta Cage during its assembly, an ultra-low-radon cleanroom is being commissioned at Syracuse University using a vacuum-swing-adsorption radon-mitigation system. The radon filter shows ˜20× reduction at its output, from 7.47±0.56 to 0.37±0.12 Bq/m3, and the cleanroom radon activity meets project requirements, with a lowest achieved value consistent with that of the filter, and levels consistently < 2 Bq/m3.

Evolutionarily conserved TCR binding sites, identification of T cells in primary lymphoid tissues, and surprising trans-rearrangements in nurse shark.

PubMed

Criscitiello, Michael F; Ohta, Yuko; Saltis, Mark; McKinney, E Churchill; Flajnik, Martin F

2010-06-15

Cartilaginous fish are the oldest animals that generate RAG-based Ag receptor diversity. We have analyzed the genes and expressed transcripts of the four TCR chains for the first time in a cartilaginous fish, the nurse shark (Ginglymostoma cirratum). Northern blotting found TCR mRNA expression predominantly in lymphoid and mucosal tissues. Southern blotting suggested translocon-type loci encoding all four chains. Based on diversity of V and J segments, the expressed combinatorial diversity for gamma is similar to that of human, alpha and beta may be slightly lower, and delta diversity is the highest of any organism studied to date. Nurse shark TCRdelta have long CDR3 loops compared with the other three chains, creating binding site topologies comparable to those of mammalian TCR in basic paratope structure; additionally, nurse shark TCRdelta CDR3 are more similar to IgH CDR3 in length and heterogeneity than to other TCR chains. Most interestingly, several cDNAs were isolated that contained IgM or IgW V segments rearranged to other gene segments of TCRdelta and alpha. Finally, in situ hybridization experiments demonstrate a conservation of both alpha/beta and gamma/delta T cell localization in the thymus across 450 million years of vertebrate evolution, with gamma/delta TCR expression especially high in the subcapsular region. Collectively, these data make the first cellular identification of TCR-expressing lymphocytes in a cartilaginous fish.

Evolution and Function of the TCR Vgamma9 Chain Repertoire: It’s Good to be Public

PubMed Central

Pauza, C. David; Cairo, Cristiana

2015-01-01

Lymphocytes expressing a T cell receptor (TCR) composed of Vgamma9 and Vdelta2 chains represent a minor fraction of human thymocytes. Extrathymic selection throughout post-natal life causes the proportion of cells with a Vgamma9-JP rearrangement to increase and elevates the capacity for responding to non-peptidic phosphoantigens. Extrathymic selection is so powerful that phosphoantigen-reactive cells comprise about 1 in 40 circulating memory T cells from healthy adults and the subset can be expanded rapidly upon infection or in response to malignancy. Skewing of the gamma delta TCR repertoire is accompanied by selection for public gamma chain sequences such that many unrelated individuals overlap extensive in their circulating repertoire. This type of selection implies the presence of a monomorphic antigen-presenting molecule that is an object of current research but remains incompletely defined. While selection on a monomorphic presenting molecule may seem unusual, similar mechanisms shape the alpha beta T cell repertoire including the extreme examples of NKT or mucosal-associated invariant T cells (MAIT) and the less dramatic amplification of public Vbeta chain rearrangements driven by individual MHC molecules and associated with resistance to viral pathogens. Selecting and amplifying public T cell receptors whether alpha beta or gamma delta, are important steps in developing an anticipatory TCR repertoire. Cell clones expressing public TCR can accelerate the kinetics of response to pathogens and impact host survival. PMID:25769734

Genotype–phenotype correlation among beta-thalassemia and beta-thalassemia/HbE disease in Thai children: predictable clinical spectrum using genotypic analysis

PubMed Central

Traivaree, Chanchai; Monsereenusorn, Chalinee; Rujkijyanont, Piya; Prasertsin, Warakorn; Boonyawat, Boonchai

2018-01-01

Introduction Beta-thalassemia is a group of inherited hemolytic anemias and one of the most common genetic disorders in Thailand. The clinical spectrum of beta-thalassemia disease ranges from mild to severe clinical symptoms including mild beta-thalassemia intermedia (TI) and severe beta-thalassemia major (TM). Objective This study aimed to determine the correlation between beta-globin gene (HBB) mutations and their phenotypic manifestations by evaluating patients’ clinical characteristics, transfusion requirements, growth and hematologic parameters, and hemoglobin typing among pediatric patients treated at Phramongkutklao Hospital. Materials and methods Seventy beta-thalassemia patients, including 63 with beta-thalassemia/hemoglobin E (HbE) and 7 with either homozygous or compound heterozygous beta-thalassemia, were enrolled in this study. Their clinical presentation, growth parameters and laboratory findings were reviewed and analyzed. The mean follow-up time was 10.52±5.62 years. Mutation analysis in each individual was performed using multiplex amplification refractory mutation system (M-ARMS), direct DNA sequencing of beta-globin gene and gap PCR for 3.4 kb deletion detection. Results All 7 homozygous and compound heterozygous beta-thalassemia patients were classified in TM. Among 63 patients with beta-thalassemia/HbE, 58 were classified in TM and 4 were classified in TI. Mean age at diagnosis was 0.8±0.49 years for homozygous or compound heterozygous beta-thalassemia and 3.43±3.5 years for beta-thalassemia/HbE. The most common HBB mutation was HBB:c.126_129delCTTT [codon 41/42 (-TCTT)] found in 34 alleles (48.6%). The height for age was also lower in homozygous beta-thalassemia patients (<3rd percentile) compared to compound heterozygous beta-thalassemia patients (25–50th percentile). Conclusion This study revealed a genotype–phenotype correlation of the most prevalent beta-thalassemia in Thai children using diagnostic capacity in genotypic analysis of HBB mutation. Our findings can provide a better prediction of clinical manifestation and severity by early identification of the type of the HBB mutations. PMID:29695942

Cytoskeleton and paclitaxel sensitivity in breast cancer: the role of beta-tubulins.

PubMed

Tommasi, Stefania; Mangia, Anita; Lacalamita, Rosanna; Bellizzi, Antonia; Fedele, Vita; Chiriatti, Annalisa; Thomssen, Christopher; Kendzierski, Nancy; Latorre, Agnese; Lorusso, Vito; Schittulli, Francesco; Zito, Francesco; Kavallaris, Maria; Paradiso, Angelo

2007-05-15

The antineoplastic effect of paclitaxel is mainly related to its ability to bind the beta subunit of tubulin, thus preventing tubulin chain depolarization and inducing apoptosis. The relevance of the Class I beta-tubulin characteristics have also been confirmed in the clinical setting where mutations of paclitaxel-binding site of beta-tubulin Class I have been related to paclitaxel resistance in non small cell lung and ovarian cancers. In the present study, we verified the hypothesis of a relationship between molecular alterations of beta-tubulin Class I and paclitaxel sensitivity in a panel of breast cell lines with different drug IC(50). The Class I beta-tubulin gene cDNA has been sequenced detecting heterozygous missense mutations (exon 1 and 4) only in MCF-7 and SK-BR-3 lines. Furthermore, the expression (at both mRNA and protein level) of the different isotypes have been analyzed demonstrating an association between low cell sensitivity to paclitaxel and Class III beta-tubulin expression increasing. Antisense oligonucleotide (ODN) experiments confirmed that the inhibition of Class III beta-tubulin could at least partially increase paclitaxel-chemosensitivity. The hypothesis of a relationship between beta-tubulin tumor expression and paclitaxel clinical response has been finally verified in a series of 92 advanced breast cancer patients treated with a first line paclitaxel-based chemotherapy. Thirty-five percent (95% CI: 45-31) of patients with high Class III beta-tubulin expression showed a disease progression vs. only 7% of patients with low expression (35% vs. 7%, p < 0.002). Our study suggests that Class III beta-tubulin tumor expression could be considered a predictive biomarker of paclitaxel-clinical resistance for breast cancer patients. (c) 2007 Wiley-Liss, Inc.

{beta}-Catenin regulates airway smooth muscle contraction.

PubMed

Jansen, Sepp R; Van Ziel, Anna M; Baarsma, Hoeke A; Gosens, Reinoud

2010-08-01

beta-Catenin is an 88-kDa member of the armadillo family of proteins that is associated with the cadherin-catenin complex in the plasma membrane. This complex interacts dynamically with the actin cytoskeleton to stabilize adherens junctions, which play a central role in force transmission by smooth muscle cells. Therefore, in the present study, we hypothesized a role for beta-catenin in the regulation of smooth muscle force production. beta-Catenin colocalized with smooth muscle alpha-actin (sm-alpha-actin) and N-cadherin in plasma membrane fractions and coimmunoprecipitated with sm-alpha-actin and N-cadherin in lysates of bovine tracheal smooth muscle (BTSM) strips. Moreover, immunocytochemistry of cultured BTSM cells revealed clear and specific colocalization of sm-alpha-actin and beta-catenin at the sites of cell-cell contact. Treatment of BTSM strips with the pharmacological beta-catenin/T cell factor-4 (TCF4) inhibitor PKF115-584 (100 nM) reduced beta-catenin expression in BTSM whole tissue lysates and in plasma membrane fractions and reduced maximal KCl- and methacholine-induced force production. These changes in force production were not accompanied by changes in the expression of sm-alpha-actin or sm-myosin heavy chain (MHC). Likewise, small interfering RNA (siRNA) knockdown of beta-catenin in BTSM strips reduced beta-catenin expression and attenuated maximal KCl- and methacholine-induced contractions without affecting sm-alpha-actin or sm-MHC expression. Conversely, pharmacological (SB-216763, LiCl) or insulin-induced inhibition of glycogen synthase kinase-3 (GSK-3) enhanced the expression of beta-catenin and augmented maximal KCl- and methacholine-induced contractions. We conclude that beta-catenin is a plasma membrane-associated protein in airway smooth muscle that regulates active tension development, presumably by stabilizing cell-cell contacts and thereby supporting force transmission between neighboring cells.

Inhibition of herpes simplex virus DNA polymerase by purine ribonucleoside monophosphates.

PubMed

Frank, K B; Cheng, Y C

1986-02-05

Purine ribonucleoside monophosphates were found to inhibit chain elongation catalyzed by herpes simplex virus (HSV) DNA polymerase when DNA template-primer concentrations were rate-limiting. Inhibition was fully competitive with DNA template-primer during chain elongation; however, DNA polymerase-associated exonuclease activity was inhibited noncompetitively with respect to DNA. Combinations of 5'-GMP and phosphonoformate were kinetically mutually exclusive in dual inhibitor studies. Pyrimidine nucleoside monophosphates and deoxynucleoside monophosphates were less inhibitory than purine riboside monophosphates. The monophosphates of 9-beta-D-arabinofuranosyladenine, Virazole (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), 9-(2-hydroxyethoxymethyl)guanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine exerted little or no inhibition. In contrast to HSV DNA polymerase, human DNA polymerase alpha was not inhibited by purine ribonucleoside monophosphates. These studies suggest the possibility of a physiological role of purine ribonucleoside monophosphates as regulators of herpesvirus DNA synthesis and a new approach to developing selective anti-herpesvirus compounds.

The etiology of community-acquired pneumonia in Australia: why penicillin plus doxycycline or a macrolide is the most appropriate therapy.

PubMed

Charles, Patrick G P; Whitby, Michael; Fuller, Andrew J; Stirling, Robert; Wright, Alistair A; Korman, Tony M; Holmes, Peter W; Christiansen, Keryn J; Waterer, Grant W; Pierce, Robert J P; Mayall, Barrie C; Armstrong, John G; Catton, Michael G; Nimmo, Graeme R; Johnson, Barbara; Hooy, Michelle; Grayson, M L

2008-05-15

Available data on the etiology of community-acquired pneumonia (CAP) in Australia are very limited. Local treatment guidelines promote the use of combination therapy with agents such as penicillin or amoxycillin combined with either doxycycline or a macrolide. The Australian CAP Study (ACAPS) was a prospective, multicenter study of 885 episodes of CAP in which all patients underwent detailed assessment for bacterial and viral pathogens (cultures, urinary antigen testing, serological methods, and polymerase chain reaction). Antibiotic agents and relevant clinical outcomes were recorded. The etiology was identified in 404 (45.6%) of 885 episodes, with the most frequent causes being Streptococcus pneumoniae (14%), Mycoplasma pneumoniae (9%), and respiratory viruses (15%; influenza, picornavirus, respiratory syncytial virus, parainfluenza virus, and adenovirus). Antibiotic-resistant pathogens were rare: only 5.4% of patients had an infection for which therapy with penicillin plus doxycycline would potentially fail. Concordance with local antibiotic recommendations was high (82.4%), with the most commonly prescribed regimens being a penicillin plus either doxycycline or a macrolide (55.8%) or ceftriaxone plus either doxycycline or a macrolide (36.8%). The 30-day mortality rate was 5.6% (50 of 885 episodes), and mechanical ventilation or vasopressor support were required in 94 episodes (10.6%). Outcomes were not compromised by receipt of narrower-spectrum beta-lactams, and they did not differ on the basis of whether a pathogen was identified. The vast majority of patients with CAP can be treated successfully with narrow-spectrum beta-lactam treatment, such as penicillin combined with doxycycline or a macrolide. Greater use of such therapy could potentially reduce the emergence of antibiotic resistance among common bacterial pathogens.

Common Cause Failure Modeling

NASA Technical Reports Server (NTRS)

Hark, Frank; Britton, Paul; Ring, Robert; Novack, Steven

2015-01-01

Space Launch System (SLS) Agenda: Objective; Key Definitions; Calculating Common Cause; Examples; Defense against Common Cause; Impact of varied Common Cause Failure (CCF) and abortability; Response Surface for various CCF Beta; Takeaways.

Removing peroxide impurities from ether improves the quality of globin chains for biosynthetic studies.

PubMed

Mirzazadeh, Roghieh; Khatami, Shohreh; Bayat, Parastoo; Zamani, Zahra; Sadeghi, Sedigheh; Roohi, Soghra; Saidi, Parinaz

2005-01-01

The diagnosis of the different forms of thalassemia is one of the important applications of analysis of globin chains. These analyses are done by high performance liquid chromatography (HPLC) using a MONO-S cation exchange column and ether is used for washing the globin powder in the final step. The presence of peroxide impurities in ether could change the structure of the globin chains. In the chromatograms, these modified globins appear as separated peaks next to the unmodified globin peaks. In these cases, the alpha/beta ratio exceed by artifact the correct value. Our study demonstrates that diagnostic centers should ensure that the ether they use is pure.

A Cuphea beta-ketoacyl-ACP synthase shifts the synthesis of fatty acids towards shorter chains in Arabidopsis seeds expressing Cuphea FatB thioesterases.

PubMed

Leonard, J M; Knapp, S J; Slabaugh, M B

1998-03-01

Acyl-acyl carrier protein (ACP) thioesterases with specificities on medium chain substrates (C8-C14) are requisite enzymes in plants that produce 8:0, 10:0, 12:0 and 14:0 seed oils, but they may not be the sole enzymatic determinants of chain length. The contribution to chain length regulation of a beta-ketoacyl-ACP synthase, Cw KAS A1, derived from Cuphea wrightii, a species that accumulates 30% 10:0 and 54% 12:0 in seed oils, was investigated. Expression of Cw KAS A1 in Arabidopsis seeds reduced 16:0 from 8.2 to 6.2 mol%, suggesting a KAS II-type activity. In the presence of the KAS I inhibitor cerulenin, however, transgenic seed extracts extended 6:0- and 8:0-ACP at a rate four- to fivefold greater than extracts from untransformed plants, whereas no difference was observed in extension of 14:0- and 16:0-ACP. The effect of KAS A1 on seed oils was tested by combining it with the C. wrightii medium chain-specific thioesterases, Cw FatB1 and Cw FatB2, in crosses of transformed plants. Fatty acid synthesis thesis shifted towards shorter chains in progeny expressing both classes of enzymes. KasA1/FatB1 homozygotes produced threefold more 12:0 than the FatB1 parent while 14:0 and 16:0 were reduced by one-third and one-half, respectively. F2 progeny expressing KasA1 and FatB2 produced twofold more 10:0 and 1.4-fold more 12:0 than the FatB2 parent, and the double-transgenic progeny produced one-quarter less 14:0 and one-half less 16:0 than the FatB2 parent. It is hypothesized that the shift towards production of shorter chains resulted from increased pools of medium chain acyl-ACP resulting from KAS A1 activity. The combined activities of KAS A1 and FatB thioesterases appear to determine the C. wrightii phenotype.

Mechanism of the protective effects of long chain n-alkyl glucopyranosides against ultrasound-induced cytolysis of HL-60 cells.

PubMed

Cheng, Jason Y; Riesz, Peter

2007-07-01

Recently it has been shown that long chain (C5-C8) n-alkyl glucopyranosides completely inhibit ultrasound-induced cytolysis [J.Z. Sostaric, N. Miyoshi, P. Riesz, W.G. DeGraff, and J.B. Mitchell, Free Radical Biol. Med., 39 (2005) 1539]. This protective effect has possible applications in HIFU (high intensity focused ultrasound) for tumor treatment, and in ultrasound assisted drug delivery and gene therapy. n-Alkyl glucopyranosides with hexyl (5mM), heptyl (3mM), octyl (2mM) n-alkyl chains protected 100% of HL-60 cells in vitro from 1.057 MHz ultrasound-induced cytolysis under a range of conditions that resulted in 35-100% cytolysis in the absence of glucopyranosides. However the hydrophilic methyl-beta-d-glucopyranoside did not protect cells. The surface active n-alkyl glucopyranosides accumulate at the gas-liquid interface of cavitation bubbles. The OH radicals and H atoms formed in collapsing cavitation bubbles react by H-atom abstraction from either the n-alkyl chain or the glucose moiety of the n-alkyl glucopyranosides. Owing to the high concentration of the long chain surfactants at the gas-liquid interface of cavitation bubbles, the initially formed carbon radicals on the alkyl chains are transferred to the glucose moieties to yield radicals which react with oxygen leading to the formation of hydrogen peroxide. In this work, we find that the sonochemically produced hydrogen peroxide yields from oxygen-saturated solutions of long chain (hexyl, octyl) n-alkyl glucopyranosides at 614 kHz and 1.057 MHz ultrasound increase with increasing n-alkyl glucopyranoside concentration but are independent of concentration for methyl-beta-D-glucopyranoside. These results are consistent with the previously proposed mechanism of sonoprotection [J.Z. Sostaric, N. Miyoshi, P. Riesz, W.G. DeGraff, and J.B. Mitchell, Free Radical Biol. Med., 39 (2005) 1539]. This sequence of events prevents sonodynamic cell killing by initiation of lipid peroxidation chain reactions in cellular membranes by peroxyl and/or alkoxyl radicals [V. Misik, P. Riesz, Ann. N.Y. Acad. Sci., 899 (2000) 335].

Secondary cell-wall assembly in flax phloem fibres: role of galactans.

PubMed

Gorshkova, Tatyana; Morvan, Claudine

2006-01-01

Non-lignified fibre cells (named gelatinous fibres) are present in tension wood and the stems of fibre crops (such as flax and hemp). These cells develop a very thick S2 layer within the secondary cell wall, which is characterised by (1) cellulose microfibrils largely parallel to the longitudinal axis of the cell, and (2) a high proportion of galactose-containing polymers among the non-cellulosic polysaccharides. In this review, we focus on the role of these polymers in the assembly of gelatinous fibres of flax. At the different stages of fibre development, we analyse in detail data based on sugar composition, linkages of pectic polymers, and immunolocalisation of the beta-(1-->4)-galactans. These data indicate that high molecular-mass gelatinous galactans accumulate in specialised Golgi-derived vesicles during fibre cell-wall thickening. They consist of RG-I-like polymers with side chains of beta-(1-->4)-linked galactose. Most of them are short, but there are also long chains containing up to 28 galactosyl residues. At fibre maturity, two types of cross-linked galactans are identified, a C-L structure that resembles the part of soluble galactan with long side chains and a C-S structure with short chains. Different possibilities for soluble galactan to give rise to C-L and C-S are analysed. In addition, we discuss the prospect for the soluble galactan in preventing the newly formed cellulose chains from completing immediate crystallisation. This leads to a hypothesis that firstly the secretion of soluble galactans plays a role in the axial orientation of cellulose microfibrils, and secondly the remodelling and cross-linking of pectic galactans are linked to the dehydration and the assembly of S2 layer.

Crystal structure of the anti-(carcinoembryonic antigen) single-chain Fv antibody MFE-23 and a model for antigen binding based on intermolecular contacts.

PubMed

Boehm, M K; Corper, A L; Wan, T; Sohi, M K; Sutton, B J; Thornton, J D; Keep, P A; Chester, K A; Begent, R H; Perkins, S J

2000-03-01

MFE-23 is the first single-chain Fv antibody molecule to be used in patients and is used to target colorectal cancer through its high affinity for carcinoembryonic antigen (CEA), a cell-surface member of the immunoglobulin superfamily. MFE-23 contains an N-terminal variable heavy-chain domain joined by a (Gly(4)Ser)(3) linker to a variable light-chain (V(L)) domain (kappa chain) with an 11-residue C-terminal Myc-tag. Its crystal structure was determined at 2.4 A resolution by molecular replacement with an R(cryst) of 19.0%. Five of the six antigen-binding loops, L1, L2, L3, H1 and H2, conformed to known canonical structures. The sixth loop, H3, displayed a unique structure, with a beta-hairpin loop and a bifurcated apex characterized by a buried Thr residue. In the crystal lattice, two MFE-23 molecules were associated back-to-back in a manner not seen before. The antigen-binding site displayed a large acidic region located mainly within the H2 loop and a large hydrophobic region within the H3 loop. Even though this structure is unliganded within the crystal, there is an unusually large region of contact between the H1, H2 and H3 loops and the beta-sheet of the V(L) domain of an adjacent molecule (strands DEBA) as a result of intermolecular packing. These interactions exhibited remarkably high surface and electrostatic complementarity. Of seven MFE-23 residues predicted to make contact with antigen, five participated in these lattice contacts, and this model for antigen binding is consistent with previously reported site-specific mutagenesis of MFE-23 and its effect on CEA binding.

Structure of a fucose-branched chondroitin sulfate from sea cucumber. Evidence for the presence of 3-O-sulfo-beta-D-glucuronosyl residues.

PubMed

Vieira, R P; Mulloy, B; Mourão, P A

1991-07-25

The structure of a unique focose-branched chondroitin sulfate isolated from the body wall of a sea cucumber was examined in detail. This glycosaminoglycan contains side chain disaccharide units of sulfated fucopyranosyl units linked to approximately one-half of the glucuronic acid moieties through the O-3 position of the acid. The intact polysaccharide is totally resistant to chondroitinase degradation, whereas, after defucosylation, it is partially degraded by the enzyme. However, only after an additional step of desulfation, the chondroitin from sea cucumber is almost totally degraded by chondroitinase AC or ABC. This result, together with the methylation and NMR studies of the native and chemically modified polysaccharide, suggest that besides the fucose branches, the sea cucumber chondroitin sulfate contains sulfate esters at position O-3 of the beta-D-glucuronic acid units. Furthermore, the proteoglycan from the sea cucumber chondroitin sulfate is recognized by anti-Leu-7 monoclonal antibody, which specifically recognizes 3-sulfoglucuronic acid residues. In analogy with the fucose branched units, the 3-O-sulfo-beta-D-glucuronosyl residues are resistant to chondroitinase degradation. Regarding the position of the glycosidic linkage and site of sulfation in the fucose branches, our results suggest high heterogeneity. Tentatively, it is possible to suggest the preponderance of disaccharide units formed by 3,4-di-O-sulfo-alpha-L-fucopyranosyl units glycosidically linked through position 1----2 to 4-O-sulfo-alpha-L-fucopyranose. Finally, the presence of unusual 4/6-disulfated disaccharide units, together with the common 6-sulfated and non-sulfated units, was detected in the chondroitin sulfate core of this polysaccharide.

Conceptually new deltanoids (vitamin D analogs) inhibit multistage skin tumorigenesis.

