Comparison of Chemical Binding to Recombinant Fathead minnow and Human Estrogen Receptor alpha (ERα) in Whole Cell and Cell-Free Assay Systems.

EPA Science Inventory

Our objectives were to assess whether binding of chemicals differs significantly between recombinant estrogen receptors from fathead minnow (fhERα) and human (hERα) and to evaluate the performance of these receptors using two different in vitro assay systems: a COS whole cell bin...

Sensitization to the mammalian oligosaccharide galactose-alpha-1,3-galactose (alpha-gal): experience in a Flemish case series.

PubMed

Ebo, D G; Faber, M; Sabato, V; Leysen, J; Gadisseur, A; Bridts, C H; De Clerck, L S

2013-01-01

Recent observations have disclosed that the galactose-alpha (1,3)-galactose (alpha-gal) moiety of non-primate glycoproteins can constitute a target for meat allergy. To describe adults with allergic reactions to mammalian meat, dairy products and gelatin. To investigate whether patients could demonstrate sensitization to activated recombinant human coagulation factor VII ectapog alpha that is produced in baby hamster kidney cells. Ten adults with mammalian meat, dairy products and gelatin allergies were examined using quantification of specific IgE and/or skin prick test for red meat, milk, milk components, gelatin, cetuximab and eptacog alpha. Most patients demonstrate quite typical clinical histories and serological profiles, with anti-alpha-gal titers varying from less than 1% to over 25% of total serum IgE. All patients demonstrate negative sIgE for gelatin, except the patient with a genuine gelatin allergy. All patients also demonstrated a negative sIgE to recombinant milk components casein, lactalbumin and lactoglobulin. Specific IgE to eptacog was positive in 5 out of the 9 patients sensitized to alpha-gal and none of the 10 control individuals. This series confirms the importance of the alpha-gal carbohydrate moiety as a potential target for allergy to mammalian meat, dairy products and gelatin (oral, topical or parenteral) in a Flemish population of meat allergic adults. It also confirms in vitro tests to mammalian meat generally to be more reliable than mammalian meat skin tests, but that diagnosis can benefit from skin testing with cetuximab. Specific IgE to gelatin is far too insensitive to diagnose alphaa-gal related gelatin allergy. IgE binding studies indicate a potential risk of alpha-gal-containing human recombinant proteins produced in mammalians.

Effects of selenium on the structure and function of recombinant human S-adenosyl-L-methionine dependent arsenic (+3 oxidation state) methyltransferase in E. coli.

PubMed

Geng, Zhirong; Song, Xiaoli; Xing, Zhi; Geng, Jinlong; Zhang, Sichun; Zhang, Xinrong; Wang, Zhilin

2009-05-01

The effects of Se(IV) on the structure and function of recombinant human arsenic (+3 oxidation state) methyltransferase (AS3MT) purified from the cytoplasm of Escherichia coli were studied. The coding region of human AS3MT complementary DNA was amplified from total RNA extracted from HepG2 cell by reverse transcription PCR. Soluble and active human AS3MT was expressed in the E. coli with a Trx fusion tag under a lower induction temperature of 25 degrees C. Spectra (UV-vis, circular dichroism, and fluorescence) were first used to probe the interaction of Se(IV) and recombinant human AS3MT and the structure-function relationship of the enzyme. The recombinant human AS3MT had a secondary structure of 29.0% alpha-helix, 23.9% beta-pleated sheet, 17.9% beta-turn, and 29.2% random coil. When Se(IV) was added, the content of the alpha-helix did not change, but that of the beta-pleated sheet increased remarkably in the conformation of recombinant human AS3MT. Se(IV) inhibited the enzymatic methylation of inorganic As(III) in a concentration-dependent manner. The IC(50) value for Se(IV) was 2.38 muM. Double-reciprocal (1/V vs. 1/[inorganic As(III)]) plots showed Se(IV) to be a noncompetitive inhibitor of the methylation of inorganic As(III) by recombinant human AS3MT with a K (i) value of 2.61 muM. We hypothesized that Se(IV) interacts with the sulfhydryl group of cysteine(s) in the structural residues rather than the cysteines of the active site (Cys156 and Cys206). When Se(IV) was combined with cysteine(s) in the structural residues, the conformation of recombinant human AS3MT changed and the enzymatic activity decreased. Considering the quenching of tryptophan fluorescence, Cys72 and/or Cys226 are deduced to be primary targets for Se(IV).

Genetic recombination as a major cause of mutagenesis in the human globin gene clusters.

PubMed

Borg, Joseph; Georgitsi, Marianthi; Aleporou-Marinou, Vassiliki; Kollia, Panagoula; Patrinos, George P

2009-12-01

Homologous recombination is a frequent phenomenon in multigene families and as such it occurs several times in both the alpha- and beta-like globin gene families. In numerous occasions, genetic recombination has been previously implicated as a major mechanism that drives mutagenesis in the human globin gene clusters, either in the form of unequal crossover or gene conversion. Unequal crossover results in the increase or decrease of the human globin gene copies, accompanied in the majority of cases with minor phenotypic consequences, while gene conversion contributes either to maintaining sequence homogeneity or generating sequence diversity. The role of genetic recombination, particularly gene conversion in the evolution of the human globin gene families has been discussed elsewhere. Here, we summarize our current knowledge and review existing experimental evidence outlining the role of genetic recombination in the mutagenic process in the human globin gene families.

Differential effects of LPS, IFN-gamma and TNF alpha on the secretion of lysozyme by individual human mononuclear phagocytes: relationship to cell maturity.

PubMed Central

Lewis, C E; McCarthy, S P; Lorenzen, J; McGee, J O

1990-01-01

Human mononuclear phagocytes can be activated to perform a variety of complex functions by exposure to the immunomodulators, lipopolysaccharide (LPS), interferon-gamma (IFN-gamma) and tumour necrosis factor alpha (TNF alpha). Although such activation often involves the release of various cytokines by monocytes and macrophages, little is known of the effects of such signals on their secretion of lysozyme (LZM). In this study, a reverse haemolytic plaque assay for LZM secretion is coupled with immunocytochemistry for the pan macrophage (CD68) marker, EBM/11. This enabled the direct effects of LPS, IFN-gamma and TNF alpha on the secretion of LZM by individual, immunoidentified human mononuclear phagocytes to be investigated. The overall secretion of this peptide by populations of freshly isolated or 3-day cultured monocytes was augmented by exposure for 6 hr to bacterial LPS, recombinant human IFN-gamma or recombinant human TNF alpha. Extension of the culture period for monocytes from 3 to 7 days prior to use in the assay resulted in higher levels of LZM secretion, which could be further increased by TNF alpha but not by LPS or IFN-gamma. Individual peritoneal macrophages activated by inflammation in vivo were uniform in their augmented LZM responses to TNF alpha, but a small subpopulation of human peritoneal macrophages, which may represent younger 'inflammatory' exudate macrophages, was seen to be preferentially responsive to the LZM-stimulating effects of LPS and IFN-gamma. These studies suggest that (i) secretion of LZM by human mononuclear phagocytes can be regulated by LPS and IFN-gamma, although the effects of these agents may be dependent upon the state of maturation and/or differentiation of the cells, and (ii) TNF alpha is a potent stimulant of LZM secretion by monocytes and macrophages irrespective of cell maturity. Images Figure 1 Figure 1 PMID:2107146

Identity of the segment of human complement C8 recognized by complement regulatory protein CD59.

PubMed

Lockert, D H; Kaufman, K M; Chang, C P; Hüsler, T; Sodetz, J M; Sims, P J

1995-08-25

CD59 antigen is a membrane glycoprotein that inhibits the activity of the C5b-9 membrane attack complex (MAC), thereby protecting human cells from lysis by human complement. The inhibitory function of CD59 derives from its capacity to interact with both the C8 and C9 components of MAC, preventing assembly of membrane-inserted C9 polymer. MAC-inhibitory activity of CD59 is species-selective and is most effective when both C8 and C9 derive from human or other primate plasma. Rabbit C8 and C9, which can substitute for human C8 and C9 in MAC, mediate virtually unrestricted lysis of human cells expressing CD59. In order to identify the segment of human C8 that is recognized by CD59, recombinant peptides containing human or rabbit C8 sequence were expressed in Escherichia coli and purified. CD59 was found to specifically bind to a peptide corresponding to residues 334-385 of the human C8 alpha-subunit, and to require a disulfide bond between Cys345 and Cys369. No specific binding was observed to the corresponding sequence from rabbit C8 alpha (residues 334-386). To obtain functional evidence that this segment of human C8 alpha is selectively recognized by CD59, recombinant C8 proteins were prepared by co-transfecting COS-7 cells with human/rabbit chimeras of the C8 alpha cDNA, and cDNAs encoding the C8 beta and C8 gamma chains. Hemolytic activity of MAC formed with chimeric C8 was analyzed using target cells reconstituted with CD59. These experiments confirmed that CD59 recognizes a conformationally sensitive epitope that is within a segment of human C8 alpha internal to residues 320-415. Our data also suggest that optimal interaction of CD59 with this segment of human C8 alpha is influenced by N-terminal flanking sequence in C8 alpha and by human C8 beta, but is unaffected by C8 gamma.

Cloning, sequence, and expression of a blood group B active recombinant alpha-D-galactosidase from pinto bean (Phaseolus vulgaris).

PubMed

Davis, M O; Hata, D J; Johnson, S A; Jones, D E; Harmata, M A; Evans, M L; Walker, J C; Smith, D S

1997-07-01

A cDNA encoding pinto bean alpha-D-galactosidase [E.C. 3.2.1.22] was obtained by amplification of cDNA using highly conserved sequences found in eucaryotic alpha-D-galactosidases. Subsequently a full length Phaseolus cDNA clone was obtained that is 1537 nt long and contains untranslated 5' and 3' sequences. The nucleotide sequence of the cDNA has a high degree of homology with other eucaryotic alpha-D-galactosidase genes. The recombinant alpha-D-galactosidase (rGal) was expressed in Escherichia coli and purified by ion exchange and affinity chromatography. Purified rGal was homogeneous by SDS-PAGE and had relative masses of 40.1 and 45.4 kDa under nonreducing and reducing conditions, respectively. The N-terminal sequence of the expressed protein contained the sequence GNGLGQTPPMG corresponding to that deduced from the cDNA sequence. The native molecular weight for rGal was determined to be 32.18 kDa by Sephacryl S-200 chromatography. The specific activity of the rGal was 349 mu moles of PNP-alpha-D-galactopyranoside hydrolyzed per mg of pure rGal per min. rGal was highly specific for alpha-D-galactosyl residues and degraded B oligosaccharide. No detectable hemagglutinin or protease activity was present in the preparations. Furthermore, rGal was active against the blood group B antigen on native human erythrocytes in cell suspension assays. The only detectable RBC phenotypic change was loss of the B and P1 epitopes. Recombinant Phaseolus vulgaris alpha-D-galactosidase may have useful biotechnical applications in the potential mass production of enzymatically converted, universally transfusable type O RBCs. alpha-D-galactosidase [E.C. 3.2.1.22] has been purified from a variety of procaryotic and eucaryotic species. Most alpha-D-galactosidases have similar low molecular weight substrate specificities, but activity against high molecular weight substrates is variable. Terminal alpha-D-galactoside residues are present in glycoproteins and glycolipids. Some alpha-D-galactosidases have activity against alpha-D-galactosyl residues on cell membrane glycoconjugates. Glycosidases with this property are useful for carbohydrate structural studies and biotechnical applications. Enzymes free of other glycosidase activities with activity near neutral pH are particularly useful for membrane modification studies on native cells. Complex sugar chains in glycolipids and glycoproteins have often been implicated in the growth and development of eucaryotes. In particular, complex sugar chains play an important role in the recognition of self in the immune system. Some alpha-D-galactosidases can modify certain carbohydrate membrane epitopes, thereby modulating the immune response. For example, the blood group B epitope expressed on erythrocytes contains a terminal alpha-D-galactosyl residue. Individuals lacking this antigen produce naturally occurring complement fixing antibodies to the B epitope. Hydrolysis of this terminal saccharide destroys the antigenic activity of the B determinant producing H antigen (blood type O) on erythrocytes. Only rare individuals produce clinically significant antibodies to the H antigen, and therefore, type O red blood cells are "universally" compatible and in great demand. Dhar purified alpha-D-galactosidase isozymes from Phaseolus vulgaris and characterized their activity. To our knowledge, our laboratory, in a brief report, is the first to describe the cloning of the gene and the use of recombinant enzyme for seroconverting blood type B to O cells. This paper describes the cloning, sequence, expression, purification, and characterization of recombinant alpha-D-galactosidase. Activity of the recombinant enzyme on the native human erythrocyte blood group B epitope is shown.

Treatment of inflammatory airway disease in young standardbreds with interferon alpha

PubMed Central

2004-01-01

Abstract The effect of oral treatment with natural or recombinant human interferon alpha (HIA) on inflammatory airway disease in young standardbreds was assessed in a double-blind, randomized clinical trial. A total of 34 horses with nasal discharge, excess mucus in the trachea, and a persistent cough of at least 2 weeks’ duration that interfered with training completed the trial. Horses were rested for 1 week and received oral treatment with either a saline placebo, recombinant human interferon alpha (rHIA; 90 U/horse/day), or natural human interferon alpha (nHIA: 50 U/horse/day) for 5 days. There was a significant decline in nasal discharge and cough scores in all groups and the apparent response rate was similar. However, significantly fewer horses relapsed within 2 weeks once treatment was ceased when interferon rather than placebo was used (P = 0.012). Seventeen of 22 horses treated with rHIA or nHIA were cough-free 4 weeks after treatment, compared with only 4 of 12 after treatment with the placebo. Treatment with oral interferon is a useful adjunct to rest in standardbreds with inflammatory airway disease. PMID:15317391

[Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].

PubMed

Inoue, Masaharu; Kikuchi, Maho; Komoriya, Tomoe; Watanabe, Kunitomo; Kouno, Hideki

2007-01-01

Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.

Preliminary in vivo efficacy studies of a recombinant rhesus anti-alpha(4)beta(7) monoclonal antibody.

PubMed

Pereira, L E; Onlamoon, N; Wang, X; Wang, R; Li, J; Reimann, K A; Villinger, F; Pattanapanyasat, K; Mori, K; Ansari, A A

2009-01-01

Recent findings established that primary targets of HIV/SIV are lymphoid cells within the gastrointestinal (GI) tract. Focus has therefore shifted to T-cells expressing alpha(4)beta(7) integrin which facilitates trafficking to the GI tract via binding to MAdCAM-1. Approaches to better understand the role of alpha(4)beta(7)+ T-cells in HIV/SIV pathogenesis include their depletion or blockade of their synthesis, binding and/or homing capabilities in vivo. Such studies can ideally be conducted in rhesus macaques (RM), the non-human primate model of AIDS. Characterization of alpha(4)beta(7) expression on cell lineages in RM blood and GI tissues reveal low densities of expression by NK cells, B-cells, naïve and TEM (effector memory) T-cells. High densities were observed on TCM (central memory) T-cells. Intravenous administration of a single 50mg/kg dose of recombinant rhesus alpha(4)beta(7) antibody resulted in significant initial decline of alpha(4)beta(7)+ lymphocytes and sustained coating of the alpha(4)beta(7) receptor in both the periphery and GI tissues.

Ancient roots for polymorphism at the HLA-DQ. alpha. locus in primates

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

Gyllensten, U.B.; Erlich, H.A.

1989-12-01

The genes encoding the human histocompatibility antigens (HLA) exhibit a remarkable degree of polymorphism as revealed by immunologic and molecular analyses. This extensive sequence polymorphism either may have been generated during the lifetime of the human species or could have arisen before speciation and been maintained in the contemporary human population by selection or, possibly, by genetic drift. These two hypotheses were examined using the polymerase chain reaction method to amplify polymorphic sequences from the DQ{alpha} locus, as well as the DX{alpha} locus, an homologous but nonexpressed locus, in a series of primates that diverged at known times. In general,more » the amino acid sequence of a specific human DQ{alpha} allelic type is more closely related to its chimpanzee or gorilla counterpart than to other human DQ{alpha} alleles. Phylogenetic analysis of the silent nucleotide position changes shows that the similarity of allelic types between species is due to common ancestry rather than convergent evolution. Thus, most of the polymorphism at the DQ{alpha} locus in the human species was already present at least 5 million years ago in the ancestral species that gave rise to the chimpanzee, gorilla, and human lineages. However, one of the DQ{alpha} alleles may have arisen after speciation by recombination between two ancestral alleles.« less

Lentiviral gene therapy of murine hematopoietic stem cells ameliorates the Pompe disease phenotype.

PubMed

van Til, Niek P; Stok, Merel; Aerts Kaya, Fatima S F; de Waard, Monique C; Farahbakhshian, Elnaz; Visser, Trudi P; Kroos, Marian A; Jacobs, Edwin H; Willart, Monique A; van der Wegen, Pascal; Scholte, Bob J; Lambrecht, Bart N; Duncker, Dirk J; van der Ploeg, Ans T; Reuser, Arnold J J; Verstegen, Monique M; Wagemaker, Gerard

2010-07-01

Pompe disease (acid alpha-glucosidase deficiency) is a lysosomal glycogen storage disorder characterized in its most severe early-onset form by rapidly progressive muscle weakness and mortality within the first year of life due to cardiac and respiratory failure. Enzyme replacement therapy prolongs the life of affected infants and supports the condition of older children and adults but entails lifelong treatment and can be counteracted by immune responses to the recombinant enzyme. We have explored the potential of lentiviral vector-mediated expression of human acid alpha-glucosidase in hematopoietic stem cells (HSCs) in a Pompe mouse model. After mild conditioning, transplantation of genetically engineered HSCs resulted in stable chimerism of approximately 35% hematopoietic cells that overexpress acid alpha-glucosidase and in major clearance of glycogen in heart, diaphragm, spleen, and liver. Cardiac remodeling was reversed, and respiratory function, skeletal muscle strength, and motor performance improved. Overexpression of acid alpha-glucosidase did not affect overall hematopoietic cell function and led to immune tolerance as shown by challenge with the human recombinant protein. On the basis of the prominent and sustained therapeutic efficacy without adverse events in mice we conclude that ex vivo HSC gene therapy is a treatment option worthwhile to pursue.

alpha-Mannosidosis in the guinea pig: cloning of the lysosomal alpha-mannosidase cDNA and identification of a missense mutation causing alpha-mannosidosis.

PubMed

Berg, Thomas; Hopwood, John J

2002-03-16

alpha-Mannosidosis is a lysosomal storage disorder caused by deficient activity of the lysosomal alpha-mannosidase. We report here the sequencing and expression of the lysosomal alpha-mannosidase cDNA from normal and alpha-mannosidosis guinea pigs. The amino acid sequence of the guinea pig enzyme displayed 82-85% identity to the lysosomal alpha-mannosidase in other mammals. The cDNA of the alpha-mannosidosis guinea pig contained a missense mutation, 679C>T, leading to substitution of arginine by tryptophan at amino acid position 227 (R227W). The R227W allele segregated with the alpha-mannosidosis genotype in the guinea pig colony and introduction of R227W into the wild-type sequence eliminated the production of recombinant alpha-mannosidase activity in heterologous expression studies. Furthermore, the guinea pig mutation has been found in human patients. Our results strongly indicate that the 679C>T mutation causes alpha-mannosidosis and suggest that the guinea pig will be an excellent model for investigation of pathogenesis and evaluation of therapeutic strategies for human alpha-mannosidosis.

Enzyme replacement therapy in alpha-mannosidosis guinea-pigs.

PubMed

Crawley, Allison C; King, Barbara; Berg, Thomas; Meikle, Peter J; Hopwood, John J

2006-01-01

alpha-Mannosidosis is a lysosomal storage disorder caused by deficient activity of lysosomal alpha-mannosidase and is characterised by massive accumulation of mannose-containing oligosaccharides in affected individuals. Patients develop behaviour and learning difficulties, skeletal abnormalities, immune deficiency and hearing impairment. Disease in alpha-mannosidosis guinea-pigs resembles the clinical, histopathological, biochemical and molecular features of the human disease. We have used the guinea-pig model to investigate efficacy of enzyme replacement therapy as a treatment for alpha-mannosidosis. Intravenous recombinant human lysosomal alpha-mannosidase, administered at a dose of 1mg/kg, was cleared from circulation with a half-life of 53 h, with significant enzyme activity (1.4x normal levels) detected in circulation one week post-injection. alpha-Mannosidase administered to alpha-mannosidosis guinea-pigs at 1mg/kg (onset at birth or approximately 30 days) and 10mg/kg (at birth) was distributed widely amongst tissues, including to capillary depleted brain. By monitoring with tandem mass spectrometry, enzyme replacement therapy was found to be effective in reducing stored substrates in peripheral tissues at both dose rates, and in brain by up to 39% at the 10mg/kg dose, compared with untreated alpha-mannosidosis controls. Reductions of up to 60% of urinary mannose containing oligosaccharides were also observed. No histological improvements were seen in the brain at either dose, however marked decreases in lysosomal vacuolation in liver, kidney, spleen and endocrine pancreas, as well as a significant reduction in trigeminal ganglion neurons were observed. Multiple injections of 1mg/kg recombinant enzyme in alpha-mannosidosis guinea-pigs induced a very rapid humoral immune response precluding long-term intravenous treatment.