PubMed

Kensler, T W; Dolan, P M; Gange, S J; Lee, J K; Wang, Q; Posner, G H

2000-07-01

Development of vitamin D analogs (deltanoids) as chemopreventive agents requires separation of desirable antiproliferative and pro-differentiating activities from the undesirable calcemic activity also found in the hormone calcitriol (1 alpha, 25-dihydroxyvitamin D(3)). Therefore, several conceptually new deltanoids were synthesized with modifications to the 1alpha- and/or 25-hydroxyl groups, positions traditionally considered essential for stimulating biological responses. In this study, 1 beta-hydroxymethyl-3-epi-25-hydroxyvitamin D(3), a non-calcemic CH(2) homolog of the natural hormone with antiproliferative activity in vitro, was ineffective as an inhibitor of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced induction of ornithine decarboxylase activity in mouse epidermis. However, a hybrid analog incorporating not only the calcemia-ablating 1 beta-hydroxymethyl alteration, but potentiating C,D ring 16-unsaturation and side chain 24,24-fluorination and 26, 27-homologation was found to be as effective as calcitriol. Several non-calcemic 24- or 25-t-butyl sulfones, some containing side chain fluorination but all lacking the 25-hydroxyl group, were also shown to be active in this assay. Three sulfones and the 1 beta-hydroxymethyl hybrid were evaluated as inhibitors of multistage carcinogenesis in mouse skin. Female CD-1 mice were initiated with a single dose of 7,12-dimethylbenz[a]anthracene and then promoted twice weekly for 20 weeks with TPA. Deltanoids were applied topically 30 min before TPA. Unlike calcitriol, none of the atypical deltanoids affected body weight gain in these animals. Minimal effects on urinary calcium excretion were observed following chronic treatment with these analogs. All deltanoids inhibited the incidence and multiplicity of papilloma formation, with the hybrid analog showing the greatest efficacy. With this deltanoid, tumor incidence was significantly reduced by 28% and tumor multiplicity by 63%. These results, coupled with the rich chemical diversity available in side chain sulfur-containing deltanoids, particularly when combined with A ring modifications such as 1 beta-hydroxylalkyl groups, provide important new advances in the fundamental understanding of chemical structure-biological activity relationships as well as more potent and safe vitamin D analogs for cancer chemoprevention and other medicinal uses.

Removal of Long-Lived Radon Daughters by Electropolishing Thin Layers of Stainless Steel

NASA Astrophysics Data System (ADS)

White, James; Schnee, Richard; Bunker, Raymond; Bowles, Michael; Cushman, Priscilla; Epland, Matthew; Pepin, Mark; Guiseppe, Vince

2012-10-01

Long-lived alpha and beta emitters in the Radon decay chain on detector surfaces may be limiting background in many experiments attempting to detect dark matter or neutrinoless double beta decay. To screen detector surfaces for this radioactive contamination, a low-radiation, multi-wire proportional chamber (the BetaCage) is under construction. Removal of Pb-210 implanted on its 25-micron stainless steel wires without causing significant variation in the diameter of the wires is critical to the BetaCage's ultimate sensitivity. An apparatus to perform electropolishing trials to remove roughly a micron of material has been assembled. These trials have shown promising results. Stainless steel square samples implanted with Pb-210 have shown counts with a reduction factor greater than 10 after electropolishing according to gamma assay. Furthermore, alpha counting has produced similar results, with a reduction factor greater than 100. Lastly, the diameters of wires after electropolishing have remained sufficiently uniform, with reduction in thickness consistent with expectations.

[Association of beta 3-adrenergic receptor gene with obesity in patients with type 2 diabetes mellitus].

PubMed

Chen, Y; Xu, Y; Zhou, L

2001-09-01

To investigate the association between the mutation of beta 3-adrenergoc receptor gene and obesity in patients with type 2 diabetes. Body mass, waist-hip ratio, blood pressure and blood lipids were measured in 154 type 2 diabetic patients. Polymerase chain reaction and the restriction fragment length polymorphism analysis were used to determine the wild, heterozygous and homozygous forms of beta 3-adrenergoc receptor gene. The frequency of the Trp64Arg mutation was 42.5% and the frequency of Arg64 allele was 22.6%. The mutation frequency of the genetic types was significantly different between the obese and non-obese type 2 diabetes mellitus patients. The body mass, systolic blood pressure, diastolic blood pressure, HDL-cholesterol were significantly different, when those with Trp64Arg heterozygous were compared with those with Trp64 homozygous. The genetic mutation of beta 3-adrenegoc receptor in patients with type 2 diabetes is probably related to obesity.

Relaxation spectra and dipolar correlations for flexible polymers with bulky side groups

DOE Office of Scientific and Technical Information (OSTI.GOV)

Diaz-Calleja, R.; Riande, E.; Roman, J.S.

1992-08-06

This paper discusses how relaxation spectra and dipolar correlations for flexible polymers with bulky side groups (PBPA chains) suggest that intermolecular correlations are not very important in this polymer and that {alpha}, {beta}, and {gamma} absorptions exist. TSDC techniques reveal that the {gamma} peak has a smaller activation energy than the {beta}, and the coupling scheme is used to interpret the complex dielectric and mechanical {alpha} relaxations. The anomalous temperature dependence of the glass-rubber relaxation is discussed in terms of the bulkiness of the side group. 23 refs., 8 figs., 3 tabs.

NMR structure and dynamics of the engineered fluorescein-binding lipocalin FluA reveal rigidification of beta-barrel and variable loops upon enthalpy-driven ligand binding.

PubMed

Mills, Jeffrey L; Liu, Gaohua; Skerra, Arne; Szyperski, Thomas

2009-08-11

The NMR structure of the 21 kDa lipocalin FluA, which was previously obtained by combinatorial design, elucidates a reshaped binding site specific for the dye fluorescein resulting from 21 side chain replacements with respect to the parental lipocalin, the naturally occurring bilin-binding protein (BBP). As expected, FluA exhibits the lipocalin fold of BBP, comprising eight antiparallel beta-strands forming a beta-barrel with an alpha-helix attached to its side. Comparison of the NMR structure of free FluA with the X-ray structures of BBP.biliverdin IX(gamma) and FluA.fluorescein complexes revealed significant conformational changes in the binding pocket, which is formed by four loops at the open end of the beta-barrel as well as adjoining beta-strand segments. An "induced fit" became apparent for the side chain conformations of Arg 88 and Phe 99, which contact the bound fluorescein in the complex and undergo concerted rearrangement upon ligand binding. Moreover, slower internal motional modes of the polypeptide backbone were identified by measuring transverse (15)N backbone spin relaxation times in the rotating frame for free FluA and also for the FluA.fluorescein complex. A reduction in the level of such motions was detected upon complex formation, indicating rigidification of the protein structure and loss of conformational entropy. This hypothesis was confirmed by isothermal titration calorimetry, showing that ligand binding is enthalpy-driven, thus overcompensating for the negative entropy associated with both ligand binding per se and rigidification of the protein. Our investigation of the solution structure and dynamics as well as thermodynamics of lipocalin-ligand interaction not only provides insight into the general mechanism of small molecule accommodation in the deep and narrow cavity of this abundant class of proteins but also supports the future design of corresponding binding proteins with novel specificities, so-called "anticalins".

THE SPATIO-TEMPORAL EVOLUTION OF SOLAR FLARES OBSERVED WITH AIA/SDO: FRACTAL DIFFUSION, SUB-DIFFUSION, OR LOGISTIC GROWTH?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aschwanden, Markus J., E-mail: aschwanden@lmsal.com

2012-09-20

We explore the spatio-temporal evolution of solar flares by fitting a radial expansion model r(t) that consists of an exponentially growing acceleration phase, followed by a deceleration phase that is parameterized by the generalized diffusion function r(t){proportional_to}{kappa}(t - t{sub 1}){sup {beta}/2}, which includes the logistic growth limit ({beta} = 0), sub-diffusion ({beta} = 0-1), classical diffusion ({beta} = 1), super-diffusion ({beta} = 1-2), and the linear expansion limit ({beta} = 2). We analyze all M- and X-class flares observed with Geostationary Operational Environmental Satellite and Atmospheric Imaging Assembly/Solar Dynamics Observatory (SDO) during the first two years of the SDO mission,more » amounting to 155 events. We find that most flares operate in the sub-diffusive regime ({beta} = 0.53 {+-} 0.27), which we interpret in terms of anisotropic chain reactions of intermittent magnetic reconnection episodes in a low plasma-{beta} corona. We find a mean propagation speed of v = 15 {+-} 12 km s{sup -1}, with maximum speeds of v{sub max} = 80 {+-} 85 km s{sup -1} per flare, which is substantially slower than the sonic speeds expected for thermal diffusion of flare plasmas. The diffusive characteristics established here (for the first time for solar flares) is consistent with the fractal-diffusive self-organized criticality model, which predicted diffusive transport merely based on cellular automaton simulations.« less

Synthetic alleles at position 121 define a functional domain of human interleukin-1 beta.

PubMed

Ambrosetti, D C; Palla, E; Mirtella, A; Galeotti, C; Solito, E; Navarra, P; Parente, L; Melli, M

1996-06-01

The non-conservative substitution of the tyrosine residue at position 121 of human interleukin-1 beta (IL-1 beta) generates protein mutants showing strong reduction of the capacity to induce (a) prostaglandin E2 (PGE2) release from fibroblasts and smooth muscle cells, (b) murine T-cells proliferation and (c) activation of interleukin-6 (IL-6) gene expression. It is generally accepted that these functions are mediated by the type-I interleukin-1 receptor (IL-1RI). However, the mutant proteins maintain the binding affinity to the types-I and II IL-1 receptors, which is the same as the control IL-1 beta, suggesting that this amino acid substitution does not alter the structure of the molecule, except locally. Thus we have identified a new functional site of IL-1 beta different from the known receptor binding region, responsible for fundamental IL-1 beta functions. Moreover, we show that the same mutants maintain at least two hypothalamic functions, that is, the in vitro short-term PGE2 release from rat hypothalamus and the induction of fever in rabbits. This result suggests that there is yet another site of the molecule responsible for the hypothalamic functions, implying that multiple active sites on the IL-1 beta molecule, possibly binding to more than one receptor chain, trigger different signals.

Calcium binding to an elastic portion of connectin/titin filaments.

PubMed

Tatsumi, R; Maeda, K; Hattori, A; Takahashi, K

2001-01-01

Alpha-connectin/titin-1 exists as an elastic filament that links a thick filament with the Z-disk, keeping thick filaments centered within the sarcomere during force generation. We have shown that the connectin filament has an affinity for calcium ions and its binding site(s) is restricted to the beta-connectin/titin-2 portion. We now report the localization and the characterization of calcium-binding sites on beta-connectin. Purified beta-connectin was digested by trypsin into 1700- and 400-kDa fragments. which were then subjected to fluorescence calcium-binding assays. The 400-kDa fragment possesses calcium-binding activity; the binding constant was 1.0 x 10(7) M(-1) and the molar ratio of bound calcium ions to the 400-kDa fragment reached a maximum of 12 at a free calcium ion concentration of approximately 1.0 microM. Antibodies against the 400-kDa fragment formed a sharp dense stripe at the boundary of the A and the I bands, indicating that the calcium-binding domain constitutes the N-terminal region of beta-connectin, that is, the elastic portion of connectin filaments. Furthermore, we estimated the N-terminal location of beta-connectin of various origins (n = 26). Myofibrils were treated with a solution containing 0.1 mM CaCl2 and 70 microM leupeptin to split connectin filaments into beta-connectin and a subfragment, and chain weights of these polypeptides were estimated according to their mobility in 2% polyacrylamide slab gels. The subfragment exhibited a similar chain weight of 1200+/-33 kDa (mean+/-SD), while alpha- and beta-connectins were variable in size according to their origin. These results suggest that the apparent length of the 1200-kDa subfragment portion is almost constant in all instances, about 0.34 microm at the slack condition, therefore that the C-terminus of the 1200-kDa subfragment, that is, the N-terminus of the calcium-binding domain, is at the N2 line region of parent filaments in situ. Because the secondary structure of the 400-kDa fragment was changed by the binding of calcium ions, connectin filaments could be expected to alter their elasticity during the contraction-relaxation cycle of skeletal muscle.

Use of eluted peptide sequence data to identify the binding characteristics of peptides to the insulin-dependent diabetes susceptibility allele HLA-DQ8 (DQ 3.2).

PubMed

Godkin, A; Friede, T; Davenport, M; Stevanovic, S; Willis, A; Jewell, D; Hill, A; Rammensee, H G

1997-06-01

HLA-DQ8 (A1*0301, B1*0302) and -DQ2 (A1*0501, B1*0201) are both associated with diseases such as insulin-dependent diabetes mellitus and coeliac disease. We used the technique of pool sequencing to look at the requirements of peptides binding to HLA-DQ8, and combined these data with naturally sequenced ligands and in vitro binding assays to describe a novel motif for HLA-DQ8. The motif, which has the same basic format as many HLA-DR molecules, consists of four or five anchor regions, in the positions from the N-terminus of the binding core of n, n + 3, n + 5/6 and n + 8, i.e. P1, P4, P6/7 and P9. P1 and P9 require negative or polar residues, with mainly aliphatic residues at P4 and P6/7. The features of the HLA-DQ8 motif were then compared to a pool sequence of peptides eluted from HLA-DQ2. A consensus motif for the binding of a common peptide which may be involved in disease pathogenesis is described. Neither of the disease-associated alleles HLA-DQ2 and -DQ8 have Asp at position 57 of the beta-chain. This Asp, if present, may form a salt bridge with an Arg at position 79 of the alpha-chain and so alter the binding specificity of P9. HLA-DQ2 and -DQ8 both appear to prefer negatively charged amino acids at P9. In contrast, HLA-DQ7 (A1*0301, B1*0301), which is not associated with diabetes, has Asp at beta 57, allowing positively charged amino acids at P9. This analysis of the sequence features of DQ-binding peptides suggests molecular characteristics which may be useful to predict epitopes involved in disease pathogenesis.

Crystal structures of the class D beta-lactamase OXA-13 in the native form and in complex with meropenem.

PubMed

Pernot, L; Frénois, F; Rybkine, T; L'Hermite, G; Petrella, S; Delettré, J; Jarlier, V; Collatz, E; Sougakoff, W

2001-07-20

The therapeutic problems posed by class D beta-lactamases, a family of serine enzymes that hydrolyse beta-lactam antibiotics following an acylation-deacylation mechanism, are increased by the very low level of sensitivity of these enzymes to beta-lactamase inhibitors. To gain structural and mechanistic insights to aid the design of new inhibitors, we have determined the crystal structure of OXA-13 from Pseudomonas aeruginosa in the apo form and in complex with the carbapenem meropenem. The native form consisted of a dimer displaying an overall organisation similar to that found in the closely related enzyme OXA-10. In the acyl-enzyme complex, the positioning of the antibiotic appeared to be ensured mainly by (i) the covalent acyl bond and (ii) a strong salt-bridge involving the carboxylate moiety of the drug. Comparison of the structures of OXA-13 in the apo form and in complex with meropenem revealed an unsuspected flexibility in the region of the essential serine 115 residue, with possible consequences for the catalytic properties of the enzyme. In the apo form, the Ser115 side-chain is oriented outside the active site, whereas the general base Lys70 adopts a conformation that seems to be incompatible with the activation of the catalytic water molecule required for the deacylation step. In the OXA-13:meropenem complex, a 3.5 A movement of the backbone of the 114-116 loop towards the side-chain of Lys70 was observed, which seems to be driven by a displacement of the neighbouring 91-104 loop and which results in the repositioning of the side-chain hydroxyl group of Ser115 toward the catalytic centre. Concomitantly, the side-chain of Lys70 is forced to curve in the direction of the deacylating water molecule, which is then strongly bound and activated by this residue. However, a distance of ca 5 A separates the catalytic water molecule from the acyl carbonyl group of meropenem, a structural feature that accounts for the inhibition of OXA-13 by this drug. Finally, the low level of penicillinase activity revealed by the kinetic analysis of OXA-13 could be related to the specific presence in position 73 of a serine residue located close to the general base Lys70, which results in a decrease of the number of hydrogen-bonding interactions stabilising the catalytic water molecule. Copyright 2001 Academic Press.

Increasing protein stability by improving beta-turns.

PubMed

Fu, Hailong; Grimsley, Gerald R; Razvi, Abbas; Scholtz, J Martin; Pace, C Nick

2009-11-15

Our goal was to gain a better understanding of how protein stability can be increased by improving beta-turns. We studied 22 beta-turns in nine proteins with 66-370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some beta-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein, Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase alpha-subunit, and Maltose binding protein. Of the 15 single proline mutations, 11 increased stability (Average = 0.8 +/- 0.3; Range = 0.3-1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. On the basis of this and our previous work, we conclude that proteins can generally be stabilized by replacing nonproline residues with proline residues at the i + 1 position of Type I and II beta-turns and at the i position in Type II beta-turns. Other turn positions can sometimes be used if the phi angle is near -60 degrees for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in beta-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in beta-turns that could be replaced by Gly to increase protein stability. Improving beta-turns by substituting Pro residues is a generally useful way of increasing protein stability. 2009 Wiley-Liss, Inc.

IL-1 beta and IL-6 in mouse parotid acinar cells: characterization of synthesis, storage, and release.

PubMed

Tanda, N; Ohyama, H; Yamakawa, M; Ericsson, M; Tsuji, T; McBride, J; Elovic, A; Wong, D T; Login, G R

1998-01-01

Synthesis, storage, and secretion of the proinflammatory cytokine interleukin-1 beta (IL-1 beta) and the anti-inflammatory cytokine IL-6 have not been established in normal exocrine gland secretory cells. Parotid glands and isolated acinar cells prepared from BALB/c mice were homogenized for RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR). IL-1 beta and IL-6 enzyme-linked immunosorbent assays (ELISAs) were done on supernatants prepared from mouse parotid acinar cell (MPAC) preparations unstimulated or stimulated between 0 and 10 min with 10(-5) M norepinephrine at 37 degrees C. MPACs were fixed in paraformaldehyde, frozen sectioned for light and electron microscopy, and labeled with antibodies to IL-1 beta and IL-6. Mouse specific riboprobes to IL-1 and IL-6 were used for in situ hybridization. RT-PCR yielded the expected IL-1 (336-bp) and IL-6 (614-bp) mRNA products. By ELISA, stimulated MPACs showed a significant increase in IL-1 beta (P < 0.03) and IL-6 (P < 0.01) release into supernatants by 10 min that paralleled the time course of amylase release. In situ hybridization showed the presence of transcripts for IL-1 and IL-6 in glandular epithelial cells. Gold-labeled IL-1 beta and IL-6 were significantly higher (P < 0.01) in granules than in the nucleus and cytoplasm. This study shows that MPACs synthesize IL-1 beta and IL-6 and release these cytokines from their granules after alpha- and beta-adrenergic stimulation.

Transforming growth factor-{beta} inhibits CCAAT/enhancer-binding protein expression and PPAR{gamma} activity in unloaded bone marrow stromal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahdjoudj, S.; Kaabeche, K.; Holy, X.

2005-02-01

The molecular mechanisms regulating the adipogenic differentiation of bone marrow stromal cells in vivo remain largely unknown. In this study, we investigated the regulatory effects of transforming growth factor beta-2 (TGF-{beta}2) on transcription factors involved in adipogenic differentiation induced by hind limb suspension in rat bone marrow stromal cells in vivo. Time course real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis of gene expression showed that skeletal unloading progressively increases the expression of CCAAT/enhancer-binding protein (C/EBP){alpha} and C/EBP{beta} {alpha} at 5 days in bone marrow stromal cells resulting in increased peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}2) transcripts at 7 days. TGF-{beta}2more » administration in unloaded rats corrected the rise in C/EBP{alpha} and C/EBP{beta} transcripts induced by unloading in bone marrow stromal cells. This resulted in inhibition of PPAR{gamma}2 expression that was associated with increased Runx2 expression. Additionally, the inhibition of C/EBP{alpha} and C/EBP{beta} expression by TGF-{beta}2 was associated with increased PPAR{gamma} serine phosphorylation in bone marrow stromal cells, a mechanism that inhibits PPAR{gamma} transactivating activity. The sequential inhibitory effect of TGF-{beta}2 on C/EBP{alpha}, C/EBP{beta}, and PPAR{gamma}2 resulted in reduced LPL expression and abolition of bone marrow stromal cell adipogenic differentiation, which contributed to prevent bone loss induced by skeletal unloading. We conclude that TGF-{beta}2 inhibits the excessive adipogenic differentiation of bone marrow stromal cells induced by skeletal unloading by inhibiting C/EBP{alpha}, C/EBP{beta}, and PPAR{gamma} expression and activity, which provides a sequential mechanism by which TGF-{beta}2 regulates adipogenic differentiation of bone marrow stromal cells in vivo.« less

Expression of beta 3-adrenoceptor mRNA in rat tissues.

PubMed

Evans, B A; Papaioannou, M; Bonazzi, V R; Summers, R J

1996-01-01

1. This study examines the expression of beta 3-adrenoceptor messenger RNA (beta 3-AR mRNA) in rat tissues to allow comparison with atypical beta-adrenoceptors determined by functional and radioligand binding techniques. 2. A reverse transcription/polymerase chain reaction protocol has been developed for determining the relative amounts of beta 3-AR mRNA in rat tissues. 3. Measurement of adipsin and uncoupling protein (UCP) mRNA was used to examine all tissues for the presence of white and brown adipose tissue which may contribute beta 3-AR mRNA. 4. The beta 3-AR mRNA is expressed at high levels in brown and white adipose tissue, stomach fundus, the longitudinal/circular smooth muscle of both colon and ileum, and colon submucosa. There was substantial expression of adipsin in colon submucosa and moderate expression in fundus, suggesting that in these regions at least some of the beta 3-AR signal may be contributed by fat. Pylorus and colon mucosa showed moderate levels of beta 3-AR mRNA with lower levels of adipsin. Ileum mucosa and submucosa showed low but readily detectable levels of beta 3-AR. 5. Expression of adipsin in rat skeletal muscles coupled to very low levels of beta 3-AR mRNA indicates that the observed beta 3-AR may be due to the presence of intrinsic fat. beta 3-AR mRNA was virtually undetectable in heart, lung and liver. These results raise the possibility that the atypical beta-AR demonstrated by functional and/or binding studies in muscle and in heart is not the beta 3-AR. 6. By use of two different sets of primers for amplification of beta 3-AR cDNA, no evidence was found for differential splicing of the mRNA in any of the tissues examined. 7. The detection of beta 3-AR mRNA in the gut mucosa and submucosa suggests that in addition to its established roles in lipolysis, thermogenesis and regulation of gut motility beta 3-AR may subserve other functions in the gastrointestinal tract. The absence of beta 3-AR mRNA in rat heart or its presence with adipsin in skeletal muscle suggests that atypical beta-adrenoceptor responses in heart and skeletal muscle are unlikely to be mediated by beta 3-AR.

Effect of polymorphic variants of GH, Pit-1, and beta-LG genes on milk production of Holstein cows.

PubMed

Heidari, M; Azari, M A; Hasani, S; Khanahmadi, A; Zerehdaran, S

2012-04-01

Effect of polymorphic variants of growth hormone (GH), beta-lactoglobulin (beta-LG), and Pit-1 genes on milk yield was analyzed in a Holstein herd. Genotypes of the cows for these genes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Allele frequencies were 0.884 and 0.116 for L and V variants of GH, 0.170 and 0.830 for A and B variants of Pit-1, and 0.529 and 0.471 for A and B variants of beta-LG, respectively. GLM procedure of SAS software was used to test the effects of these genes on milk yield. Results indicated significant effects of these genes on milk yield (P < 0.05). Cows with LL genotype of GH produced more milk than cows with LVgenotype (P < 0.05). Also, for Pit-1 gene, animals with AB genotype produced more milk than BB genotype (P < 0.05). In the case of beta-LG gene, milk yield of animals with AA genotype was more than BB genotype (P < 0.01). Therefore, it might be concluded that homozygote genotypes of GH (LL) and beta-LG (AA) were superior compared to heterozygote genotypes, whereas, the heterozygote genotype of Pit-1 gene (AB) was desirable.

[Effects of lipopolysaccharides extracted from Porphyromonas endodontalis on the expression of IL-1beta mRNA and IL-6 mRNA in osteoblasts].

PubMed

Yang, Di; Li, Ren; Qiu, Li-Hong; Li, Chen

2009-04-01

To quantify the IL-1 beta mRNA and IL-6 mRNA expression induced by lipopolysaccharides (LPS)extracted from Porphyromonas endodontalis(P.e) in osteoblasts, and to relate P.e-LPS to bone absorption pathogenesis in lesions of chronical apical periodontitis. MG63 was treated with different concentrations of P.e-LPS(0-50 microg/mL) for different hours(0-24h). The expression of IL-1 beta mRNA and IL-6 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR).Statistical analysis was performed using one- way ANOVA and Dunnett t test with SPSS11.0 software package. The level of IL-1 beta mRNA and IL-6 mRNA increased significantly after treatment with P.e-LPS at more than 5 microg/mL (P<0.01)and for more than 1 hour (P<0.01), which indicated that P.e-LPS induced osteoblasts to express IL-1 beta mRNA and IL-6 mRNA in dose and time dependent manners. P.e-LPS may promote bone resorption in lesions of chronical apical periodontitis by inducing IL-1 beta mRNA and IL-6 mRNA expression in osteoblasts.

Molecular cloning and characterization of two mouse peroxisome proliferator-activated receptor alpha (PPARalpha)-regulated peroxisomal acyl-CoA thioesterases.