[Engineering of a System for the Production of Mutant Human Alpha-Fetoprotein in the Methylotrophic Yeast Pichia pastoris].

PubMed

Morozkina, E V; Vavilova, E A; Zatsepin, S S; Klyachko, E V; Yagudin, T A; Chulkin, A M; Dudich, I V; Semenkova, L N; Churilova, I V; Benevolenskii, S V

2016-01-01

A system for the production of mutant recombinant human alpha-fetoprotein (rhAFPO) lacking the glycosylation site has been engineered in the yeast Pichia pastoris. A strain of the methylotrophic yeast Pichia pastoris GS 115/pPICZ?A/rhAFP0, which produces unglycosylated rhAFPO and secretes it to the culture medium, has been constructed. Optimization and scale-up of the fermentation technology have resulted in an increase in the rhAFP0 yield to 20 mg/L. A scheme of isolation and purification of biologically active rhAFP0 has been developed. The synthesized protein has the antitumor activity, which is analogous to the activity of natural human embryonic alpha-fetoprotein.

Rapid, Automated, and Specific Immunoassay to Directly Measure Matrix Metalloproteinase-9–Tissue Inhibitor of Metalloproteinase-1 Interactions in Human Plasma Using AlphaLISA Technology: A New Alternative to Classical ELISA

PubMed Central

Pulido-Olmo, Helena; Rodríguez-Sánchez, Elena; Navarro-García, José Alberto; Barderas, María G.; Álvarez-Llamas, Gloria; Segura, Julián; Fernández-Alfonso, Marisol; Ruilope, Luis M.; Ruiz-Hurtado, Gema

2017-01-01

The protocol describes a novel, rapid, and no-wash one-step immunoassay for highly sensitive and direct detection of the complexes between matrix metalloproteinases (MMPs) and their tissue inhibitor of metalloproteinases (TIMPs) based on AlphaLISA® technology. We describe two procedures: (i) one approach is used to analyze MMP-9–TIMP-1 interactions using recombinant human MMP-9 with its corresponding recombinant human TIMP-1 inhibitor and (ii) the second approach is used to analyze native or endogenous MMP-9–TIMP-1 protein interactions in samples of human plasma. Evaluating native MMP-9–TIMP-1 complexes using this approach avoids the use of indirect calculations of the MMP-9/TIMP-1 ratio for which independent MMP-9 and TIMP-1 quantifications by two conventional ELISAs are needed. The MMP-9–TIMP-1 AlphaLISA® assay is quick, highly simplified, and cost-effective and can be completed in less than 3 h. Moreover, the assay has great potential for use in basic and preclinical research as it allows direct determination of native MMP-9–TIMP-1 complexes in circulating blood as biofluid. PMID:28791014

HPV-18 confers resistance to TNF-{alpha} in organotypic cultures of human keratinocytes

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

Boccardo, Enrique; Noya, Francisco; Broker, Thomas R.

2004-10-25

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-{alpha}) inhibits normal keratinocytes proliferation. However, many human papillomavirus (HPV)-immortalized or transformed cell lines are resistant to TNF-{alpha} antiproliferative effect. The present study analyzes the effects of TNF-{alpha} on organotypic cultures of primary human keratinocytes (PHKs) that express HPV-18 oncogenes. Raft cultures prepared with PHKs acutely transfected with HPV-18 whole genome or infected with recombinant retroviruses containing only E6/E7 or E7 were treated with 2 nM TNF-{alpha}. While BrdU incorporation into basal/parabasal cells of normal PHKs cultures was markedly inhibited by TNF-{alpha} cultures transfected with HPV-18 whole genome showed proliferation in all cell strata.more » Furthermore, BrdU incorporation into cultures expressing E6/E7 or E7 was not significantly reduced, indicating that E7 alone confers partial resistance to TNF-{alpha}. Besides, TNF-{alpha} treatment did not alter p16{sup ink4a}, p21{sup cip1}, p27{sup kip1}, or cyclin E levels, but did reduce cyclin A and PCNA levels in sensitive cells.« less

IFN-beta1b augments glucocorticoid-induced suppression of tumor necrosis factor-alpha production by increasing the number of glucocorticoid receptors on a human monocytic cell line.

PubMed

Uitdehaag, B M; Hoekstra, K; Koper, J W; Polman, C H; Dijkstra, C D

2001-03-01

We studied the effect of recombinant interferon-beta1b (IFN-beta1b) on the sensitivity to glucocorticoids (GC) and on the number of GC receptors (GCR) in the human monocytic cell line THP-1. We found that IFN-beta1b augments the suppressive effect that dexamethasone has on the stimulated production of tumor necrosis factor-alpha (TNF-alpha), most likely related to the increased number of GCR observed after exposure to IFN-beta1b. This provides a possible clue to the mechanism of action of IFN-beta in multiple sclerosis.

Treatment Strategies for Human Arboviral Infections Applicable to Veterinary Medicine

DTIC Science & Technology

1992-06-16

16. MORILL , J. C., G. B. JENNINGS, T. M. COSGRIFF, P. H. GIBBS & C. J. PETERS. 1989. Prevention of Rift Valley fever in rhesus monkeys with interferon...alpha. Rev. Infect. Dis. 2(Suppl. 4): 815. 17. MORILL , J. C., C. W. CZARNECKI & C. J. PETERS. 1991. Recombinant human interferon- gamma modulates

[Immune diagnostics of disorders in the IFN-alpha/NK-cell system in patients with frequently recurrent herpes simplex].

PubMed

Karsonova, A V; Shulzhenko, A E; Karaulov, A V

2014-01-01

Study of features of NK-cell response to the effect of recombinant IFN-alpha in complex with evaluation of the ability to synthesize inherent IFN-alpha in patients with frequently recurrent herpes simplex (FRHS). 48 patients with genital (n = 31), labial (n = 10) and mixed localization (n = 7) FRHS diagnosis were observed. 31 healthy donors composed the control group. MC were cultivated in the presence of a recombinant human IFN-alpha2b at the concentration of 10, 100 and 1000 U/ml for 24 hours. NK-cell response to the effect of IFN-alpha was evaluated after 24 hours using flow cytometry by degranulation reaction and in the NK-activity test. IFN-alpha synthesis was evaluated in HSV-1, HSV-2 and Newcastle disease virus stimulated cell supernatants by EIA method. Patients with FRHS were established to be a heterogeneous group by parameters in the IFN-alpha/NK-cell cytotoxicity system. 2 types of NK-cell response to the stimulation by recombinant IFN-alpha were identified. Type A is characterized by a decrease of NK-cell response to IFN-alpha in the remission phase and does not have this defect in the exacerbation phase. Synthesis of inherent IFN-alpha in response to viral inductors for type A was comparable with the response in healthy donors in both phases. On the contrary type B having normal sensitivity of NK-cells to IFN-alpha in the remission phase is characterized by a decrease of this parameter in the exacerbation phase for more than 3 times. Synthesis of inherent IFN-alpha in response to viral inductors during type B is increased in the remission phase and decreased in the exacerbation phase. During immune-correcting therapy of FRHS a personalized approach taking into account features of NK-cell response to IFN-alpha is necessary, because types A and B have principal differences by cytotoxicity parameters of NK-cells and their change under the effect of IFN-alpha, as well as by parameters of IFN-alpha synthesis in response to viral inductors at various phases of the clinical process.

New approaches to the treatment of inflammatory disorders small molecule inhibitors of p38 MAP kinase.

PubMed

Peifer, Christian; Wagner, Gerd; Laufer, Stefan

2006-01-01

The therapy of chronic inflammatory diseases like rheumatoid arthritis (RA) and inflammatory bowel disease (IBD) has recently been enriched by the successful launch of the anti-cytokine biologicals Etanercept (tumor necrosis factor (TNF) receptor-p75 Fc fusion protein), Infliximab (chimeric anti-human TNF-alpha monoclonal antibody), Adalimumab (recombinant human anti-human TNF-alpha monoclonal antibody) and Anakinra (recombinant form of human interleukin 1beta (IL-1) receptor antagonist). The success of these novel treatments has impressively demonstrated the clinical benefit that can be gained from therapeutic intervention in cytokine signalling, highlighting the central role of proinflammatory cytokine systems like IL-1alpha and TNF-alpha to be validated targets. However, all of the anti-cytokine biologicals available to date are proteins, and therefore suffering to a varying degree from the general disadvantages associated with protein drugs. Therefore, small molecular, orally active anti-cytokine agents, which target specific pathways of proinflammatory cytokines, would offer an attractive alternative to anti-cytokine biologicals. A number of molecular targets have been identified for the development of such small molecular agents but p38 mitogen-activated protein (MAP) kinase occupies a central role in the regulation of IL-1beta and TNF-alpha signalling network at both the transcriptional and translational level. Since the mid-1990s, an immense number of inhibitors of p38 MAP kinase has been characterised in vitro, and to date several compounds have been advanced into clinical trials. This review will highlight the correlation between effective inhibition of p38 MAP kinase at the molecular target and cellular activity in functional assays of cytokine, particularly TNF-alpha and IL-1beta production. SAR will be discussed regarding activity at the enzyme target, but also with regard to properties required for efficient in vitro and in vivo activity.

Comparison of the effects of agalsidase alfa and agalsidase beta on cultured human Fabry fibroblasts and Fabry mice.

PubMed

Sakuraba, Hitoshi; Murata-Ohsawa, Mai; Kawashima, Ikuo; Tajima, Youichi; Kotani, Masaharu; Ohshima, Toshio; Chiba, Yasunori; Takashiba, Minako; Jigami, Yoshifumi; Fukushige, Tomoko; Kanzaki, Tamotsu; Itoh, Kohji

2006-01-01

We compared two recombinant alpha-galactosidases developed for enzyme replacement therapy for Fabry disease, agalsidase alfa and agalsidase beta, as to specific alpha-galactosidase activity, stability in plasma, mannose 6-phosphate (M6P) residue content, and effects on cultured human Fabry fibroblasts and Fabry mice. The specific enzyme activities of agalsidase alfa and agalsidase beta were 1.70 and 3.24 mmol h(-1) mg protein(-1), respectively, and there was no difference in stability in plasma between them. The M6P content of agalsidase beta (3.6 mol/mol protein) was higher than that of agalsidase alfa (1.3 mol/mol protein). The administration of both enzymes resulted in marked increases in alpha-galactosidase activity in cultured human Fabry fibroblasts, and Fabry mouse kidneys, heart, spleen and liver. However, the increase in enzyme activity in cultured fibroblasts, kidneys, heart and spleen was higher when agalsidase beta was used. An immunocytochemical analysis revealed that the incorporated recombinant enzyme degraded the globotriaosyl ceramide accumulated in cultured Fabry fibroblasts in a dose-dependent manner, with the effect being maintained for at least 7 days. Repeated administration of agalsidase beta apparently decreased the number of accumulated lamellar inclusion bodies in renal tubular cells of Fabry mice.

Reduction of adenovirus E1A mRNA by RNAi results in enhanced recombinant protein expression in transiently transfected HEK293 cells.

PubMed

Hacker, David L; Bertschinger, Martin; Baldi, Lucia; Wurm, Florian M

2004-10-27

Human embryonic kidney 293 (HEK293) cells, a widely used host for large-scale transient expression of recombinant proteins, are transformed with the adenovirus E1A and E1B genes. Because the E1A proteins function as transcriptional activators or repressors, they may have a positive or negative effect on transient transgene expression in this cell line. Suspension cultures of HEK293 EBNA (HEK293E) cells were co-transfected with a reporter plasmid expressing the GFP gene and a plasmid expressing a short hairpin RNA (shRNA) targeting the E1A mRNAs for degradation by RNA interference (RNAi). The presence of the shRNA in HEK293E cells reduced the steady state level of E1A mRNA up to 75% and increased transient GFP expression from either the elongation factor-1alpha (EF-1alpha) promoter or the human cytomegalovirus (HCMV) immediate early promoter up to twofold. E1A mRNA depletion also resulted in a twofold increase in transient expression of a recombinant IgG in both small- and large-scale suspension cultures when the IgG light and heavy chain genes were controlled by the EF-1alpha promoter. Finally, transient IgG expression was enhanced 2.5-fold when the anti-E1A shRNA was expressed from the same vector as the IgG light chain gene. These results demonstrated that E1A has a negative effect on transient gene expression in HEK293E cells, and they established that RNAi can be used to enhance recombinant protein expression in mammalian cells.

Effects of combined treatment with interferon and mezerein on melanogenesis and growth in human melanoma cells.

PubMed

Fisher, P B; Prignoli, D R; Hermo, H; Weinstein, I B; Pestka, S

1985-01-01

We have analyzed the effects of various human interferons produced in bacteria and the antileukemic compound mezerein (MEZ) on growth and melanogenesis in human melanoma cells. In four human melanoma cell lines, recombinant human fibroblast interferon (IFN-beta) was more active than recombinant human leukocyte interferons (IFN-alpha A, IFN-alpha D, or IFN-alpha A/D (Bgl] in inhibiting cellular proliferation. When monolayer cultures were exposed to 1000 IU/ml IFN-beta for four days the degree of growth inhibition in the different melanoma cell lines varied between 94 and 26%. Similarly, four days growth in medium containing 10 ng/ml MEZ resulted in either no inhibition of growth or as much as 53% inhibition of growth, depending on the specific melanoma cell line tested. MEZ induced dendrite-like processes, cytoplasmic projections morphologically similar to those normally found in neurons and melanocytes, in all four melanoma cell lines, whereas none of the interferons tested had this effect. The combination of interferon and MEZ resulted in a dramatic inhibition in cellular proliferation in all four melanoma cell lines. When cell extracts were assayed for melanin content, a marker of melanoma cell differentiation, the combination of IFN-beta and MEZ resulted in higher levels of melanin than with either agent alone. Dendrite-like formation was also prominent in the cultures treated with this combination. These results indicate that the antiproliferative effect of interferon toward human melanoma dells can be enhanced by treatment with MEZ and that this effect is associated with an enhancement of terminal differentiation.

[AVIAN RECOMBINANT VIRUS H5N1 INFLUENZA (A/VIETNAM/1203/04) AND ITS ESCAPE-MUTANT m13(13) INDUCE EARLY SIGNALING REACTIONS OF THE IMMUNITY IN HUMAN LYMPHOCYTES].

PubMed

Sokolova, T M; Poloskov, V V; Shuvalov, A N; Rudneva, I A; Ershov, F I

2016-01-01

The innate immune receptors TLR4, TLR7, TLR8, and RIG1 recognized the structures of the influenza viruses in human lymphocytes and were activated by the recombinant avian influenza virus A/Vietnam/1203/04 and its escape-mutant m13(13) during early period of interaction. The stimulated levels are not connected with viral reproduction. Donor cells with the low constitutive immune receptors gene expression levels showed higher stimulation. Inflammation virus effects resulted in. increasing production of TNF-alpha and IFN-gamma by lymphocytes. Signaling gene reactions of the parent and mutant viruses endosomal as well as cytoplasmic receptors are very similar. The mutant virus A/Vietnam/1203/04 (HA S145F), stimulated an increase in the transcription level of the membrane receptor gene TLR4 and a decrease in the level of activation of TNF-alpha gene. Further studies of natural influenza virus isolates are necessary to estimate the role of HA antigenic changes on immune reactions in humans.

[Effects of recombinant human alpha-2b and gamma interferons on bone marrow megakaryocyte progenitors (CFU-Meg) from patients with chronic myelocytic leukemia].

PubMed

Tanabe, Y; Dan, K; Kuriya, S; Nomura, T

1989-10-01

The effects of recombinant human interferon (IFN) alpha-2b and gamma on the bone marrow megakaryocyte progenitors (CFU-Meg) were compared between eight patients in the chronic phase of Ph1-positive chronic myelocytic leukemia (CML) and five hematologically normal patients. CFU-Meg was assayed in plasma clot culture added with phytohemagglutinin-stimulated leukocyte-conditioned medium as a source of colony stimulating activity. The average count of CFU-Meg colonies formed from the bone marrow of CML patients was 5.5 times that of normal controls. Spontaneous CFU-Meg colonies were grown in seven of eight CML patients, but in none of five controls. Colony formation by CFU-Meg in CML as well as normal bone marrow was suppressed by the two preparations of IFN in a dose dependent fashion. Their suppressive influence on colonies from CFU-Meg was comparable between CML and normal bone marrow at lower concentrations, but was less marked for CML than normal bone marrow at higher concentrations. The formation of CFU-Meg colonies from CML bone marrow was more severely suppressed by IFN-gamma than IFN-alpha-2b. Depletion of either T lymphocytes or adherent cells from the CML bone marrow cells diminished the suppressive effects of IFN-gamma, but had no influence on the effects of IFN-alpha-2b.

Molecular cloning of rock bream (Oplegnathus fasciatus) tumor necrosis factor-alpha and its effect on the respiratory burst activity of phagocytes.

PubMed

Kim, Min Sun; Hwang, Yoon Jung; Yoon, Ki Joon; Zenke, Kosuke; Nam, Yoon Kwon; Kim, Sung Koo; Kim, Ki Hong

2009-11-01

Rock bream (Oplegnathus fasciatus) tumor necrosis factor-alpha (rbTNF-alpha) gene was cloned, recombinantly produced, and the effect of the recombinant rbTNF-alpha on the respiratory burst activity of rock bream phagocytes was analyzed. Structurally, genomic DNA of rbTNF-alpha was comprised with four exons and three introns, and deduced amino acid sequence of its cDNA possessed the TNF family signature, a transmembrane domain, a protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF-alpha gene in mammals and fish. The chemiluminescent (CL) response of rock bream phagocytes was significantly enhanced by pre-incubation with recombinant rbTNF-alpha, when opsonized zymosan was used as a stimulant of the respiratory burst. However, CL enhancing effect of the recombinant rbTNF-alpha was very weak when the respiratory burst activity of phagocytes was triggered with phorbol-12-myristate-13-acetate (PMA) instead of zymosan. These results suggest that rock bream TNF-alpha might have an ability to prime the respiratory burst activity of phagocytes against receptor-mediated phagocytosis inducing stimulants, such as zymosan, but have little ability against stimulants not accompanying receptor-mediated phagocytosis.

A yeast-based genetic screening to identify human proteins that increase homologous recombination.

PubMed

Collavoli, Anita; Comelli, Laura; Rainaldi, Giuseppe; Galli, Alvaro

2008-05-01

To identify new human proteins implicated in homologous recombination (HR), we set up 'a papillae assay' to screen a human cDNA library using the RS112 strain of Saccharomyces cerevisiae containing an intrachromosomal recombination substrate. We isolated 23 cDNAs, 11 coding for complete proteins and 12 for partially deleted proteins that increased HR when overexpressed in yeast. We characterized the effect induced by the overexpression of the complete human proteasome subunit beta 2, the partially deleted proteasome subunits alpha 3 and beta 8, the ribosomal protein L12, the brain abundant membrane signal protein (BASP1) and the human homologue to v-Ha-RAS (HRAS), which elevated HR by 2-6.5-fold over the control. We found that deletion of the RAD52 gene, which has a key role in most HR events, abolished the increase of HR induced by the proteasome subunits and HRAS; by contrast, the RAD52 deletion did not affect the high level of HR due to BASP1 and RPL12. This suggests that the proteins stimulated yeast HR via different mechanisms. Overexpression of the complete beta 2 human proteasome subunit or the partially deleted alpha 3 and beta 8 subunits increased methyl methanesulphonate (MMS) resistance much more in the rad52 Delta mutant than in the wild-type. Overexpression of RPL12 and BASP1 did not affect MMS resistance in both the wild-type and the rad52 Delta mutant, whereas HRAS decreased MMS resistance in the rad52 Delta mutant. The results indicate that these proteins may interfere with the pathway(s) involved in the repair of MMS-induced DNA damage. Finally, we provide further evidence that yeast is a helpful tool to identify human proteins that may have a regulatory role in HR.

Identification of an antagonist that selectively blocks the activity of prostamides (prostaglandin-ethanolamides) in the feline iris.

PubMed

Woodward, D F; Krauss, A H; Wang, J W; Protzman, C E; Nieves, A L; Liang, Y; Donde, Y; Burk, R M; Landsverk, K; Struble, C

2007-02-01

The prostamides (prostaglandin-ethanolamides) and prostaglandin (PG) glyceryl esters are biosynthesized by COX-2 from the respective endocannabinoids anandamide and 2-arachidonyl glycerol. Agonist studies suggest that their pharmacologies are unique and unrelated to prostanoid receptors. This concept was further investigated using antagonists. The isolated feline iris was used as a key preparation, where prostanoid FP receptors and prostamide activity co-exist. Activity at human recombinant FP and other prostanoid receptors was determined using stable transfectants. In the feline iris, AGN 204396 produced a rightward shift of the dose-response curves for prostamide F2alpha and the prostamide F2alpha analog bimatoprost but did not block the effects of PGF2alpha and synthetic FP receptor agonists. Studies on human recombinant prostanoid receptors confirmed that AGN 204396 did not behave as a prostanoid FP receptor antagonist. AGN 204396 exhibited no antagonism at DP and EP1-4, but was a highly effective TP receptor antagonist. Contrary to expectation, the FP receptor antagonist AL-8810 efficaciously contracted the cat iris. AGN 204396 did not affect AL-8810 induced contractions, demonstrating that AL-8810 and AGN 204396 are pharmacologically distinct. Unlike AL-8810, the ethylamide derivate of AL-8810 was not an agonist. Al-8810 did not block prostamide F2alpha activity. Finally, AGN 204396 did not block PGE2-glyceryl ester activity. The ability of AGN 204396 to selectively block prostamide responses suggests the existence of prostamide sensitive receptors as entities distinct from receptors recognizing PGF2alpha and PGE2-glyceryl ester.