PubMed

Westin, Maria A K; Alexson, Stefan E H; Hunt, Mary C

2004-05-21

Peroxisomes are organelles that function in the beta-oxidation of long- and very long-chain acyl-CoAs, bile acid-CoA intermediates, prostaglandins, leukotrienes, thromboxanes, dicarboxylic fatty acids, pristanic acid, and xenobiotic carboxylic acids. The very long- and long-chain acyl-CoAs are mainly chain-shortened and then transported to mitochondria for further metabolism. We have now identified and characterized two peroxisomal acyl-CoA thioesterases, named PTE-Ia and PTE-Ic, that hydrolyze acyl-CoAs to the free fatty acid and coenzyme A. PTE-Ia and PTE-Ic show 82% sequence identity at the amino acid level, and a putative peroxisomal type 1 targeting signal of -AKL was identified at the carboxyl-terminal end of both proteins. Localization experiments using green fluorescent fusion protein showed PTE-Ia and PTE-Ic to be localized in peroxisomes. Despite their high level of sequence identity, we show that PTE-Ia is mainly active on long-chain acyl-CoAs, whereas PTE-Ic is mainly active on medium-chain acyl-CoAs. Lack of regulation of enzyme activity by free CoASH suggests that PTE-Ia and PTE-Ic regulate intraperoxisomal levels of acyl-CoA, and they may have a function in termination of beta-oxidation of fatty acids of different chain lengths. Tissue expression studies revealed that PTE-Ia is highly expressed in kidney, whereas PTE-Ic is most highly expressed in spleen, brain, testis, and proximal and distal intestine. Both PTE-Ia and PTE-Ic were highly up-regulated in mouse liver by treatment with the peroxisome proliferator WY-14,643 and by fasting in a peroxisome proliferator-activated receptor alpha-dependent manner. These data show that PTE-Ia and PTE-Ic have different functions based on different substrate specificities and tissue expression.

Continental rifting and the origin of Beta Regio, Venus

NASA Technical Reports Server (NTRS)

Mcgill, G. E.; Steenstrup, S. J.; Barton, C.; Ford, P. G.

1981-01-01

Topographic maps based on Pioneer Venus altimetry suggest that Beta Regio, an elevated feature centered at 27 deg N, 282 deg E, is analogous to domes associated with continental rift systems on earth. This interpretation is consistent with the commonly quoted analogy between the East African rift system and the topography of the region from Beta Regio southward to Phoebe Regio. If Beta Regio is a dome, major structural uplift of the crust of Venus is implied, suggesting a more dynamic upper mantle than would be the case if Beta Regio were simply a large volcanic construct.

Deciphering the Biophysical Effects of Oxidizing Sulfur-Containing Amino Acids in Interferon-beta-1a using MS and HDX-MS

NASA Astrophysics Data System (ADS)

Houde, Damian J.; Bou-Assaf, George M.; Berkowitz, Steven A.

2017-05-01

Introduction of a chemical change to one or more amino acids in a protein's polypeptide chain can result in various effects on its higher-order structure (HOS) and biophysical behavior (or properties). These effects range from no detectable change to significant structural or conformational alteration that can greatly affect the protein's biophysical properties and its resulting biological function. The ability to reliably detect the absence or presence of such changes is essential to understanding the structure-function relationship in a protein and in the successful commercial development of protein-based drugs (biopharmaceuticals). In this paper, we focus our attention on the latter by specifically elucidating the impact of oxidation on the HOS, structural dynamics, and biophysical properties of interferon beta-1a (IFNβ-1a). Oxidation is a common biochemical modification that occurs in many biopharmaceuticals, specifically in two naturally-occurring sulfur-containing amino acids, methionine and cysteine. To carry out this work, we used combinations of hydrogen peroxide and pH to differentially oxidize IFNβ-1a (to focus on only methionine oxidation versus methionine and cysteine oxidation). We then employed several analytical and biophysical techniques to acquire information about the differential impact of these two oxidation scenarios on IFNβ-1a. In particular, the use of MS-based techniques, especially HDX-MS, play a dominant role in revealing the differential effects.

Identification of suitable reference genes in bone marrow stromal cells from osteoarthritic donors.

PubMed

Schildberg, Theresa; Rauh, Juliane; Bretschneider, Henriette; Stiehler, Maik

2013-11-01

Bone marrow stromal cells (BMSCs) are key cellular components for musculoskeletal tissue engineering strategies. Furthermore, recent data suggest that BMSCs are involved in the development of Osteoarthritis (OA) being a frequently occurring degenerative joint disease. Reliable reference genes for the molecular evaluation of BMSCs derived from donors exhibiting OA as a primary co-morbidity have not been reported on yet. Hence, the aim of the study was to identify reference genes suitable for comparative gene expression analyses using OA-BMSCs. Passage 1 bone marrow derived BMSCs were isolated from n=13 patients with advanced stage idiopathic hip osteoarthritis and n=15 age-matched healthy donors. The expression of 31 putative reference genes was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using a commercially available TaqMan(®) assay. Calculating the coefficient of variation (CV), mRNA expression stability was determined and afterwards validated using geNorm and NormFinder algorithms. Importin 8 (IPO8), TATA box binding protein (TBP), and cancer susceptibility candidate 3 (CASC3) were identified as the most stable reference genes. Notably, commonly used reference genes, e.g. beta-actin (ACTB) and beta-2-microglobulin (B2M) were among the most unstable genes. For normalization of gene expression data of OA-BMSCs the combined use of IPO8, TBP, and CASC3 gene is recommended. © 2013.

MetroBeta: Beta Spectrometry with Metallic Magnetic Calorimeters in the Framework of the European Program of Ionizing Radiation Metrology

NASA Astrophysics Data System (ADS)

Loidl, M.; Beyer, J.; Bockhorn, L.; Enss, C.; Györi, D.; Kempf, S.; Kossert, K.; Mariam, R.; Nähle, O.; Paulsen, M.; Rodrigues, M.; Schmidt, M.

2018-05-01

MetroBeta is a European project aiming at the improvement of the knowledge of the shapes of beta spectra, both in terms of theoretical calculations and measurements. It is part of a common European program of ionizing radiation metrology. Metallic magnetic calorimeters (MMCs) with the beta emitter embedded in the absorber have in the past proven to be among the best beta spectrometers, in particular for low-energy beta transitions. Within this project, new designs of MMCs optimized for five different beta energy ranges were developed. A new detector module with thermal decoupling of MMC and SQUID chips was designed. An important aspect of the research and development concerns the source/absorber preparation techniques. Four beta spectra with maximum energies ranging from 76 to 709 keV will be measured. Improved theoretical calculation methods and complementary measurement techniques complete the project.

Heterogeneity in acute undifferentiated leukemia.

PubMed

LeMaistre, A; Childs, C C; Hirsch-Ginsberg, C; Reuben, J; Cork, A; Trujillo, J M; Andersson, B; McCredie, K B; Freireich, E; Stass, S A

1988-01-01

From January 1985 to May 1987, we studied 256 adults with newly diagnosed acute leukemia. Acute undifferentiated leukemia (AUL) was diagnosed in 12 of the 256 (4.6%) cases when lineage could not be delineated by light microscopy and light cytochemistry. To further characterize the blasts, immunophenotyping, ultrastructural myeloperoxidase (UMPO), and ultrastructural platelet peroxidase parameters were examined in 10, 11, and 6 of the 12 cases, respectively. Five cases demonstrated UMPO and were reclassified as acute myeloblastic leukemia (AML). Of the six UMPO-negative cases, three had a myeloid and one had a mixed immunophenotype. One UMPO-negative patient with a myeloid immunophenotype was probed for the immunoglobulin heavy chain gene (JH) and the beta chain of the T-cell receptor gene (Tcr beta) with no evidence of rearrangement. Six cases were treated with standard acute lymphoblastic leukemia (ALL) chemotherapy and failed to achieve complete remission (CR). Various AML chemotherapeutic regimens produced CR in only 3 of the 12 cases. One case was treated with gamma interferon and the other 2 with high-dose Ara-C. Our findings indicate a myeloid lineage can be detected by UMPO (5/12) in some cases of AUL. A germline configuration with JH and Tcr beta in one case as well as a myeloid immunophenotype in 3 UMPO-negative cases raises the possibility that myeloid lineage commitment may occur in the absence of myeloid peroxidase (MPO) cytochemical positivity.

Molecular basis of maple syrup urine disease: Novel mutations at the E1[alpha] locus that impair E1([alpha][sub 2][beta][sub 2]) assembly or decrease steady-state E1[alpha] mRNA levels of branched-chain [alpha]-keto acid dehydrogenase complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chuang, J.L.; Fisher, C.R.; Chuang, D.T.

1994-08-01

The authors report the occurrence of three novel mutations in the E1[alpha] (BCKDHA) locus of the branched-chain [alpha]-keto acid dehydrogenase (BCKAD) complex that cause maple syrup urine disease (MSUD). An 8-bp deletion in exon 7 is present in one allele of a compound-heterozygous patient (GM-649). A single C nucleotide insertion in exon 2 occurs in one allele of an intermediate-MSUD patient (Lo). The second allele of patient Lo carries an A-to-G transition in exon 9 of the E1[alpha] gene. This missense mutation changes Tyr-368 to Cys (Y368C) in the E1[alpha] subunit. Both the 8-bp deletion and the single C insertionmore » generate a downstream nonsense codon. Both mutations appear to be associated with a low abundance of the mutant E1[alpha] mRNA, as determined by allele-specific oligonucleotide probing. Transfection studies strongly suggest that the Y368C substitution in the E1[alpha] subunit impairs its proper assembly with the normal E1[beta]. Unassembled as well as misassembled E1[alpha] and E1[beta] subunits are degraded in the cell. 32 refs., 8 figs.« less

Construction and measurements of a vacuum-swing-adsorption radon-mitigation system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schnee, R. W.; Bunker, R.; Ghulam, G.

Long-lived alpha and beta emitters in the {sup 222}Rn decay chain on (and near) detector surfaces may be the limiting background in many experiments attempting to detect dark matter or neutrinoless double beta decay, and in screening detectors. In order to reduce backgrounds from radon-daughter plate-out onto the wires of the Beta Cage during its assembly, an ultra-low-radon cleanroom is being commissioned at Syracuse University using a vacuum-swing-adsorption radon-mitigation system. The radon filter shows ∼20× reduction at its output, from 7.47±0.56 to 0.37±0.12 Bq/m{sup 3}, and the cleanroom radon activity meets project requirements, with a lowest achieved value consistent withmore » that of the filter, and levels consistently < 2 Bq/m{sup 3}.« less

Hb Potomac (101 Glu replaced by Asp): speculations on placental oxygen transport in carriers of high-affinity hemoglobins.

PubMed

Charache, S; Jacobson, R; Brimhall, B; Murphy, E A; Hathaway, P; Winslow, R; Jones, R; Rath, C; Simkovich, J

1978-02-01

Blood from a woman with unexplained erythrocytosis had increased oxygen affinity, but no abnormality could be detected by electrophoresis or chromatography of her hemolysate. Separation of the tryptic peptides of her beta chains disclosed two half-sized peaks in the regions of beta T-11. The faster of these was abnormal, with the structure beta 101 Glu replaced by Asp. The new hemoglobin was called "Potomac." Three of the proband's four surviving siblings and both of her children were carriers. Differences in the ratio of carrier: normal children born to male of female carriers of 23 other high-affinity hemoglobins were not significant. The high proportion of carriers in this kindred was probably due to chance alone, and not because high maternal oxygen affinity interfered with oxygen transport to fetuses with normal hemoglobin.

Hemoglobin J-Korat in Thais.

PubMed

Blackwell, R Q; Blackwell, B N; Huang, J T; Chien, L C; Samaharn, A; Thephusdin, C; Borvornsin, C

1965-12-17

Hemoglobin J(Korat), a "fast" hemoglobin with an anomaly in its beta chain different from the anomalies previously reported, was the major hemoglobin component in the blood of nine subjects among 1923 Thais from northeastern Thailand. After hemoglobin E, J(Korat) is the second most frequent of the anomalous hemoglobins among Thais.

Acylcarnitines activate pro-inflammatory signaling pathways

USDA-ARS?s Scientific Manuscript database

Incomplete beta-oxidation of fatty acids in mitochondria is a feature of insulin resistance and type 2 diabetes mellitus (T2DM) and the resulting metabolic by-products, medium- and long-chain acylcarnitines are shown to be elevated. In preliminary studies, mixed isomers of C12- or C14-carnitine act...

Comparative Genomics of Regulation of Fatty Acid and Branched-chain Amino Acid Utilization in Proteobacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kazakov, Alexey E.; Rodionov, Dmitry A.; Arkin, Adam Paul

2008-10-31

Bacteria can use branched-chain amino acids (ILV, i.e. isoleucine, leucine, valine) and fatty acids (FA) as sole carbon and energy sources convering ILV into acetyl-CoA, propanoyl-CoA and propionyl-CoA, respectively. In this work, we used the comparative genomic approach to identify candidate transcriptional factors and DNA motifs that control ILV and FA utilization pathways in proteobacteria. The metabolic regulons were characterized based on the identification and comparison of candidate transcription factor binding sites in groups of phylogenetically related genomes. The reconstructed ILV/FA regulatory network demonstrates considerable variability and involves six transcriptional factors from the MerR, TetR and GntR families binding tomore » eleven distinct DNA motifs. The ILV degradation genes in gamma- and beta-proteobacteria are mainly regulated by anovel regulator from the MerR family (e.g., LiuR in Pseudomonas aeruginosa) (40 species), in addition, the TetR-type regulator LiuQ was identified in some beta-proteobacteria (8 species). Besides the core set of ILV utilization genes, the LiuR regulon in some lineages is expanded to include genes from other metabolic pathways, such as the glyoxylate shunt and glutamate synthase in the Shewanella species. The FA degradation genes are controlled by four regulators including FadR in gamma-proteobacteria (34 species), PsrA in gamma- and beta-proteobacteria (45 species), FadP in beta-proteobacteria (14 species), and LiuR orthologs in alpha-proteobacteria (22 species). The remarkable variability of the regulatory systems associated with the FA degradation pathway is discussed from the functional and evolutionary points of view.« less

Role of water molecules in structure and energetics of Pseudomonas aeruginosa lectin I interacting with disaccharides.

PubMed

Nurisso, Alessandra; Blanchard, Bertrand; Audfray, Aymeric; Rydner, Lina; Oscarson, Stefan; Varrot, Annabelle; Imberty, Anne

2010-06-25

Calcium-dependent lectin I from Pseudomonas aeruginosa (PA-IL) binds specifically to oligosaccharides presenting an alpha-galactose residue at their nonreducing end, such as the disaccharides alphaGal1-2betaGalOMe, alphaGal1-3betaGalOMe, and alphaGal1-4betaGalOMe. This provides a unique model for studying the effect of the glycosidic linkage of the ligands on structure and thermodynamics of the complexes by means of experimental and theoretical tools. The structural features of PA-IL in complex with the three disaccharides were established by docking and molecular dynamics simulations and compared with those observed in available crystal structures, including PA-IL.alphaGal1-2betaGalOMe complex, which was solved at 2.4 A resolution and reported herein. The role of a structural bridge water molecule in the binding site of PA-IL was also elucidated through molecular dynamics simulations and free energy calculations. This water molecule establishes three very stable hydrogen bonds with O6 of nonreducing galactose, oxygen from Pro-51 main chain, and nitrogen from Gln-53 main chain of the lectin binding site. Binding free energies for PA-IL in complex with the three disaccharides were investigated, and the results were compared with the experimental data determined by titration microcalorimetry. When the bridge water molecule was included in the free energy calculations, the simulations predicted the correct binding affinity trends with the 1-2-linked disaccharide presenting three times stronger affinity ligand than the other two. These results highlight the role of the water molecule in the binding site of PA-IL and indicate that it should be taken into account when designing glycoderivatives active against P. aeruginosa adhesion.

Increased flow of fatty acids toward beta-oxidation in developing seeds of Arabidopsis deficient in diacylglycerol acyltransferase activity or synthesizing medium-chain-length fatty acids.

PubMed

Poirier, Y; Ventre, G; Caldelari, D

1999-12-01

Synthesis of polyhydroxyalkanoates (PHAs) from intermediates of fatty acid beta-oxidation was used as a tool to study fatty acid degradation in developing seeds of Arabidopsis. Transgenic plants expressing a peroxisomal PHA synthase under the control of a napin promoter accumulated PHA in developing seeds to a final level of 0. 06 mg g(-1) dry weight. In plants co-expressing a plastidial acyl-acyl carrier protein thioesterase from Cuphea lanceolata and a peroxisomal PHA synthase, approximately 18-fold more PHA accumulated in developing seeds. The proportion of 3-hydroxydecanoic acid monomer in the PHA was strongly increased, indicating a large flow of capric acid toward beta-oxidation. Furthermore, expression of the peroxisomal PHA synthase in an Arabidopsis mutant deficient in the enzyme diacylglycerol acyltransferase resulted in a 10-fold increase in PHA accumulation in developing seeds. These data indicate that plants can respond to the inadequate incorporation of fatty acids into triacylglycerides by recycling the fatty acids via beta-oxidation and that a considerable flow toward beta-oxidation can occur even in a plant tissue primarily devoted to the accumulation of storage lipids.

Potential therapeutic impact of omega-3 long chain-polyunsaturated fatty acids on inflammation markers in Duchenne muscular dystrophy: A double-blind, controlled randomized trial.

PubMed

Rodríguez-Cruz, Maricela; Cruz-Guzmán, Oriana Del Rocío; Almeida-Becerril, Tomás; Solís-Serna, Alan Donovan; Atilano-Miguel, Salvador; Sánchez-González, Juan Raúl; Barbosa-Cortés, Lourdes; Ruíz-Cruz, Eugenia Dolores; Huicochea, Juan Carlos; Cárdenas-Conejo, Alan; Escobar-Cedillo, Rosa Elena; Yam-Ontiveros, Carlos Alberto; Ricárdez-Marcial, Edgar F

2017-09-23

Duchenne Muscular Dystrophy (DMD) is the most frequent dystrophy in childhood generated by a deficiency in dystrophin. DMD is a neuromuscular disease and its clinical course comprises chronic inflammation and gradual muscle weakness. Supplementation of omega-3 long chain-Polyunsaturated Fatty Acids (ω-3 long chain-PUFA) reduces inflammatory markers in various disorders. The goal of this research was to analyze the influence of ω-3 long chain-PUFA intake on gene expression and blood inflammatory markers in boys with DMD. In a placebo-controlled, double. Blind, randomized trial, boys with DMD (n = 36) consumed 2.9 g/day of ω-3 long chain-PUFA or sunflower oil as control, in capsules, for a period of 6 months. Blood was analyzed at baseline and at months 1, 2, 3, and 6 of supplementation for expression of inflammatory markers in leukocytes and serum. There was high adherence to capsule intake (control: 95.3% ± 7.2%, and ω-3 long chain-PUFA: 97.4% ± 3.7% at month 6). Enrichment of EicosaPentaenoic Acid (EPA) and DocosaHexaenoic Acid (DHA) in erythrocytes increased significantly in patients supplemented with ω-3 long chain-PUFA compared with the placebo group during the 6 months of supplementation. Messenger RNA (mRNA) of the Nuclear Factor kappa beta (NF-κB) and its target genes InterLeukin 1 beta (IL-1β) and IL-6 was downregulated significantly (p < 0.05) in leukocytes from DMD boys supplemented with ω-3 long chain-PUFA for 6 months, compared to the placebo group. Omega-3 long chain-PUFA intake decreased the serum IL-1β (-59.5%; p = 0.011) and IL-6 (-54.8%; p = 0.041), and increased the serum IL-10 (99.9%, p < 0.005), in relation to those with placebo treatment. Supplementation with ω-3 long chain-PUFA 2.9 g/day is well-tolerated, has a beneficial reductive effect on proinflammatory markers, and increases an anti-inflammatory marker, indicating that ω-3 long chain-PUFA could have a potential therapeutic impact on chronic inflammation in DMD. This research is registered at clinicaltrials.gov (NCT018264229). Copyright © 2017 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

A STUDY OF BACILLUS PYOGENES

PubMed Central

Brown, J. Howard; Orcutt, Marion L.

1920-01-01

Bacillus pyogenes is probably quite common in this country, as it is known to be in Europe. A careful study of twelve strains from cattle and one from a hog has disclosed the following characteristics which have not been reported or have been in dispute. Bacillus pyogenes is Gram-positive and pleomorphic, producing forms ranging from short chains of streptococcoid elements to branching filaments. It is hemolytic, producing the beta type of hemolysis in blood agar. It is not hemoglobinophilic, though its growth is greatly favored by some higher protein material such as egg albumin, serum, or blood. It ferments xylose in addition to the substances previously reported. The coagulation of milk by Bacillus pyogenes is primarily an enzyme coagulation and the subsequent digestion of the curd takes place in an acid medium. The intravenous injection of rabbits was invariably fatal. The lesions most commonly developed were those of the bones. Paralysis was frequently produced, and in each case was caused by lesions in the vertebrae exerting pressure against the ventral columns of the spinal cord. Muscle abscesses were also frequently produced. The authors regard the organism as belonging to the Corynebacteria rather than to the influenza group. PMID:19868442

Prenatal programming of metabolic syndrome in the common marmoset is associated with increased expression of 11beta-hydroxysteroid dehydrogenase type 1.

PubMed

Nyirenda, Moffat J; Carter, Roderick; Tang, Justin I; de Vries, Annick; Schlumbohm, Christina; Hillier, Stephen G; Streit, Frank; Oellerich, Michael; Armstrong, Victor W; Fuchs, Eberhard; Seckl, Jonathan R

2009-12-01

Recent studies in humans and animal models of obesity have shown increased adipose tissue activity of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which amplifies local tissue glucocorticoid concentrations. The reasons for this 11beta-HSD1 dysregulation are unknown. Here, we tested whether 11beta-HSD1 expression, like the metabolic syndrome, is "programmed" by prenatal environmental events in a nonhuman primate model, the common marmoset monkey. We used a "fetal programming" paradigm where brief antenatal exposure to glucocorticoids leads to the metabolic syndrome in the offspring. Pregnant marmosets were given the synthetic glucocorticoid dexamethasone orally for 1 week in either early or late gestation, or they were given vehicle. Tissue 11beta-HSD1 and glucocorticoid receptor mRNA expression were examined in the offspring at 4 and 24 months of age. Prenatal dexamethasone administration, selectively during late gestation, resulted in early and persistent elevations in 11beta-HSD1 mRNA expression and activity in the liver, pancreas, and subcutaneous-but not visceral-fat. The increase in 11beta-HSD1 occurred before animals developed obesity or overt features of the metabolic syndrome. In contrast to rodents, in utero dexamethasone exposure did not alter glucocorticoid receptor expression in metabolic tissues in marmosets. These data suggest that long-term upregulation of 11beta-HSD1 in metabolically active tissues may follow prenatal "stress" hormone exposure and indicates a novel mechanism for fetal origins of adult obesity and the metabolic syndrome.

Expression of the leukemia-associated CBF{beta}/SMMHC chimeric gene causes transformation of 3T3 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hajra, A.; Liu, P.; Collins, E.S.

1994-09-01

A pericentric inversion of chromosome 16 (inv(16)(p13;q22)) is consistently seen in acute myeloid leukemia of the M4Eo subtype. This inversion fuses almost the entire coding region of the gene encoding of the {beta} subunit of the heterodimeric transcription factor CBF/PEBP2 to the region of the MYH11 gene encoding the rod domain for the smooth muscle myosin heavy chain (SMMHC). To investigate the biological properties of the CBF{beta}/SMMHC fusion protein, we have generated 3T3 cell lines that stably express the CBF{beta}/SMMHC chimeric cDNA or the normal, nonchimeric CBF{beta} and SMMHC cDNAs. 3T3 cells expressing CBF{beta}/SMMHC acquire a transformed phenotype, as indicatedmore » by altered cell morphology, formation of foci, and growth in soft agar. Cells constitutively overexpressing the normal CBF{beta} cDNA or the rod region of SMMHC remain nontransformed. Western blot analysis using antibodies to CBF{beta} and the SMMHC rod demonstrates that stably transfected cells express the appropriate chimeric or normal protein. Electrophoretic mobility shift assays reveal that cells transformed by the chimeric cDNA do not have a CBF-DNA complex of the expected mobility, but instead contain a large complex with CBF DNA-binding activity that fails to migrate out of the gel wells. In order to define the regions of CBF{beta}/SMMHC necessary for 3T3 transformation, we have stably transfected cells with mutant CBF{beta}/SMMHC cDNAs containing various deletions of the coding region. Analysis of these cell lines indicates that the transformation property of CBF{beta}/SMMHC requires regions of CBF{beta} known to be necessary for association with the DNA-binding CBF{alpha} subunit, and also requires an intact SMMHC carboxyl terminus, which is necessary for formation of the coiled coil domain of the myosin rod.« less

Effects of beta-blockers and nicardipine on oxotremorine-induced tremor in common marmosets.

PubMed

Mitsuda, M; Nomoto, M; Iwata, S

1999-10-01

Effects of beta-blockers (propranolol, arotinolol and nipradilol) and a Ca2+ channel blocker (nicardipine) on oxotremorine-induced tremor were studied in common marmosets. Generalized tremor was elicited by an intraperitoneal administration of 0.25 mg/kg oxotremorine. Intensity of the tremor was classified into 7 degrees, and it was evaluated every 10 min. The total intensity of oxotremorine-induced tremor for each drug was expressed as "points", which were the sum of tremor intensity scores evaluated every 10 min up to 190 min following the administration of oxotremorine. Beta-blockers significantly suppressed the tremor. On the other hand, the Ca2+ channel blocker exacerbated the tremor.