Enhanced homologous recombination is induced by alpha-particle radiation in somatic cells of Arabidopsis thaliana

NASA Astrophysics Data System (ADS)

Bian, Po; Liu, Ping; Wu, Yuejin

Almost 9 percent of cosmic rays which strike the earth's atmosphere are alpha particles. As one of the ionizing radiations (IR), its biological effects have been widely studied. However, the plant genomic instability induced by alpha-particle radiation was not largely known. In this research, the Arabidopsis thaliana transgenic for GUS recombination substrate was used to evaluate the genomic instability induced by alpha-particle radiation (3.3MeV). The pronounced effects of systemic exposure to alpha-particle radiation on the somatic homologous recombination frequency (HRF) were found at different doses. The 10Gy dose of radiation induced the maximal HRF which was 1.9-fold higher than the control. The local radiation of alpha-particle (10Gy) on root also resulted in a 2.5-fold increase of somatic HRF in non-radiated aerial plant, indicating that the signal(s) of genomic instability was transferred to non-radiated parts and initiated their genomic instability. Concurrent treatment of seedlings of Arabidopsis thaliana with alpha-particle and DMSO(ROS scavenger) both in systemic and local radiation signifi- cantly suppressed the somatic HR, indicating that the free radicals produced by alpha-particle radiation took part in the production of signal of genomic instability rather than the signal transfer. Key words: alpha-particle radiation, somatic homologous recombination, genomic instability

Interleukin-9 enhances interleukin-5 receptor expression, differentiation, and survival of human eosinophils.

PubMed

Gounni, A S; Gregory, B; Nutku, E; Aris, F; Latifa, K; Minshall, E; North, J; Tavernier, J; Levit, R; Nicolaides, N; Robinson, D; Hamid, Q

2000-09-15

Interleukin-9 (IL-9) has been implicated in the pathogenesis of allergic disorders. To examine the interaction between IL-9 and eosinophils, we evaluated mature peripheral blood eosinophils for their expression of the specific alpha-subunit of the IL-9 receptor (IL-9R-alpha). The expression of IL-9R-alpha by human eosinophils was detected at the messenger RNA (mRNA) and protein levels by reverse transcriptase-polymerase chain reaction (RT-PCR), flow cytometry, and immunocytochemical analysis, respectively. Functional analyses demonstrated that recombinant human (rh)IL-9 inhibited in vitro peripheral blood human eosinophil apoptosis in a concentration-dependent manner. We then examined the role of IL-9 in eosinophil differentiation using the human cord blood CD34(+) cells and human promyelocytic leukemia cells (HL-60). The addition of IL-9 to CD34(+) cells cultured in IL-3 and IL-5 enhanced eosinophil development, and IL-9 alone induced the expression of IL-5R-alpha. IL-9 also up-regulated the IL-5R-alpha chain cell surface expression during terminal eosinophil differentiation of the HL-60 cell line. Our findings suggest that IL-9 may potentiate in vivo eosinophil function by increasing their survival and IL-5-mediated differentiation and maturation. Taken together, these results suggest a mechanism by which IL-9 potentiates airway and tissue eosinophilia.

Functional assessment of recombinant human alpha(2)-adrenoceptor subtypes with cytosensor microphysiometry.

PubMed

Pihlavisto, M; Scheinin, M

1999-12-03

We applied the Cytosensor Microphysiometry system to study the three human alpha(2)-adrenoceptor subtypes, alpha(2A), alpha(2B) and alpha(2C), expressed in Chinese hamster ovary (CHO) cells, and assessed its potential in the quantitative monitoring of agonist activity. The natural full agonist, (-)-noradrenaline, was used to define agonist efficacy. The imidazole derivative dexmedetomidine was a potent full agonist of all three receptor subtypes. The imidazolines clonidine and UK 14,304 (5-bromo-N-(4, 5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine) appeared to be partial agonists at alpha(2B)-adrenoceptors (E(max) approximately 60% of (-)-noradrenaline) but full agonists at alpha(2A)- and alpha(2C)-adrenoceptors. The responses mediated by all three alpha(2)-adrenoceptor subtypes were partly inhibited by the sodium-hydrogen (Na(+)/H(+)) exchange inhibitor, MIA (5-(N-methyl-N-isobutyl)-amiloride). The agonist responses were totally abolished by pretreatment with pertussis toxin in cells with alpha(2A)- and alpha(2C)-adrenoceptors, and partly abolished in cells with alpha(2B)-adrenoceptors. The residual signal in alpha(2B)-cells was sensitive to the intracellular Ca(2+)chelator, BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester). Cholera toxin (which acts on G(s)-proteins) had no effect on the agonist responses. The results suggest that the extracellular acidification responses mediated by all three human alpha(2)-adrenoceptor subtypes are dependent on Na(+)/H(+)exchange and G(i/o) pathways, and that alpha(2B)-adrenoceptors are capable of coupling to another, G(i/o)-independent and Ca(2+)-dependent signaling pathway.

Preparation and characterization of human interleukin-5 expressed in recombinant Escherichia coli.

PubMed Central

Proudfoot, A E; Fattah, D; Kawashima, E H; Bernard, A; Wingfield, P T

1990-01-01

The gene coding for human interleukin-5 was synthesized and expressed in Escherichia coli under control of a heat-inducible promoter. High-level expression, 10-15% of total cellular protein, was achieved in E. coli. The protein was produced in an insoluble state. A simple extraction, renaturation and purification scheme is described. The recombinant protein was found to be a homodimer, similar to the natural murine-derived protein. Despite the lack of glycosylation, high specific activities were obtained in three 'in vitro' biological assays. Physical characterization of the protein showed it to be mostly alpha-helical, supporting the hypothesis that a conformational similarity exists among certain cytokines. Images Fig. 1. Fig. 3. PMID:2205201

Treatment in Fabry disease.

PubMed

López Rodríguez, M

2018-04-13

Fabry disease is an X-linked inborn disease caused by deficit of alpha-galactosidaseA. This results in accumulation of glycosphingolipids in all cells and tissues. All males should receive enzyme replacement treatment in case of very low or undetectable levels of alpha-galactosidaseA. Female carriers and males with marginally levels of alpha-galactosidaseA should be treated in case of renal, neurologic o cardiac manifestations. There are two intravenous formulations of human recombinant enzyme, agalsidase alpha and agalsidase beta, showing similar efficacy and safety. Patients with amenable mutations of alpha-galactosidase can be treated with oral migalastat hydrochloride. Migalastat hydrochloride is a pharmacological chaperone that facilitates trafficking of alpha-galactosidaseA to lysosomes increasing enzyme activity. Patients treated with migalastat hydrochloride had significant improvements in left ventricular mass and gastrointestinal symptoms. Copyright © 2018 Elsevier España, S.L.U. and Sociedad Española de Medicina Interna (SEMI). All rights reserved.

Receptor protection studies comparing recombinant and native nicotinic receptors: Evidence for a subpopulation of mecamylamine-sensitive native alpha3beta4* nicotinic receptors.

PubMed

Free, R Benjamin; Kaser, Daniel J; Boyd, R Thomas; McKay, Dennis B

2006-01-09

Studies involving receptor protection have been used to define the functional involvement of specific receptor subtypes in tissues expressing multiple receptor subtypes. Previous functional studies from our laboratory demonstrate the feasibility of this approach when applied to neuronal tissues expressing multiple nicotinic acetylcholine receptors (nAChRs). In the current studies, the ability of a variety of nAChR agonists and antagonists to protect native and recombinant alpha3beta4 nAChRs from alkylation were investigated using nAChR binding techniques. Alkylation of native alpha3beta4* nAChRs from membrane preparations of bovine adrenal chromaffin cells resulted in a complete loss of specific [(3)H]epibatidine binding. This loss of binding to native nAChRs was preventable by pretreatment with the agonists, carbachol or nicotine. The partial agonist, cytisine, produced partial protection. Several nAChR antagonists were also tested for their ability to protect. Hexamethonium and decamethonium were without protective activity while mecamylamine and tubocurarine were partially effective. Addition protection studies were performed on recombinant alpha3beta4 nAChRs. As with native alpha3beta4* nAChRs, alkylation produced a complete loss of specific [(3)H]epibatidine binding to recombinant alpha3beta4 nAChRs which was preventable by pretreatment with nicotine. However, unlike native alpha3beta4* nAChRs, cytisine and mecamylamine, provide no protection for alkylation. These results highlight the differences between native alpha3beta4* nAChRs and recombinant alpha3beta4 nAChRs and support the use of protection assays to characterize native nAChR subpopulations.

[Cloning of Chinese Banna minipig inbred-line alpha1,3-galactosyltransferase gene and construction of its recombinant eukaryotic expression vector].

PubMed

Zhu, Shengming; Wang, Yanping; Zheng, Hong; Cheng, Jingqiu; Lu, Yanrong; Zeng, Yangzhi; Wang, Yu; Wang, Zhu

2009-04-01

This study sought to clone Chinese Banna minipig inbred-line (BMI) alpha1,3-galactosyltransferase (alpha1,3-GT) gene and construct its recombinant eukaryotic expression vector. Total RNA was isolated from BMI liver. Full length cDNA of alpha1,3-GT gene was amplified by RT-PCR and cloned into pMD18-T vector to sequence. Subsequently, alpha1,3-GT gene was inserted into pEGFP-N1 to construct eukaryotic expression vector pEGFP-N1-GT. Then the reconstructed plasmid pEGFP-N1-GT was transiently transfected into human lung cancer cell line A549. The expression of alpha1,3-GT mRNA in transfected cells was detected by RT-PCR. FITC-BS-IB4 lectin was used in the direct immunofluorescence method, which was performed to observe the alpha-Gal synthesis function of BMI alpha1,3-GT in transfected cells. The results showed that full length of BMI alpha1,3-GT cDNA was 1116 bp. BMI alpha1,3-GT cDNA sequence was highly homogenous with those of mouse and bovine, and was exactly the same as the complete sequence of those of swine, pEGFP-N1-GT was confirmed by enzyme digestion and PCR. The expression of alpha1,3-GT mRNA was detected in A549 cells transfected by pEGFP-N1-GT. The expression of alpha-Gal was observed on the membrane of A549 cells transfected by pEGFP-N1-GT. Successful cloning of BMI alpha1,3-GT cDNA and construction of its eukaryotic expression vector have established a foundation for further research and application of BMI alpha1,3-GT in the fields of xenotransplantation and immunological therapy of cancer.

Effect of the combination of fibrin glue and growth hormone on intestinal anastomoses in a pig model of traumatic shock associated with peritonitis.

PubMed

Wang, Pengfei; Wang, Jian; Zhang, Wenbo; Li, Yousheng; Li, Jieshou

2009-03-01

Intra-abdominal sepsis and hemorrhagic shock have been found to impair the healing of intestinal anastomoses. The present study examined whether fibrin glue (FG) and recombinant human growth hormone (GH) can improve intestinal primary anastomotic healing in a pig model of traumatic shock associated with peritonitis. Further, the study was designed to investigate the probable mechanism of these agents. Female anesthetized pigs were divided into five groups. Group sham (n = 7), pigs without traumatic shock had small bowel resection anastomoses; group control (n = 14), pigs had bowel resection anastomoses 24 h after abdominal gunshot plus exsanguination/resuscitation; group FG (n = 14); group GH (n = 14); group FG/GH (n = 14), pigs received FG, recombinant GH, or both, respectively. Recombinant GH was given daily for 7 days. Blood samples were collected daily for measurement of interleukin-6 (IL-6) and tumor necrosis factor (TNF)-alpha levels. Investigations also included adhesion formation, anastomotic bursting pressure, tensile strength, hydroxyproline (HP) content, myeloperoxidase (MPO), tumor necrosis factor (NF)-kappaB activity, and histology analysis 10 days later. A second experiment (n = 20 subjects assigned to each of the five groups) was designed to study survival during the first 20 postoperative days. Traumatic shock associated with peritonitis led to significant decreases in intestinal anastomotic bursting pressures, tensile strengths, and tissue hydroxyproline content, along with severe adhesion formation, increases in MPO activity and NF-kappaB activity, and plasma levels of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). Both FG and recombinant GH treatment led to early significant increases in plasma levels of TNF-alpha and IL-6. At the same time, FG alone, unlike recombinant GH alone, led to significant increases in anastomotic bursting pressures, tensile strength, and tissue HP content, along with decreases in anastomotic MPO and NF-kappaB activity and later plasma levels of TNF-a and IL-6. The FG group also developed more marked neoangiogenesis and collagen deposition on histology analysis. However, FG and recombinant GH synergistically effected improved anastomotic healing, abolishing the infaust effects promoted by recombinant GH. Adhesion formation after intestinal anastomosis could not be lowered by FG alone or by the combination of FG and recombinant GH. Both FG alone and FG/GH, in contrast to GH alone and control treatment, significantly prolonged the survival time of experimental animals. We found that FG, but not recombinant GH, could lower the risk of anastomotic leakage, improve intestinal anastomotic healing, and prolong survival in a pig model of traumatic shock associated with peritonitis. Both FG and recombinant GH synergistically effected improved intestinal anastomotic healing. It was suggested that GH could be used locally to promote intestinal anastomotic healing in intra-abdominal peritonitis.

3D cultured immortalized human hepatocytes useful to develop drugs for blood-borne HCV

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

Aly, Hussein Hassan; Shimotohno, Kunitada; Hijikata, Makoto

2009-02-06

Due to the high polymorphism of natural hepatitis C virus (HCV) variants, existing recombinant HCV replication models have failed to be effective in developing effective anti-HCV agents. In the current study, we describe an in vitro system that supports the infection and replication of natural HCV from patient blood using an immortalized primary human hepatocyte cell line cultured in a three-dimensional (3D) culture system. Comparison of the gene expression profile of cells cultured in the 3D system to those cultured in the existing 2D system demonstrated an up-regulation of several genes activated by peroxisome proliferator-activated receptor alpha (PPAR{alpha}) signaling. Furthermore,more » using PPAR{alpha} agonists and antagonists, we also analyzed the effect of PPAR{alpha} signaling on the modulation of HCV replication using this system. The 3D in vitro system described in this study provides significant insight into the search for novel anti-HCV strategies that are specific to various strains of HCV.« less

A retrospective analysis of the potential impact of IgG antibodies to agalsidase beta on efficacy during enzyme replacement therapy for Fabry disease.

PubMed

Bénichou, Bernard; Goyal, Sunita; Sung, Crystal; Norfleet, Andrea M; O'Brien, Fanny

2009-01-01

Fabry disease results from a genetic deficiency of alpha-galactosidase A (alpha GAL) and the impaired catabolism of globotriasoylceramide (GL-3) and other glycosphingolipid substrates, which then accumulate pathogenically within most cells. Enzyme replacement therapy (ERT) with agalsidase beta (Fabrazyme), one of two available forms of recombinant human alpha GAL, involves regular intravenous infusions of the therapeutic protein. Immunoglobulin G (IgG) antibodies to recombinant alpha GAL develop in the majority of patients upon repeated infusion. To explore whether anti-alpha GAL IgG interferes with therapeutic efficacy, retrospective analyses were conducted using data obtained from a total of 134 adult male and female patients with Fabry disease who were treated with agalsidase beta at 1mg/kg every 2 weeks for up to 5 years during placebo-controlled trials and the corresponding open-label extension studies. The analyses did not reveal a correlation between anti-alpha GAL IgG titers and the onset of clinical events or the rate of change in estimated GFR during treatment, and no statistically significant association was found between anti-alpha GAL IgG titers and abnormal elevations in plasma GL-3 during treatment. However, a statistically significant association was found between anti-alpha GAL IgG titers and observation of some GL-3 deposition in the dermal capillary endothelial cells of skin during treatment, suggesting that GL-3 clearance may be partially impaired in some patients with high antibody titers. Determination of the long-term impact of circulating anti-alpha GAL IgG antibodies on clinical outcomes will require continued monitoring, and serology testing is recommended as part of the routine care of Fabry disease patients during ERT.

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.

Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis

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

Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J.

Highlights: Black-Right-Pointing-Pointer Non-infectious and protease-deficient Bacillus anthracis protein expression system. Black-Right-Pointing-Pointer Successful expression and purification of a tumor-targeted fusion protein drug. Black-Right-Pointing-Pointer Very low endotoxin contamination of purified protein. Black-Right-Pointing-Pointer Efficient protein secretion simplifies purification. Black-Right-Pointing-Pointer Functional anti-tumor fusion protein purified. -- Abstract: Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression hostmore » offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGF{alpha}). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGF{alpha}). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.« less

Methamphetamine enhances Hepatitis C virus replication in human hepatocytes

PubMed Central

Ye, L.; Peng, J. S.; Wang, X.; Wang, Y. J.; Luo, G. X.; Ho, W. Z.

2009-01-01

SUMMARY Very little is known about the interactions between hepatitis C virus (HCV) and methamphetamine, which is a highly abused psychostimulant and a known risk factor for human immunodeficiency virus (HIV)/HCV infection. This study examined whether methamphetamine has the ability to inhibit innate immunity in the host cells, facilitating HCV replication in human hepatocytes. Methamphetamine inhibited intracellular interferon alpha expression in human hepatocytes, which was associated with the increase in HCV replication. In addition, methamphetamine also compromised the anti-HCV effect of recombinant interferon alpha. Further investigation of mechanism(s) responsible for the methamphetamine action revealed that methamphetamine was able to inhibit the expression of the signal transducer and activator of transcription 1, a key modulator in interferon-mediated immune and biological responses. Methamphetamine also down-regulated the expression of interferon regulatory factor-5, a crucial transcriptional factor that activates the interferon pathway. These in vitro findings that methamphetamine compromises interferon alpha-mediated innate immunity against HCV infection indicate that methamphetamine may have a cofactor role in the immunopathogenesis of HCV disease. PMID:18307590

Control of erythropoietin gene expression and its use in medicine.

PubMed

Jelkmann, Wolfgang

2007-01-01

Erythropoietin (EPO) gene expression is under the control of inhibitory (GATA-2, NF-kappaB) and stimulatory (hypoxia-inducible transcription factor [HIF]-2, hepatocyte nuclear factor [HNF]-4alpha [alpha]) transcription factors. EPO deficiency is the main cause of the anemia in chronic kidney disease (CKD) and a contributing factor in the anemias of inflammation and cancer. Small, orally active compounds capable of stimulating endogenous EPO production are in preclinical or clinical trials for treatment of anemia. These agents include stabilizers of the HIFs that bind to the EPO enhancer and GATA inhibitors which prevent GATA from suppressing the EPO promoter. While HIF stabilizing drugs may prove useful as inexpensive second-line choices, at present, their side effects--particularly tumorigenicity--preclude their use as first-choice therapy. As an alternative, EPO gene therapy has been explored in animal studies and in trials on CKD patients. Here, a major problem is immunogenicity of ex vivo transfected implanted cells and of the recombinant protein produced after ex vivo or in vivo EPO complementary DNA (cDNA) transfer. Recombinant human EPO (rhEPO) engineered in Chinese hamster ovary (CHO) cell cultures (epoetin alpha and epoetin beta [beta]) and its hyperglycosylated analogue darbepoetin alpha are established and safe drugs to avoid allogeneic red blood cell transfusion. Gene-activated EPO (epoetin delta [delta]) from human fibrosarcoma cells (HT-1080) has recently been launched for use in CKD. It is important to know the basics of the technologies, production processes, and structural properties of the novel anti-anemic strategies and drugs.

The alpha form of human tryptase is the predominant type present in blood at baseline in normal subjects and is elevated in those with systemic mastocytosis.

PubMed Central

Schwartz, L B; Sakai, K; Bradford, T R; Ren, S; Zweiman, B; Worobec, A S; Metcalfe, D D

1995-01-01

Tryptase, a protease produced by all mast cells, was evaluated as a clinical marker of systemic mastocytosis. Two sandwich immunoassays were evaluated, one which used the mAb G5 for capture, the other which used B12 for capture. The B12 capture assay measured both recombinant alpha- and beta-tryptase, whereas the G5 capture assay measured primarily recombinant beta-tryptase. G5 binds with low affinity to both recombinant alpha-tryptase and tryptase in blood from normal and nonacute mastocytosis subjects, and binds with high affinity to recombinant beta-tryptase, tryptase in serum during anaphylaxis, and tryptase stored in mast cell secretory granules. B12 recognizes all of these forms of tryptase with high affinity. As reported previously, during systemic anaphylaxis in patients without known mastocytosis, the ratio of B12- to G5-measured tryptase was always < 5 and approached unity (Schwartz L.B., T.R. Bradford, C. Rouse, A.-M. Irani, G. Rasp, J.K. Van der Zwan and P.-W.G. Van der Linden, J. Clin. Immunol. 14:190-204). In this report, most mastocytosis patients with systemic disease have B12-measured tryptase levels that are elevated (> 20 ng/ml) and are at least 10-fold greater than the corresponding G5-measured tryptase level. Most of those subjects with B12-measured tryptase levels of < 20 ng/ml had only cutaneous manifestations. The B12 assay for alpha-tryptase and beta-tryptase, particularly when performed in conjunction with the G5 assay for beta-tryptase, provides a more precise measure of mast cell involvement than currently available assessments, a promising potential screening test for systemic mastocytosis and may provide an improved means to follow disease progression and response to therapy. Images PMID:8675637

The alpha form of human tryptase is the predominant type present in blood at baseline in normal subjects and is elevated in those with systemic mastocytosis.

PubMed

Schwartz, L B; Sakai, K; Bradford, T R; Ren, S; Zweiman, B; Worobec, A S; Metcalfe, D D

1995-12-01

Tryptase, a protease produced by all mast cells, was evaluated as a clinical marker of systemic mastocytosis. Two sandwich immunoassays were evaluated, one which used the mAb G5 for capture, the other which used B12 for capture. The B12 capture assay measured both recombinant alpha- and beta-tryptase, whereas the G5 capture assay measured primarily recombinant beta-tryptase. G5 binds with low affinity to both recombinant alpha-tryptase and tryptase in blood from normal and nonacute mastocytosis subjects, and binds with high affinity to recombinant beta-tryptase, tryptase in serum during anaphylaxis, and tryptase stored in mast cell secretory granules. B12 recognizes all of these forms of tryptase with high affinity. As reported previously, during systemic anaphylaxis in patients without known mastocytosis, the ratio of B12- to G5-measured tryptase was always < 5 and approached unity (Schwartz L.B., T.R. Bradford, C. Rouse, A.-M. Irani, G. Rasp, J.K. Van der Zwan and P.-W.G. Van der Linden, J. Clin. Immunol. 14:190-204). In this report, most mastocytosis patients with systemic disease have B12-measured tryptase levels that are elevated (> 20 ng/ml) and are at least 10-fold greater than the corresponding G5-measured tryptase level. Most of those subjects with B12-measured tryptase levels of < 20 ng/ml had only cutaneous manifestations. The B12 assay for alpha-tryptase and beta-tryptase, particularly when performed in conjunction with the G5 assay for beta-tryptase, provides a more precise measure of mast cell involvement than currently available assessments, a promising potential screening test for systemic mastocytosis and may provide an improved means to follow disease progression and response to therapy.

Purification of target proteins from intracellular inclusions mediated by intein cleavable polyhydroxyalkanoate synthase fusions.

PubMed

Du, Jinping; Rehm, Bernd H A

2017-11-02

Recombinant protein production and purification from Escherichia coli is often accompanied with expensive and complicated procedures, especially for therapeutic proteins. Here it was demonstrated that, by using an intein cleavable polyhydroxyalkanoate synthase fusion, recombinant proteins can be first produced and sequestered on a natural resin, the polyhydroxyalkanoate (PHA) inclusions, then separated from contaminating host proteins via simple PHA bead isolation steps, and finally purified by specific release into the soluble fraction induced by a pH reduction. By translationally fusing a target protein to PHA synthase using a self-cleaving intein as linker, intracellular production of PHA beads was achieved. Upon isolation of respective PHA beads the soluble pure target protein was released by a simple pH shift to 6. The utility of this approach was exemplified by producing six target proteins, including Aequorea victoria green fluorescent protein (GFP), Mycobacterium tuberculosis vaccine candidate Rv1626, the immunoglobulin G (IgG) binding ZZ domain of protein A derived from Staphylococcus aureus, human tumor necrosis factor alpha (TNFα), human granulocyte colony-stimulating factor (G-CSF), and human interferon alpha 2b (IFNα2b). Here a new method for production and purification of a tag-less protein was developed through intein cleavable polyhydroxyalkanoate synthase fusion. Pure target protein could be easily obtained without laborious downstream processing.

Characterization of N-linked oligosaccharides assembled on secretory recombinant glucose oxidase and cell wall mannoproteins from the methylotrophic yeast Hansenula polymorpha.

PubMed

Kim, Moo Woong; Rhee, Sang Ki; Kim, Jeong-Yoon; Shimma, Yoh-ichi; Chiba, Yasunori; Jigami, Yoshifumi; Kang, Hyun Ah

2004-03-01

Presently almost no information is available on the oligosaccharide structure of the glycoproteins secreted from the methylotrophic yeast Hansenula polymorpha, a promising host for the production of recombinant proteins. In this study, we analyze the size distribution and structure of N-linked oligosaccharides attached to the recombinant glycoprotein glucose oxidase (GOD) and the cell wall mannoproteins obtained from H. polymorpha. Oligosaccharide profiling showed that the major oligosaccharide species derived from the H. polymorpha-secreted recombinant GOD (rGOD) had core-type structures (Man(8-12)GlcNAc(2)). Analyses using anti-alpha 1,3-mannose antibody and exoglycosidases specific for alpha 1,2- or alpha 1,6-mannose linkages revealed that the mannose outer chains of N-glycans on the rGOD have very short alpha 1,6 extensions and are mainly elongated in alpha 1,2-linkages without a terminal alpha 1,3-linked mannose addition. The N-glycans released from the H. polymorpha mannoproteins were shown to contain mostly mannose in their outer chains, which displayed almost identical size distribution and structure to those of H. polymorpha-derived rGOD. These results strongly indicate that the outer chain processing of N-glycans by H. polymorpha significantly differs from that by Saccharomyces cerevisiae, thus generating much shorter mannose outer chains devoid of terminal alpha 1,3-linked mannoses.

Inefficiency in GM2 ganglioside elimination by human lysosomal beta-hexosaminidase beta-subunit gene transfer to fibroblastic cell line derived from Sandhoff disease model mice.

PubMed

Itakura, Tomohiro; Kuroki, Aya; Ishibashi, Yasuhiro; Tsuji, Daisuke; Kawashita, Eri; Higashine, Yukari; Sakuraba, Hitoshi; Yamanaka, Shoji; Itoh, Kohji

2006-08-01

Sandhoff disease (SD) is an autosomal recessive GM2 gangliosidosis caused by the defect of lysosomal beta-hexosaminidase (Hex) beta-subunit gene associated with neurosomatic manifestations. Therapeutic effects of Hex subunit gene transduction have been examined on Sandhoff disease model mice (SD mice) produced by the allelic disruption of Hexb gene encoding the murine beta-subunit. We demonstrate here that elimination of GM2 ganglioside (GM2) accumulated in the fibroblastic cell line derived from SD mice (FSD) did not occur when the HEXB gene only was transfected. In contrast, a significant increase in the HexB (betabeta homodimer) activity toward neutral substrates, including GA2 (asialo-GM2) and oligosaccharides carrying the terminal N-acetylglucosamine residues at their non-reducing ends (GlcNAc-oligosaccharides) was observed. Immunoblotting with anti-human HexA (alphabeta heterodimer) serum after native polyacrylamide gel electrophoresis (Native-PAGE) revealed that the human HEXB gene product could hardly form the chimeric HexA through associating with the murine alpha-subunit. However, co-introduction of the HEXA encoding the human alpha-subunit and HEXB genes caused significant corrective effect on the GM2 degradation by producing the human HexA. These results indicate that the recombinant human HexA could interspeciesly associate with the murine GM2 activator protein to degrade GM2 accumulated in the FSD cells. Thus, therapeutic effects of the recombinant human HexA isozyme but not human HEXB gene product could be evaluated by using the SD mice.

Reprocessing of Soft X-ray Emission Lines in Black Hole Accretion Disks

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

Mauche, C W; Liedahl, D A; Mathiesen, B F

By means of a Monte Carlo code that accounts for Compton scattering and photoabsorption followed by recombination, we have investigated the radiation transfer of Ly{alpha}, He{alpha}, and recombination continua photons of H- and He-like C, N, O, and Ne produced in the photoionized atmosphere of a relativistic black hole accretion disk. We find that photoelectric opacity causes significant attenuation of photons with energies above the O VIII K-edge; that the conversion efficiencies of these photons into lower-energy lines and recombination continua are high; and that accounting for this reprocessing significantly (by factors of 21% to 105%) increases the flux ofmore » the Ly{alpha} and He{alpha} emission lines of H- and He-like C and O escaping the disk atmosphere.« less

Structure, organization, and sequence of alpha satellite DNA from human chromosome 17: evidence for evolution by unequal crossing-over and an ancestral pentamer repeat shared with the human X chromosome.

PubMed

Waye, J S; Willard, H F

1986-09-01

The centromeric regions of all human chromosomes are characterized by distinct subsets of a diverse tandemly repeated DNA family, alpha satellite. On human chromosome 17, the predominant form of alpha satellite is a 2.7-kilobase-pair higher-order repeat unit consisting of 16 alphoid monomers. We present the complete nucleotide sequence of the 16-monomer repeat, which is present in 500 to 1,000 copies per chromosome 17, as well as that of a less abundant 15-monomer repeat, also from chromosome 17. These repeat units were approximately 98% identical in sequence, differing by the exclusion of precisely 1 monomer from the 15-monomer repeat. Homologous unequal crossing-over is suggested as a probable mechanism by which the different repeat lengths on chromosome 17 were generated, and the putative site of such a recombination event is identified. The monomer organization of the chromosome 17 higher-order repeat unit is based, in part, on tandemly repeated pentamers. A similar pentameric suborganization has been previously demonstrated for alpha satellite of the human X chromosome. Despite the organizational similarities, substantial sequence divergence distinguishes these subsets. Hybridization experiments indicate that the chromosome 17 and X subsets are more similar to each other than to the subsets found on several other human chromosomes. We suggest that the chromosome 17 and X alpha satellite subsets may be related components of a larger alphoid subfamily which have evolved from a common ancestral repeat into the contemporary chromosome-specific subsets.

Human recombinant soluble guanylyl cyclase: expression, purification, and regulation

NASA Technical Reports Server (NTRS)

Lee, Y. C.; Martin, E.; Murad, F.

2000-01-01

The alpha1- and beta1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5'-hydroxymethyl-2'furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2, 4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.

Potential role of recombinant secretory leucoprotease inhibitor in the prevention of neutrophil mediated matrix degradation.

PubMed

Llewellyn-Jones, C G; Lomas, D A; Stockley, R A

1994-06-01

Neutrophil elastase is able to degrade connective tissue matrices and is thought to be involved in the pathogenesis of destructive lung diseases. The ability of recombinant secretory leucoprotease inhibitor (rSLPI) to inhibit neutrophil mediated degradation of fibronectin in vitro is demonstrated and its efficacy compared with native alpha-1-proteinase inhibitor (n alpha 1-PI), recombinant alpha-1-proteinase inhibitor (r alpha 1-PI), and the chemical elastase inhibitor ICI 200,355. When preincubated with neutrophils both rSLPI and r alpha 1-PI were effective inhibitors of fibronectin degradation although n alpha 1-PI and ICI 200,355 were less effective. Recombinant SLPI was the most effective inhibitor when the cells were allowed to adhere to fibronectin before the addition of the inhibitors. Preincubation of rSLPI (0.1 mumol/l) with the fibronectin plate resulted in almost total inhibition of fibronectin degradation (reduced to 3.3 (SE 0.9)% of control). Pretreating the fibronectin plate with 1 mumol/l rSLPI, r alpha 1-PI and ICI 200,355 followed by thorough washing before the addition of cells resulted in no inhibition of fibronectin degradation with r alpha 1-PI and the ICI inhibitor, but rSLPI retained its inhibitory effect. This effect could be reduced by adding rSLPI in high pH buffer or 2 mol/1 NaCl. It is postulated that rSLPI binds to fibronectin to form a protective layer which prevents its degradation by neutrophil elastase. It may prove to be the most useful therapeutic agent in the prevention of neutrophil mediated lung damage.

Mid Infrared Hydrogen Recombination Line Emission from the Maser Star MWC 349A

NASA Technical Reports Server (NTRS)

Smith, Howard A.; Strelnitski, V.; Miles, J. W.; Kelly, D. M.; Lacy, J. H.

1997-01-01

We have detected and spectrally resolved the mid-IR hydrogen recombination lines H6(alpha)(12.372 micrometers), H7(alpha)(19.062 micrometers), H7(beta)(l1.309 micrometers) and H8(gamma)(12.385 micrometers) from the star MWC349A. This object has strong hydrogen maser emission (reported in the millimeter and submillimeter hydrogen recombination lines from H36(alpha) to H21(alpha)) and laser emission (reported in the H15(alpha), H12(alpha) and H10(alpha) lines). The lasers/masers are thought to arise predominantly in a Keplerian disk around the star. The mid-IR lines do not show evident signs of lasing, and can be well modeled as arising from the strong stellar wind, with a component arising from a quasi-static atmosphere around the disk, similar to what is hypothesized for the near IR (less than or equal to 4 micrometers) recombination lines. Since populations inversions in the levels producing these mid-IR transitions are expected at densities up to approximately 10(exp 11)/cu cm, these results imply either that the disk does not contain high-density ionized gas over long enough path lengths to produce a gain approximately 1, and/or that any laser emission from such regions is small compared to the spontaneous background emission from the rest of the source as observed with a large beam. The results reinforce the interpretation of the far-IR lines as true lasers.

Competitive antagonism of recombinant P2X(2/3) receptors by 2', 3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP).

PubMed

Burgard, E C; Niforatos, W; van Biesen, T; Lynch, K J; Kage, K L; Touma, E; Kowaluk, E A; Jarvis, M F

2000-12-01

TNP-ATP has become widely recognized as a potent and selective P2X receptor antagonist, and is currently being used to discriminate between subtypes of P2X receptors in a variety of tissues. We have investigated the ability of TNP-ATP to inhibit alpha,beta-methylene ATP (alpha,beta-meATP)-evoked responses in 1321N1 human astrocytoma cells expressing recombinant rat or human P2X(2/3) receptors. Pharmacological responses were measured using electrophysiological and calcium imaging techniques. TNP-ATP was a potent inhibitor of P2X(2/3) receptors, blocking both rat and human receptors with IC(50) values of 3 to 6 nM. In competition studies, 10 to 1000 microM alpha,beta-meATP was able to overcome TNP-ATP inhibition. Schild analysis revealed that TNP-ATP was a competitive antagonist with pA(2) values of -8.7 and -8.2. Inhibition of P2X(2/3) receptors by TNP-ATP was rapid in onset, reversible, and did not display use dependence. Although the onset kinetics of inhibition were concentration-dependent, the TNP-ATP off-kinetics were concentration-independent and relatively slow. Full recovery from TNP-ATP inhibition did not occur until >/=5 s after removal of the antagonist. Because of the slow off-kinetics of TNP-ATP, full competition with alpha,beta-meATP for receptor occupancy could be seen only after both ligands had reached a steady-state condition. It is proposed that the slowly desensitizing P2X(2/3) receptor allowed this competitive interaction to be observed over time, whereas the rapid desensitization of other P2X receptors (P2X(3)) may mask the detection of competitive inhibition by TNP-ATP.

Cross-reactivity of antibodies against leptospiral recurrent uveitis-associated proteins A and B (LruA and LruB) with eye proteins.

PubMed

Verma, Ashutosh; Kumar, Pawan; Babb, Kelly; Timoney, John F; Stevenson, Brian

2010-08-03

Infection by Leptospira interrogans has been causally associated with human and equine uveitis. Studies in our laboratories have demonstrated that leptospiral lipoprotein LruA and LruB are expressed in the eyes of uveitic horses, and that antibodies directed against LruA and LruB react with equine lenticular and retinal extracts, respectively. These reactivities were investigated further by performing immunofluorescent assays on lenticular and retinal tissue sections. Incubation of lens tissue sections with LruA-antiserum and retinal sections with LruB-antiserum resulted in positive fluorescence. By employing two-dimensional gel analyses followed by immunoblotting and mass spectrometry, lens proteins cross-reacting with LruA antiserum were identified to be alpha-crystallin B and vimentin. Similarly, mass spectrometric analyses identified beta-crystallin B2 as the retinal protein cross-reacting with LruB-antiserum. Purified recombinant human alpha-crystallin B and vimentin were recognized by LruA-directed antiserum, but not by control pre-immune serum. Recombinant beta-crystallin B2 was likewise recognized by LruB-directed antiserum, but not by pre-immune serum. Moreover, uveitic eye fluids contained significantly higher levels of antiibodies that recognized alpha-crystallin B, beta-crystallin B2 and vimentin than did normal eye fluids. Our results indicate that LruA and LruB share immuno-relevant epitopes with eye proteins, suggesting that cross-reactive antibody interactions with eye antigens may contribute to immunopathogenesis of Leptospira-associated recurrent uveitis.

Does granulocyte colony-stimulating factor ameliorate the proinflammatory response in human meningococcal septic shock?

PubMed

Rojahn, Astrid; Brusletto, Berit; Øvstebø, Reidun; Haug, Kari B F; Kierulf, Peter; Brandtzaeg, Petter

2008-09-01

To test the hypothesis that granulocyte colony-stimulating factor acts cooperatively with interleukin-10 in down-regulating monocyte function in severe meningococcal septic shock. 1) We quantified the plasma levels of granulocyte colony-stimulating factor, interleukin-10, Neisseria meningitidis lipopolysaccharide and the number of N. meningitidis DNA copies in 28 patients with systemic meningococcal disease. 2) We studied the inhibitory effect of recombinant human granulocyte colony-stimulating factor on normal human monocytes stimulated with purified meningococcal lipopolysaccaride. 3) We monitored the inhibitory effects of endogenously produced granulocyte colony-stimulating factor and interleukin-10 in meningococcal shock plasmas on monocytes. Comparative, experimental study. University Hospital and laboratory. Twenty-eight patients with systemic meningococcal disease, 13 with persistent shock, 7 died, and 15 without shock. The median levels of granulocyte colony-stimulating factor in shock and nonshock patients were 1.7 x 10(6) and 8.1 x 10(2) pg/mL; interleukin-10, 2.1 x 10(4) and 4 x 10(1) pg/mL; number of N. meningitidis DNA copies, 2.9 x 10(7) and <10(3)/mL; and lipopolysaccharide, 105 and <0.04 endotoxin units/mL, respectively. The plasma levels of granulocyte colony-stimulating factor were reduced by 50% within 4 to 6 hrs after initiation of antibiotic treatment. In model experiments with lipopolysaccharide-stimulated human monocytes, recombinant human granulocyte colony-stimulating factor and interleukin-10 reduced the release of tumor necrosis factor-alpha by mean 30% and 92%, respectively. When plasmas from three shock patients were depleted of native granulocyte colony-stimulating factor or interleukin-10 by immunoprecipitation, no increase in tumor necrosis factor-alpha release occurred after removal of granulocyte colony-stimulating factor, whereas removal of interleukin-10 increased the tumor necrosis factor-alpha release eight-fold. Although granulocyte colony-stimulating factor in plasma increases by five orders of magnitude in patients with meningococcal shock, the anti-inflammatory effect on patients' monocytes is uncertain.

Increased proliferation of endothelial cells with overexpression of soluble TNF-alpha receptor I gene.

PubMed

Sugano, Masahiro; Tsuchida, Keiko; Tomita, Hideharu; Makino, Naoki

2002-05-01

Vascular endothelial growth factor (VEGF) can overcome a potential anti-angiogenic effect of TNF-alpha by inhibiting endothelial apoptosis induced by this cytokine. Soluble TNF-alpha receptor I (sTNFRI) is an extracellular domain of TNFRI and antagonizes the activity of TNF-alpha. Here we report that sTNFRI is able to stimulate the growth of endothelial cells not by antagonizing TNF-alpha. Exogenously added recombinant human sTNFRI stimulated significantly more cell growth of human umbilical venous endothelial cells (HUVEC) with a low dose (50-200 pg/ml) compared with smooth muscle cells. In contrast, monoclonal antibody against TNF-alpha did not stimulate growth of human HUVEC. The sTNFRI expression plasmid (pcDNA3.1 plasmid) was introduced into the cell culture using OPTI-MEM, lipofectin and transferrin. Growth of HUVEC transfected with sTNFRI vector also increased significantly compared with those transfected with control vector. HUVEC transfected with sTNFRI vector increased the extracellular domain of TNFRI mRNA levels, but did not affect the intracellular domain of TNFRI mRNA levels. Accumulation of sTNFRI significantly increased in conditioned medium from HUVEC transfected with sTNFRI vector compared with those transfected with control vector. HUVEC transfected with sTNFRI vector not only increased sTNFRI but also prevented shedding of sTNFRI from TNFRI. The TNF-alpha -induced internucleosomic fragmentation was also significantly prevented in HUVEC transfected with sTNFRI vector compared with those transfected with control vector. These results suggest that instead of growth factors such as VEGF, local transfection of the sTNFRI gene may have potential therapeutic value in vascular diseases in which TNF-alpha is also usually highly expressed.

Carbon recombination lines as a diagnostic of photodissociation regions

NASA Technical Reports Server (NTRS)

Natta, A.; Walmsley, C. M.; Tielens, A. G. G. M.