Classical sickle beta-globin haplotypes exhibit a high degree of long-range haplotype similarity in African and Afro-Caribbean populations.

PubMed

Hanchard, Neil; Elzein, Abier; Trafford, Clare; Rockett, Kirk; Pinder, Margaret; Jallow, Muminatou; Harding, Rosalind; Kwiatkowski, Dominic; McKenzie, Colin

2007-08-10

The sickle (betas) mutation in the beta-globin gene (HBB) occurs on five "classical" betas haplotype backgrounds in ethnic groups of African ancestry. Strong selection in favour of the betas allele - a consequence of protection from severe malarial infection afforded by heterozygotes - has been associated with a high degree of extended haplotype similarity. The relationship between classical betas haplotypes and long-range haplotype similarity may have both anthropological and clinical implications, but to date has not been explored. Here we evaluate the haplotype similarity of classical betas haplotypes over 400 kb in population samples from Jamaica, The Gambia, and among the Yoruba of Nigeria (Hapmap YRI). The most common betas sub-haplotype among Jamaicans and the Yoruba was the Benin haplotype, while in The Gambia the Senegal haplotype was observed most commonly. Both subtypes exhibited a high degree of long-range haplotype similarity extending across approximately 400 kb in all three populations. This long-range similarity was significantly greater than that seen for other haplotypes sampled in these populations (P < 0.001), and was independent of marker choice and marker density. Among the Yoruba, Benin haplotypes were highly conserved, with very strong linkage disequilibrium (LD) extending a megabase across the betas mutation. Two different classical betas haplotypes, sampled from different populations, exhibit comparable and extensive long-range haplotype similarity and strong LD. This LD extends across the adjacent recombination hotspot, and is discernable at distances in excess of 400 kb. Although the multi-centric geographic distribution of betas haplotypes indicates strong subdivision among early Holocene sub-Saharan populations, we find no evidence that selective pressures imposed by falciparum malaria varied in intensity or timing between these subpopulations. Our observations also suggest that cis-acting loci, which may influence outcomes in sickle cell disease, could lie considerable distances away from beta-globin.

BetaTPred: prediction of beta-TURNS in a protein using statistical algorithms.

PubMed

Kaur, Harpreet; Raghava, G P S

2002-03-01

beta-turns play an important role from a structural and functional point of view. beta-turns are the most common type of non-repetitive structures in proteins and comprise on average, 25% of the residues. In the past numerous methods have been developed to predict beta-turns in a protein. Most of these prediction methods are based on statistical approaches. In order to utilize the full potential of these methods, there is a need to develop a web server. This paper describes a web server called BetaTPred, developed for predicting beta-TURNS in a protein from its amino acid sequence. BetaTPred allows the user to predict turns in a protein using existing statistical algorithms. It also allows to predict different types of beta-TURNS e.g. type I, I', II, II', VI, VIII and non-specific. This server assists the users in predicting the consensus beta-TURNS in a protein. The server is accessible from http://imtech.res.in/raghava/betatpred/

Diacyltransferase Activity and Chain Length Specificity of Mycobacterium tuberculosis PapA5 in the Synthesis of Alkyl β-Diol Lipids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Touchette, Megan H.; Bommineni, Gopal R.; Delle Bovi, Richard J.

Although classified as Gram-positive bacteria, Corynebacterineae possess an asymmetric outer membrane that imparts structural and thereby physiological similarity to more distantly related Gram-negative bacteria. Like lipopolysaccharide in Gram-negative bacteria, lipids in the outer membrane of Corynebacterineae have been associated with the virulence of pathogenic species such as Mycobacterium tuberculosis (Mtb). For example, Mtb strains that lack long, branched-chain alkyl esters known as dimycocerosates (DIMs) are significantly attenuated in model infections. The resultant interest in the biosynthetic pathway of these unusual virulence factors has led to the elucidation of many of the steps leading to the final esterification of the alkylmore » beta-diol, phthiocerol, with branched-chain fatty acids know as mycocerosates. PapA5 is an acyltransferase implicated in these final reactions. We here show that PapA5 is indeed the terminal enzyme in DIM biosynthesis by demonstrating its dual esterification activity and chain-length preference using synthetic alkyl beta-diol substrate analogues. Applying these analogues to a series of PapA5 mutants, we also revise a model for the substrate binding within PapA5. Finally, we demonstrate that the Mtb Ser/Thr kinase PknB modifies PapA5 on three Thr residues, including two (T196, T198) located on an unresolved loop. These results clarify the DIM biosynthetic pathway and suggest possible mechanisms by which DIM biosynthesis may be regulated by the post-translational modification of PapA5.« less

Processing of N-linked oligosaccharides from precursor- to mature-form herpes simplex virus type 1 glycoprotein gC.

PubMed

Serafini-Cessi, F; Dall'Olio, F; Pereira, L; Campadelli-Fiume, G

1984-09-01

Immature and mature forms of glycoprotein gC were purified by immunoadsorbent from herpes simplex virus type 1-infected BHK cells labeled with [3H]mannose for a 20-min pulse or for 11 h followed by a 3-h chase. The nature of N-asparagine-linked oligosaccharides carried by the immature form, pgC (molecular weight = 92,000), and the mature gC (molecular weight = 120,000) has been investigated. All pronase-digested glycopeptides of pgC were susceptible to endo-beta-N-acetylglucosaminidase H treatment; thus they have a high-mannose structure. Using thin-layer chromatography to separate endo-beta-N-acetylglucosaminidase H-cleaved oligosaccharides, polymannosyl chains of different sizes, ranging from Man9GlcNAc to Man5GlcNAc, were separated. The major components were Man8GlcNAc and Man7GlcNAc, suggesting that pgC labeled in a 20-min pulse represents the form of glycoprotein already routed to the Golgi apparatus. Analysis of glycopeptides of mature gC showed that the majority (95%) of N-linked glycans were converted to complex-type glycans. Ion-exchange chromatography and affinity chromatography on concanavalin A-Sepharose and leucoagglutinin-agarose revealed that diantennary and triantennary glycans predominated, whereas tetrantennary chains were not present. Parts of the di- and triantennary chains were not fully sialylated. The high heterogeneity of complex-type chains found in mature gC may be related to the high number of N-glycosylation sites of the glycoprotein as predicted by DNA sequencing studies (Frink et al., J. Virol. 45:634-647, 1983).

Cerebrospinal fluid and neuroimaging biomarker abnormalities suggest early neurological injury in a subset of individuals during primary HIV infection.

PubMed

Peluso, Michael J; Meyerhoff, Dieter J; Price, Richard W; Peterson, Julia; Lee, Evelyn; Young, Andrew C; Walter, Rudy; Fuchs, Dietmar; Brew, Bruce J; Cinque, Paola; Robertson, Kevin; Hagberg, Lars; Zetterberg, Henrik; Gisslén, Magnus; Spudich, Serena

2013-06-01

Cerebrospinal fluid (CSF) and neuroimaging abnormalities demonstrate neuronal injury during chronic AIDS, but data on these biomarkers during primary human immunodeficiency virus (HIV) infection is limited. We compared CSF concentrations of neurofilament light chain, t-tau, p-tau, amyloid precursor proteins, and amyloid-beta 42 in 92 subjects with primary HIV infection and 25 controls. We examined relationships with disease progression and neuroinflammation, neuropsychological testing, and proton-magnetic resonance spectroscopy (MRS)-based metabolites. Neurofilament light chain was elevated in primary HIV infection compared with controls (P = .0004) and correlated with CSF neopterin (r = 0.38; P = .0005), interferon gamma-induced protein 10 (r = 0.39; P = .002), white blood cells (r = 0.32; P = .004), protein (r = 0.59; P < .0001), and CSF/plasma albumin ratio (r = 0.60; P < .0001). Neurofilament light chain correlated with decreased N-acteylaspartate/creatine and glutamate/creatine in the anterior cingulate (r = -0.35, P = .02; r = -0.40, P = .009, respectively), frontal white matter (r = -0.43, P = .003; r = -0.30, P = .048, respectively), and parietal gray matter (r = -0.43, P = .003; r = -0.47, P = .001, respectively). Beta-amyloid was elevated in the primary infection group (P = .0005) and correlated with time infected (r = 0.34; P = .003). Neither marker correlated with neuropsychological abnormalities. T-tau and soluble amyloid precursor proteins did not differ between groups. Elevated neurofilament light chain and its correlation with MRS-based metabolites suggest early neuronal injury in a subset of participants with primary HIV infection through mechanisms involving central nervous system inflammation.

Analysis of betaS and betaA genes in a Mexican population with African roots.

PubMed

Magaña, María Teresa; Ongay, Zoyla; Tagle, Juan; Bentura, Gilberto; Cobián, José G; Perea, F Javier; Casas-Castañeda, Maricela; Sánchez-López, Yoaly J; Ibarra, Bertha

2002-01-01

To investigate the origin of the beta(A) and beta(S) genes in a Mexican population with African roots and a high frequency of hemoglobin S, we analyzed 467 individuals (288 unrelated) from different towns in the states of Guerrero and Oaxaca in the Costa Chica region. The frequency of the sickle-cell trait was 12.8%, which may represent a public health problem. The frequencies of the beta-haplotypes were determined from 350 nonrelated chromosomes (313 beta(A) and 37 beta(S)). We observed 15 different beta(A) haplotypes, the most common of which were haplotypes 1 (48.9%), 2 (13.4%), and 3 (13.4%). The calculation of pairwise distributions and Nei's genetic distance analysis using 32 worldwide populations showed that the beta(A) genes are more closely related to those of Mexican Mestizos and North Africans. Bantu and Benin haplotypes and haplotype 9 were related to the beta(S) genes, with frequencies of 78.8, 18.2, and 3.0%, respectively. Comparison of these haplotypes with 17 other populations revealed a high similitude with the population of the Central African Republic. These data suggest distinct origins for the beta(A) and beta(S) genes in Mexican individuals from the Costa Chica region.

Recombination and mutation of class II histocompatibility genes in wild mice.

PubMed

Wakeland, E K; Darby, B R

1983-12-01

We have compared the tryptic peptide fingerprints of the A alpha, A beta, E alpha, and E beta subunits encoded by four wild-derived H-2 complexes expressing A molecules closely related to Ak. The A molecules encoded by these Ak-related mice have A alpha and A beta subunits that differ from A alpha k and A beta k by less than 10% of their tryptic peptides. Comparisons among the four wild-derived A molecules suggested that these contemporary A alpha and A beta alleles arose by sequential mutational events from common ancestor A alpha and A beta alleles. These results suggest that A alpha and A beta may co-evolve as an A beta A alpha gene duplex in wild mice. Tryptic peptide fingerprint comparisons of the E beta gene linked to these Ak-related A beta A alpha gene duplexes indicate that two encode E beta d-like subunits, whereas another encodes an E beta s-like subunit. These results strongly suggest that the A beta A alpha duplex and E beta recombine in wild mouse populations. The significantly different evolutionary patterns exhibited by the class II genes encoding A vs E molecules are discussed.

Time-patterns of antibiotic exposure in poultry production--a Markov chains exploratory study of nature and consequences.

PubMed

Chauvin, C; Clement, C; Bruneau, M; Pommeret, D

2007-07-16

This article describes the use of Markov chains to explore the time-patterns of antimicrobial exposure in broiler poultry. The transition in antimicrobial exposure status (exposed/not exposed to an antimicrobial, with a distinction between exposures to the different antimicrobial classes) in extensive data collected in broiler chicken flocks from November 2003 onwards, was investigated. All Markov chains were first-order chains. Mortality rate, geographical location and slaughter semester were sources of heterogeneity between transition matrices. Transitions towards a 'no antimicrobial' exposure state were highly predominant, whatever the initial state. From a 'no antimicrobial' exposure state, the transition to beta-lactams was predominant among transitions to an antimicrobial exposure state. Transitions between antimicrobial classes were rare and variable. Switches between antimicrobial classes and repeats of a particular class were both observed. Application of Markov chains analysis to the database of the nation-wide antimicrobial resistance monitoring programme pointed out that transition probabilities between antimicrobial exposure states increased with the number of resistances in Escherichia coli strains.

Family disintegration: one fusarium verticillioides beta-lactamase at a time

USDA-ARS?s Scientific Manuscript database

Fusarium verticillioides is a mycotoxigenic fungus found commonly on maize, where it primarily exhibits asymptomatic endophytic growth. The F. verticillioides genome possesses approximately 30 regions that potentially encode beta-lactamase enzymatic domains. These enzymes are classically involved ...

Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.

PubMed

Tinkhauser, Gerd; Pogosyan, Alek; Tan, Huiling; Herz, Damian M; Kühn, Andrea A; Brown, Peter

2017-11-01

Exaggerated basal ganglia beta activity (13-35 Hz) is commonly found in patients with Parkinson's disease and can be suppressed by dopaminergic medication, with the degree of suppression being correlated with the improvement in motor symptoms. Importantly, beta activity is not continuously elevated, but fluctuates to give beta bursts. The percentage number of longer beta bursts in a given interval is positively correlated with clinical impairment in Parkinson's disease patients. Here we determine whether the characteristics of beta bursts are dependent on dopaminergic state. Local field potentials were recorded from the subthalamic nucleus of eight Parkinson's disease patients during temporary lead externalization during surgery for deep brain stimulation. The recordings took place with the patient quietly seated following overnight withdrawal of levodopa and after administration of levodopa. Beta bursts were defined by applying a common amplitude threshold and burst characteristics were compared between the two drug conditions. The amplitude of beta bursts, indicative of the degree of local neural synchronization, progressively increased with burst duration. Treatment with levodopa limited this evolution leading to a relative increase of shorter, lower amplitude bursts. Synchronization, however, was not limited to local neural populations during bursts, but also, when such bursts were cotemporaneous across the hemispheres, was evidenced by bilateral phase synchronization. The probability of beta bursts and the proportion of cotemporaneous bursts were reduced by levodopa. The percentage number of longer beta bursts in a given interval was positively related to motor impairment, while the opposite was true for the percentage number of short duration beta bursts. Importantly, the decrease in burst duration was also correlated with the motor improvement. In conclusion, we demonstrate that long duration beta bursts are associated with an increase in local and interhemispheric synchronization. This may compromise information coding capacity and thereby motor processing. Dopaminergic activity limits this uncontrolled beta synchronization by terminating long duration beta bursts, with positive consequences on network state and motor symptoms. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain.

Cooperative alpha-helix formation of beta-lactoglobulin induced by sodium n-alkyl sulfates.

PubMed

Chamani, J; Moosavi-Movahedi, A A; Rajabi, O; Gharanfoli, M; Momen-Heravi, M; Hakimelahi, G H; Neamati-Baghsiah, A; Varasteh, A R

2006-01-01

It is generally assumed that folding intermediates contain partially formed native-like secondary structures. However, if we consider the fact that the conformational stability of the intermediate state is simpler than that of the native state, it would be expected that the secondary structures in a folding intermediate would not necessarily be similar to those of the native state. beta-Lactoglobulin is a predominantly beta-sheet protein, although it has a markedly high intrinsic preference for alpha-helical structure. The formation of non-native alpha-helical intermediate of beta-lactoglobulin was induced by n-alkyl sulfates including sodium octyl sulfate, SOS; sodium decyl sulfate, SDeS; sodium dodecyl sulfate, SDS; and sodium tetradecyl sulfate, STS at special condition. The effect of n-alkyl sulfates on the structure of native beta-lactoglobulin at pH 2 was utilized to investigate the contribution of hydrophobic interactions to the stability of non-native alpha-helical intermediate. The addition of various concentrations of n-alkyl sulfates to the native state of beta-lactoglobulin (pH 2) appears to support the stabilized form of non-native alpha-helical intermediate at pH 2. The m values of the intermediate state of beta-lactoglobulin by SOS, SDeS, SDS and STS showed substantial variation. The enhancement of m values as the stability criterion of non-native alpha-helical intermediate state corresponded with increasing chain length of the cited n-alkyl sulfates. The present results suggest that the folding reaction of beta-lactoglobulin follows a non-hierarchical mechanism and hydrophobic interactions play important roles in stabilizing the non-native alpha-helical intermediate state.

Accumulation of nerve growth factor and its receptors in the uterus and dorsal root ganglia in a mouse model of adenomyosis.

PubMed

Li, Yan; Zhang, Shao-fen; Zou, Shi-en; Xia, Xian; Bao, Lei

2011-03-08

Adenomyosis is a common gynecological disease, which is accompanied by a series of immunological and neuroendocrinological changes. Nerve growth factor (NGF) plays a critical role in producing pain, neural plasticity, immunocyte aggregation and release of inflammatory factors. This study aimed to investigate the expression of NGF and its two receptors in uteri and dorsal root ganglia (DRG) in an adenomyosis mouse model, as well as their relationship with the severity of adenomyosis. Forty newborn ICR mice were randomly divided into the adenomyosis model group and control group (n = 20 in each group). Mice in the adenomyosis model group were orally dosed with 2.7 μmol/kg tamoxifen on days 2-5 after birth. Experiments were conducted to identify the expression of NGF- beta and its receptors, tyrosine kinase receptor (trkA) and p75 neurotrophin receptor (p75NTR), in the uterus and DRG in four age groups (90+/-5 d, 140+/-5 d, 190+/-5 d and 240+/-5 d; n = 5 mice in each group) by western bolt, immunochemistry and real time reverse transcription-polymerase chain reaction. Adenomyosis, which became more serious as age increased, was successfully induced in dosed ICR mice. NGF-beta, trkA and p75NTR protein levels in the uterus and trkA mRNA levels in DRG were higher in the older aged adenomyosis model group than those in controls (190+/-5 d and 240+/-5 d groups, P < 0.05). The expression of NGF-beta and its receptors in the uterus increased gradually as age increased for adenomyosis mice (190+/-5 d and 240+/-5 d, P < 0.05, compared with 90+/-5 d) but it showed little change in control mice. The mRNA level of trkA in DRG also increased as age increased in the adenomyosis model group (190+/-5 d and 240+/-5 d, P < 0.05, compared with 90+/-5 d) but was unchanged in controls. The mRNA level of p75NTR in DRG was not different between the adenomyosis and control groups and was stable from young to old mice. NGF- beta can be used as an indicator for the severity of adenomyosis. The gradually increasing level of NGF- beta and its receptors while the disease becomes more severe suggests an effect of NGF- beta on pathogenic mechanisms of adenomyosis.

Expression of estrogen receptors-alpha and -beta in the pregnant ovine uterine artery endothelial cells in vivo and in vitro.

PubMed

Liao, Wu Xiang; Magness, Ronald R; Chen, Dong-Bao

2005-03-01

Estrogen is recognized to be one of the driving forces in increases in uterine blood flow through both rapid and delayed actions via binding to its receptors, ER alpha and ER beta at the uterine artery (UA) wall, and especially in UA endothelium (UAE). However, information regarding estrogen receptor (ER) expression in UAE is limited. This study was designed to test whether ERs are expressed in UAE in vivo, and if they are, whether these receptors are maintained in cultured UA endothelial cells (UAECs) in vitro. By using immunohistochemical and Western blot analyses, we clearly demonstrated ER alpha and ER beta protein expression in pregnant (Days 120-130) sheep UA and UAE in vivo and as well as cultured UAECs in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) amplified both ER alpha and ER beta mRNAs in UA, UAE, and UAECs. Of interest, a truncated ER beta (ER beta2) variant due to a splicing deletion of exon 5 of the ER beta gene was detected in these cells. Quantitative RT-PCR analysis revealed that ER alpha mRNA levels are approximately 8-fold (P < 0.01) higher than that of ER beta in UAECs, indicating that ER alpha may play a more important role than ER beta in the UAEC responses to estrogen. Fluorescence immunolabeling analysis showed that ER alpha is present in both nuclei and plasma membranes in UAECs, and the latter is also colocalized with caveolin-1. The membrane and nuclear ER alpha presumably participate in rapid and delayed responses, respectively, to estrogen on UAE. Taken together, our data demonstrated that UAE is a direct target of estrogen actions and that the UAEC culture model we established is suitable for dissecting estrogen actions on UAE.

Pituitary resistance to thyroid hormone associated with a base mutation in the hormone-binding domain of the human 3,5,3'-triiodothyronine receptor-beta.

PubMed

Sasaki, S; Nakamura, H; Tagami, T; Miyoshi, Y; Nogimori, T; Mitsuma, T; Imura, H

1993-05-01

Point mutations in the human T3 receptor-beta (TR beta) gene causing single amino acid substitutions have been identified in several different kindreds with generalized resistance to thyroid hormone. Until now, no study has been reported on the TR gene in cases of pituitary resistance (PRTH). In the present study, we analyzed the TR beta gene in a 30-yr-old Japanese female with PRTH. She exhibited clinical features of hyperthyroidism, elevated serum thyroid hormone levels accompanied by inappropriately increased secretion of TSH, mildly elevated basal metabolic rate, and increased urinary excretion of hydroxyproline. No pituitary tumor was detected. DNA fragments of exons 3-8 of the genomic TR beta gene were generated by the polymerase chain reaction and analyzed by a single stranded conformation polymorphism method. Exon 7 of the patient's TR beta gene showed an abnormal band, suggesting the existence of mutation(s). By subcloning and sequencing the DNA, a point mutation was identified in one allele at nucleotide 1297 (C to T), which altered the 333rd amino acid, arginine, to tryptophan. Neither of her apparently normal parents had any mutations of the TR beta gene. In vitro translation products of the mutant TR beta gene showed remarkably decreased T3-binding activity (Ka, 2.1 x 10(8) M-1; normal TR beta Ka, 1.1 x 10(10) M-1). Since the molecular defect detected in a patient with PRTH is similar to that seen in subjects with generalized resistance to thyroid hormone, both types of the syndrome may represent a continuous spectrum of the same etiological defect with variable tissue resistance to thyroid hormone.

Molecular dynamics studies of protein folding and aggregation

NASA Astrophysics Data System (ADS)

Ding, Feng

This thesis applies molecular dynamics simulations and statistical mechanics to study: (i) protein folding; and (ii) protein aggregation. Most small proteins fold into their native states via a first-order-like phase transition with a major free energy barrier between the folded and unfolded states. A set of protein conformations corresponding to the free energy barrier, Delta G >> kBT, are the folding transition state ensemble (TSE). Due to their evasive nature, TSE conformations are hard to capture (probability ∝ exp(-DeltaG/k BT)) and characterize. A coarse-grained discrete molecular dynamics model with realistic steric constraints is constructed to reproduce the experimentally observed two-state folding thermodynamics. A kinetic approach is proposed to identify the folding TSE. A specific set of contacts, common to the TSE conformations, is identified as the folding nuclei which are necessary to be formed in order for the protein to fold. Interestingly, the amino acids at the site of the identified folding nuclei are highly conserved for homologous proteins sharing the same structures. Such conservation suggests that amino acids that are important for folding kinetics are under selective pressure to be preserved during the course of molecular evolution. In addition, studies of the conformations close to the transition states uncover the importance of topology in the construction of order parameter for protein folding transition. Misfolded proteins often form insoluble aggregates, amyloid fibrils, that deposit in the extracellular space and lead to a type of disease known as amyloidosis. Due to its insoluble and non-crystalline nature, the aggregation structure and, thus the aggregation mechanism, has yet to be uncovered. Discrete molecular dynamics studies reveal an aggregate structure with the same structural signatures as in experimental observations and show a nucleation aggregation scenario. The simulations also suggest a generic aggregation mechanism that globular proteins under a denaturing environment partially unfold and aggregate by forming stabilizing hydrogen bonds between the backbones of the partial folded substructures. Proteins or peptides rich in alpha-helices also aggregate into beta-rich amyloid fibrils. Upon aggregation, the protein or peptide undergoes a conformational transition from alpha-helices to beta-sheets. The transition of alpha-helix to beta-hairpin (two-stranded beta-sheet) is studied in an all-heavy-atom discrete molecular dynamics model of a polyalanine chain. An entropical driving scenario for the alpha-helix to beta-hairpin transition is discovered.

Tuning the conformational properties of the prion peptide.