1994-01-01

We have observed the C91 alpha radio recombination line toward the Orion H II region. This narrow (approximately 3-5 km per sec full width at half maximum (FWHM)) line is spatially very extended (approximately 8 arcmin or 1 pc). These charateristics compare well with the observed characteristics of the C II fine structure line at 158 microns. Thus, the C91 alpha line originates in the predominantly neutral photodissociation regions separating the H II region from the molecular cloud. We have developed theoretical models for the C II radio recombination lines from photodissociation regions. The results show that the I(C91 alpha)/I(C158) intensity ratio is a sensitive function of the temperature and density of the emitting gas. We have also extended theoretical models for photodissociation regions to include the C II recombination lines. Comparison with these models show that, in the central portion of the Orion region, the C91 alpha line originates in dense (10(exp 6) per cu cm), warm (500-1000 K) gas. Even at large projected distances (approximately 1 pc), the inferred density is still high (10(exp 5) per cu cm) and implies extremely high thermal pressures. As in the case of the (C II) 158 microns line, the large extent of the C91 alpha line shows that (FUV) photons can penetrate to large distances from the illuminating source. The decline of the intensity of the incident radiation field with distance from Theta(sup 1) C seems to be dominated by geometrical dilution, rather than dust extinction. Finally, we have used our models to calculate the intensity of the 9850 A recombination line of C II. The physical conditions inferred from this line are in good agreement with those determined from the radio recombination and the far-infrared fine-structure lines. We show that the ratio of the 9850 A to the C91 alpha lines is a very good probe of very high density clumps.

Interleukin-2-dependent long-term cultures of low-density lymphocytes allow the proliferation of lymphokine-activated killer cells with natural killer, Ti gamma/delta or TNK phenotype.

PubMed

Testa, U; Care, A; Montesoro, E; Fossati, C; Giannella, G; Masciulli, R; Fagioli, M; Bulgarini, D; Habetswallner, D; Isacchi, G

1990-01-01

We have developed a culture system for "long-term" growth of human lymphokine-activated killer (LAK) cells exhibiting an elevated, wide-spectrum antitumor cytotoxicity. The system allows the exponential growth of monocyte-depleted low-density lymphocytes in the presence of human serum and recombinant human interleukin-2 (10(3) U/ml), alone or in combination with interleukin-1 alpha or beta (both at 10 U/ml). Eighteen cultures were established from 18 normal adult donors. The membrane phenotypes of the final LAK cell population, assessed by a panel of monoclonal antibodies (mAb), consist of three main types: (a) NKH-1+, Ti alpha/beta-, Ti gamma/delta-, and CD3- lymphocytes; (b) NKH-1+, Ti alpha/beta-, Ti gamma/delta+, and CD3+ lymphocytes and (c) NKH-1+, Ti alpha/beta+, Ti gamma/delta- and CD3+ lymphocytes. Northern blot analysis showed that all these cell populations express relatively high levels of perforin RNA, particularly cells exhibiting the first phenotype. This culture system may provide a tool for cellular and molecular studies on the mechanisms of antitumor cytotoxicity, as well as the basis for new adoptive immunotherapy protocols in advanced center.

Potential role of recombinant secretory leucoprotease inhibitor in the prevention of neutrophil mediated matrix degradation.

PubMed Central

Llewellyn-Jones, C. G.; Lomas, D. A.; Stockley, R. A.

1994-01-01

BACKGROUND--Neutrophil elastase is able to degrade connective tissue matrices and is thought to be involved in the pathogenesis of destructive lung diseases. METHODS--The ability of recombinant secretory leucoprotease inhibitor (rSLPI) to inhibit neutrophil mediated degradation of fibronectin in vitro is demonstrated and its efficacy compared with native alpha-1-proteinase inhibitor (n alpha 1-PI), recombinant alpha-1-proteinase inhibitor (r alpha 1-PI), and the chemical elastase inhibitor ICI 200,355. RESULTS--When preincubated with neutrophils both rSLPI and r alpha 1-PI were effective inhibitors of fibronectin degradation although n alpha 1-PI and ICI 200,355 were less effective. Recombinant SLPI was the most effective inhibitor when the cells were allowed to adhere to fibronectin before the addition of the inhibitors. Preincubation of rSLPI (0.1 mumol/l) with the fibronectin plate resulted in almost total inhibition of fibronectin degradation (reduced to 3.3 (SE 0.9)% of control). Pretreating the fibronectin plate with 1 mumol/l rSLPI, r alpha 1-PI and ICI 200,355 followed by thorough washing before the addition of cells resulted in no inhibition of fibronectin degradation with r alpha 1-PI and the ICI inhibitor, but rSLPI retained its inhibitory effect. This effect could be reduced by adding rSLPI in high pH buffer or 2 mol/1 NaCl. CONCLUSIONS--It is postulated that rSLPI binds to fibronectin to form a protective layer which prevents its degradation by neutrophil elastase. It may prove to be the most useful therapeutic agent in the prevention of neutrophil mediated lung damage. Images PMID:7912452

Hyperexpression of the gene for a Bacillus alpha-amylase in Bacillus subtilis cells: enzymatic properties and crystallization of the recombinant enzyme.

PubMed

Ikawa, K; Araki, H; Tsujino, Y; Hayashi, Y; Igarashi, K; Hatada, Y; Hagihara, H; Ozawa, T; Ozaki, K; Kobayashi, T; Ito, S

1998-09-01

We have constructed a new excretion vector, pHSP64, to develop a hyperexcretion system for Bacillus subtilis [Sumitomo et al., Biosci. Biotech. Biochem., 59, 2172-2175 (1995)]. The structural gene for a novel liquefying semi-alkaline alpha-amylase from the alkaliphilic Bacillus sp. KSM-1378 was amplified by PCR. It was cloned into a SalI-SmaI site of pHSP64 and the recombinant plasmid obtained was introduced into B. subtilis. The transformed B. subtilis hyperproduced the alpha-amylase activity extracellularly, corresponding to approximately 1.0 g (5 x 10(6) units) per liter of an optimized liquid culture. The recombinant enzyme was purified to homogeneity by a simple purification procedure with very high yield. No significant differences in physiochemical and catalytic properties were observed between the recombinant enzyme and the native enzyme produced by Bacillus sp. KSM-1378. The enzymatic properties of the recombinant enzyme were further examined with respect to the responses to various metal ions. The recombinant enzyme could easily be crystallized at room temperature within one day in a buffered solution of 10% (w/v) ammonium sulfate (pH 6.5).

Cloning and expression of a novel UDP-GlcNAc:alpha-D-mannoside beta1,2-N-acetylglucosaminyltransferase homologous to UDP-GlcNAc:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I.

PubMed Central

Zhang, Wenli; Betel, Doron; Schachter, Harry

2002-01-01

A TBLASTN search with human UDP-GlcNAc:alpha-3-d-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101) as a probe identified human and mouse Unigenes encoding a protein similar to human GnT I (34% identity over 340 amino acids). The recombinant protein converted Man(alpha1-6)[Man(alpha1-3)]Man(beta1-)O-octyl to Man(alpha1-6)[GlcNAc(beta1-2)Man(alpha1-3)]Man(beta1-)O-octyl, the reaction catalysed by GnT I. The enzyme also added GlcNAc to Man(alpha1-6)[GlcNAc(beta1-2)Man(alpha1-3)]Man(beta1-)O-octyl (the substrate for beta-1,2-N-acetylglucosaminyltransferase II), Man(alpha1-)O-benzyl [with K(m) values of approximately 0.3 and >30 mM for UDP-GlcNAc and Man(alpha1-)O-benzyl respectively] and the glycopeptide CYA[Man(alpha1-)O-T]AV (K(m) approximately 12 mM). The product formed with Man(alpha1-)O-benzyl was identified as GlcNAc(beta1-2)Man(alpha1-)O-benzyl by proton NMR spectroscopy. The enzyme was named UDP-GlcNAc:alpha-d-mannoside beta-1,2-N-acetylglucosaminyltransferase I.2 (GnT I.2). The human gene mapped to chromosome 1. Northern-blot analysis showed a 3.3 kb message with a wide tissue distribution. The cDNA has a 1980 bp open reading frame encoding a 660 amino acid protein with a type-2 domain structure typical of glycosyltransferases. Man(beta1-)O-octyl, Man(beta1-)O-p-nitrophenyl and GlcNAc(beta1-2)Man(alpha1-6)[GlcNAc(beta1-2)Man(alpha1-3)]Man(beta1-4)GlcNAc(beta1-4)GlcNAc(beta1-)O-Asn were not acceptors, indicating that GnT I.2 is specific for alpha-linked terminal Man and does not have N-acetylglucosaminyltransferase III, IV, V, VII or VIII activities. CYA[Man(alpha1-)O-T]AV was between three and seven times more effective as an acceptor than the other substrates, suggesting that GnT I.2 may be responsible for the synthesis of the GlcNAc(beta1-2)Man(alpha1-)O-Ser/Thr moiety on alpha-dystroglycan and other O-mannosylated proteins. PMID:11742540

Rescue of a recombinant Machupo virus from cloned cDNAs and in vivo characterization in interferon (αβ/γ) receptor double knockout mice.

PubMed

Patterson, Michael; Seregin, Alexey; Huang, Cheng; Kolokoltsova, Olga; Smith, Jennifer; Miller, Milagros; Smith, Jeanon; Yun, Nadezhda; Poussard, Allison; Grant, Ashley; Tigabu, Bersabeh; Walker, Aida; Paessler, Slobodan

2014-02-01

Machupo virus (MACV) is the etiological agent of Bolivian hemorrhagic fever (BHF), a reemerging and neglected tropical disease associated with high mortality. The prototypical strain of MACV, Carvallo, was isolated from a human patient in 1963, but minimal in vitro and in vivo characterization has been reported. To this end, we utilized reverse genetics to rescue a pathogenic MACV from cloned cDNAs. The recombinant MACV (rMACV) had in vitro growth properties similar to those of the parental MACV. Both viruses caused similar disease development in alpha/beta and gamma interferon receptor knockout mice, including neurological disease development and high mortality. In addition, we have identified a novel murine model with mortality and neurological disease similar to BHF disease reported in humans and nonhuman primates.

[Building immune microsphere against tumor necrosis factor-alpha (TNF-alpha)].

PubMed

Wang, Qin; Wu, Xiongfei; Wang, Junxia; Liu, Hong; Li, Lian; Jin, Xiyu

2005-12-01

We have constructed the immune microsphere against tumor necrosis factor-alpha (TNF-alpha) prospectively, hoping to establish the experiment groundwork in more researches which could be used in specific elimination of the TNF-alpha by blood purification method for the future. The recombinant human tumor necrosis factor-alpha monoclonal antibody (rHTNF-alpha McAb) was wrapped on the polystyrene microsphere (PSM) carrier connecting poly-L-lysine (PLL) beforehand. They were earmarked by the fluorescein isothiocyanate (FITC) respectively. The packing conditions were examined using the inversted and fluorescence microscopes and the spectrophotometer. The results showed that the best conditions for wrapping were 20 degrees C, pH9.5 and 60 minutes. The PLL content was not changed in the washing fluid after coating, which indicated the wrapping was quite firm. At the same temperature and same coating time, the rHTNF-alpha McAb coated on the PLL was obviously substantial when the concentration of glutaraldehyde solution was 0.2%. The findings demonstrated that the built immune microsphers can be used as a novel adsorption material. This method is simple and economic, and it offers a new approach to the related studies.

Heterologous expression of bovine lactoferricin in Pichia methanolica.

PubMed

Wang, Haikuan; Zhao, Xinhuai; Lu, Fuping

2007-06-01

According to the bias of codon utilization of Pichia methanolica, a fragment encoding bovine lactoferricin has been cloned and expressed in the P. methanolica under the control of the alcohol oxidase promoter, which was followed by the Saccharomyces cerevisiae alpha-factor signal peptide. The alpha-factor signal peptide efficiently directed the secretion of bovine lactoferricin from the recombinant yeast cell. The recombinant bovine lactoferricin appears to be successfully expressed, as it displays antibacterial activity (antibacterial assay). Moreover, the identity of the recombinant product was estimated by Tricine-SDS-PAGE.

Optimization of the primary recovery of human interferon alpha2b from Escherichia coli inclusion bodies.

PubMed

Valente, C A; Monteiro, G A; Cabral, J M S; Fevereiro, M; Prazeres, D M F

2006-01-01

The human interferon alpha2b (hu-IFNalpha2b) gene was cloned in Escherichia coli JM109(DE3) and the recombinant protein was expressed as cytoplasmic inclusion bodies (IB). The present work discusses the recovery of hu-IFNalpha2b IB from the E. coli cells. An optimized protocol is proposed based on the sequential evaluation of recovery steps and parameters: (i) cell disruption, (ii) IB recovery and separation from cell debris, (iii) IB washing, and (iv) IB solubilization. Parameters such as hu-IFNalpha2b purity and recovery yield were measured after each step. The optimized recovery protocol yielded 60% of hu-IFNalpha2b with a purity of up to 80%. The protein was renatured at high concentration after recovery and it was found to display biological activity.

Differential processing of the two subunits of human choriogonadotropin (hCG) by granulosa cells. I. Preparation and characterization of selectively labeled hCG

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

Landefeld, T.D.; Byrne, M.D.; Campbell, K.L.

1981-12-01

The alpha- and beta-subunits of hCG were radioiodinated and recombined with unlabeled complementary subunits. The resultant recombined hormones, selectively labeled in either the alpha- or beta-subunit, were separated from unrecombined subunit by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, extracted with Triton X-100, and characterized by binding analysis. The estimates of maximum binding (active fraction) of the two resultant selectively labeled, recombined hCG preparations, determined with excess receptor were 0.41 and 0.59. These values are similar to those obtained when hCG is labeled as an intact molecule. The specific activities of the recombined preparations were estimated by four different methods, and themore » resulting values were used in combination with the active fraction estimates to determine the concentrations of active free and bound hormone. Binding analyses were run using varying concentrations of both labeled and unlabeled hormone. Estimates of the equilibrium dissociation binding constant (Kd) and receptor capacity were calculated in three different ways. The mean estimates of capacity (52.6 and 52.7 fmol/mg tissue) and Kd (66.6 and 65.7 pM) for the two preparations were indistinguishable. Additionally, these values were similar to values reported previously for hCG radioiodinated as an intact molecule. The availability of well characterized, selectively labeled hCG preparations provides new tools for studying the mechanism of action and the target cell processing of the subunits of this hormone.« less

Recombinant GDNF: Tetanus toxin fragment C fusion protein produced from insect cells

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

Li, Jianhong; Chian, Ru-Ju; Ay, Ilknur

2009-07-31

Glial cell line-derived neurotrophic factor (GDNF) has potent survival-promoting effects on CNS motor neurons in experimental animals. Its therapeutic efficacy in humans, however, may have been limited by poor bioavailability to the brain and spinal cord. With a view toward improving delivery of GDNF to CNS motor neurons in vivo, we generated a recombinant fusion protein comprised of rat GDNF linked to the non-toxic, neuron-binding fragment of tetanus toxin. Recombinant GDNF:TTC produced from insect cells was a soluble homodimer like wild-type GDNF and was bi-functional with respect to GDNF and TTC activity. Like recombinant rat GDNF, the fusion protein increasedmore » levels of immunoreactive phosphoAkt in treated NB41A3-hGFR{alpha}-1 neuroblastoma cells. Like TTC, GDNF:TTC bound to immobilized ganglioside GT1b in vitro with high affinity and selectivity. These results support further testing of recombinant GDNF:TTC as a non-viral vector to improve delivery of GDNF to brain and spinal cord in vivo.« less

Roles of ikB-alpha Protein Kinases in Activation of NF-kB in Breast Cancer

DTIC Science & Technology

2005-07-01

observed previously that treatment with the selective pharmacological inhibitors of CK2, apigenin or emodin , inhibited NF-B activity in human breast...mM apigenin or 1–25 mg/ml emodin (both from Sigma Chemical Co.) dissolved in DMSO or similar dilution of DMSO as control. MCF-10F is a human mammary... emodin , or 0.58–1.46 mM CK2-specific peptide substrate RRREEETEEE (Sigma Genosys Inc.) was added to the kinase reaction. Alternatively, recombinant CK2

Stability of nonaqueous suspension formulations of plasma derived factor IX and recombinant human alpha interferon at elevated temperatures.

PubMed

Knepp, V M; Muchnik, A; Oldmark, S; Kalashnikova, L

1998-07-01

To identify a suitable nonaqueous, parenterally acceptable suspending vehicle whereby a therapeutic protein is delivered as a stable flowable powder, making it amenable to delivery from sustained delivery systems maintained at body temperature. Formulations of plasma derived Factor IX (pdFIX) and recombinant human alpha interferon (rhalpha-IFN) were formulated as dry powders, suspended in various vehicles (perfluorodecalin, perfluorotributylamine, methoxyflurane, polyethylene glycol 400, soybean oil, tetradecane or octanol) and stored at 37 degrees C. Stability was assessed by size exclusion chromatography, reverse phase chromatography, ion exchange chromatography, and bioassay, and was compared to the stability of dry powder formulations stored at 37 degrees C and -80 degrees C. PdFIX was stable when stored at 37 degrees C as a dry powder, or when the dry powder was suspended in the pharmaceutically acceptable vehicles perfluorodecalin or perfluorotributylamine. Suspensions of the powder in other pharmaceutically/parenterally acceptable vehicles such as soybean oil or PEG 400 resulted in aggregation and loss of bioactivity. A dry powder formulation of rhalpha-IFN suspended in perfluorodecalin was also stable at 37 degrees C. This study shows the potential utility of perfluorinated hydrocarbons as nonaqueous suspending vehicles for long term in-vivo delivery of therapeutic proteins.

Identification of Diverse Alphacoronaviruses and Genomic Characterization of a Novel Severe Acute Respiratory Syndrome-Like Coronavirus from Bats in China

PubMed Central

He, Biao; Zhang, Yuzhen; Xu, Lin; Yang, Weihong; Yang, Fanli; Feng, Yun; Xia, Lele; Zhou, Jihua; Zhen, Weibin; Feng, Ye; Guo, Huancheng

2014-01-01

ABSTRACT Although many severe acute respiratory syndrome-like coronaviruses (SARS-like CoVs) have been identified in bats in China, Europe, and Africa, most have a genetic organization significantly distinct from human/civet SARS CoVs in the receptor-binding domain (RBD), which mediates receptor binding and determines the host spectrum, resulting in their failure to cause human infections and making them unlikely progenitors of human/civet SARS CoVs. Here, a viral metagenomic analysis of 268 bat rectal swabs collected from four counties in Yunnan Province has identified hundreds of sequences relating to alpha- and betacoronaviruses. Phylogenetic analysis based on a conserved region of the RNA-dependent RNA polymerase gene revealed that alphacoronaviruses had diversities with some obvious differences from those reported previously. Full genomic analysis of a new SARS-like CoV from Baoshan (LYRa11) showed that it was 29,805 nucleotides (nt) in length with 13 open reading frames (ORFs), sharing 91% nucleotide identity with human/civet SARS CoVs and the most recently reported SARS-like CoV Rs3367, while sharing 89% with other bat SARS-like CoVs. Notably, it showed the highest sequence identity with the S gene of SARS CoVs and Rs3367, especially in the RBD region. Antigenic analysis showed that the S1 domain of LYRa11 could be efficiently recognized by SARS-convalescent human serum, indicating that LYRa11 is a novel virus antigenically close to SARS CoV. Recombination analyses indicate that LYRa11 is likely a recombinant descended from parental lineages that had evolved into a number of bat SARS-like CoVs. IMPORTANCE Although many severe acute respiratory syndrome-like coronaviruses (SARS-like CoVs) have been discovered in bats worldwide, there are significant different genic structures, particularly in the S1 domain, which are responsible for host tropism determination, between bat SARS-like CoVs and human SARS CoVs, indicating that most reported bat SARS-like CoVs are not the progenitors of human SARS CoV. We have identified diverse alphacoronaviruses and a close relative (LYRa11) to SARS CoV in bats collected in Yunnan, China. Further analysis showed that alpha- and betacoronaviruses have different circulation and transmission dynamics in bat populations. Notably, full genomic sequencing and antigenic study demonstrated that LYRa11 is phylogenetically and antigenically closely related to SARS CoV. Recombination analyses indicate that LYRa11 is a recombinant from certain bat SARS-like CoVs circulating in Yunnan Province. PMID:24719429

Integrated Summary Report: Validation of Two Binding Assays ...

EPA Pesticide Factsheets

This Integrated Summary Report (ISR) summarizes, in a single document, the results from an international multi-laboratory validation study conducted for two in vitro estrogen receptor (ER) binding assays. These assays both use human recombinant estrogen receptor, alpha subtype (hrERα), to identify chemicals that may impact estrogen signaling through binding to the ER. The purpose of the ISR is to support the peer review of the findings obtained during the validation process.The two assays evaluated during this validation process are: The Freyberger-Wilson Assay (FW) using a full length human ER, and The Chemical Evaluation and Research Institute (CERI) Assay using a ligand-binding domain of the human ER.The two assays are mechanistically and functionally similar in that each measures the ability of a test chemical to competitively inhibit binding of [3H]17β-estradiol to the human recombinant ER. The essential elements of the FW and the CERI assays were developed at the laboratories of Bayer Pharma AG, Wuppertal, Germany (Freyberger et al., 2010) and CERI, Tokyo, Japan (Akahori et al., 2008), respectively.The ER competitive binding assay has long been in use, and is a well characterized approach, but historically uses rodent or other animal tissues as a source of the ER. Validation of the FW and CERI assays using human recombinant estrogen receptors ( subtype) will provide an updated alternative for the Agency’s current test guideline (OPPTS 89

Decreased agonist sensitivity of human GABA(A) receptors by an amino acid variant, isoleucine to valine, in the alpha1 subunit.