PubMed

Ho, Chai-Chi; Lee, Lily Y-L; Huang, Kuo-Ting; Lin, Chun-Cheng; Ku, Mei-Yun; Yang, Chien-Chih; Chan, Sunney I; Hsu, Ruei-Lin; Chen, Rita P-Y

2009-07-01

Previously, we disclosed that O-linked glycosylation of Ser-132 or Ser-135 could dramatically change the amyloidogenic property of the hamster prion peptide (sequence 108-144). This peptide, which corresponds to the flexible loop and the first beta-strand in the structure of the prion protein, is a random coil when it is initially dissolved in buffer, but amyloid fibrils are formed with time. Thus, it offers a convenient model system to observe and compare how different chemical modifications and sequence mutations alter the amyloidogenic property of the peptide within a reasonable experimental time frame. In our earlier study, aside from uncovering a site-specificity of the glycosylation on the fibrillogenesis, different effects of alpha-GalNAc and beta-GlcNAc were observed. In this work, we explore further how different sugar configurations affect the conformational property of the polypeptide chain. We compare the effects of O-linked glycosylation by the common sugars alpha-GalNAc, beta-GlcNAc with their non-native analogs beta-GalNAc, alpha-GlcNAc in an effort to uncover the origin of the sugar-specificity on the fibril formation. We find that the anomeric configuration of the sugar is the most important factor affecting the fibrillogenesis. Sugars with the glycosidic bond in the alpha-configuration at Ser-135 have a dramatic inhibitory effect on the structural conversion of the glycosylated peptide. Because O-glycosylation of Ser-135 with alpha-linked sugars also promote the formation of three slowly converting conformations at the site of glycosylation, we surmise that the amyloidogenic property of the peptide is related to its conformational flexibility, and the proclivity of this region of the peptide to undergo the structural conversion from the random coil to form the beta-structure. Upon O-glycosylation with an alpha-linked sugar, this conversion is inhibited and the nucleation of fibril formation is largely retarded. Consistent with this scenario, Arg-136 is the residue most affected in the TOCSY NMR spectra of the glycosylated peptides, other than the serine site modified. In addition, when Arg-136 is substituted by Gly, a mutation that should provide higher structural flexibility in this part of the peptide, the amyloidogenic property of the peptide is greatly enhanced, and the inhibition effect of glycosylation is largely diminished. These results are consistent with Ser-135 and Arg-136 being part of the kink region involved in the structural conversion.

Inhibition of laminin alpha 1-chain expression leads to alteration of basement membrane assembly and cell differentiation

PubMed Central

1996-01-01

The expression of the constituent alpha 1 chain of laminin-1, a major component of basement membranes, is markedly regulated during development and differentiation. We have designed an antisense RNA strategy to analyze the direct involvement of the alpha 1 chain in laminin assembly, basement membrane formation, and cell differentiation. We report that the absence of alpha 1-chain expression, resulting from the stable transfection of the human colonic cancer Caco2 cells with an eukaryotic expression vector comprising a cDNA fragment of the alpha 1 chain inserted in an antisense orientation, led to (a) an incorrect secretion of the two other constituent chains of laminin-1, the beta 1/gamma 1 chains, (b) the lack of basement membrane assembly when Caco2-deficient cells were cultured on top of fibroblasts, assessed by the absence of collagen IV and nidogen deposition, and (c) changes in the structural polarity of cells accompanied by the inhibition of an apical digestive enzyme, sucrase-isomaltase. The results demonstrate that the alpha 1 chain is required for secretion of laminin-1 and for the assembly of basement membrane network. Furthermore, expression of the laminin alpha 1-chain gene may be a regulatory element in determining cell differentiation. PMID:8609173

Discovery of the beta-form crystal structure in electrospun nanofibers of bio-based poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] and its implication on properties

NASA Astrophysics Data System (ADS)

Gong, Liang

Bacterially produced poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] (PHBHx) is a new type of bioplastic which not only inherits the excellent biodegradability and biocompatibility of its parent homopolymer, polyhydroxybutyrate (PHB), but also overcomes PHB’s brittleness and stiffness with the incorporation of 3-hydroxyhexanoate (Hx) comonomer units with medium-chain-length (mcl) side chains. The tough and ductile PHBHx, with a much lower crystallinity and melting temperature, is well-suited for many practical applications. Efforts have been made to broaden the application range of PHBHx by introducing the beta-form crystalline structure, where the molecular chains adopt a planar zig-zag conformation. However, it is extremely difficult to produce this beta-form in PHBHx due to its much lower crystallinity and much more flexible molecular chains. In this study, we report an approach using the technique of electrospinning. The strain-induced metastable β-form crystalline structure was successfully introduced in PHBHx by collecting the macroscopically aligned electrospun PHBHx nanofibers across the air gap on a piece of aluminum foil and on the tapered edge of a high-speed rotary disk. The presence of the β-form crystal structure in electrospun fiber mats was confirmed by wide-angle X-ray diffraction (WAXD) and Fourier transform infrared spectroscopy (FTIR), with molecular orientation of the polymer chains along the fiber axis revealed by polarized FTIR. Selected area electron diffraction (SAED) and AFM-IR were utilized to investigate the morphological and structural details of individual PHBHx nanofibers. The results demonstrated a coexistence of the thermodynamically stable α-form crystalline structure, where molecular chains adopt a left-handed 21 helical conformation, and the β-form in single fibers. The molecular orientation level and the relative amounts of the two crystalline polymorphs were found to be highly dependent on fiber collection methods and fiber diameter. Moreover, the α and β-form were revealed to be spatially distributed as a core-shell structure consisting of an α-form-rich core and a β-form-rich shell, with the thickness of the shell remaining constant despite the variation of fiber diameter. According to these observations, a possible mechanism for the generation of the β-form was proposed. The effects of electrospinning parameters on the formation of the beta-form were systematically investigated. The results indicated that more β-crystals can be produced when 1) a higher fiber take-up is used, so that the polymer chains are further stretched before fiber solidification; 2) an optimal solution concentration is chosen, so that a balance between polymer chain deformation and relaxation is maintained throughout the whole electrospinning process; and 3) a more volatile solvent is used, so that more planar zig-zag chains are kinetically frozen in the fibers without being converted to the helical conformation as the fibers solidify. These experimental results indicate that the β-content in PHBHx nanofibers can be easily regulated by modifying the electrospinning conditions. Finally, the influence of the presence of the β-form on the piezoelectric response of the electrospun PHBHx nanofibers was studied. It was observed that the fibers containing the β-form exhibited an obvious piezoelectric response to the applied pressure, possibly due to the planar zig-zag conformation of the chains which gives rise to a significant dipole moment change when subjected to mechanical deformation. In addition, the sensitivity of the piezoelectric PHBHx nanofibers to mechanical pressure was measured to be 7.46 mV/kPa. These preliminary investigations indicate that the piezoelectric performance of PHBHx can be largely improved by increasing the concentration of the piezoelectric-active β-form crystalline structure. The piezoelectric PHBHx distinguishes itself from all the other piezoelectric polymers with its excellent biodegradability and biocompatibility, environmental-friendliness and most importantly, low manufacturing cost. It is a promising piezoelectric polymer which can be applied in advanced areas including portable/foldable electronic devices, artificial electronic skins and implantable sensors.

Identification of an active acidic residue in the catalytic site of beta-hexosaminidase.

PubMed

Tse, R; Vavougios, G; Hou, Y; Mahuran, D J

1996-06-11

Human beta-hexosaminidases A and B (EC 3.2.1.52) are dimeric lysosomal glycosidases composed of evolutionarily related alpha and/or beta subunits. Both isozymes hydrolyze terminal beta-linked GalNAc or GlcNAc residues from numerous artificial and natural substrates; however, in vivo GM2 ganglioside is a substrate for only the heterodimeric A isozyme. Thus, mutations in either gene encoding its alpha or beta subunits can result in GM2 ganglioside storage and Tay-Sachs or Sandhoff disease, respectively. All glycosyl hydrolases ae believed to have one or more acidic residues in their catalytic site. We demonstrate that incubation of hexosaminidase with a chemical modifier specific for carboxyl side chains produces a time-dependent loss of activity, and that this effect can be blocked by the inclusion of a strong competitive inhibitor in the reaction mix. We hypothesized that the catalytic acid residue(s) should be located in a region of overall homology and be invariant within the aligned deduced primary sequences of the human alpha and beta subunits, as well as hexosaminidases from other species, including bacteria. Such a region is encoded by exons 5-6 of the HEXA and HEXB genes. This region includes beta Arg211 (invariant in 15 sequences), which we have previously shown to be an active residue. This region also contains two invariant and one conserved acidic residues. A fourth acidic residue, Asp alpha 258, beta 290, in exon 7 was also investigated because of its association with the B1 variant of Tay-Sachs disease. Conservative substitutions were made at each candidate residue by in vitro mutagenesis of a beta cDNA, followed by cellular expression. Of these, only the beta Asp196Asn substitution decreased the kcat (350-910-fold) without any noticeable effect on the K(m). Mutagenesis of either beta Asp240 or beta Asp290 to Asn decreased kcat by 10- or 1.4-fold but also raised the K(m) of the enzyme 11- of 3-fold, respectively. The above results strongly suggest that beta Asp196 is a catalytic acid residue in beta-hexosaminidase.

Engineering ..beta..-Oxidation in Yarrowia lipolytica for Methyl Ketone Production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sanchez i Nogue, Violeta; Ramirez, Kelsey J; Singer, Christine

Medium- and long-chain methyl ketones are fatty acid-derived compounds that can be used as biofuel blending agents, flavors and fragrances. However, their large-scale production from sustainable feedstocks is currently limited due to the lack of robust microbial biocatalysts. The oleaginous yeast Yarrowia lipolytica is a promising biorefinery platform strain for the production of methyl ketones from renewable lignocellulosic biomass due to its natively high flux towards fatty acid biosynthesis. In this study, we report the metabolic engineering of Y. lipolytica to produce long- and very long-chain methyl ketones. Truncation of peroxisomal ..beta..-oxidation by chromosomal deletion of pot1 resulted in themore » biosynthesis of saturated, mono-, and diunsaturated methyl ketones in the C13-C23 range. Additional overexpression and peroxisomal targeting of a heterologous bacterial methyl ketone biosynthesis pathway yielded an initial titer of 151.5 mg/L of saturated methyl ketones. Dissolved oxygen concentrations in the cultures were found to substantially impact cell morphology and methyl ketone biosynthesis. Bioreactor cultivation under optimized conditions resulted in a titer of 314.8 mg/L of total methyl ketones, representing more than a 6000-fold increase over the parental strain. This work highlights the potential of Y. lipolytica to serve as chassis organism for the biosynthesis of acyl-thioester derived long- and very long-chain methyl ketones.« less

Characterization of human DHRS6, an orphan short chain dehydrogenase/reductase enzyme: a novel, cytosolic type 2 R-beta-hydroxybutyrate dehydrogenase.

PubMed

Guo, Kunde; Lukacik, Petra; Papagrigoriou, Evangelos; Meier, Marc; Lee, Wen Hwa; Adamski, Jerzy; Oppermann, Udo

2006-04-14

Human DHRS6 is a previously uncharacterized member of the short chain dehydrogenases/reductase family and displays significant homologies to bacterial hydroxybutyrate dehydrogenases. Substrate screening reveals sole NAD(+)-dependent conversion of (R)-hydroxybutyrate to acetoacetate with K(m) values of about 10 mm, consistent with plasma levels of circulating ketone bodies in situations of starvation or ketoacidosis. The structure of human DHRS6 was determined at a resolution of 1.8 A in complex with NAD(H) and reveals a tetrameric organization with a short chain dehydrogenases/reductase-typical folding pattern. A highly conserved triad of Arg residues ("triple R" motif consisting of Arg(144), Arg(188), and Arg(205)) was found to bind a sulfate molecule at the active site. Docking analysis of R-beta-hydroxybutyrate into the active site reveals an experimentally consistent model of substrate carboxylate binding and catalytically competent orientation. GFP reporter gene analysis reveals a cytosolic localization upon transfection into mammalian cells. These data establish DHRS6 as a novel, cytosolic type 2 (R)-hydroxybutyrate dehydrogenase, distinct from its well characterized mitochondrial type 1 counterpart. The properties determined for DHRS6 suggest a possible physiological role in cytosolic ketone body utilization, either as a secondary system for energy supply in starvation or to generate precursors for lipid and sterol synthesis.

mRNA Expression of Platelet-Derived Growth Factor Receptor-{beta} and C-KIT: Correlation With Pathologic Response to Cetuximab-Based Chemoradiotherapy in Patients With Rectal Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Erben, Philipp; Horisberger, Karoline; Muessle, Benjamin

2008-12-01

Purpose: Deviant expression of platelet-derived growth factor receptor-{beta} (PDGFR{beta}) and c-kit was shown in patients with colorectal cancer. In the present study, mRNA expression of PDGFR{beta} and c-kit in 33 patients with locally advanced rectal cancer undergoing preoperative chemoradiotherapy with cetuximab/capecitabine/irinotecan in correlation with the tumor regression rate was investigated. Methods and Materials: Pretherapeutic biopsy cores and tumor material from the resected specimens were collected in parallel with normal rectal mucosa. The expression levels of PDGFR{beta} and c-kit were measured by quantitative polymerase chain reaction. Tumors were classified as good responders (tumor regression grade [TRG], 2-3) or poor responders (TRG,more » 0-1). Results: The TRG evaluation of the resected specimen was TRG 0-1 in 11 and TRG 2-3 in 22. The median normalized ratios in the pretreatment mucosa vs. tumor biopsy cores was as follows: PDGFR{beta} ratio of 15.2 vs. 49.5 (p <0.0001) and c-kit ratio of 0.94 vs. 0.67 (p = 0.014). The same tendency was observed for the median PDGFR{beta} ratios after chemoradiotherapy completion: 34.2 vs. 170.0 (p <0.0001). The PDGFR{beta} and c-kit mRNA expression values in the pretreatment tumor biopsy cores were lower than the values in the resected specimens: PDGFR{beta} ratio 49.5 vs. 170.0 (p = 0.0002) and c-kit ratio 0.67 vs. 1.1 (p = 0.0003). Nevertheless, no correlation was seen between the pretherapeutic PDGFR{beta} and c-kit mRNA expression and the pathologic regression rate. Conclusion: Cetuximab-based chemoradiotherapy increased PDGFR{beta} levels even further compared with the pretreatment samples and deserves further investigation.« less

Increased expression of mineralocorticoid receptor and 11beta-hydroxysteroid dehydrogenase type 2 in human atria during atrial fibrillation.

PubMed

De-An, Pei; Li, Li; Zhi-Yun, Xu; Jin-Yu, Huang; Zheng-Ming, Xu; Min, Wang; Qiang, Yao; Shi-Eng, Huang

2010-01-01

Atrialfibrillation (AF) is associated with the activation of the renin-angiotensin-aldosterone system in the atria. It is not clear whether the expression of a mineralocorticoid receptor (MR), or 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2), conferring aldosterone specificity to the MR, in patients with AF is altered. Patients with AF may be associated with increased expression of MR and 11betaHSD2 in the atria. Atrial tissue samples of 25 patients with rheumatic heart valve disease undergoing a valve replacement operation were examined. A total of 13 patients had chronic persistent AF (>6 mo) and 12 patients had no history of AF. The MR and 11betaHSD2 expression were analyzed at the mRNA and protein level. The localization of MR and 11betaHSD2 in atrial tissue was performed using specific immunohistochemistry staining. The results of real-time quantitative polymerase chain reaction (PCR) showed that AF groups, in comparison with sinus rhythm, had a higher mRNA expression level of MR or 11betaHSD2 (all P < 0.01). Both the MR and 11betaHSD2 protein expression level in atrial tissue were also significantly increased in patients with AF compared with patients with sinus rhythm (P < 0.05 or P < 0.01). The immunohistochemical staining of MR or 11betaHSD2 demonstrated that MR and 11betaHSD2 predominately located in the cytoplasm of myocardial cells in the atrium and the intensity and density of immunostaining appeared to be increased in the atria of patients with AF compared to those without AF. Increasing expression of MR and 11betaHSD2 in the atria during AF is one of the molecular mechanisms for development of atrial interstitial fibrosis in patients with AF. These findings may have an important impact on the treatment of AF with aldosterone antagonists. Copyright 2010 Wiley Periodicals, Inc.

Structure and expression of the rat CYP3A1 gene: isolation of the gene (P450/6betaB) and characterization of the recombinant protein.

PubMed

Nagata, K; Ogino, M; Shimada, M; Miyata, M; Gonzalez, F J; Yamazoe, Y

1999-02-15

A P450 gene (P450/6betaB) of the CYP3A subfamily was isolated from a rat genomic library. Nucleotide sequencing of the exons revealed a high similarity with P450PCN1 cDNA (Gonzalez et al. (1985), J. Biol. Chem. 260, 7345-7441), but differed in 41 nucleotides, resulting in 11 changes and 2 deletions of amino acid residues. The P450/6betaB spanned about 30 kbp and consisted of 13 exons, and was in exon number and size identical with CYP3A2 gene except in the 6th exon, which was shorter than that of CYP3A2. 6beta-B mRNA, which may be transcribed from P450/6betaB, was detected on Northern blotting and by reverse transcription-polymerase chain reaction (RT-PCR). Profiles of the developmental change and induction by a treatment with several chemicals were very similar to those of P450PCN1 mRNA reported previously. P450PCN1 mRNA and gene, however, were not detected by PCR in rats. To determine whether P450/6betaB encodes an active protein, a cDNA was isolated and expressed. Expression of 6beta-B cDNA in COS-1 cells was carried out and revealed that the recombinant protein comigrated with purified P4506beta-4 previously identified as CYP3A1. The recombinant 6beta-B protein showed similar turnover rate and regioselectivity for testosterone with purified P4506beta-4 by the simultaneous addition of NADPH-cytochrome P450 reductase and cytochrome b5. These data suggest that P450/6betaB encodes an active P450 form corresponding to CYP3A1 and P450PCN1 reported previously does not exist in rats. Copyright 1999 Academic Press.

Electrocardiographic Presentation, Cardiac Arrhythmias, and Their Management in β-Thalassemia Major Patients.

PubMed

Russo, Vincenzo; Rago, Anna; Papa, Andrea Antonio; Nigro, Gerardo

2016-07-01

Beta-thalassemia major (β-TM) is a genetic hemoglobin disorder characterized by an absent synthesis of globin chains that are essential for hemoglobin formation, causing chronic hemolytic anemia. Clinical management of thalassemia major consists in regular long-life red blood cell transfusions and iron chelation therapy to remove iron introduced in excess with transfusions. Iron deposition in combination with inflammatory and immunogenic factors is involved in the pathophysiology of cardiac dysfunction in these patients. Heart failure and arrhythmias, caused by myocardial siderosis, are the most important life-limiting complications of iron overload in beta-thalassemia patients. Cardiac complications are responsible for 71% of global death in the beta-thalassemia major patients. The aim of this review was to describe the most frequent electrocardiographic abnormalities and arrhythmias observed in β-TM patients, analyzing their prognostic impact and current treatment strategies. © 2016 Wiley Periodicals, Inc.

Zinc(II) binds to the neuroprotective peptide humanin.

PubMed

Armas, Ambar; Sonois, Vanessa; Mothes, Emmanuelle; Mazarguil, Honoré; Faller, Peter

2006-10-01

The abnormal accumulation of the peptide amyloid-beta in the form of senile (or amyloid) plaques is one of the hallmarks of Alzheimer's disease (AD). Zinc ions have been implicated in AD and plaques formation. Recently, the peptide humanin has been discovered. Humanin showed neuroprotective activity against amyloid-beta insults. Here the question investigated is if humanin could interact directly with Zn(II). It is shown that Zn(II) and its substitutes Cd(II)/Co(II) bind to humanin via a thiolate bond from the side chain of the single cysteine at position 8. The low intensity of the d-d bands of Co(II)-humanin indicated an octahedral coordination geometry. Titration experiments suggest that Zn(II) binds to humanin with an apparent affinity in the low muM range. This apparent Zn-binding affinity is in the same order as for amyloid-beta and glutathione and could thus be of physiological relevance.

Structural and energetic consequences of mutations in a solvated hydrophobic cavity

NASA Technical Reports Server (NTRS)

Adamek, D. H.; Guerrero, L.; Blaber, M.; Caspar, D. L. D.

2005-01-01

The structural and energetic consequences of modifications to the hydrophobic cavity of interleukin 1-beta (IL-1beta) are described. Previous reports demonstrated that the entirely hydrophobic cavity of IL-1beta contains positionally disordered water. To gain a better understanding of the nature of this cavity and the water therein, a number of mutant proteins were constructed by site-directed mutagenesis, designed to result in altered hydrophobicity of the cavity. These mutations involve the replacement of specific phenylalanine residues, which circumscribe the cavity, with tyrosine, tryptophan, leucine and isoleucine. Using differential scanning calorimetry to determine the relative stabilities of the wild-type and mutant proteins, we found all of the mutants to be destabilizing. X-ray crystallography was used to identify the structural consequences of the mutations. No clear correlation between the hydrophobicities of the specific side-chains introduced and the resulting stabilities was found.

Polyhydroxyalkanoate synthesis in plants

DOEpatents

Srienc, Friedrich; Somers, David A.; Hahn, J. J.; Eschenlauer, Arthur C.

2000-01-01

Novel transgenic plants and plant cells are capable of biosynthesis of polyhydroxyalkanoate (PHA). Heterologous enzymes involved in PHA biosynthesis, particularly PHA polymerase, are targeted to the peroxisome of a transgenic plant. Transgenic plant materials that biosynthesize short chain length monomer PHAs in the absence of heterologous .beta.-ketothiolase and acetoacetyl-CoA reductase are also disclosed.

Redox mediators modify end product distribution in biomass fermentations by mixed ruminal microbes in vitro

USDA-ARS?s Scientific Manuscript database

The fermentation system of mixed ruminal bacteria is capable of generating large amounts of short-chain volatile fatty acids (VFA) via the carboxylate platform in vitro. These VFAs are subject to elongation to larger, more energy-dense products through reverse beta-oxidation. This study examined the...

Shape coexistence, shape evolution and Gamow-Teller {beta}-decay of neutron-rich A Asymptotically-Equal-To 100 nuclei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petrovici, A.; Schmid, K. W.; Faessler, A.

The structure of neutron-rich nuclei in the A Asymptotically-Equal-To 100 mass region relevant for the astrophysical r process manifests drastic changes in some isotopic chains and often sudden variations of particular nuclear properties have been identified. For a realistic description of the evolution in structure with increasing energy, spin, and isospin determined by shape coexistence and mixing beyond-mean-field approaches are required. Our recent studies represent an attempt to the self-consistent description of the shape coexistence phenomena in neutron-rich A Asymptotically-Equal-To 100 nuclei within the complex Excited Vampir variational model with symmetry projection before variation using a realistic effective interaction basedmore » on the Bonn A potential in a large model space. Results concerning the triple shape coexistence and the shape evolution in the N=58 Sr and Zr isotopes, the shape evolution in a chain of Zr nuclei, as well as the Gamow-Teller {beta}-decay properties of neutron-rich Zr and Tc nuclei are presented.« less

Characterization of epoxy carotenoids by fast atom bombardment collision-induced dissociation MS/MS.

PubMed

Maoka, Takashi; Fujiwara, Yasuhiro; Hashimoto, Keiji; Akimoto, Naoshige

2004-02-01

The characterization and structure of epoxy carotenoids possessing 5,6-epoxy, 5,8-epoxy and 3,6-epoxy end groups conjugated to the polyene chain were investigated using high-energy fast atom bombardment collision-induced dissociation MS/MS methods. In addition to [M - 80](+*), a characteristic fragment ion of an epoxy carotenoid, product ions resulting from the cleavage of C-C bonds in the polyene chain from the epoxy end group, such as m/z 181 (b ion) and 121 (c ion), were detected. On the other hand, diagnostic ions of m/z 286 (e-H ion) and 312 (f-H ion) were observed, not in the 5,6-epoxy or 5,8-epoxy carotenoid but in the 3,6-epoxy carotenoid. These fragmentation patterns can be used to distinguish 3,6-epoxy carotenoids from 5,6-epoxy or 5,8-epoxy carotenoids. The structure of an epoxy carotenoid, 3,6-epoxy-5,6-dihydro-7',8'-didehydro-beta,beta-carotene-5,3'-diol (8), isolated from oyster, was characterized using FAB CID-MS/MS by comparing fragmentation patterns with those of related known compounds.

Transfer factor - hypotheses for its structure and function.

PubMed

Shifrine, M; Scibienski, R

1975-01-01

Transfer factor (TF) is a dialyzable extract from primed lymphocytes that is able to transfer specific delayed hypersensitivity from one animal to another. On the basis of available data we suggest that TF is a polypeptide with a molecular weight below 15,000 daltons. We hypothesize that TF is the variable light or heavy chain domain of immunoglobulin: such a molecule conforms with the accepted properties of TF and also has the necessary specificity requirements. We also hypothesize that TF is part of a receptor site. beta-2-microglobulin, a molecule that is an integral part of cell surfaces, could be the anchor for TF. beta-2-microglobulin has homologies with the constant portion of immunoglobulin light or heavy chain and thus would combine with the variable domain (TF) to form a complete receptor site for a specific antigen. The properties of TF suggest its mode of action, which is discussed in detail in the text. The biologic advantages of TF is its ability to confer immediate (immunologie specific) protection while the 'normal' immune response develops.

Antibiotics in the human food chain: establishing no effect levels of tetracycline, neomycin, and erythromycin using a chemostat model of the human colonic microflora.