PubMed

Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nabekura, J; Noguchi, K; Akaike, N; Witt, M R; Nielsen, M

1997-06-25

Recombinant human GABA(A) receptors were investigated in vitro by coexpression of cDNAs coding for alpha1, beta2, and gamma2 subunits in the baculovirus/Sf-9 insect cell system. We report that a single amino acid exchange (isoleucine 121 to valine 121) in the N-terminal, extracellular part of the alpha1 subunit induces a marked decrease in agonist GABA(A) receptor ligand sensitivity. The potency of muscimol and GABA to inhibit the binding of the GABA(A) receptor antagonist [3H]SR 95531 (2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide) was higher in receptor complexes of alpha1(ile 121) beta2gamma2 than in those of alpha1(val 121) beta2gamma2 (IC50 values were 32-fold and 26-fold lower for muscimol and GABA, respectively). The apparent affinity of the GABA(A) receptor antagonist bicuculline methiodide to inhibit the binding of [3H]SR 95531 did not differ between the two receptor complex variants. Electrophysiological measurements of GABA induced whole-cell Cl- currents showed a ten-fold decrease in the GABA(A) receptor sensitivity of alpha1 (val 121) beta2gamma2 as compared to alpha1(ile 121) beta2gamma2 receptor complexes. Thus, a relatively small change in the primary structure of the alpha1 subunit leads to a decrease selective for GABA(A) receptor sensitivity to agonist ligands, since no changes were observed in a GABA(A) receptor antagonist affinity and benzodiazepine receptor binding.

Characteristics of recombinantly expressed rat and human histamine H3 receptors.

PubMed

Wulff, Birgitte S; Hastrup, Sven; Rimvall, Karin

2002-10-18

Human and rat histamine H(3) receptors were recombinantly expressed and characterized using receptor binding and a functional cAMP assay. Seven of nine agonists had similar affinities and potencies at the rat and human histamine H(3) receptor. S-alpha-methylhistamine had a significantly higher affinity and potency at the human than rat receptor, and for 4-[(1R*,2R*)-2-(5,5-dimethyl-1-hexynyl)cyclopropyl]-1H-imidazole (Perceptin) the preference was the reverse. Only two of six antagonists had similar affinities and potencies at the human and the rat histamine H(3) receptor. Ciproxifan, thioperamide and (1R*,2R*)-trans-2-imidazol-4 ylcyclopropyl) (cyclohexylmethoxy) carboxamide (GT2394) had significantly higher affinities and potencies at the rat than at the human histamine H(3) receptor, while for N-(4-chlorobenzyl)-N-(7-pyrrolodin-1-ylheptyl)guanidine (JB98064) the preference was the reverse. All antagonists also showed potent inverse agonism properties. Iodoproxyfan, Perceptin, proxyfan and GR175737, compounds previously described as histamine H(3) receptor antagonists, acted as full or partial agonists at both the rat and the human histamine H(3) receptor. Copyright 2002 Elsevier Science B.V.

A complex of serine protease genes expressed preferentially in cytotoxic T-lymphocytes is closely linked to the T-cell receptor alpha- and delta-chain genes on mouse chromosome 14.

PubMed

Crosby, J L; Bleackley, R C; Nadeau, J H

1990-02-01

A complex of genes encoding serine proteases that are preferentially expressed in cytotoxic T-cells was shown to be closely linked to the T-cell receptor alpha- and delta-chain genes on mouse chromosome 14. A striking difference in recombination frequencies among linkage crosses was reported. Two genes, Np-1 and Tcra, which fail to recombine in crosses involving conventional strains of mice, were shown to recombine readily in interspecific crosses involving Mus spretus. This difference in recombination frequency suggests chromosomal rearrangements that suppress recombination in conventional crosses, recombination hot spots in interspecific crosses, or selection against recombinant haplotypes during development of recombinant inbred strains. Finally, a mutation called disorganization, which is located near the serine protease complex, is of considerable interest because it causes an extraordinarily wide variety of congenital defects. Because of the involvement of serine protease loci in several homeotic mutations in Drosophila, disorganization must be considered a candidate for a mutation in a serine protease-encoding gene.

The effect of N2/+/ recombination on the aeronomic determination of the charge exchange rate coefficient of O/+//2D/ with N2

NASA Technical Reports Server (NTRS)

Torr, D. G.; Orsini, N.

1978-01-01

The Atmosphere Explorer (AE) data are reexamined in the light of new laboratory measurements of the N2(+) recombination rate coefficient alpha. The new measurements support earlier measurements which yielded values of alpha significantly lower than the AE values. It is found that the values for alpha determined from the satellite data can be reconciled with the laboratory measurements, if the charge exchange rate coefficient for O(+)(2D) with N2 is less than one-quarter of that derived in the laboratory by Rutherford and Vroom (1971).

Encapsulated Hsp70 decreases endotoxin-induced production of ROS and TNFα in human phagocytes.

PubMed

Yurinskaya, M M; Kochetkova, O Yu; Shabarchina, L I; Antonova, O Yu; Suslikov, A V; Evgen'ev, M B; Vinokurov, M G

2017-01-01

Human heat shock protein Hsp70 was experimentally inserted into polyelectrolyte microcapsules. Encapsulated recombinant Hsp70 was studied in terms of its effects on neutrophil apoptosis, the production of reactive oxygen species, and the secretion of tumor necrosis factor alpha by promonocytic THP-1 cells. It was found that encapsulated Hsp70 effectively inhibits neutrophil apoptosis, unlike free exogenous protein used in solution. In THP-1 cells, encapsulated and free Hsp70 reduced LPS-induced tumor necrosis factor alpha production with a similar efficiency. Encapsulated Hsp70 reduces LPS-induced reactive oxygen species production by neutrophils in the course of its release from the microcapsules but not as much as free Hsp70. Thus, the polyelectrolyte microcapsules can be used as containers for the effective delivery of Hsp70 to neutrophils and monocytes to significantly improve the functioning of the innate immune system.

Assessment of a robust model protocol with accelerated throughput for a human recombinant full length estrogen receptor-alpha binding assay: protocol optimization and intralaboratory assay performance as initial steps towards validation.

PubMed

Freyberger, Alexius; Wilson, Vickie; Weimer, Marc; Tan, Shirlee; Tran, Hoai-Son; Ahr, Hans-Jürgen

2010-08-01

Despite about two decades of research in the field of endocrine active compounds, still no validated human recombinant (hr) estrogen receptor-alpha (ERalpha) binding assay is available, although hr-ERalpha is available from several sources. In a joint effort, US EPA and Bayer Schering Pharma with funding from the EU-sponsored 6th framework project, ReProTect, developed a model protocol for such a binding assay. Important features of this assay are the use of a full length hr-ERalpha and performance in a 96-well plate format. A full length hr-ERalpha was chosen, as it was considered to provide the most accurate and human-relevant results, whereas truncated receptors could perform differently. Besides three reference compounds [17beta-estradiol, norethynodrel, dibutylphthalate] nine test compounds with different affinities for the ERalpha [diethylstilbestrol (DES), ethynylestradiol, meso-hexestrol, equol, genistein, o,p'-DDT, nonylphenol, n-butylparaben, and corticosterone] were used to explore the performance of the assay. Three independent experiments per compound were performed on different days, and dilutions of test compounds from deep-frozen stocks, solutions of radiolabeled ligand and receptor preparation were freshly prepared for each experiment. The ERalpha binding properties of reference and test compounds were well detected. As expected dibutylphthalate and corticosterone were non-binders in this assay. In terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using a human recombinant ERalpha ligand binding domain. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.5. Our data demonstrate that the assay was robust and reliably ranked compounds with strong, weak, and no affinity for the ERalpha with high accuracy. It avoids the manipulation and use of animals, i.e., the preparation of uterine cytosol as receptor source from ovariectomized rats, as a recombinant protein is used and thus contributes to the 3R concept (reduce, replace, and refine). Furthermore, in contrast to other assays, this assay could be adjusted to an intermediate/high throughput format. On the whole, this assay is a promising candidate for further validation. Copyright 2010 Elsevier Inc. All rights reserved.

α-Fetoprotein as a modulator of the pro-inflammatory response of human keratinocytes

PubMed Central

Potapovich, AI; Pastore, S; Kostyuk, VA; Lulli, D; Mariani, V; De Luca, C; Dudich, EI; Korkina, LG

2009-01-01

Background and purpose: The immunomodulatory effects of α-fetoprotein (AFP) on lymphocytes and macrophages have been described in vitro and in vivo. Recombinant forms of human AFP have been proposed as potential therapeutic entities for the treatment of autoimmune diseases. We examined the effects of embryonic and recombinant human AFP on the spontaneous, UVA- and cytokine-induced pro-inflammatory responses of human keratinocytes. Experimental approach: Cultures of primary and immortalized human keratinocytes (HaCaT) and human blood T lymphocytes were used. The effects of AFP on cytokine expression were studied by bioplexed elisa and quantitative reverse transcriptase polymerase chain reaction assay. Kinase and nuclear factor kappa B (NFκB) phosphorylation were quantified by intracellular elisa. Nuclear activator protein 1 and NFκB DNA binding activity was measured by specific assays. Nitric oxide and H2O2 production and redox status were assessed by fluorescent probe and biochemical methods. Key results: All forms of AFP enhanced baseline expression of cytokines, chemokines and growth factors. AFP dose-dependently increased tumour necrosis factor alpha-stimulated granulocyte macrophage colony stimulating factor and interleukin 8 expression and decreased tumour necrosis factor alpha-induced monocyte chemotactic protein 1 and IP-10 (interferon gamma-produced protein of 10 kDa) expression. AFP induced a marked activator protein 1 activation in human keratinocytes. AFP also increased H2O2 and modulated nitrite/nitrate levels in non-stimulated keratinocytes whereas it did not affect these parameters or cytokine release from UVA-stimulated cells. Phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Akt1 but not NFκB was activated by AFP alone or by its combination with UVA. Conclusions and implications: Exogenous AFP induces activation of human keratinocytes, with de novo expression of a number of pro-inflammatory mediators and modulation of their pro-inflammatory response to cytokines or UVA. AFP may modulate inflammatory events in human skin. PMID:19785658

13-cis-retinoic acid competitively inhibits 3 alpha-hydroxysteroid oxidation by retinol dehydrogenase RoDH-4: a mechanism for its anti-androgenic effects in sebaceous glands?

PubMed

Karlsson, Teresa; Vahlquist, Anders; Kedishvili, Natalia; Törmä, Hans

2003-03-28

Retinol dehydrogenase-4 (RoDH-4) converts retinol and 13-cis-retinol to corresponding aldehydes in human liver and skin in the presence of NAD(+). RoDH-4 also converts 3 alpha-androstanediol and androsterone into dihydrotestosterone and androstanedione, which may stimulate sebum secretion. This oxidative 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) activity of RoDH-4 is competitively inhibited by retinol and 13-cis-retinol. Here, we further examine the substrate specificity of RoDH-4 and the inhibition of its 3 alpha-HSD activity by retinoids. Recombinant RoDH-4 oxidized 3,4-didehydroretinol-a major form of vitamin A in the skin-to its corresponding aldehyde. 13-cis-retinoic acid (isotretinoin), 3,4-didehydroretinoic acid, and 3,4-didehydroretinol, but not all-trans-retinoic acid or the synthetic retinoids acitretin and adapalene, were potent competitive inhibitors of the oxidative 3 alpha-HSD activity of RoDH-4, i.e., reduced the formation of dihydrotestosterone and androstandione in vitro. Extrapolated to the in vivo situation, this effect might explain the unique sebosuppressive effect of isotretinoin when treating acne.

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.

Toxoplasma gondii: biochemical and biophysical characterization of recombinant soluble dense granule proteins GRA2 and GRA6.

PubMed

Bittame, Amina; Effantin, Grégory; Pètre, Graciane; Ruffiot, Pauline; Travier, Laetitia; Schoehn, Guy; Weissenhorn, Winfried; Cesbron-Delauw, Marie-France; Gagnon, Jean; Mercier, Corinne

2015-03-27

The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressed in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6-8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8-15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

[Erythropoietin influence on oxygen transport function of blood and prooxidant/antioxidant balance in rabbits under lipopolysaccharide injection].

PubMed

Zinchuk, V V; Shul'ga, E V; Guliaĭ, I E

2010-01-01

We aimed to study the erythropoietin influence on oxygen transport function of blood and prooxidant/antioxidant balance in rabbits under lipopolysaccharide injection. Recombinant human erythropoietin-alpha was administered intraperitoneally in the dose 1000 U/kg 30 minutes before intravenous injection of 500 mkg/kg lipopolysaccharide from E. coli. After 12 hours, blood samples were collected for the assessment of oxygen transport function of blood; nitrate/nitrite levels and tissue samples were collected for measurement of conjugated dienes, malondialdehyde, alpha-tocopherol and catalase. Erythropoietin improves parameters of oxygen transport function of blood, increases hemoglobin-oxygen affinity through the NO-dependent mechanism, reduces activity of free radical processes, and increases antioxidant protection under lipopolysaccharide injection.

Sequence of the cDNA of a human dihydrodiol dehydrogenase isoform (AKR1C2) and tissue distribution of its mRNA.

PubMed Central

Shiraishi, H; Ishikura, S; Matsuura, K; Deyashiki, Y; Ninomiya, M; Sakai, S; Hara, A

1998-01-01

Human liver contains three isoforms (DD1, DD2 and DD4) of dihydrodiol dehydrogenase with 20alpha- or 3alpha-hydroxysteroid dehydrogenase activity; the dehydrogenases belong to the aldo-oxo reductase (AKR) superfamily. cDNA species encoding DD1 and DD4 have been identified. However, four cDNA species with more than 99% sequence identity have been cloned and are compatible with a partial amino acid sequence of DD2. In this study we have isolated a cDNA clone encoding DD2, which was confirmed by comparison of the properties of the recombinant and hepatic enzymes. This cDNA showed differences of one, two, four and five nucleotides from the previously reported four cDNA species for a dehydrogenase of human colon carcinoma HT29 cells, human prostatic 3alpha-hydroxysteroid dehydrogenase, a human liver 3alpha-hydroxysteroid dehydrogenase-like protein and chlordecone reductase-like protein respectively. Expression of mRNA species for the five similar cDNA species in 20 liver samples and 10 other different tissue samples was examined by reverse transcriptase-mediated PCR with specific primers followed by diagnostic restriction with endonucleases. All the tissues expressed only one mRNA species corresponding to the newly identified cDNA for DD2: mRNA transcripts corresponding to the other cDNA species were not detected. We suggest that the new cDNA is derived from the principal gene for DD2, which has been named AKR1C2 by a new nomenclature for the AKR superfamily. It is possible that some of the other cDNA species previously reported are rare allelic variants of this gene. PMID:9716498

Cloning and characterization of the mouse alpha1C/A-adrenergic receptor gene and analysis of an alpha1C promoter in cardiac myocytes: role of an MCAT element that binds transcriptional enhancer factor-1 (TEF-1).

PubMed

O'Connell, T D; Rokosh, D G; Simpson, P C

2001-05-01

alpha1-Adrenergic receptor (AR) subtypes in the heart are expressed by myocytes but not by fibroblasts, a feature that distinguishes alpha1-ARs from beta-ARs. Here we studied myocyte-specific expression of alpha1-ARs, focusing on the subtype alpha1C (also called alpha1A), a subtype implicated in cardiac hypertrophic signaling in rat models. We first cloned the mouse alpha1C-AR gene, which consisted of two exons with an 18 kb intron, similar to the alpha1B-AR gene. The receptor coding sequence was >90% homologous to that of rat and human. alpha1C-AR transcription in mouse heart was initiated from a single Inr consensus sequence at -588 from the ATG; this and a putative polyadenylation sequence 8.5 kb 3' could account for the predominant 11 kb alpha1C mRNA in mouse heart. A 5'-nontranscribed fragment of 4.4 kb was active as a promoter in cardiac myocytes but not in fibroblasts. Promoter activity in myocytes required a single muscle CAT (MCAT) element, and this MCAT bound in vitro to recombinant and endogenous transcriptional enhancer factor-1. Thus, alpha1C-AR transcription in cardiac myocytes shares MCAT dependence with other cardiac-specific genes, including the alpha- and beta-myosin heavy chains, skeletal alpha-actin, and brain natriuretic peptide. However, the mouse alpha1C gene was not transcribed in the neonatal heart and was not activated by alpha1-AR and other hypertrophic agonists in rat myocytes, and thus differed from other MCAT-dependent genes and the rat alpha1C gene.

Characterization of cognitive deficits in rats overexpressing human alpha-synuclein in the ventral tegmental area and medial septum using recombinant adeno-associated viral vectors.

PubMed

Hall, Hélène; Jewett, Michael; Landeck, Natalie; Nilsson, Nathalie; Schagerlöf, Ulrika; Leanza, Giampiero; Kirik, Deniz

2013-01-01

Intraneuronal inclusions containing alpha-synuclein (a-syn) constitute one of the pathological hallmarks of Parkinson's disease (PD) and are accompanied by severe neurodegeneration of A9 dopaminergic neurons located in the substantia nigra. Although to a lesser extent, A10 dopaminergic neurons are also affected. Neurodegeneration of other neuronal populations, such as the cholinergic, serotonergic and noradrenergic cell groups, has also been documented in PD patients. Studies in human post-mortem PD brains and in rodent models suggest that deficits in cholinergic and dopaminergic systems may be associated with the cognitive impairment seen in this disease. Here, we investigated the consequences of targeted overexpression of a-syn in the mesocorticolimbic dopaminergic and septohippocampal cholinergic pathways. Rats were injected with recombinant adeno-associated viral vectors encoding for either human wild-type a-syn or green fluorescent protein (GFP) in the ventral tegmental area and the medial septum/vertical limb of the diagonal band of Broca, two regions rich in dopaminergic and cholinergic neurons, respectively. Histopathological analysis showed widespread insoluble a-syn positive inclusions in all major projections areas of the targeted nuclei, including the hippocampus, neocortex, nucleus accumbens and anteromedial striatum. In addition, the rats overexpressing human a-syn displayed an abnormal locomotor response to apomorphine injection and exhibited spatial learning and memory deficits in the Morris water maze task, in the absence of obvious spontaneous locomotor impairment. As losses in dopaminergic and cholinergic immunoreactivity in both the GFP and a-syn expressing animals were mild-to-moderate and did not differ from each other, the behavioral impairments seen in the a-syn overexpressing animals appear to be determined by the long term persisting neuropathology in the surviving neurons rather than by neurodegeneration.

Role of alpha-synuclein in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism in mice.

PubMed

Schlüter, O M; Fornai, F; Alessandrí, M G; Takamori, S; Geppert, M; Jahn, R; Südhof, T C

2003-01-01

In humans, mutations in the alpha-synuclein gene or exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produce Parkinson's disease with loss of dopaminergic neurons and depletion of nigrostriatal dopamine. alpha-Synuclein is a vertebrate-specific component of presynaptic nerve terminals that may function in modulating synaptic transmission. To test whether MPTP toxicity involves alpha-synuclein, we generated alpha-synuclein-deficient mice by homologous recombination, and analyzed the effect of deleting alpha-synuclein on MPTP toxicity using these knockout mice. In addition, we examined commercially available mice that contain a spontaneous loss of the alpha-synuclein gene. As described previously, deletion of alpha-synuclein had no significant effects on brain structure or composition. In particular, the levels of synaptic proteins were not altered, and the concentrations of dopamine, dopamine metabolites, and dopaminergic proteins were unchanged. Upon acute MPTP challenge, alpha-synuclein knockout mice were partly protected from chronic depletion of nigrostriatal dopamine when compared with littermates of the same genetic background, whereas mice carrying the spontaneous deletion of the alpha-synuclein gene exhibited no protection. Furthermore, alpha-synuclein knockout mice but not the mice with the alpha-synuclein gene deletion were slightly more sensitive to methamphetamine than littermate control mice. These results demonstrate that alpha-synuclein is not obligatorily coupled to MPTP sensitivity, but can influence MPTP toxicity on some genetic backgrounds, and illustrate the need for extensive controls in studies aimed at describing the effects of mouse knockouts on MPTP sensitivity.

Preparation of fluorescent tocopherols for use in protein binding and localization with the alpha-tocopherol transfer protein.

PubMed

Nava, Phillip; Cecchini, Matt; Chirico, Sara; Gordon, Heather; Morley, Samantha; Manor, Danny; Atkinson, Jeffrey

2006-06-01

Sixteen fluorescent analogues of the lipid-soluble antioxidant vitamin alpha-tocopherol were prepared incorporating fluorophores at the terminus of omega-functionalized 2-n-alkyl-substituted chromanols (1a-d and 4a-d) that match the methylation pattern of alpha-tocopherol, the most biologically active form of vitamin E. The fluorophores used include 9-anthroyloxy (AO), 7-nitrobenz-2-oxa-1,3-diazole (NBD), N-methyl anthranilamide (NMA), and dansyl (DAN). The compounds were designed to function as fluorescent reporter ligands for protein-binding and lipid transfer assays. The fluorophores were chosen to maximize the fluorescence changes observed upon moving from an aqueous environment (low fluorescence intensity) to an hydrophobic environment such as a protein's binding site (high fluorescence intensity). Compounds 9d (anthroyloxy) and 10d (nitrobenzoxadiazole), having a C9-carbon chain between the chromanol and the fluorophore, were shown to bind specifically and reversibly to recombinant human tocopherol transfer protein (alpha-TTP) with dissociation constants of approximately 280 and 60 nM, respectively, as compared to 25 nM for the natural ligand 2R,4'R,8'R-alpha-tocopherol. Thus, compounds have been prepared that allow the investigation of the rate of alpha-TTP-mediated inter-membrane transfer of alpha-tocopherol and to investigate the mechanism of alpha-TTP function at membranes of different composition.

Recombinant human erythropoietin (rHuEPO): cross-linking with disuccinimidyl esters and identification of the interfacing domains in EPO.

PubMed Central

Haniu, M.; Narhi, L. O.; Arakawa, T.; Elliott, S.; Rohde, M. F.

1993-01-01

Several amino groups of recombinant human erythropoietin are selectively cross-linked by specific cross-linkers including disuccinimidyl suberate or dithiobis(succinimidyl propionate). Intramolecular cross-linkings are obtained without significant change of the protein conformation using appropriate concentrations (0.2 mM) of the cross-linkers, which possess an 11-12-A length of a spacer between two reacting groups. Intramolecularly cross-linked peptides obtained suggest that several amino groups in erythropoietin (EPO) are positioned at a distance of near 12 A in the solution state. These interfacing amino groups include Lys 20-Lys 154, Lys 45-Lys 140, Lys 52-Lys 154, Lys 52-Lys 140, and Ala 1-Lys 116. A comparison of the cross-linking results between nonglycosylated EPO and glycosylated EPO suggests that both proteins retain high similarity regarding protein conformation. These results fit a structural model similar to that of human growth hormone, in which four alpha-helical bundles and a long stretch of beta-sheet structure are involved in the active protein. PMID:8401229

Electron-Temperature Dependence of the Recombination of NH4(+)((NH3)(sub n) Ions with Electrons

NASA Technical Reports Server (NTRS)

Skrzypkowski, M. P.; Johnson, R.

1997-01-01

The two-body recombination of NH4(+)(NH3)(sub 2,3) cluster-ions with electrons has been studied in an afterglow experiment in which the electron temperature T, was elevated by radio-frequency heating from 300 K up to 900 K. The recombination coefficients for the n = 2 and n = 3 cluster ions were found to be equal, alpha(sub 2, sup(2)) = alpha(sub 3, sup(2)) = (4.8 +/- 0.5) x 10(exp - 6)cu cm/s, and to vary with electron temperature as T(sub c, sup -0.65) rather than to be nearly temperature-independent as had been inferred from measurements in microwave-heated plasmas.

Development of Real-Time Reverse Transcriptase PCR Assays for the Detection of Punta Toro Virus and Pichinde Virus

DTIC Science & Technology

2016-09-09

Gowen et al., 2006c; Smee et al., 1993) and guinea pigs (Jahrling et al., 1981; Lucia et al., 1989) 91 as LASV infection in humans. Both PICV and...Moe, J.B., 1981. Pathogenesis of a pichinde virus 281 strain adapted to produce lethal infections in guinea pigs . Infect Immun 32, 872-880. 282... guinea pig model: antiviral 286 therapy with recombinant interferon-alpha, the immunomodulator CL246,738 and ribavirin. Antiviral 287 Res 12, 279-292

The production of O(1S) from dissociative recombination of O2(+). [in earth upper atmosphere

NASA Technical Reports Server (NTRS)

Guberman, Steven L.

1987-01-01

The first theoretical calculations of the rate coefficient alpha for dissociative recombination of O2(+) leading to O(1S) are reported for a wide range of temperatures. The findings are discussed in terms of the potential energy curves for the ground state of O2(+) and for the dissociative 1Sigma(u) state calculated here. Values of alpha for the equilibrium case in which the electron and vibrational temperatures are identical are shown.

In vitro hCG and human recombinant FSH actions on testicular steroidogenesis in the toad Bufo arenarum.

PubMed

Canosa, L F; Ceballos, N R

2002-05-01

In order to study the regulation of testicular steroidogenesis in the toad Bufo arenarum, the effect of gonadotropins (hCG and hrFSH) on steroidogenic enzymes was determined using an in vitro system. 3beta-Hydroxysteroid dehydrogenase/isomerase activity was not affected by any of the gonadotropins, at any of the concentrations used. In contrast, 5alpha-reductase activity was strongly reduced by both hCG and hrFSH. Human chorionic gonadotropin inhibited the activity of cytochrome P450 17alpha-hydroxylase-C(17-20) lyase (P450(c17)), only at the highest concentration used, while hrFSH strongly reduced P450(c17) activity at all the doses assayed. In conclusion, these data suggest that LH (hCG) and FSH regulate steroidogenic enzymes such as 5alphaRed and P450(c17). The results also suggest that FSH could be involved in the regulation of the change in steroidogenesis undergone by the testis during the breeding season. In turn, the inhibition of P450(c17) activity could result in a reduction of androgen production and an increment of C21 steroids. (c) 2002 Elsevier Science (USA).

L-arginine mediated renaturation enhances yield of human, α6 type IV collagen non-collagenous domain from bacterial inclusion bodies

PubMed Central

Gunda, Venugopal; Boosani, Chandra Shekhar; Verma, Raj Kumar; Guda, Chittibabu; Akul Sudhakar, Yakkanti

2012-01-01

The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility in purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated. PMID:22512648

L-arginine mediated renaturation enhances yield of human, α6 Type IV collagen non-collagenous domain from bacterial inclusion bodies.

PubMed

Gunda, Venugopal; Boosani, Chandra Shekhar; Verma, Raj Kumar; Guda, Chittibabu; Sudhakar, Yakkanti Akul

2012-10-01

The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility in purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated.

Human T-cell lymphotropic virus type 1-infected T lymphocytes impair catabolism and uptake of glutamate by astrocytes via Tax-1 and tumor necrosis factor alpha.

PubMed

Szymocha, R; Akaoka, H; Dutuit, M; Malcus, C; Didier-Bazes, M; Belin, M F; Giraudon, P

2000-07-01

Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of a chronic progressive myelopathy called tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). In this disease, lesions of the central nervous system (CNS) are associated with perivascular infiltration by lymphocytes. We and others have hypothesized that these T lymphocytes infiltrating the CNS may play a prominent role in TSP/HAM. Here, we show that transient contact of human or rat astrocytes with T lymphocytes chronically infected by HTLV-1 impairs some of the major functions of brain astrocytes. Uptake of extracellular glutamate by astrocytes was significantly decreased after transient contact with infected T cells, while the expression of the glial transporters GLAST and GLT-1 was decreased. In two-compartment cultures avoiding direct cell-to-cell contact, similar results were obtained, suggesting possible involvement of soluble factors, such as cytokines and the viral protein Tax-1. Recombinant Tax-1 and tumor necrosis factor alpha (TNF-alpha) decreased glutamate uptake by astrocytes. Tax-1 probably acts by inducing TNF-alpha, as the effect of Tax-1 was abolished by anti-TNF-alpha antibody. The expression of glutamate-catabolizing enzymes in astrocytes was increased for glutamine synthetase and decreased for glutamate dehydrogenase, the magnitudes of these effects being correlated with the level of Tax-1 transcripts. In conclusion, Tax-1 and cytokines produced by HTLV-1-infected T cells impair the ability of astrocytes to manage the steady-state level of glutamate, which in turn may affect neuronal and oligodendrocytic functions and survival.

Inhibition of macrophage proinflammatory cytokine expression by steroids and recombinant IL-10.

PubMed

Li, Y H; Brauner, A; Jonsson, B; Van der Ploeg, I; Söder, O; Holst, M; Jensen, J S; Lagercrantz, H; Tullus, K

2001-08-01

Chronic lung disease (CLD) of prematurity is a prolonged respiratory failure in very-low-birth-weight neonates. Proinflammatory cytokines have been implicated in the development of CLD. Steroids have been shown to produce some improvement in neonates with this disease. The purpose of this study was to evaluate the downregulation of these proinflammatory cytokines by dexamethasone, budesonide and recombinant IL-10 (rIL-10) in order to elucidate the mechanism of the clinical benefit of steroids in babies. Our results showed that dexamethasone, budesonide and rIL-10 significantly inhibited both IL-6 and TNF-alpha production in the THP-1 cell line stimulated by lipopolysaccharide and Ureaplasma urealyticum antigen. Similar effects were found in macrophages from tracheobronchial aspirate fluid from newborn infants. In the rat alveolar macrophage cell line, steroids inhibited IL-6 and TNF-alpha production, while rat rIL-10 did not significantly decrease production. In conclusion, steroids and human rIL-10 were able to downregulate proinflammatory cytokine production, which may explain the beneficial effect of steroids and suggests that rIL-10 could be tried as an anti-inflammatory agent in neonates with a high risk of CLD.

Diffusion tensor imaging and myelin composition analysis reveal abnormal myelination in corpus callosum of canine mucopolysaccharidosis I

PubMed Central

Provenzale, James M.; Nestrasil, Igor; Chen, Steven; Kan, Shih-hsin; Le, Steven Q.; Jens, Jacqueline K.; Snella, Elizabeth M.; Vondrak, Kristen N.; Yee, Jennifer K.; Vite, Charles H.; Elashoff, David; Duan, Lewei; Wang, Raymond Y.; Ellinwood, N. Matthew; Guzman, Miguel A.; Shapiro, Elsa G.; Dickson, Patricia I.

2015-01-01

Children with mucopolysaccharidosis I (MPS I) develop hyperintense white matter foci on T2-weighted brain magnetic resonance (MR) imaging that are associated clinically with cognitive impairment. We report here a diffusion tensor imaging (DTI) and tissue evaluation of white matter in a canine model of MPS I. We found that two DTI parameters, fractional anisotropy (a measure of white matter integrity) and radial diffusivity (which reflects degree of myelination) were abnormal in the corpus callosum of MPS I dogs compared to carrier controls. Tissue studies of the corpus callosum showed reduced expression of myelin-related genes and an abnormal composition of myelin in MPS I dogs. We treated MPS I dogs with recombinant alpha-l-iduronidase, which is the enzyme that is deficient in MPS I disease. The recombinant alpha-l-iduronidase was administered by intrathecal injection into the cisterna magna. Treated dogs showed partial correction of corpus callosum myelination. Our findings suggest that abnormal myelination occurs in the canine MPS I brain, that it may underlie clinically-relevant brain imaging findings in human MPS I patients, and that it may respond to treatment. PMID:26222335

Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein.

PubMed

Jagadish, Nirmala; Rana, Ritu; Selvi, Ramasamy; Mishra, Deepshikha; Garg, Manoj; Yadav, Shikha; Herr, John C; Okumura, Katsuzumi; Hasegawa, Akiko; Koyama, Koji; Suri, Anil

2005-07-01

We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561-565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an alpha-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38alpha or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa-egg interaction.

Fusion protein of CDR mimetic peptide with Fc inhibit TNF-alpha induced cytotoxicity.

PubMed

Qin, Weisong; Feng, Jiannan; Li, Yan; Lin, Zhou; Shen, Beifen

2006-02-01

The variable regions of antibodies play central roles in the binding with antigens. Based on the model of a tumour necrosis factor-alpha (TNF-alpha) neutralizing monoclonal antibody (named as Z12) with TNF-alpha, heavy chain CDR2 (HCDR2) and light chain CDR3 (LCDR3) of Z12 were found to be the most responsible to bind with TNF-alpha. A mimetic peptide (PT) was designed based on the sequence derived from HCDR2 and LCDR3. Fusion protein PT-Fc was constructed by linking PT with Fc of human IgG1 through a flexible linker (GGGGGS). The primary structural characteristics of Fc and PT-Fc were analyzed, including the flexibility, hydrophilicity and epitopes. It was demonstrated that PT and Fc in the fusion protein possessed bio-function properly and non-interfering with each other. Furthermore, PT-Fc was expressed in Escherichia coli by fusion with thioredoxin (Trx). After trx-PT-Fc was cleaved with recombinant enterokinase, PT-Fc was obtained. The results of in vitro cytotoxic assays showed that both PT and PT-Fc could efficiently inhibit TNF-alpha induced apoptosis on L929 cells. At the same micromole concentration, the inhibition activity of PT-Fc was significantly higher than PT.

NKG2D and CD94 bind to multimeric alpha2,3-linked N-acetylneuraminic acid.

PubMed

Imaizumi, Yuzo; Higai, Koji; Suzuki, Chiho; Azuma, Yutaro; Matsumoto, Kojiro

2009-05-08

Killer lectin-like receptors on natural killer cells mediate cytotoxicity through glycans on target cells including the sialyl Lewis X antigen (sLeX). We investigated whether NK group 2D (NKG2D) and CD94 can bind to sialylated N-linked glycans, using recombinant glutathione S-transferase-fused extracellular lectin-like domains of NKG2D (rNKG2Dlec) and CD94 (rCD94lec). Both rNKG2Dlec and rCD94lec bound to plates coated with high-sLeX-expressing transferrin secreted by HepG2 cells (HepTF). The binding of rNKG2Dlec and rCD94lec to HepTF was markedly suppressed by treatment of HepTF with neuraminidase and in the presence of N-acetylneuraminic acid. Moreover, rNKG2Dlec and rCD94lec bound to alpha2,3-sialylated human alpha(1)-acid glycoprotein (AGP) but not to alpha2,6-sialylated AGP. Mutagenesis revealed that (152)Y of NKG2D and (144)F and (160)N of CD94 were critical for HepTF binding. This is the first report that NKG2D and CD94 bind to alpha2,3-sialylated but not to alpha2,6-sialylated multi-antennary N-glycans.

Toxoplasma gondii: Biochemical and biophysical characterization of recombinant soluble dense granule proteins GRA2 and GRA6

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

Bittame, Amina; Université Grenoble Alpes, 38042 Grenoble; Effantin, Grégory

2015-03-27

The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressedmore » in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6–8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8–15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed. - Highlights: • Toxoplasma gondii: soluble GRA2 forms 2 populations of particles. • T. gondii: the dense granule protein GRA2 folds intrinsically as an alpha-helix. • T. gondii: monomeric soluble GRA6 forms particles of 6–8 nm in diameter. • T. gondii: monomeric soluble GRA6 is random coiled. • Unusual biophysical properties of the dense granule protein GRA2 from T. gondii.« less

Cloning and expression of an alpha-1,3-glucanase gene from Bacillus circulans KA-304: the enzyme participates in protoplast formation of Schizophyllum commune.

PubMed

Yano, Shigekazu; Wakayama, Mamoru; Tachiki, Takashi

2006-07-01

A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, which were isolated from the filtrate, brings about the protoplast-forming activity. The gene of alpha-1,3-glucanase was cloned from B. circulans KA-304. It consists of 3,879 nucleotides, which encodes 1,293 amino acids including a putative signal peptide (31 amino acid residues), and the molecular weight of alpha-1,3-glucanase without the putative signal peptide was calculated to be 132,184. The deduced amino acid sequence of alpha-1,3-glucanase of B. circulans KA-304 showed approximately 80% similarity to that of mutanase (alpha-1,3-glucanase) of Bacillus sp. RM1, but no significant similarity to those of fungal mutanases. The recombinant alpha-1,3-glucanase was expressed in Escherichia coli Rosetta-gami B (DE 3), and significant alpha-1,3-glucanase activity was detected in the cell-free extract of the organism treated with isopropyl-beta-D-thiogalactopyranoside. The recombinant alpha-1,3-glucanase showed protoplast-forming activity when the enzyme was combined with chitinase I.

Characterization of antibodies that selectively detect alpha-synuclein in pathological inclusions.

PubMed

Waxman, Elisa A; Duda, John E; Giasson, Benoit I

2008-07-01

Sensitive detection of alpha-synuclein (alpha-syn) pathology is important in the diagnosis of disorders like Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy and in providing better insights into the etiology of these diseases. Several monoclonal antibodies that selectively react with aggregated alpha-syn in pathological inclusions and reveal extensive and underappreciated alpha-syn pathology in the brains of diseased patients were previously reported by Duda et al. (Ann Neurol 52:205-210, 2002). We sought to characterize the specificity of some of these antibodies (Syn 505, Syn 506 and Syn 514); using C-terminal and N-terminal truncations of alpha-syn, all three antibodies were determined to require N-terminal epitopes that minimally comprise amino acids 2-4, but possibly extend to amino acid 12 of alpha-syn. The selectivity of these antibodies was further assessed using biochemical analysis of human brains and reactivity to altered recombinant alpha-syn proteins with duplication variants of amino acids 1-12. In addition, by expressing wild-type or a double mutant (E46K/A53T) of alpha-syn in cultured cells and by comparing their immunoreactivities to another antibody (SNL-4), which has a similar primary epitope, it was determined that Syn 505, Syn 506 and Syn 514 recognize conformational variants of alpha-syn that is enhanced by the presence of the double mutations. These studies indicate that antibodies Syn 505, Syn 506 and Syn 514 preferentially recognize N-terminal epitopes in complex conformations, consistent with the dramatic conformational change associated with the polymerization of alpha-synuclein into amyloid fibrils that form pathological inclusions.

Activity, splice variants, conserved peptide motifs, and phylogeny of two new alpha1,3-fucosyltransferase families (FUT10 and FUT11).

PubMed

Mollicone, Rosella; Moore, Stuart E H; Bovin, Nicolai; Garcia-Rosasco, Marcela; Candelier, Jean-Jacques; Martinez-Duncker, Iván; Oriol, Rafael

2009-02-13

We report the cloning of three splice variants of the FUT10 gene, encoding for active alpha-l-fucosyltransferase-isoforms of 391, 419, and 479 amino acids, and two splice variants of the FUT11 gene, encoding for two related alpha-l-fucosyltransferases of 476 and 492 amino acids. The FUT10 and FUT11 appeared 830 million years ago, whereas the other alpha1,3-fucosyltransferases emerged 450 million years ago. FUT10-391 and FUT10-419 were expressed in human embryos, whereas FUT10-479 was cloned from adult brain and was not found in embryos. Recombinant FUT10-419 and FUT10-479 have a type II trans-membrane topology and are retained in the endoplasmic reticulum (ER) by a membrane retention signal at their NH(2) termini. The FUT10-479 has, in addition, a COOH-ER membrane retention signal. The FUT10-391 is a soluble protein without a trans-membrane domain or ER retention signal that transiently localizes to the Golgi and then is routed to the lysosome. After transfection in COS7 cells, the three FUT10s and at least one FUT11, link alpha-l-fucose onto conalbumin glycopeptides and biantennary N-glycan acceptors but not onto short lactosaminyl acceptor substrates as do classical monoexonic alpha1,3-fucosyltransferases. Modifications of the innermost core GlcNAc of the N-glycan, by substitution with ManNAc or with an opened GlcNAc ring or by the addition of an alpha1,6-fucose, suggest that the FUT10 transfer is performed on the innermost GlcNAc of the core chitobiose. We can exclude alpha1,3-fucosylation of the two peripheral GlcNAcs linked to the trimannosyl core of the acceptor, because the FUT10 fucosylated biantennary N-glycan product loses both terminal GlcNAc residues after digestion with human placenta alpha-N-acetylglucosaminidase.

Unusual features of a recombinant apple alpha-farnesene synthase.

PubMed

Green, Sol; Friel, Ellen N; Matich, Adam; Beuning, Lesley L; Cooney, Janine M; Rowan, Daryl D; MacRae, Elspeth

2007-01-01

A recombinant alpha-farnesene synthase from apple (Malus x domestica), expressed in Escherichia coli, showed features not previously reported. Activity was enhanced 5-fold by K(+) and all four isomers of alpha-farnesene, as well as beta-farnesene, were produced from an isomeric mixture of farnesyl diphosphate (FDP). Monoterpenes, linalool, (Z)- and (E)-beta-ocimene and beta-myrcene, were synthesised from geranyl diphosphate (GDP), but at 18% of the optimised rate for alpha-farnesene synthesis from FDP. Addition of K(+) reduced monoterpene synthase activity. The enzyme also produced alpha-farnesene by a reaction involving coupling of GDP and isoprenyl diphosphate but at <1% of the rate with FDP. Mutagenesis of active site aspartate residues removed sesquiterpene, monoterpene and prenyltransferase activities suggesting catalysis through the same active site. Phylogenetic analysis clusters this enzyme with isoprene synthases rather than with other sesquiterpene synthases, suggesting that it has evolved differently from other plant sesquiterpene synthases. This is the first demonstration of a sesquiterpene synthase possessing prenyltransferase activity.

Interferon-alpha 2b quantification in inclusion bodies using reversed phase-ultra performance liquid chromatography (RP-UPLC).