PubMed

Carman, Robert J; Simon, Mary Alice; Petzold, H Earl; Wimmer, Robert F; Batra, Monica R; Fernández, A Haydée; Miller, Margaret A; Bartholomew, Mary

2005-11-01

A chemostat model of the healthy human large bowel ecosystem was used to establish no effect levels for tetracycline, neomycin, and erythromycin. For each compound, the equivalent to four oral doses (0, 1.5, 15, and 150 mg/60 kg person/d) was studied. Concentrations of the test compounds in the chemostat medium were intended to simulate fecal levels that might be expected following consumption of food containing antibiotic residue and were based on published oral doses and fecal levels. We monitored the following parameters: short chain fatty acids, bile acids, sulfate reduction, azoreductase and nitroreductase activities, beta-glucosidase and beta-glucuronidase activities, a range of bacterial counts and, lastly, the susceptibility among sentinel bacteria to each test compound. Neomycin and erythromycin reduced bile acid metabolism. Neomycin elevated propionate levels and caused a marginal diminution in azoreductase activity. Based on our results, the no observed effect level (NOEL) of both tetracycline and erythromycin was 15 mg/60 kg person/d. The NOEL for neomycin was 1.5 mg/60 kg person/d.

Structural determination of the acidic exopolysaccharide produced by a Pseudomonas sp. strain 1.15.

PubMed

Cescutti, P; Toffanin, R; Pollesello, P; Sutherland, I W

1999-01-31

Pseudomonas strain 1.15 was isolated from a freshwater biofilm and shown to produce considerable amounts of an acidic polysaccharide which was investigated by methylation analysis, NMR spectroscopy and ionspray mass spectrometry (ISMS). The polysaccharide was depolymerised by a bacteriophage-associated endoglucosidase and by autohydrolysis, and the resulting oligosaccharides were investigated by NMR spectroscopy and mass spectrometry. The resulting data showed that the parent repeating unit of the 1.15 exopolysaccharide (EPS) is a branched hexasaccharide. The main chain is constituted of the trisaccharide -->4)-alpha-L-Fucp-(1-->4)-alpha-L-Fucp-(1-->3)-beta-D-Glcp- (1--> and the side chain alpha-D-Galp-(1-->4)-beta-D-GlcAp-(1-->3)-alpha-D-Galp-(1-->is linked to O-3 of the first Fuc residue. The terminal non-reducing Gal carries a 1-carboxyethylidene acetal in the R configuration at the positions 4 and 6. Of the four different O-acetyl groups present in non-stoichiometric amounts, two were established to be on O-2 of the 3-linked Gal and on O-2 of the 4-linked Fuc.

First report of a lyase for cepacian, the polysaccharide produced by Burkholderia cepacia complex bacteria.

PubMed

Cescutti, Paola; Scussolin, Silvia; Herasimenka, Yury; Impallomeni, Giuseppe; Bicego, Massimiliano; Rizzo, Roberto

2006-01-20

Bacteria belonging to the Burkholderia cepacia complex (Bcc) are interesting for their involvement in pulmonary infections in patients affected by cystic fibrosis (CF) or chronic granulomatous disease. Many Bcc strains isolated from CF patients produce high amounts of exopolysaccharides (EPS). Although different strains sometimes biosynthesise different EPS, the majority of Bcc bacteria produce only one type of polysaccharide, which is called cepacian. The polymer has a unique heptasaccharidic repeating unit, containing three side chains, and up to three O-acetyl substituents.. We here report for the first time the isolation and characterisation of a lyase active towards cepacian produced by a Bacillus sp., which was isolated in our laboratory. The enzyme molecular mass, evaluated by size-exclusion chromatography, is 32,700+/-1500Da. The enzyme catalyses a beta-elimination reaction of the disaccharide side chain beta-d-Galp-(1-->2)-alpha-d-Rhap-(1--> from the C-4 of the glucuronic acid residue present in the polymer backbone. Although active on both native and de-acetylated cepacian, the enzyme showed higher activity on the latter polymer.

Cellular Fatty Acid Composition, Soluble-Protein Profile, and Antimicrobial Resistance Pattern of Eubacterium lentum

PubMed Central

Mosca, Adriana; Summanen, Paula; Finegold, Sydney M.; De Michele, Giampiero; Miragliotta, Giuseppe

1998-01-01

Phenotypic heterogeneity among isolates of Eubacterium lentum has been recognized for many years. To better delineate their taxonomic relatedness, 29 clinical isolates of E. lentum were examined for soluble-protein content, cellular fatty acid profile, and antimicrobial resistance pattern in order to ascertain whether differences in these characteristics could be correlated with differences in biochemical activities. Among 29 isolates we could identify 6 that were different from all the others. These strains were coccobacilli with translucent colonies; they were catalase and H2S negative, not fluorescent under UV light, and susceptible to beta-lactam drugs; growth was not stimulated by arginine; and fatty acid analysis revealed the presence of straight-chain fatty acids. The remainder of the strains, including the type species, were pleomorphic bacilli with speckled colonies and were catalase and H2S positive; all but two were fluorescent under UV light; they were resistant to beta-lactam antibiotics; growth was greatly stimulated by arginine; and they demonstrated saturated branched-chain fatty acids. Our data suggest that E. lentum can be further differentiated into different types. PMID:9508307

[The Val606Met mutation of human beta myosin heavy chain in a Chinese familial hypertrophic cardiomyopathy family].

PubMed

Yuan, Jian-song; Qiao, Shu-bin; Wang, Shu-xia; Teng, Si-yong; You, Shi-jie; Yang, Wei-xian; Gao, Run-lin; Chen, Ji-lin; Yang, Yue-jin

2008-04-01

To explore the disease-causing gene mutation in Chinese families with hypertrophic cardiomyopathy (HCM) and to analyze the correlation between the genotype and phenotype. Samples of peripheral blood were collected from three Chinese families with HCM (at least two HCM patients existed/family). The exons in the functional regions of the beta myosin heavy chain gene (MYH7) were amplified with PCR and the products were sequenced. A Val606Met missen mutation was identified in the exon 16 of MYH7 gene in a Chinese family and this mutation was identified in all HCM patients (n = 4) and there was also a 15-years-old young mutation carrier who was not HCM patient now (penetrance of 80%). This mutation was not identified in other healthy family members in this family, in other 2 Chinese familiar HCM families and in 120 non-HCM control patients. The Val606Met missen mutation is closely associated with familiar HCM in a Chinese family which is associated with clinical phenotype with a penetrance of 80%.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thein, S.L.; Hesketh, C.; Wood, W.G.

Analysis of the molecular basis of dominantly inherited {beta}-thalassemia in four families has revealed different mutations involving exon 3 of the {beta}-globin gene. It is suggested that the phenotypic difference between this condition and the more common recessive forms of {beta}-thalassemia lies mainly in the length and stability of the abnormal translation products that are synthesized and, in particular, whether they are capable of binding heme and producing aggregations that are relatively resistant to proteolytic degradation.

Fat and Sugar Metabolism During Exercise in Patients With Metabolic Myopathy

ClinicalTrials.gov

2017-08-31

Metabolism, Inborn Errors; Lipid Metabolism, Inborn Errors; Carbohydrate Metabolism, Inborn Errors; Long-Chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency; Glycogenin-1 Deficiency (Glycogen Storage Disease Type XV); Carnitine Palmitoyl Transferase 2 Deficiency; VLCAD Deficiency; Medium-chain Acyl-CoA Dehydrogenase Deficiency; Multiple Acyl-CoA Dehydrogenase Deficiency; Carnitine Transporter Deficiency; Neutral Lipid Storage Disease; Glycogen Storage Disease Type II; Glycogen Storage Disease Type III; Glycogen Storage Disease Type IV; Glycogen Storage Disease Type V; Muscle Phosphofructokinase Deficiency; Phosphoglucomutase 1 Deficiency; Phosphoglycerate Mutase Deficiency; Phosphoglycerate Kinase Deficiency; Phosphorylase Kinase Deficiency; Beta Enolase Deficiency; Lactate Dehydrogenase Deficiency; Glycogen Synthase Deficiency

Differential usage of T-cell receptor V beta gene families by CD4+ and CD8+ T cells in patients with CD8hi common variable immunodeficiency: evidence of a post-thymic effect.

PubMed Central

Duchmann, R; Jaffe, J; Ehrhardt, R; Alling, D W; Strober, W

1996-01-01

In this study, we report that differences between T-cell receptor (TCR) V beta gene family usage in CD4+ and CD8+ T cells are significantly greater in a subgroup of patients with common variable immunodeficiency (CVI) and high levels of activated CD8+ T cells (CD8hi CVI) than in controls (P < 0.001). In CD8hi CVI patients, such differences were also significantly greater for V beta 12 than for other V beta families. As the causes of the differential usage of V beta gene families by CD4+ and CD8+ T cells are under investigation, it was interesting that the combined differences between V beta gene family usage in the CD4+ and CD8+ T-cell subpopulations as a whole were significantly lower than the combined differences between individual V beta gene family usage in either CD4+ or CD8+ T-cell subpopulations (P < 0.001 in both control and CD8hi CVI patients). Further, the pattern of V beta gene family usage in CD4+ T cells was remarkably similar to that in CD8+ T cells in both groups. These data strongly suggest that differences in V beta gene family usage arising from coselection by major histocompatibility complex (MHC) class I versus MHC class II restriction elements do not fundamentally distort 'basic' V beta gene family usage patterns. They also support the concept that differences in CD4+ and CD8+ T-cell V beta gene family usage, which were increased in CD8hi CVI, can arise from high-affinity interactions between disease-associated antigens or superantigens and T cells in the post-thymic T-cell compartment. Images Figure 6 PMID:8666443

Novel estrogens and their radical scavenging effects, iron-chelating, and total antioxidative activities: 17 alpha-substituted analogs of delta 9(11)-dehydro-17 beta-estradiol.

PubMed

Römer, W; Oettel, M; Menzenbach, B; Droescher, P; Schwarz, S

1997-11-01

Antioxidant effects of N,N-dimethyl-p-toluidine, p-cresol, and p-(hydroxy)thioanisol 17 alpha-substituted analogs of 17 beta-estradiol and their delta 9(11)-dehydro homologs were investigated using four different in vitro models: rat synaptosomal lipid peroxidation induced by Fenton's reagent, Fe(II)-chelating activities, the formation of superoxide anion radicals, and total antioxidative activity. Whereas the classical estrogen 17 beta-estradiol as well as selected phenolic compounds was only moderately inhibiting iron-dependent lipid peroxidation and stimulating total antioxidative activity, besides delta 9(11)-dehydro-17 beta-estradiol (J 1213), novel estrogens such as C-17-oriented side chain analogs of 17 beta-estradiol (J 843, J 872, and J 897) and delta 9(11)-dehydro homologs (J 844, J 864, and J 898) directly altered the iron redox chemistry and diminished the formation of superoxide anion radicals generated by a xanthine/xanthine oxidase-dependent luminescence reaction to a great extent. These results suggest that definite modifications in the chemical structure of 17 beta-estradiol, e.g., the introduction of a delta 9(11)-double bond and/or p-cresol as well as p-(hydroxy)thioanisol C-17 substitution, may result in substantial changes in their antioxidant behavior. These compounds may be drug candidates for treating pathologies related to free radical formation.

Effect of in vitro degradation of poly(D,L-lactide)/beta-tricalcium composite on its shape-memory properties.

PubMed

Zheng, Xiaotong; Zhou, Shaobing; Yu, Xiongjun; Li, Xiaohong; Feng, Bo; Qu, Shuxin; Weng, Jie

2008-07-01

The in vitro degradation characteristic and shape-memory properties of poly(D,L-lactide) (PDLLA)/beta-tricalcium phosphate (beta-TCP) composites were investigated because of their wide application in biomedical fields. In this article, PDLLA and crystalline beta-TCP were compounded and interesting shape-memory behaviors of the composite were first investigated. Then, in vitro degradation of the PDLLA/beta-TCP composites with weight ratios of 1:1, 2:1, and 3:1 was performed in phosphate buffer saline solution (PBS) (154 mM, pH 7.4) at 37 degrees C. The effect of in vitro degradation time for PDLLA/beta-TCP composites on shape-memory properties was studied by scanning electron microscopy, differential scanning calorimetry, gel permeation chromatography, X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The changes of structural morphology, glass transition temperature (T(g)), molecular weight, and weight loss of composites matrix and pH change of degradation medium indicated that shape-memory effects at different degradation time were nonlinearly influenced because of the breaking down of polymer chain and the formation of degradation products. Furthermore, the results from XRD and FTIR implied that the degradation products, for example, hydroxyapatite (HA), calcium hydrogen phosphate (CaHPO(4)), and calcium pyrophosphate (Ca(2)P(2)O(7)) phases also had some effects on shape-memory properties during the degradation. 2007 Wiley Periodicals, Inc.

Association between leukocyte telomere length and serum carotenoid in US adults.

PubMed

Min, Kyoung-Bok; Min, Jin-Young

2017-04-01

Telomere length is a biomarker for aging. It is known that oxidative stress can accelerate telomere shortening, whereas antioxidants can delay their shortening. Carotenoids as antioxidants are favorably associated with health- and aging-related diseases caused by oxidative stress, but their association with telomere length is less certain. We investigated the association between blood carotenoid levels and leukocyte telomere length in a representative sample of US adults. We analyzed 3660 participants aged 20 years and older in the 1999-2002 National Health and Nutrition Examination Survey. The levels of carotenoids-alpha-carotene, beta-carotene (trans + cis), beta-cryptoxanthin, combined lutein/zeaxanthin, and trans-lycopene-were measured using high-performance liquid chromatography. The leukocyte telomere length (T/S ratio) was assayed using the quantitative polymerase chain reaction method. A doubling of blood alpha-carotene, beta-carotene (trans + cis), and beta-cryptoxanthin was associated with approximately 2 % longer telomeres. Compared with the lowest carotenoid quartile of alpha-carotene, beta-carotene (trans + cis), and beta-cryptoxanthin, telomere length for adults with the highest quartiles was significantly increased by 5-8 %. We found that increasing levels of blood carotenoid were significantly associated with longer leukocyte telomeres in US adults. High intake of carotenoid-rich food may play a role in protecting telomeres and regulating telomere length.

Activation of PPAR{delta} up-regulates fatty acid oxidation and energy uncoupling genes of mitochondria and reduces palmitate-induced apoptosis in pancreatic {beta}-cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wan, Jun; Jiang, Li; Lue, Qingguo

2010-01-15

Recent evidence indicates that decreased oxidative capacity, lipotoxicity, and mitochondrial aberrations contribute to the development of insulin resistance and type 2 diabetes. The goal of this study was to investigate the effects of peroxisome proliferator-activated receptor {delta} (PPAR{delta}) activation on lipid oxidation, mitochondrial function, and insulin secretion in pancreatic {beta}-cells. After HIT-T15 cells (a {beta}-cell line) were exposed to high concentrations of palmitate and GW501516 (GW; a selective agonist of PPAR{delta}), we found that administration of GW increased the expression of PPAR{delta} mRNA. GW-induced activation of PPAR{delta} up-regulated carnitine palmitoyltransferase 1 (CPT1), long-chain acyl-CoA dehydrogenase (LCAD), pyruvate dehydrogenase kinase 4more » (PDK4), and uncoupling protein 2 (UCP2); alleviated mitochondrial swelling; attenuated apoptosis; and reduced basal insulin secretion induced by increased palmitate in HIT cells. These results suggest that activation of PPAR{delta} plays an important role in protecting pancreatic {beta}-cells against aberrations caused by lipotoxicity in metabolic syndrome and diabetes.« less

Folding cooperativity in a three-stranded beta-sheet model.

PubMed

Roe, Daniel R; Hornak, Viktor; Simmerling, Carlos

2005-09-16

The thermodynamic behavior of a previously designed three-stranded beta-sheet was studied via several microseconds of standard and replica exchange molecular dynamics simulations. The system is shown to populate at least four thermodynamic minima, including two partially folded states in which only a single hairpin is formed. Simulated melting curves show different profiles for the C and N-terminal hairpins, consistent with differences in secondary structure content in published NMR and CD/FTIR measurements, which probed different regions of the chain. Individual beta-hairpins that comprise the three-stranded beta-sheet are observed to form cooperatively. Partial folding cooperativity between the component hairpins is observed, and good agreement between calculated and experimental values quantifying this cooperativity is obtained when similar analysis techniques are used. However, the structural detail in the ensemble of conformations sampled in the simulations permits a more direct analysis of this cooperativity than has been performed on the basis of experimental data. The results indicate the actual folding cooperativity perpendicular to strand direction is significantly larger than the lower bound obtained previously.

Beta/gamma and alpha backgrounds in CRESST-II Phase 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strauss, R.; Angloher, G.; Ferreiro Iachellini, N.

2015-06-01

The experiment CRESST-II aims at the detection of dark matter with scintillating CaWO{sub 4} crystals operated as cryogenic detectors. Recent results on spin-independent WIMP-nucleon scattering from the CRESST-II Phase 2 allowed to probe a new region of parameter space for WIMP masses below 3 GeV/c{sup 2}. This sensitivity was achieved after background levels were reduced significantly. We present extensive background studies of a CaWO{sub 4} crystal, called TUM40, grown at the Technische Universität München. The average beta/gamma rate of 3.51/[kg keV day] (1-40 keV) and the total intrinsic alpha activity from natural decay chains of 3.08±0.04 mBq/kg are the lowestmore » reported for CaWO{sub 4} detectors. Contributions from cosmogenic activation, surface-alpha decays, external radiation and intrinsic alpha/beta emitters are investigated in detail. A Monte-Carlo based background decomposition allows to identify the origin of the majority of beta/gamma events in the energy region relevant for dark matter search.« less

Laboratory modeling of multiple zonal jets on the polar beta-plane

NASA Astrophysics Data System (ADS)

Afanasyev, Y.

2011-12-01

Zonal jets observed in the oceans and atmospheres of planets are studied in a laboratory rotating tank. The fluid layer in the rotating tank has parabolic free surface and dynamically simulates the polar beta-plane where the Coriolis parameter varies quadratically with distance from the pole. Velocity and surface elevation fields are measured with an optical altimetry method (Afanasyev et al., Exps Fluids 2009). The flows are induced by a localized buoyancy source along radial direction. The baroclinic flow consisting of a field of eddies propagates away from the source due West and forms zonal jets (Fig. 1). Barotropic jets ahead of the baroclinic flow are formed by radiation of beta plumes. Inside the baroclinic flow the jets flow between the chains of eddies. Experimental evidence of so-called noodles (baroclinic instability mode with motions in the radial, North-South direction) theoretically predicted by Berloff et al. (JFM, JPO 2009) was found in our experiments. Beta plume radiation mechanism and the mechanism associated with the instability of noodles are likely to contribute to formation of jets in the baroclinic flow.

Molecular models of NS3 protease variants of the Hepatitis C virus.

PubMed

da Silveira, Nelson J F; Arcuri, Helen A; Bonalumi, Carlos E; de Souza, Fátima P; Mello, Isabel M V G C; Rahal, Paula; Pinho, João R R; de Azevedo, Walter F

2005-01-21

Hepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed. The atomic coordinates of crystallographic structure 1CU1 and 1DY9 were used as starting model for modeling of the NS3 protease variant structures. The NS3 protease variant structures are composed of six subdomains, which occur in sequence along the polypeptide chain. The protease domain exhibits the dual beta-barrel fold that is common among members of the chymotrypsin serine protease family. The helicase domain contains two structurally related beta-alpha-beta subdomains and a third subdomain of seven helices and three short beta strands. The latter domain is usually referred to as the helicase alpha-helical subdomain. The rmsd value of bond lengths and bond angles, the average G-factor and Verify 3D values are presented for NS3 protease variant structures. This project increases the certainty that homology modeling is an useful tool in structural biology and that it can be very valuable in annotating genome sequence information and contributing to structural and functional genomics from virus. The structural models will be used to guide future efforts in the structure-based drug design of a new generation of NS3 protease variants inhibitors. All models in the database are publicly accessible via our interactive website, providing us with large amount of structural models for use in protein-ligand docking analysis.

FcepsilonRI-alpha siRNA inhibits the antigen-induced activation of mast cells.

PubMed

Safaralizadeh, Reza; Soheili, Zahra-Soheila; Deezagi, Abdolkhaleg; Pourpak, Zahra; Samiei, Shahram; Moin, Mostafa

2009-12-01

FcepsilonRI, The high affinity receptor for IgE plays a critical role in triggering the allergic reactions. It is responsible for inducing mast cell degranulation and deliberation of allergy mediators when it is aggregated by allergen and IgE complexes. FcepsilonRI on the mast cells consists of three subunits; alpha chain directly binds IgE, beta chain and dimmer of gamma chains together mediate intracellular signaling. Cross-linking of IgE-bound FcepsilonRI on the surface of mast cells and basophils by the multivalent antigen induces release of chemical mediators. The present in vitro study was designed to investigate the effect of synthetic FcepsilonRI-alpha siRNA on the antigen-induced activation of MC/9 cells. MC/9 cells which are murine mast cells were transfected by FcepsilonRI-alpha siRNA and negative control siRNA. After 6 h, anti-DNP (Dinitrophenyl) IgE was used for the cells sensitization. Then the cells were challenged with Dinitrophenyl-Human Serum Albumin (DNP-HSA) for mast cell degranulation induction before collection of supernatants. The amount of mRNA and protein expression was measured by Real Time PCR and western blot analysis, respectively. Determination of the expression rate of FcepsilonRI-alpha on cell surface was achieved by flow cytometry. ELISA and spectrophotometry methods were used subsequently for measuring the effects of FcepsilonRI-alpha siRNA on antigen-induced histamine and beta-hexosaminidase release. FcepsilonRI-alpha siRNA treated cells showed significant decrease in FcepsilonRI-alpha mRNA and protein expression in comparison to control cells. FcepsilonRI-mediated mast cell release of beta-hexosaminidase and histamine were also inhibited. In this study it was shown that FcepsilonRI-alpha siRNA could suppress FcepsilonRI-alpha expression and inhibited degranulation and histamine release in antigen-stimulated MC/9 cells. In conclusion, knock-down of FcepsilonRI-alpha by siRNA could be a promising method for inhibition of the mast cell-mediated allergic reactions.

Evidence for a role for the phosphotyrosine-binding domain of Shc in interleukin 2 signaling.

PubMed Central

Ravichandran, K S; Igras, V; Shoelson, S E; Fesik, S W; Burakoff, S J

1996-01-01

Stimulation via the T-cell growth factor interleukin 2 (IL-2) leads to tyrosine phosphorylation of Shc, the interaction of Shc with Grb2, and the Ras GTP/GDP exchange factor, mSOS. Shc also coprecipitates with the IL-2 receptor (IL-2R), and therefore, may link IL-2R to Ras activation. We have further characterized the Shc-IL-2R interaction and have made the following observations. (i) Among the two phosphotyrosine-interaction domains present in Shc, the phosphotyrosine-binding (PTB) domain, rather than its SH2 domain, interacts with the tyrosine-phosphorylated IL-2R beta chain. Moreover, the Shc-PTB domain binds a phosphopeptide derived from the IL-2R beta chain (corresponding to residues surrounding Y338, SCFTNQGpYFF) with high affinity. (ii) In vivo, mutant IL-2R beta chains lacking the acidic region of IL-2Rbeta (which contains Y338) fail to phosphorylate Shc. Furthermore, when wild type or mutant Shc proteins that lack the PTB domain were expressed in the IL-2-dependent CTLL-20 cell line, an intact Shc-PTB domain was required for Shc phosphorylation by the IL-2R, which provides further support for a Shc-PTB-IL-2R interaction in vivo. (iii) PTB and SH2 domains of Shc associate with different proteins in IL-2- and T-cell-receptor-stimulated lysates, suggesting that Shc, through the concurrent use of its two different phosphotyrosine-binding domains, could assemble multiple protein complexes. Taken together, our in vivo and in vitro observations suggest that the PTB domain of Shc interacts with Y338 of the IL-2R and provide evidence for a functional role for the Shc-PTB domain in IL-2 signaling. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8643566

Hemoglobin Hiroshima (β143 histidine → aspartic acid): a newly identified fast moving beta chain variant associated with increased oxygen affinity and compensatory erythremia

PubMed Central

Hamilton, Howard B.; Iuchi, Iwao; Miyaji, Takaoki; Shibata, Susumu

1969-01-01

During a survey for hemoglobinopathies in over 9000 residents of Hiroshima Prefecture, Japan, a fast moving hemoglobin was identified in eight members of three generations in a Japanese family. The abnormal hemoglobin, named Hb Hiroshima, constitutes about 50% of the total hemoglobin in hemolysates from the carriers who have a mild erythremia but are otherwise apparently clinically unaffected. All preparations of Hb Hiroshima have increased affinity for oxygen, by either tonometric or oxygen electrode determinations. At pH 7.0, the oxygen pressure, P50 required to half saturate an unfractionated hemolysate from a carrier was one-half that of Hb A, and the P50 of a purified sample containing no Hb A was one-fourth that of Hb A. The pH dependence of the oxygen equilibrium (Bohr effect) is below normal, as shown by the absolute value of the Bohr effect factor which is about half that of Hb A, in the pH range between 7.0 and 7.4. The Hill constant, n, for Hb Hiroshima between pH 7.0 and 7.4 is 2-2.4, compared to 2.8-3 for Hb A under the same conditions, indicating reduction of, but not complete abolition of heme-heme interaction. Urea dissociation and canine hybridization tests located the biochemical lesion in the beta chain. Fingerprints (Ingram), carboxypeptidase digestion, and amino acid analysis demonstrated that the substitution was at residue 143 in the beta chain, where histidine was replaced by aspartic acid. In contrast to other recently described high oxygen affinity mutants that show intact Bohr effects, all three of the major characteristics of the reversible combination of hemoglobin with oxygen (oxygen equilibrium, heme-heme interaction, and pH dependence) are affected in Hb Hiroshima. A tentative interpretation of these effects, relating structure to function, is offered in terms of recently developed models of normal hemoglobin. Images PMID:5773089

Functional characterization of the beta-adrenergic receptor subtypes expressed by CA1 pyramidal cells in the rat hippocampus.

PubMed

Hillman, Kristin L; Doze, Van A; Porter, James E

2005-08-01

Recent studies have demonstrated that activation of the beta-adrenergic receptor (AR) using the selective beta-AR agonist isoproterenol (ISO) facilitates pyramidal cell long-term potentiation in the cornu ammonis 1 (CA1) region of the rat hippocampus. We have previously analyzed beta-AR genomic expression patterns of 17 CA1 pyramidal cells using single cell reverse transcription-polymerase chain reaction, demonstrating that all samples expressed the beta2-AR transcript, with four of the 17 cells additionally expressing mRNA for the beta1-AR subtype. However, it has not been determined which beta-AR subtypes are functionally expressed in CA1 for these same pyramidal neurons. Using cell-attached recordings, we tested the ability of ISO to increase pyramidal cell action potential (AP) frequency in the presence of subtype-selective beta-AR antagonists. ICI-118,551 [(+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol] and butoxamine [alpha-[1-(t-butylamino)ethyl]-2,5-dimethoxybenzyl alcohol) hydrochloride], agents that selectively block the beta2-AR, produced significant parallel rightward shifts in the concentration-response curves for ISO. From these curves, apparent equilibrium dissociation constant (K(b)) values of 0.3 nM for ICI-118,551 and 355 nM for butoxamine were calculated using Schild regression analysis. Conversely, effective concentrations of the selective beta1-AR antagonists CGP 20712A [(+/-)-2-hydroxy-5-[2-([2-hydroxy-3-(4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy)propyl]amino)ethoxy]-benzamide methanesulfonate] and atenolol [4-[2'-hydroxy-3'-(isopropyl-amino)propoxy]phenylacetamide] did not significantly affect the pyramidal cell response to ISO. However, at higher concentrations, atenolol significantly decreased the potency for ISO-mediated AP frequencies. From these curves, an apparent atenolol K(b) value of 3162 nM was calculated. This pharmacological profile for subtype-selective beta-AR antagonists indicates that beta2-AR activation is mediating the increased AP frequency. Knowledge of functional AR expression in CA1 pyramidal neurons will aid future long-term potentiation studies by allowing selective manipulation of specific beta-AR subtypes.

A binding energy study of the Atomic Mass Evaluation 2012 and an updated beta-decay study of neutron-rich 74Cu

NASA Astrophysics Data System (ADS)

Tracy, James L., Jr.

A study of ground state binding energy values listed in the Atomic Mass Evaluation 2012 (AME2012) using an interpretive approach, as opposed to the exploratory methods of previous models, is presented. This model is based on a postulate requiring all protons to pair with available neutrons to form bound alpha clusters as the ground state for an N = Z core upon which excess neutrons are added. For each core, the trend of the binding energy as a function of excess neutrons in the isotopic chain can be fit with a three-term quadratic function. The quadratic parameter reveals a smooth decaying exponential function. By re-envisioning the determination of mass excess, the constant-term fit parameters, representing N = Z nuclei, reveal a near-symmetry around Z = 50. The linear fit parameters exhibit trends which are linear functions of core size. A neutron drip-line prediction is compared against current models. By considering the possibility of an alpha-cluster core, a new ground-state structure grouping scheme is presented; nucleon-nucleon pairing is shown to have a greater role in level filling. This model, referred to as the Alpha-Deuteron-Neutron Model, yields promising first results when considering root-mean-square variances from the AME2012. The beta-decay of the neutron-rich isotope 74Cu has been studied using three high-purity Germanium clover detectors at the Holifield Radioactive Ion Beam Facility at Oak Ridge National Laboratory. A high-resolution mass separator greatly improved the purity of the 74Cu beam by removing isobaric contaminants, thus allowing decay through its isobar chain to the stable 74Ge at the center of the LeRIBSS detector array without any decay chain member dominating. Using coincidence gating techniques, 121 gamma-rays associated with 74Cu were isolated from the collective singles spectrum. Eighty-seven of these were placed in an expanded level scheme, and updated beta-feeding level intensities and log( ft) values are presented based on multiple newly-placed excited states up to 6.8 MeV. The progression of simulated Total Absorption gamma-ray Spectroscopy (TAGS) based on known levels and beta feeding values from previous measurements to this evaluation are presented and demonstrate the need for a TAGS measurement of this isotope to gain a more complete understanding of its decay scheme.

S-allylcysteine ameliorates isoproterenol-induced cardiac toxicity in rats by stabilizing cardiac mitochondrial and lysosomal enzymes.

PubMed

Padmanabhan, M; Mainzen Prince, P Stanely

2007-02-13

This study was aimed to evaluate the preventive role of S-allylcysteine (SAC) on mitochondrial and lysosomal enzymes in isoproterenol (ISO)-induced rats. Male albino Wistar rats were pretreated with SAC (50, 100 and 150 mg/kg) daily for a period of 45 days. After the treatment period, ISO (150 mg/kg) was subcutaneously injected to rats at an interval of 24 h for two days. The activities of heart mitochondrial enzymes (isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase and alpha-ketoglutarate dehydrogenase) and respiratory chain enzymes (NADH dehydrogenase and cytochrome C oxidase) were decreased significantly (p<0.05) in ISO-induced rats. The activities of lysosomal enzymes (beta-glucuronidase, beta-N-acetyl glucosaminidase, beta-galactosidase, cathepsin-D and acid phosphatase) were increased significantly (p<0.05) in serum and heart of ISO-induced rats. Pretreatment with SAC (100 mg/kg and 150 mg/kg) for a period of 45 days increased significantly (p<0.05) the activities of mitochondrial and respiratory chain enzymes and decreased the activities of lysosomal enzymes significantly (p<0.05) in ISO-induced rats. Oral administration of SAC (50, 100 and 150 mg/kg) for a period of 45 days to normal rats did not show any significant (p<0.05) effect in all the parameters studied. The altered electrocardiogram (ECG) of ISO-treated rats was also restored to near normal by treatment with SAC (100 and 150 mg/kg). These results confirm the efficacy of SAC in alleviating ISO-induced cardiac damage.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kasid, A.; Morecki, S.; Aebersold, P.

Tumor-infiltrating lymphocytes (TILs) are cells generated from tumor suspensions cultured in interleukin 2 that can mediate cancer regression when adoptively transferred into mice or humans. Since TILs proliferate rapidly in vitro, recirculate, and preferentially localize at the tumor site in vivo, they provide an attractive model for delivery of exogenous genetic material into man. To determine whether efficient gene transfer into TILs is feasible. The authors transduced human TILs with the bacterial gene for neomycin-resistance (Neo{sup R}) using the retroviral vector N2. The transduced TIL populations were stable and polyclonal with respect to the intact Neo{sup R} gene integration andmore » expressed high levels of neomycin phosphotransferase activity. The Neo{sup R} gene insertion did not alter the in vitro growth pattern and interleukin 2 dependence of the transduced TILs. Analyses of T-cell receptor gene rearrangement for {beta}- and {gamma}-chain genes revealed the oligoclonal nature of the TIL populations with no major change in the DNA rearrangement patterns or the levels of mRNA expression of the {beta} and {gamma} chains following transduction and selection of TILs in the neomycin analog G418. Human TILs expressed mRNA for tumor necrosis factors ({alpha} and {beta}) and interleukin 2 receptor P55. This pattern of cytokine-mRNA expression was not significantly altered following the transduction of TILs. The studies demonstrate the feasibility of TILs as suitable cellular vehicles for the introduction of therapeutic genes into patients receiving autologous TILs.« less

The effects of dan-shen root on cardiomyogenic differentiation of human placenta-derived mesenchymal stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Kun; Li, Shi-zheng, E-mail: ychozon@yahoo.com.cn; Zhang, Yun-li

2011-11-11

Highlights: Black-Right-Pointing-Pointer Conditional medium and dan-shen root were used for cardiomyogenic differentiation. Black-Right-Pointing-Pointer They all could induce hPDMSCs to differentiate into cardiomyocytes. Black-Right-Pointing-Pointer The induction effect of the latter was slightly higher compared to that of the former. Black-Right-Pointing-Pointer Dan-shen root could be a good inducer for cardiomyogenic differentiation. -- Abstract: The aim of this study was to search for a good inducer agent using for cardiomyogenic differentiation of stem cells. Human placenta-derived mesenchymal stem cells (hPDMSCs) were isolated and incubated in enriched medium. Fourth passaged cells were treated with 10 mg/L dan-shen root for 20 days. Morphologic characteristics weremore » analyzed by confocal and electron microscopy. Expression of {alpha}-sarcomeric actin was analyzed by immunohistochemistry. Expression of cardiac troponin-I (TnI) was analyzed by immunohistofluorescence. Atrial natriuretic factor (ANF) and beta-myocin heavy chain ({beta}-MHC) were detected by reverse transcriptase polymerase chain reaction (RT-PCR). hPDMSCs treated with dan-shen root gradually formed a stick-like morphology and connected with adjoining cells. On the 20th day, most of the induced cells stained positive with {alpha}-sarcomeric actin and TnI antibody. ANF and {beta}-MHC were also detected in the induced cells. Approximately 80% of the cells were successfully transdifferentiated into cardiomyocytes. In conclusion, dan-shen root is a good inducer agent used for cardiomyogenic differentiation of hPDMSCs.« less

Organocatalysed asymmetric beta-amination and multicomponent syn-selective diamination of alpha,beta-unsaturated aldehydes.

PubMed

Jiang, Hao; Nielsen, Johanne B; Nielsen, Martin; Jørgensen, Karl Anker

2007-01-01

An easy and affordable route for obtaining chiral beta-aminated- and alpha,beta-diaminated aldehydes, 1,3-aminoalcohols, and related compounds by using organocatalysis is presented. The chiral secondary amine (S)-2-[bis(3,5-bistrifluoromethylphenyl)trimethylsilanyloxymethyl]pyrrolidine is used as the catalyst to activate alpha,beta-unsaturated aldehydes, which allows succinimide to add in a 1,4-regio- and stereoselective fashion thereby forming N-protected 1,3-aminoaldehydes in good yields and enantioselectivities. This is followed by two easy transformations giving rise to optically active 1,3-aminoalcohols, a common motif in many biologically active compounds, for example, fibrinogen receptor antagonists. Furthermore, optically active alpha,beta-syn-diaminated aldehydes were obtained by the addition of diethyl azodicarboxylate in a one-pot reaction.

Comparison of Observed Beta Cloth Interactions with Simulated and Actual Space Environment

NASA Technical Reports Server (NTRS)

Kamenetzy, R. R.; Finckenor, M. M.

1999-01-01

A common component of multilayer insulation blankets is beta cloth, a woven fiberglass cloth impregnated with Teflon(TM). It is planned for extensive use on the International Space Station. The Environmental Etl'ects Group of the Marshall Space Flight Center Materials, Processing, and Manufacturing Department has investigated the impact of atomic oxygen (AO) and ultraviolet (UV) radiation on the optical properties of plain and aluminized beta cloth. both in the laboratory and as part of long-duration flight experiments. These investigations indicate that beta cloth is susceptible to darkening in the presence of UV radiation, dependent on the additives used. AO interactions resulted in bleaching of the beta cloth.

Maternal breast milk transforming growth factor beta and feeding intolerance in preterm infants

PubMed Central

Frost, Brandy L.; Jilling, Tamas; Lapin, Brittany; Maheshwari, Akhil; Caplan, Michael S.

2015-01-01

Background Feeding intolerance occurs commonly in the NICU. Breast milk contains a large pool of transforming growth factor-beta (TGF-beta). Few studies describe TGF-beta levels in preterm milk, and the relationship to feeding intolerance (FI) remains unexplored. We measured TGF-beta levels in preterm breast milk to investigate a correlation with FI in preterm infants. Methods Prospective observational trial of 100 mother-infant pairs, enrolling infants born below 32 weeks gestation and less than 1500 grams, and mothers who planned to provide breast milk. TGF-beta levels were measured using ELISA. Infant charts were reviewed for outcomes. Results TGF-beta declined postnatally, most elevated in colostrum (p<0.01). TGF-beta 2 levels were higher than TGF-beta 1 at all time points (p<0.01). Colostrum TGF-beta levels correlated inversely with birth weight (p<0.01) and gestational age (p<0.05). One week TGF-beta 2 levels were reduced in growth-restricted infants with FI (p<0.01). Of infants with NEC, TGF-beta 2 levels appeared low, but small sample size precluded meaningful statistical comparisons. Conclusions TGF-beta levels decline temporally in preterm milk. TGF-beta 1 colostrum levels correlate inversely with birth weight and gestational age. TGF-beta 2 may play a role in FI in growth-restricted infants. The relationship of TGF-beta 2 and NEC merits future investigation. PMID:24995914

Generalized Jastrow Variational Method for Liquid HELIUM-3-HELIUM-4 Mixtures at T = 0 K.

NASA Astrophysics Data System (ADS)

Mirabbaszadeh, Kavoos

Microscopic theory of dilute liquid { ^3 He}-{^4 He} mixtures is of great interest, because it provides a physical realization of a nearly degenerate weakly interacting Fermion system. An understanding of properties of the mixtures has received considerable attention both theoretically and experimentally over the past thirty years. We present here a variational procedure based on the Jastrow function for the ground state of {^3 He}- {^4 He} mixtures by minimizing the total energy of the mixture using the hypernetted-chain (HNC) approximation and the Percus-Yevick (PY) approximation for the two body correlation functions. Our goal is to compute from first principles the internal energy of the system and the various two body correlation functions at various densities and compare the results with experiment. The Jastrow variational method for the ground state energy of liquid {^4 He} consists of the following ansatz for the wave function Psi_alpha {rm(vec r_{1 alpha},} {vec r_{2alpha},} dots, {vec r_{N _alpha})} = prod _{rm i < j} {rm f_ {alphaalpha}(r_{ij}). } For a {^3 He } system the corresponding ansatz is Psi_beta {rm( vec r_{1beta},} {vec r_{2beta },} dots, {vec r_{N_beta})} = {[prod _{i < j} f_{betabeta }(r_{ij})]} Phi {rm( vec r_{1beta},} {vec r_{2beta },} dots, {vec r_{Nbeta}),} where Phi is a Slater determinant of plane waves for the ground state of the Fermion system. The total energy per particle can be written in the form: E = x_sp{alpha}{2} E_{alphaalpha} + x_sp{beta}{2 }E_{betabeta } + 2x_{alpha} x_{beta}E _{alphabeta}, where E_{alphaalpha} , E_{betabeta} , E_{alphabeta} are unknown parameters to be determined from a microscopic theory. Using the Jastrow wave function Psi for the mixture, a general expression is given for the ground state energy in terms of the two body potential and two and three body correlation functions. The Kirkwood Super-position Approximation (KSA) is used for the three-body correlation functions. The antisymmetry of the wave function for Fermions is incorporated following the procedure given earlier by Lado, Inguva and Smith. This procedure for treating the antisymmetry of the wave function simplifies the equations for the two-body correlation functions considerably. The equations for the correlation functions are solved in the hypernetted-chain approximation. Once the two-particle correlation functions for the mixture ( ^3He-^4He) have been obtained, the energy is minimized with respect to the variational parameters involved in the Jastrow wave function. The binding energy and the optimal correlation functions are then obtained as a function of the concentration of ^3He atoms in the mixture. (Abstract shortened with permission of author.).

Limitations of commonly used internal controls for real-time RT-PCR analysis of renal epithelial-mesenchymal cell transition.

PubMed

Elberg, Gerard; Elberg, Dorit; Logan, Charlotte J; Chen, Lijuan; Turman, Martin A

2006-01-01

Progressive renal fibrotic disease is accompanied by the massive accumulation of myofibroblasts as defined by alpha smooth muscle actin (alphaSMA) expression. We quantitated gene expression using real-time RT-PCR analysis during conversion of primary cultured human renal tubular cells (RTC) to myofibroblasts after treatment with transforming growth factor-beta1 (TGF-beta1). We report herein the limitations of commonly used reference genes for mRNA quantitation. We determined the expression of alphaSMA and megakaryoblastic leukemia-1 (MKL1), a transcriptional regulator of alphaSMA, by quantitative real-time PCR using three common internal controls, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A and 18S rRNA. Expression of GAPDH mRNA and cyclophilin A mRNA, and to a lesser extent, 18S rRNA levels varied over time in culture and with exposure to TGF-beta1. Thus, depending on which reference gene was used, TGF-beta1 appeared to have different effects on expression of MKL1 and alphaSMA. RTC converting to myofibroblasts in primary culture is a valuable system to study renal fibrosis in humans. However, variability in expression of reference genes with TGF-beta1 treatment illustrates the need to validate mRNA quantitation with multiple reference genes to provide accurate interpretation of fibrosis studies in the absence of a universal internal standard for mRNA expression. 2006 S. Karger AG, Basel.

Psoriasis is associated with increased beta-defensin genomic copy number

PubMed Central

Hollox, Edward J.; Huffmeier, Ulrike; Zeeuwen, Patrick L.J.M.; Palla, Raquel; Lascorz, Jesús; Rodijk-Olthuis, Diana; van de Kerkhof, Peter C.M.; Traupe, Heiko; de Jongh, Gys; den Heijer, Martin; Reis, André; Armour, John A.L.; Schalkwijk, Joost

2008-01-01

Psoriasis is a common inflammatory skin disease with a strong genetic component. We have analysed the genomic copy number polymorphism of the beta-defensin region on human chromosome 8 in 179 Dutch psoriasis patients and 272 controls, and in 319 German psoriasis patients and 305 controls. Comparisons in both cohorts show a significant association between higher genomic copy number for beta-defensin genes and the risk of psoriasis. PMID:18059266

Deconstructing Quinoline-Class Antimalarials to Identify Fundamental Physicochemical Properties of Beta-Hematin Crystal Growth Inhibitors.

PubMed

Olafson, Katy N; Nguyen, Tam Q; Vekilov, Peter G; Rimer, Jeffrey D

2017-10-04

A versatile approach to control crystallization involves the use of modifiers, which are additives that interact with crystal surfaces and alter their growth rates. Elucidating a modifier's binding specificity to anisotropic crystal surfaces is a ubiquitous challenge that is critical to their design. In this study, we select hematin, a byproduct of malaria parasites, as a model system to examine the complementarity of modifiers (i.e., antimalarial drugs) to β-hematin crystal surfaces. We divide two antimalarials, chloroquine and amodiaquine, into segments consisting of a quinoline base, common to both drugs, and side chains that differentiate their modes of action. Using a combination of scanning probe microscopy, bulk crystallization, and analytical techniques, we show that the base and side chain work synergistically to reduce the rate of hematin crystallization. In contrast to general observations that modifiers retain their function upon segmentation, we show that the constituents do not act as modifiers. A systematic study of quinoline isomers and analogues shows how subtle rearrangement and removal of functional moieties can create effective constituents from previously ineffective modifiers, along with tuning their inhibitory modes of action. These findings highlight the importance of specific functional moieties in drug compounds, leading to an improved understanding of modifier-crystal interactions that could prove to be applicable to the design of new antimalarials. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

IL-4 function can be transferred to the IL-2 receptor by tyrosine containing sequences found in the IL-4 receptor alpha chain.

PubMed

Wang, H Y; Paul, W E; Keegan, A D

1996-02-01

IL-4 binds to a cell surface receptor complex that consists of the IL-4 binding protein (IL-4R alpha) and the gamma chain of the IL-2 receptor complex (gamma c). The receptors for IL-4 and IL-2 have several features in common; both use the gamma c as a receptor component, and both activate the Janus kinases JAK-1 and JAK-3. In spite of these similarities, IL-4 evokes specific responses, including the tyrosine phosphorylation of 4PS/IRS-2 and the induction of CD23. To determine whether sequences within the cytoplasmic domain of the IL-4R alpha specify these IL-4-specific responses, we transplanted the insulin IL-4 receptor motif (I4R motif) of the huIL-4R alpha to the cytoplasmic domain of a truncated IL-2R beta. In addition, we transplanted a region that contains peptide sequences shown to block Stat6 binding to DNA. We analyzed the ability of cells expressing these IL-2R-IL-4R chimeric constructs to respond to IL-2. We found that IL-4 function could be transplanted to the IL-2 receptor by these regions and that proliferative and differentiative functions can be induced by different receptor sequences.

Unexpectedly low mutation rates in beta-myosin heavy chain and cardiac myosin binding protein genes in Italian patients with hypertrophic cardiomyopathy.

PubMed

Roncarati, Roberta; Latronico, Michael V G; Musumeci, Beatrice; Aurino, Stefania; Torella, Annalaura; Bang, Marie-Louise; Jotti, Gloria Saccani; Puca, Annibale A; Volpe, Massimo; Nigro, Vincenzo; Autore, Camillo; Condorelli, Gianluigi

2011-11-01

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease. Fourteen sarcomeric and sarcomere-related genes have been implicated in HCM etiology, those encoding β-myosin heavy chain (MYH7) and cardiac myosin binding protein C (MYBPC3) reported as the most frequently mutated: in fact, these account for around 50% of all cases related to sarcomeric gene mutations, which are collectively responsible for approximately 70% of all HCM cases. Here, we used denaturing high-performance liquid chromatography followed by bidirectional sequencing to screen the coding regions of MYH7 and MYBPC3 in a cohort (n = 125) of Italian patients presenting with HCM. We found 6 MHY7 mutations in 9/125 patients and 18 MYBPC3 mutations in 19/125 patients. Of the three novel MYH7 mutations found, two were missense, and one was a silent mutation; of the eight novel MYBPC3 mutations, one was a substitution, three were stop codons, and four were missense mutations. Thus, our cohort of Italian HCM patients did not harbor the high frequency of mutations usually found in MYH7 and MYBPC3. This finding, coupled to the clinical diversity of our cohort, emphasizes the complexity of HCM and the need for more inclusive investigative approaches in order to fully understand the pathogenesis of this disease. Copyright © 2011 Wiley-Liss, Inc.

Formation of thermally induced aggregates of the soya globulin beta-conglycinin.

PubMed

Mills, E N; Huang, L; Noel, T R; Gunning, A P; Morris, V J

2001-06-11

The effect of ionic strength (I) on the formation of thermally induced aggregates by the 7S globular storage protein of soya, beta-conglycinin, has been studied using atomic force microscopy. Aggregates were only apparent when I> or =0.1, and had a fibrous appearance, with a height (diameter) of 8-11 nm. At high ionic strength (I=1.0) the aggregates appeared to associate into clumps. When aggregate formation was studied at I=0.2, it was clear that aggregation only began at temperatures above the main thermal transition for the protein at 75 degrees C, as determined by differential scanning calorimetry. This coincided with a small change in secondary structure, as indicated by circular dichroism spectroscopy, suggesting that a degree of unfolding was necessary for aggregation to proceed. Despite prolonged heating the size of the aggregates did not increase indefinitely, suggesting that certain beta-conglycinin isoforms were able to act as chain terminators. At higher protein concentrations (1% w/v) the linear aggregates appeared to form large macroaggregates, which may be the precursors of protein gel formation. The ability of beta-conglycinin to form such distinctive aggregates is discussed in relation to the presence of acidic inserts in certain of the beta-conglycinin subunits, which may play an important role in limiting aggregate length.

A program to generate simulated radioxenon beta–gamma data for concentration verification and validation and training exercises

DOE Office of Scientific and Technical Information (OSTI.GOV)

McIntyre, Justin I.; Schrom, Brian T.; Cooper, Matthew W.

2016-03-08

Abstract Several hundred simulated radioxenon beta-gamma data files were developed to assist in evaluating the performance and results from radioxenon concentration calculation analysis at the International Data Center (IDC) and other National Data Centers (NDC). PNNL developed a Beta-Gamma Simulator (BGSim) that incorporated GEANT-modeled data sets from radioxenon decay chains, as well as functionality to use nuclear detector-acquired data sets to create new beta-gamma spectra with varying amounts of background, 133Xe, 131mXe, 133mXe, 135Xe, and 222Rn and its decay products. The program has been implemented on a web-based applications platform and allows the user to create very specific data setsmore » that incorporate most of the operational parameters for the current beta-gamma systems deployed in the International Monitoring System (IMS) and the On-site Inspection (OSI) equipment. After an initial beta-gamma simulations program was developed, additional uses began to be identified for the program output: training sets of two-dimensional spectra for data analysts at the IDC and other NDC, spectra for exercises such as the Integrated Field Exercise 2014 (IFE14) held in Jordan at the Dead Sea, and testing new analysis methods and algorithms« less

[Study of signal transduction pathway in the expression of inflammatory factors stimulated by lipopolysaccharides from Porphyromonas endodontalis in osteoblasts].

PubMed

Yang, Di; Qiu, Li-hong; Li, Ren; Li, Zi-mu; Li, Chen

2010-04-01

To quantify the interleukin (IL)-1beta mRNA and IL-6 mRNA expression induced by lipopolysaccharides ([PS) extracted from Porphyromonoas endodontalis (P. endodontalis) in osteoblasts, and to relate P. endodontalis LPS to the bone resorptive pathogenesis in the lesions of chronic apical periodontitis. MG63 cells was pretreated with PD98059 or SB203580 for 1 h and then treated with P. endodontolis LPS for 6 h. The expression of IL-1beta mRNA and IL-6 mRNA were detected by reverse transcription polymerase chain reaction (RT-PCR) technique. The production of IL-1beta mRNA induced by P. endodontalis LPS decreased in osteoblasts pretreated with PD98059. Both of the production of IL-1beta mRNA and JL-6 mRNA induced by P. endodontalis LPS decreased in osteoblasts pretreated with SB203580. The synthesis of IL-1beta mRNA stimulated by Pendodontalis LPS in MG63 probably occur via extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen activated protein kinase (MAPK) signal transduction system. The synthesis of IL-6 mRNA stimulated by P.endodontalis LPS in MG63 probahly occur via p38MAPK signal transduction system.

Polymorphism of the MHC class II Eb gene determines the protection against collagen-induced arthritis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gonzalez-Gay, M.A.; Zanelli, E.; Krco, C.J.

1995-05-01

Collagen-induced arthritis (CIA) is an animal model of auto immune polyarthritis, sharing similarities with rheumatoid arthritis (RA). Paradoxally, susceptibility to mouse CIA is controlled by the H2A loci (DQ homologous) while RA is linked to HLA.DR genes (H2E homologous). We recently showed that the E{beta}{sup d} molecule prevents CIA development in susceptible H2{sup q} mice. We addressed the question of whether H2Eb polymorphism will influence CIA incidence as HLA.DRB1 polymorphism does in RA. In F{sub 1} mice, only H2Eb{sup d} and H2Eb{sup s} molecules showed protection. Using recombinant B10.RDD (Eb{sup d/b}) mice, we found that CIA protection was mediated bymore » the first domain of the E{beta}{sup d} molecule. Using peptides covering the third hypervariable region of the E{beta} chain, we found a perfect correlation between presentation of E{beta} peptides by the H2A{sup q} molecule and protection on CIA. Therefore, the mechanism by which H2Eb protects against CIA seems to rely on the affinity of E{beta} peptides for the H2A{sup q} molecule. 35 refs., 2 figs., 3 tabs.« less

Analysis of correlated domain motions in IgG light chain reveals possible mechanisms of immunological signal transduction.

PubMed

Król, Marcin; Roterman, Irena; Piekarska, Barbara; Konieczny, Leszek; Rybarska, Janina; Stopa, Barbara; Spólnik, Paweł

2005-05-15

It was shown experimentally that binding of a micelle composed of Congo red molecules to immunological complexes leads to the enhanced stability of the latter, and simultaneously prevents binding of a complement molecule (C1q). The dye binds in a cavity created by the removal of N-terminal polypeptide chain, as observed experimentally in a model system-immunoglobulin G (IgG) light chain dimer. Molecular Dynamics (MD) simulations of three forms of IgG light chain dimer, with and without the dye, were performed to investigate the role of N-terminal fragment and self-assembled ligand in coupling between V and C domains. Root-mean-square distance (RMSD) time profiles show that removal of N-terminal fragment leads to destabilization of V domain. A micelle composed of four self-assembled dye molecules stabilizes and fixes the domain. Analysis of root-mean-square fluctuation (RMSF) values and dynamic cross-correlation matrices (DCCM) reveals that removal of N-terminal fragment results in complete decoupling between V and C domains. Binding of self-assembled Congo red molecules improves the coupling, albeit slightly. The disruption of a small beta-sheet composed of N- and C-terminal fragments of the domain (NC sheet) is the most likely reason for the decoupling. Self-assembled ligand, bound in the place originally occupied by N-terminal fragment, is not able to take over the function of the beta-sheet. Lack of correlation of motions between residues in V and C domains denotes that light chain-Congo red complexes have hampered ability to transmit conformational changes between domains. This is a likely explanation of the lack of complement binding by immunological complexes, which bind Congo red, and supports the idea that the NC sheet is the key structural fragment taking part in immunological signal transduction. Copyright 2005 Wiley-Liss, Inc.

Hydrothermal synthesis, crystal structure and properties of 2-D and 3-D lanthanide sulfates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu Yan; Ding Shaohua; Zheng Xuefang

2007-07-15

Two new lanthanum sulfates DySO{sub 4}(OH) 1 and Eu{sub 2}(SO{sub 4}){sub 3}(H{sub 2}O){sub 8} 2 have been hydrothermally synthesized. The colorless crystals were characterized by IR, TGA, ICP and XRD. The structure was determined by single-crystal X-ray diffraction. 1 crystallizes with monoclinic symmetry, space group P2(1)/n [a=7.995(4) A, b=10.945(5) A, c=8.164(4) A, {alpha}=90{sup o}, {beta}=93.619(6){sup o}, {gamma}=90{sup o}, V=713.0(5) A{sup 3}, Z=8]. It displays a three-dimensional framework, based on the novel Dy-O chains connected by the sulfate groups through helical chains. 2 crystallizes with monoclinic symmetry, space group C2/c, [a=13.5605(17) A, b=6.7676(8) A, c=18.318(2) A, {alpha}=90{sup o}, {beta}=102.265(2){sup o}, {gamma}=90{supmore » o}, V=1642.7 (4) A{sup 3}, Z=4]. Its layered framework is attained by the europium atoms connected by the sulfate groups arranged in a helical manner. - Graphical abstract: Two new lanthanum sulfates DySO{sub 4}(OH) 1 and Eu{sub 2} (SO{sub 4}){sub 3} (H{sub 2}O){sub 8} 2 have been hydrothermally synthesized. The colorless crystals were characterized by IR, TGA, ICP and XRD. The structure was determined by single-crystal X-ray diffraction. It displays a three dimensional framework, based on the novel Dy-O chains connected by the sulfate groups through helical chains.« less

Involvement of activator protein 1 complexes in the epithelium-specific activation of the laminin gamma2-chain gene promoter by hepatocyte growth factor (scatter factor).

PubMed Central

Olsen, J; Lefebvre, O; Fritsch, C; Troelsen, J T; Orian-Rousseau, V; Kedinger, M; Simon-Assmann, P

2000-01-01

Laminin-5 is a trimer of laminin alpha3, beta3 and gamma2 chains that is found in the intestinal basement membrane. Deposition of the laminin gamma2 chain at the basement membrane is of great interest because it undergoes a developmental shift in its cellular expression. Here we study the regulatory elements that control basal and cytokine-activated transcriptional expression of the LAMC2 gene, which encodes the laminin gamma2 chain. By using transient transfection experiments we demonstrated the presence of constitutive and cytokine-responsive cis-elements. Comparison of the transcriptional activity of the LAMC2 promoter in the epithelial HT29mtx cells with that in small-intestinal fibroblastic cells (C20 cells) led us to conclude that two regions with constitutive epithelium-specific activity are present between positions -1.2 and -0.12 kb. This was further validated by transfections of primary foetal intestinal endoderm and mesenchyme. A 2.5 kb portion of the LAMC2 5' flanking region was equally responsive to PMA and hepatocyte growth factor (HGF), whereas it was less responsive to transforming growth factor beta1. A minimal promoter limited to the initial 120 bp upstream of the transcriptional start site maintained inducibility by PMA and HGF. This short promoter fragment contains two activator protein 1 (AP-1) elements and the 5'-most of these is a composite AP-1/Sp1 element. The 5'AP-1 element is crucial to the HGF-mediated activity of the promoter; analysis of interacting nuclear proteins demonstrated that AP-1 proteins containing JunD mediate the response to HGF. PMID:10749670

Processing of N-linked oligosaccharides from precursor- to mature-form herpes simplex virus type 1 glycoprotein gC.

PubMed Central

Serafini-Cessi, F; Dall'Olio, F; Pereira, L; Campadelli-Fiume, G

1984-01-01

Immature and mature forms of glycoprotein gC were purified by immunoadsorbent from herpes simplex virus type 1-infected BHK cells labeled with [3H]mannose for a 20-min pulse or for 11 h followed by a 3-h chase. The nature of N-asparagine-linked oligosaccharides carried by the immature form, pgC (molecular weight = 92,000), and the mature gC (molecular weight = 120,000) has been investigated. All pronase-digested glycopeptides of pgC were susceptible to endo-beta-N-acetylglucosaminidase H treatment; thus they have a high-mannose structure. Using thin-layer chromatography to separate endo-beta-N-acetylglucosaminidase H-cleaved oligosaccharides, polymannosyl chains of different sizes, ranging from Man9GlcNAc to Man5GlcNAc, were separated. The major components were Man8GlcNAc and Man7GlcNAc, suggesting that pgC labeled in a 20-min pulse represents the form of glycoprotein already routed to the Golgi apparatus. Analysis of glycopeptides of mature gC showed that the majority (95%) of N-linked glycans were converted to complex-type glycans. Ion-exchange chromatography and affinity chromatography on concanavalin A-Sepharose and leucoagglutinin-agarose revealed that diantennary and triantennary glycans predominated, whereas tetrantennary chains were not present. Parts of the di- and triantennary chains were not fully sialylated. The high heterogeneity of complex-type chains found in mature gC may be related to the high number of N-glycosylation sites of the glycoprotein as predicted by DNA sequencing studies (Frink et al., J. Virol. 45:634-647, 1983). Images PMID:6088806

Cerebrospinal Fluid and Neuroimaging Biomarker Abnormalities Suggest Early Neurological Injury in a Subset of Individuals During Primary HIV Infection

PubMed Central

Peluso, Michael J.; Meyerhoff, Dieter J.; Price, Richard W.; Peterson, Julia; Lee, Evelyn; Young, Andrew C.; Walter, Rudy; Fuchs, Dietmar; Brew, Bruce J.; Cinque, Paola; Robertson, Kevin; Hagberg, Lars; Zetterberg, Henrik; Gisslén, Magnus; Spudich, Serena

2013-01-01

Background. Cerebrospinal fluid (CSF) and neuroimaging abnormalities demonstrate neuronal injury during chronic AIDS, but data on these biomarkers during primary human immunodeficiency virus (HIV) infection is limited. Methods. We compared CSF concentrations of neurofilament light chain, t-tau, p-tau, amyloid precursor proteins, and amyloid-beta 42 in 92 subjects with primary HIV infection and 25 controls. We examined relationships with disease progression and neuroinflammation, neuropsychological testing, and proton-magnetic resonance spectroscopy (MRS)–based metabolites. Results. Neurofilament light chain was elevated in primary HIV infection compared with controls (P = .0004) and correlated with CSF neopterin (r = 0.38; P = .0005), interferon gamma-induced protein 10 (r = 0.39; P = .002), white blood cells (r = 0.32; P = .004), protein (r = 0.59; P < .0001), and CSF/plasma albumin ratio (r = 0.60; P < .0001). Neurofilament light chain correlated with decreased N-acteylaspartate/creatine and glutamate/creatine in the anterior cingulate (r = −0.35, P = .02; r = −0.40, P = .009, respectively), frontal white matter (r = −0.43, P = .003; r = −0.30, P = .048, respectively), and parietal gray matter (r = −0.43, P = .003; r = −0.47, P = .001, respectively). Beta-amyloid was elevated in the primary infection group (P = .0005) and correlated with time infected (r = 0.34; P = .003). Neither marker correlated with neuropsychological abnormalities. T-tau and soluble amyloid precursor proteins did not differ between groups. Conclusions. Elevated neurofilament light chain and its correlation with MRS-based metabolites suggest early neuronal injury in a subset of participants with primary HIV infection through mechanisms involving central nervous system inflammation. PMID:23460748

Butyrate modulates TGF-beta1 generation and function: potential renal benefit for Acacia(sen) SUPERGUM (gum arabic)?

PubMed

Matsumoto, N; Riley, S; Fraser, D; Al-Assaf, S; Ishimura, E; Wolever, T; Phillips, G O; Phillips, A O

2006-01-01

Anecdotal evidence suggests that high fibre supplementation of dietary intake may have health benefits in renal disease related to alterations in circulating levels of short-chain fatty acids. The aim of the study was to examine the hypothesis that dietary manipulation may increase serum butyrate and thus have potential beneficial effects in renal disease. We examined the effect of dietary supplementation with a gum arabic sample of standardized molecular characteristics, Acacia(sen) SUPERGUM EM2 (SUPERGUM), on systemic levels of butyrate in normal human subjects. In an in vitro study, we also examined the potential role of butyrate in modifying the generation of the profibrotic cytokine transforming growth factor-beta (TGF-beta1) by renal epithelial cells. Following 8 weeks of dietary supplementation with 25 g/day of SUPERGUM, there was a two-fold increase in serum butyrate (n=7, P=0.03). In vitro work demonstrated that exposure of renal epithelial cells to elevated concentrations of butyrate suppressed both basal and stimulated TGF-beta1 synthesis. The action of butyrate was mediated by suppression of the extracellular signal-regulated kinase/mitogen-activated protein kinase signalling pathway. In addition, butyrate exposures reduced the response of renal epithelial cells to TGF-beta1 as assessed by luciferase activity of a TGF-beta-responsive reporter construct. Attenuation of TGF-beta1 signalling was associated with reduced phosphorylation of Smad 3 and decreased trafficking of TGF-beta1 receptors into signalling, non-lipid raft-associated membrane fractions. In conclusion, the data demonstrate that dietary supplementation with SUPERGU increased serum butyrate, which at least in vitro has beneficial effects on renal pro-fibrotic cytokine generation.

Effect of doxycycline on transforming growth factor-beta-1-induced matrix metalloproteinase 2 expression, migration, and collagen contraction in nasal polyp-derived fibroblasts.

PubMed

Shin, Jae-Min; Park, Joo-Hoo; Kang, Byungjin; Lee, Seoung-Ae; Park, Il-Ho; Lee, Heung-Man

2016-11-01

It is well known that doxycycline has antibacterial and anti-inflammatory effects. In this study, we aimed to investigate the effects of doxycycline on the transforming growth factor (TGF) beta 1-induced matrix metalloproteinase (MMP) 2 expression, migration, and collagen contraction, and to determine its molecular mechanism on nasal polyp-derived fibroblasts (NPDF). NPDFs were isolated from the nasal polyps of six patients. Doxycycline was used to pretreat TGF-beta-1-induced NPDFs and ex vivo organ cultures of nasal polyps. Cytotoxicity was evaluated by using a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. Smad2/3 is one of the major transcription factors of TGF-beta signaling. The expression levels of MMP2 and Smad2/3 were measured by using Western blotting, reverse transcription-polymerase chain reaction, and immunofluorescence staining. The enzymic activity of MMP2 was analyzed by using gelatin zymography. Fibroblast migration was evaluated by using transwell migration assays. Contractile activity was measured by a collagen gel contraction assay. The expression level of MMP2 in nasal polyp tissues increased in comparison with inferior turbinate tissues. TGF-beta-1-induced NPDFs were not affected by doxycycline (0-40 μg/mL). The expression levels of MMP2 and activation of Smad2/3 in TGF-beta-1-induced NPDFs and in organ cultures of nasal polyps were significantly downregulated with doxycycline pretreatment. Doxycycline also reduced TGF-beta-1-induced fibroblast migration and collagen contraction in NPDFs. Doxycycline inhibited TGF-beta-1-induced MMP2 expression, migration, and collagen contraction via the Smad2/3 signal pathways in NPDFs.

Antibacterial properties of (2,3)-alpha- and (2,3)-beta-methylene analogs of penicillin G.

PubMed Central

Christenson, J G; Pruess, D L; Talbot, M K; Keith, D D

1988-01-01

The penam nucleus can assume two conformations; these are designated open and closed. The synthetic (2,3)-alpha- and (2,3)-beta-methylenepenams can be regarded as analogs of the open and closed conformations, respectively. It has been shown that the beta-methylenepenams are essentially inactive, suggesting that the closed conformation of penams is also inactive. In this study, we investigated a series of beta-lactams, all of which contained phenylacetamido side chains: penicillin G, the (2,3)-alpha- and (2,3)-beta-methylenepenams, and the 3-acetoxymethyl- and 3-methylcephalosporins. The alpha-methylenepenam and penicillin G were the most active compounds, while the beta-methylene isomer was only poorly active. Results with permeability mutants suggested that the alpha-methylene compound penetrated the outer membrane somewhat more readily than penicillin G did. The intrinsic potency of the alpha-methylenepenam appeared to be similar to that of penicillin G, on the basis of their affinities for penicillin-binding proteins and their abilities to inhibit peptidoglycan synthesis in ether-permeabilized Escherichia coli, while the beta-methylene analog had very poor intrinsic potency. The alpha-methylene analog was about 10-fold more efficient (Vmax/Km) than penicillin G as a substrate for the cephalosporinases from Enterobacter cloacae and Proteus vulgaris, but it was about 40-fold less efficient with penicillinase from Staphylococcus aureus. These results strongly support the hypothesis that the active conformation of penams is the open conformation and suggest that the position in space of the carboxyl group relative to the beta-lactam carbonyl is an important determinant of cephalosporinlike character, as distinct from penicillinlike character. Images PMID:3190190

Defining the carbohydrate specificities of Abrus precatorius agglutinin as T (Gal beta 1----3GalNAc) greater than I/II (Gal beta 1----3/4GlcNAc).

PubMed

Wu, A M; Lin, S R; Chin, L K; Chow, L P; Lin, J Y

1992-09-25

The combining site of the nontoxic carbohydrate binding protein (Abrus precatorius agglutinin, APA) purified from the needs of Abrus precatorius (Jequirity bean), was studied by quantitative precipitin and precipitin-inhibition assays. Of 26 glycoproteins and polysaccharides tested, all, except sialic acid-containing glycoproteins and desialized ovine salivary glycoproteins, reacted strongly with the lectin, and precipitated over 70% of the lectin added, indicating that APA has a broad range of affinity and recognizes (internal) Gal beta 1----sequences of carbohydrate chains. The strong reaction with desialized porcine and rat salivary glycoproteins as well as pneumococcus type XIV polysaccharide suggests that APA has affinity for one or more of the following carbohydrate sequences: Thomsen-Friedenreich (T, Gal beta 1----3GalNAc), blood group precursor type I and/or type II (Gal beta 1----3/4GlcNAc) disaccharide determinants of complex carbohydrates. Among the oligosaccharides tested, the T structure was the best inhibitor; it was 2.4 and 3.2 times more active than type II and type I sequences, respectively. The blood group I Ma-active trisaccharide, Gal beta 1----4GlcNAc beta 1----6Gal, was about as active as the corresponding disaccharide (II). From the above results, we conclude that the size of the combining site of the A. precatorius agglutinin is probably as large as a disaccharide and most strongly complementary to the Gal beta 1----3GalNAc (T determinant) sequence. The carbohydrate specificities of this lectin will be further investigated once the related oligosaccharide structures become available.

Decreased hepatocyte membrane potential differences and GABAA-beta3 expression in human hepatocellular carcinoma.

PubMed

Minuk, Gerald Y; Zhang, Manna; Gong, Yuewen; Minuk, Leonard; Dienes, Hans; Pettigrew, Norman; Kew, Michael; Lipschitz, Jeremy; Sun, Dongfeng

2007-03-01

To determine whether hepatocyte membrane potential differences (PDs) are depolarized in human HCC and whether depolarization is associated with changes in GABAA receptor expression, hepatocyte PDs and gamma-aminobutyric acid (GABA)A receptor messenger RNA (mRNA) and protein expression were documented in HCC tissues via microelectrode impalement, real-time reverse-transcriptase polymerase chain reaction, and Western blot analysis, respectively. HCC tissues were significantly depolarized (-19.8+/-1.3 versus -25.9+/-3.2 mV, respectively [P<0.05]), and GABAA-beta3 expression was down-regulated (GABAA-beta3 mRNA and protein expression in HCC; 5,693+/-1,385 and 0.29+/-0.11 versus 11,046+/-4,979 copies/100 mg RNA and 0.62+/-0.16 optical density in adjacent tumor tissues, respectively [P=0.002 and P<0.0001, respectively]) when compared with adjacent nontumor tissues. To determine the physiological relevance of the down-regulation, human malignant hepatocytes deficient in GABAA-beta3 receptor expression (Huh-7 cells) were transfected with GABAA-beta3 complementary DNA (cDNA) or vector alone and injected into nu/nu nude mice (n=16-17 group). Tumors developed after a mean (+/-SD) of 51+/-6 days (range: 41-60 days) in 7/16 (44%) mice injected with vector-transfected cells and 70+/-12 days (range: 59-86 days) in 4/17 (24%) mice injected with GABAA-beta3 cDNA-transfected cells (P<0.005). The results of this study indicate that (1) human HCC tissues are depolarized compared with adjacent nontumor tissues, (2) hepatic GABAA-beta3 receptor expression is down-regulated in human HCC, and (3) restoration of GABAA-beta3 receptor expression results in attenuated in vivo tumor growth in nude mice.

Inflammation induction of Dickkopf-1 mediates chondrocyte apoptosis in osteoarthritic joint.

PubMed

Weng, L-H; Wang, C-J; Ko, J-Y; Sun, Y-C; Su, Y-S; Wang, F-S

2009-07-01

Dysregulated Wnt signaling appears to modulate chondrocyte fate and joint disorders. Dickkopf-1 (DKK1) regulates the pathogenesis of skeletal tissue by inhibiting Wnt actions. This study examined whether DKK1 expression is linked to chondrocyte fate in osteoarthritis (OA). Articular cartilage specimens harvested from nine patients with knee OA and from six controls with femoral neck fracture were assessed for DKK1, interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), Bad, Bax, Bcl2 and caspase-3 expression by real time-polymerase chain reaction (RT-PCR) and immunohistochemistry. Apoptotic chondrocytes were detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labelling (TUNEL) and 4', 6-dianidino-2-phenylindole dihydrochloride (DAPI) staining. Human chondrocyte cultures were treated with recombinant IL-1beta and monoclonal DKK1 antibody to determine whether DKK1 impairs chondrocyte survival. Expression of DKK1 correlated with inflammatory cytokine levels (IL-1beta and TNF-alpha expressions), proapoptosis regulators (Bad and caspase-3 expressions) and TUNEL staining in OA cartilage tissues. The IL-1beta induced expressions of DKK1, Bax, Bad and caspase-3-dependent apoptosis of chondrocyte cultures. Neutralization of DKK1 by monoclonal DKK1 antibody significantly abrogated IL-1beta-mediated caspase-3 cleavage and apoptosis and reversed chondrocyte proliferation. Recombinant DKK1 treatment impaired chondrocyte growth and promoted apoptosis. By suppressing nuclear beta-catenin accumulation and Akt phosphorylation, DKK1 mediated IL-1beta promotion of chondrocyte apoptosis. Chondrocyte apoptosis correlates with joint OA. Expression of DKK1 contributes to cartilage deterioration and is a potent factor in OA pathogenesis. Attenuating DKK1 may reduce cartilage deterioration in OA.

Role for transforming growth factor-beta1 in alport renal disease progression.

PubMed

Sayers, R; Kalluri, R; Rodgers, K D; Shield, C F; Meehan, D T; Cosgrove, D

1999-11-01

Alport syndrome results from mutations in either the alpha3(IV), alpha4(IV), or alpha5(IV) collagen genes. The disease is characterized by a progressive glomerulonephritis usually associated with a high-frequency sensorineural hearing loss. A mouse model for an autosomal form of Alport syndrome [collagen alpha3(IV) knockout] was produced and characterized. In this study, the model was exploited to demonstrate a potential role for transforming growth factor-beta1 (TGF-beta1) in Alport renal disease pathogenesis. Kidneys from normal and Alport mice, taken at different stages during the course of renal disease progression, were analyzed by Northern blot, in situ hybridization, and immunohistology for expression of TGF-beta1 and components of the extracellular matrix. Normal and Alport human kidney was examined for TGF-beta1 expression using RNase protection. The mRNAs encoding TGF-beta1 (in both mouse and human), entactin, fibronectin, and the collagen alpha1(IV) and alpha2(IV) chains were significantly induced in total kidney as a function of Alport renal disease progression. The induction of these specific mRNAs was observed in the glomerular podocytes of animals with advanced disease. Type IV collagen, laminin-1, and fibronectin were markedly elevated in the tubulointerstitium at 10 weeks, but not at 6 weeks, suggesting that elevated expression of specific mRNAs on Northern blots reflects events associated with tubulointerstitial fibrosis. The concomitant accumulation of mRNAs encoding TGF-beta1 and extracellular matrix components in the podocytes of diseased kidneys may reflect key events in Alport renal disease progression. These data suggest a role for TGF-beta1 in both glomerular and tubulointerstitial damage associated with Alport syndrome.