PubMed

Cueto-Rojas, H F; Pérez, N O; Pérez-Sánchez, G; Ocampo-Juárez, I; Medina-Rivero, E

2010-04-15

Interferon-alpha 2b (IFN-alpha 2b) is a recombinant therapeutic cytokine produced as inclusion bodies using a strain of Escherichia coli as expression system. After fermentation and recovery, it is necessary to know the amount of recombinant IFN-alpha 2b, in order to determine the yield and the load for solubilization, and chromatographic protein purification steps. The present work details the validation of a new short run-time and fast sample-preparation method to quantify IFN-alpha 2b in inclusion bodies using Reversed Phase-Ultra Performance Liquid Chromatography (RP-UPLC). The developed method demonstrated an accuracy of 100.28%; the relative standard deviations for method precision, repeatability and inter-day precision tests were found to be 0.57%, 1.54% and 1.83%, respectively. Linearity of the method was assessed in the range of concentrations from 0.05 mg/mL to 0.5 mg/mL, the curve obtained had a determination coefficient (r(2)) of 0.9989. Detection and quantification limits were found to be 0.008 mg/mL and 0.025 mg/mL, respectively. The method also demonstrated robustness for changes in column temperature, and specificity against host proteins and other recombinant protein expressed in the same E. coli strain. Copyright 2010 Elsevier B.V. All rights reserved.

Regulation of CD93 cell surface expression by protein kinase C isoenzymes.

PubMed

Ikewaki, Nobunao; Kulski, Jerzy K; Inoko, Hidetoshi

2006-01-01

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell-lines. Whereas recombinant tumor necrosis factor-alpha (rTNF-alpha) slightly up-regulated CD93 expression on the U937 cells, recombinant interleukin-1beta (rIL-1beta), recombinant interleukin-2 (rIL-2), recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS) had no effect. Taken together, these findings indicate that the regulation of CD93 expression on these cells involves the PKC isoenzymes.

Cost-saving treatment strategies in in vitro fertilization: a combined economic evaluation of two large randomized clinical trials comparing highly purified human menopausal gonadotropin and recombinant follicle-stimulating hormone alpha.

PubMed

Wechowski, Jaroslaw; Connolly, Mark; Schneider, Dirk; McEwan, Philip; Kennedy, Richard

2009-04-01

To assess the cost-effectiveness of two gonadotropin treatments that are available in the United Kingdom in light of limited public funding and the fundamental role of costs in IVF treatment decisions. An economic evaluation based on two large randomized clinical trials in IVF patients using a simulation model. Fifty-three fertility clinics in 13 European countries and Israel. Women indicated for treatment with IVF (N = 986), aged 18-38, participating in double-blind, randomized controlled trials. Highly purified menotropin (HP-hMG, Menopur) or recombinant follitropin alpha (rFSH, Gonal-F). Cost per IVF cycle and cost per live birth for HP-hMG and rFSH alpha. HP-hMG was more effective and less costly versus rFSH for both IVF cost per live birth and for IVF cost per baby (incremental cost-effectiveness ratio was negative). The mean costs per IVF treatment for HP-hMG and rFSH were 2408 pounds (95% confidence interval [CI], 2392 pounds, 2421 pounds) and 2660 pounds (95% CI 2644 pounds, 2678 pounds), respectively. The mean cost saving of 253 pounds per cycle using HP-hMG allows one additional cycle to be delivered for every 9.5 cycles. Treatment with HP-hMG was dominant compared with rFSH in the United Kingdom. Gonadotropin costs should be considered alongside live-birth rates to optimize outcomes using scarce health-care resources.

Production of interferon-alpha in high cell density cultures of recombinant Escherichia coli and its single step purification from refolded inclusion body proteins.

PubMed

Babu, K R; Swaminathan, S; Marten, S; Khanna, N; Rinas, U

2000-06-01

Escherichia coli TG1 transformed with a temperature-regulated interferon-alpha expression vector was grown to high cell density in defined medium containing glucose as the sole carbon and energy source, utilizing a simple fed-batch process. Feeding was carried out to achieve an exponential increase in biomass at growth rates which minimized acetate production. Thermal induction of such high cell density cultures resulted in the production of approximately 4 g interferon-alpha/l culture broth. Interferon-alpha was produced exclusively in the form of insoluble inclusion bodies and was solubilized under denaturing conditions, refolded in the presence of arginine and purified to near homogeneity, utilizing single-step ion-exchange chromatography on Q-Sepharose. The yield of purified interferon-alpha was approximately 300 mg/l with respect to the original high cell density culture broth (overall yield of approximately 7.5% active interferon-alpha). The purified recombinant interferon-alpha was found by different criteria to be predominantly monomeric and possessed a specific bioactivity of approximately 2.5 x 10(8) IU/mg based on viral cytopathic assay.

Spotlight on agalsidase beta in Fabry disease.

PubMed

Keating, Gillian M; Simpson, Dene

2007-01-01

Agalsidase beta (Fabrazyme) is a recombinant human alpha-galactosidase A enzyme approved for intravenous use in the treatment of Fabry disease. Fabry disease is a progressive, multisystemic, potentially life-threatening disorder caused by a deficiency of alpha-galactosidase A. This deficiency results in accumulation of glycosphingolipids, particularly globotriaosylceramide (GL-3), in the lysosomes of various tissues. This accumulation is the underlying driver of disease progression. Agalsidase beta provides an exogenous source of alpha-galactosidase A. Intravenous agalsidase beta is effective and well tolerated in patients with Fabry disease. In a phase III trial, agalsidase beta was shown to clear GL-3 from various target cells and, in a subsequent extension of this trial, prevent GL-3 reaccumulation. In a post-approval trial, agalsidase beta was shown to provide significant clinical benefit by reducing the risk of a major clinical event. Thus, agalsidase beta represents an important advance in the treatment of Fabry disease, and agalsidase beta therapy should be strongly considered in patients with Fabry disease who are suitable candidates.

Agalsidase Beta: a review of its use in the management of Fabry disease.

PubMed

Keating, Gillian M; Simpson, Dene

2007-01-01

Agalsidase beta (Fabrazyme) is a recombinant human alpha-galactosidase A enzyme approved for intravenous use in the treatment of Fabry disease. Fabry disease is a progressive, multisystemic, potentially life threatening disorder caused by a deficiency of alpha-galactosidase A. This deficiency results in accumulation of glycosphingolipids, particularly globotriaosylceramide (GL-3), in the lysosomes of various tissues. This accumulation is the underlying driver of disease progression. Agalsidase beta provides an exogenous source of alpha-galactosidase A.Intravenous agalsidase beta is effective and well tolerated in patients with Fabry disease. In a phase III trial, agalsidase beta was shown to clear GL-3 from various target cells and, in a subsequent extension of this trial, prevent GL-3 reaccumulation. In a post-approval trial, agalsidase beta was shown to provide significant clinical benefit by reducing the risk of a major clinical event. Thus, agalsidase beta represents an important advance in the treatment of Fabry disease, and agalsidase beta therapy should be strongly considered in patients with Fabry disease who are suitable candidates.

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

Adenoviral mediated interferon-alpha 2b gene therapy suppresses the pro-angiogenic effect of vascular endothelial growth factor in superficial bladder cancer.

PubMed

Adam, Liana; Black, Peter C; Kassouf, Wassim; Eve, Beryl; McConkey, David; Munsell, Mark F; Benedict, William F; Dinney, Colin P N

2007-05-01

Intravesical adenovirus mediated interferon-alpha gene transfer has a potent therapeutic effect against superficial human bladder carcinoma xenografts growing in the bladder of athymic nude mice. We determined whether the inhibition of angiogenesis might contribute to the antitumor effect. We treated several human urothelial carcinoma cells with adenovirus mediated interferon-alpha 2b and monitored its effects on the production of angiogenic factors using real-time reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemical analysis and a gel shift based transcription factor array. To assess the role of adenovirus mediated interferon 2b in angiogenic activity we used in vitro invasion assays and evaluated the anti-angiogenic effects of adenovirus mediated interferon gene therapy in an orthotopic murine model of human superficial bladder cancer. In adenovirus mediated interferon-alpha infected 253J B-V cells vascular endothelial growth factor was decreased and anti-angiogenic interferon-gamma inducible protein 10 was up-regulated. In contrast, the addition of as much as 100,000 IU recombinant interferon had no apparent effect on vascular endothelial growth factor production. Conditioned medium derived from adenovirus mediated interferon 2b infected 253J B-V cells greatly decreased the invasive potential of human endothelial cells and down-regulated their matrix metalloproteinase 2 expression compared to controls. Furthermore, adenovirus mediated interferon 2b blocked pro-angiogenic nuclear signals, such as the transcription factors activating protein-1 and 2, stimulating protein-1, nuclear factor kappaB and c-myb. In vivo experiments revealed significant vascular endothelial growth factor down-regulation and decreased tumor vessel density in the adenovirus mediated interferon 2b treated group compared to controls. Treatment with adenovirus mediated interferon 2b increases the angiostatic activity of the bladder cancer microenvironment. This inhibition may prove beneficial for treating superficial bladder cancer with adenovirus mediated interferon-alpha and hopefully contribute to a decreased recurrence rate of this neoplasm.

Electron-temperature dependence of dissociative recombination of electrons with CO(+)-(CO)n-series ions

NASA Technical Reports Server (NTRS)

Whitaker, M.; Biondi, M. A.; Johnsen, R.

1980-01-01

A microwave afterglow mass spectrometer apparatus is used to determine the dependence on electron temperature T sub e of the recombination coefficients alpha sub n of the dimer and trimer ions of the series CO+.(CO) sub n. It is found that alpha sub 1 = (1.3 + or - 0.3)x 0.000001 (T sub e(K)/300) to the -0.34; and alpha sub 2 = (1.9 + or - 0.4)x 0.000001 (T sub e(K)/300) to the -0.33 cu cm/sec. These dependences on T sub e are quite different from those obtained previously for polar-cluster ions of the hydronium and ammonium series but are similar to that for simple diatomic ions.

Influence of major structural features of tocopherols and tocotrienols on their omega-oxidation by tocopherol-omega-hydroxylase.

PubMed

Sontag, Timothy J; Parker, Robert S

2007-05-01

Human cytochrome P450 4F2 (CYP4F2) catalyzes the initial omega-hydroxylation reaction in the metabolism of tocopherols and tocotrienols to carboxychromanols and is, to date, the only enzyme shown to metabolize vitamin E. The objective of this study was to characterize this activity, particularly the influence of key features of tocochromanol substrate structure. The influence of the number and positions of methyl groups on the chromanol ring, and of stereochemistry and saturation of the side chain, were explored using HepG2 cultures and microsomal reaction systems. Human liver microsomes and microsomes selectively expressing recombinant human CYP4F2 exhibited substrate activity patterns similar to those of HepG2 cells. Although activity was strongly associated with substrate accumulation by cells or microsomes, substantial differences in specific activities between substrates remained under conditions of similar microsomal membrane substrate concentration. Methylation at C5 of the chromanol ring was associated with markedly low activity. Tocotrienols exhibited much higher Vmax values than their tocopherol counterparts. Side chain stereochemistry had no effect on omega-hydroxylation of alpha-tocopherol (alpha-TOH) by any system. Kinetic analysis of microsomal CYP4F2 activity revealed Michaelis-Menten kinetics for alpha-TOH but allosteric cooperativity for other vitamers, especially tocotrienols. Additionally, alpha-TOH was a positive effector of omega-hydroxylation of other vitamers. These results indicate that CYP4F2-mediated tocopherol-omega-hydroxylation is a central feature underlying the different biological half-lives, and therefore biopotencies, of the tocopherols and tocotrienols.

Metabolism of endosulfan-alpha by human liver microsomes and its utility as a simultaneous in vitro probe for CYP2B6 and CYP3A4.

PubMed

Casabar, Richard C T; Wallace, Andrew D; Hodgson, Ernest; Rose, Randy L

2006-10-01

Endosulfan-alpha is metabolized to a single metabolite, endosulfan sulfate, in pooled human liver microsomes (Km = 9.8 microM, Vmax = 178.5 pmol/mg/min). With the use of recombinant cytochrome P450 (P450) isoforms, we identified CYP2B6 (Km = 16.2 microM, Vmax = 11.4 nmol/nmol P450/min) and CYP3A4 (Km = 14.4 microM, Vmax = 1.3 nmol/nmol P450/min) as the primary enzymes catalyzing the metabolism of endosulfan-alpha, although CYP2B6 had an 8-fold higher intrinsic clearance rate (CL(int) = 0.70 microl/min/pmol P450) than CYP3A4 (CL(int) = 0.09 microl/min/pmol P450). Using 16 individual human liver microsomes (HLMs), a strong correlation was observed with endosulfan sulfate formation and S-mephenytoin N-demethylase activity of CYP2B6 (r(2) = 0.79), whereas a moderate correlation with testosterone 6 beta-hydroxylase activity of CYP3A4 (r(2) = 0.54) was observed. Ticlopidine (5 microM), a potent CYP2B6 inhibitor, and ketoconazole (10 microM), a selective CYP3A4 inhibitor, together inhibited approximately 90% of endosulfan-alpha metabolism in HLMs. Using six HLM samples, the percentage total normalized rate (% TNR) was calculated to estimate the contribution of each P450 in the total metabolism of endosulfan-alpha. In five of the six HLMs used, the percentage inhibition with ticlopidine and ketoconazole in the same incubation correlated with the combined % TNRs for CYP2B6 and CYP3A4. This study shows that endosulfan-alpha is metabolized by HLMs to a single metabolite, endosulfan sulfate, and that it has potential use, in combination with inhibitors, as an in vitro probe for CYP2B6 and 3A4 catalytic activities.

Schistosome egg antigens, including the glycoprotein IPSE/alpha-1, trigger the development of regulatory B cells

PubMed Central

Veninga, Henrike; van der Vlugt, Luciën E. P. M.; Voskamp, Astrid; Boon, Louis; Westerhof, Lotte B.; Smits, Hermelijn H.

2017-01-01

Infection with the helminth Schistosoma (S.) mansoni drives the development of interleukin (IL)-10-producing regulatory B (Breg) cells in mice and man, which have the capacity to reduce experimental allergic airway inflammation and are thus of high therapeutic interest. However, both the involved antigen and cellular mechanisms that drive Breg cell development remain to be elucidated. Therefore, we investigated whether S. mansoni soluble egg antigens (SEA) directly interact with B cells to enhance their regulatory potential, or act indirectly on B cells via SEA-modulated macrophage subsets. Intraperitoneal injections of S. mansoni eggs or SEA significantly upregulated IL-10 and CD86 expression by marginal zone B cells. Both B cells as well as macrophages of the splenic marginal zone efficiently bound SEA in vivo, but macrophages were dispensable for Breg cell induction as shown by macrophage depletion with clodronate liposomes. SEA was internalized into acidic cell compartments of B cells and induced a 3-fold increase of IL-10, which was dependent on endosomal acidification and was further enhanced by CD40 ligation. IPSE/alpha-1, one of the major antigens in SEA, was also capable of inducing IL-10 in naïve B cells, which was reproduced by tobacco plant-derived recombinant IPSE. Other major schistosomal antigens, omega-1 and kappa-5, had no effect. SEA depleted of IPSE/alpha-1 was still able to induce Breg cells indicating that SEA contains more Breg cell-inducing components. Importantly, SEA- and IPSE-induced Breg cells triggered regulatory T cell development in vitro. SEA and recombinant IPSE/alpha-1 also induced IL-10 production in human CD1d+ B cells. In conclusion, the mechanism of S. mansoni-induced Breg cell development involves a direct targeting of B cells by SEA components such as the secretory glycoprotein IPSE/alpha-1. PMID:28753651

Coordinate regulation of estrogen-mediated fibronectin matrix assembly and epidermal growth factor receptor transactivation by the G protein-coupled receptor, GPR30.

PubMed

Quinn, Jeffrey A; Graeber, C Thomas; Frackelton, A Raymond; Kim, Minsoo; Schwarzbauer, Jean E; Filardo, Edward J

2009-07-01

Estrogen promotes changes in cytoskeletal architecture not easily attributed to the biological action of estrogen receptors, ERalpha and ERbeta. The Gs protein-coupled transmembrane receptor, GPR30, is linked to specific estrogen binding and rapid estrogen-mediated release of heparin-bound epidermal growth factor. Using marker rescue and dominant interfering mutant strategies, we show that estrogen action via GPR30 promotes fibronectin (FN) matrix assembly by human breast cancer cells. Stimulation with 17beta-estradiol or the ER antagonist, ICI 182, 780, results in the recruitment of FN-engaged integrin alpha5beta1 conformers to fibrillar adhesions and the synthesis of FN fibrils. Concurrent with this cellular response, GPR30 promotes the formation of Src-dependent, Shc-integrin alpha5beta1 complexes. Function-blocking antibodies directed against integrin alpha5beta1 or soluble Arg-Gly-Asp peptide fragments derived from FN specifically inhibited GPR30-mediated epidermal growth factor receptor transactivation. Estrogen-mediated FN matrix assembly and epidermal growth factor receptor transactivation were similarly disrupted in integrin beta1-deficient GE11 cells, whereas reintroduction of integrin beta1 into GE11 cells restored these responses. Mutant Shc (317Y/F) blocked GPR30-induced FN matrix assembly and tyrosyl phosphorylation of erbB1. Interestingly, relative to recombinant wild-type Shc, 317Y/F Shc was more readily retained in GPR30-induced integrin alpha5beta1 complexes, yet this mutant did not prevent endogenous Shc-integrin alpha5beta1 complex formation. Our results suggest that GPR30 coordinates estrogen-mediated FN matrix assembly and growth factor release in human breast cancer cells via a Shc-dependent signaling mechanism that activates integrin alpha5beta1.

Generation of cell lines for drug discovery through random activation of gene expression: application to the human histamine H3 receptor.

PubMed

Song, J; Doucette, C; Hanniford, D; Hunady, K; Wang, N; Sherf, B; Harrington, J J; Brunden, K R; Stricker-Krongrad, A

2005-06-01

Target-based high-throughput screening (HTS) plays an integral role in drug discovery. The implementation of HTS assays generally requires high expression levels of the target protein, and this is typically accomplished using recombinant cDNA methodologies. However, the isolated gene sequences to many drug targets have intellectual property claims that restrict the ability to implement drug discovery programs. The present study describes the pharmacological characterization of the human histamine H3 receptor that was expressed using random activation of gene expression (RAGE), a technology that over-expresses proteins by up-regulating endogenous genes rather than introducing cDNA expression vectors into the cell. Saturation binding analysis using [125I]iodoproxyfan and RAGE-H3 membranes revealed a single class of binding sites with a K(D) value of 0.77 nM and a B(max) equal to 756 fmol/mg of protein. Competition binding studies showed that the rank order of potency for H3 agonists was N(alpha)-methylhistamine approximately (R)-alpha- methylhistamine > histamine and that the rank order of potency for H3 antagonists was clobenpropit > iodophenpropit > thioperamide. The same rank order of potency for H3 agonists and antagonists was observed in the functional assays as in the binding assays. The Fluorometic Imaging Plate Reader assays in RAGE-H3 cells gave high Z' values for agonist and antagonist screening, respectively. These results reveal that the human H3 receptor expressed with the RAGE technology is pharmacologically comparable to that expressed through recombinant methods. Moreover, the level of expression of the H3 receptor in the RAGE-H3 cells is suitable for HTS and secondary assays.

Production, purification, and characterization of human alpha1 proteinase inhibitor from Aspergillus niger.

PubMed

Chill, Liat; Trinh, Loc; Azadi, Parastoo; Ishihara, Mayumi; Sonon, Roberto; Karnaukhova, Elena; Ophir, Yakir; Golding, Basil; Shiloach, Joseph

2009-02-15

Human alpha one proteinase inhibitor (alpha1-PI) was cloned and expressed in Aspergillus niger, filamentious fungus that can grow in defined media and can perform glycosylation. Submerged culture conditions were established using starch as carbon source, 30% dissolved oxygen concentration, pH 7.0 and 28 degrees C. Eight milligrams per liter of active alpha1-PI were secreted to the growth media in about 40 h. Controlling the protein proteolysis was found to be an important factor in the production. The effects of various carbon sources, pH and temperature on the production and stability of the protein were tested and the product was purified and characterized. Two molecular weights variants of the recombinant alpha1-PI were produced by the fungus; the difference is attributed to the glycosylated part of the molecule. The two glycoproteins were treated with PNGAse F and the released glycans were analyzed by HPAEC, MALDI/TOF-MS, NSI-MS(n), and GC-MS. The MALDI and NSI- full MS spectra of permethylated N-glycans revealed that the N-glycans of both variants contain a series of high-mannose type glycans with 5-20 hexose units. Monosaccharide analysis showed that these were composed of N-acetylglucos-amine, mannose, and galactose. Linkage analysis revealed that the galactosyl component was in the furanoic conformation, which was attaching in a terminal non-reducing position. The Galactofuranose-containing high-mannnose type N-glycans are typical structures, which recently have been found as part of several glycoproteins produced by Aspergillus niger.

Dissociative recombination of N2/+/ in the ionosphere

NASA Technical Reports Server (NTRS)

Torr, M. R.; Torr, D. G.

1979-01-01

N2(+) ion measurements are examined which were made with the Atmospheric Explorer-C satellite during a phase of solar activity in 1978 that was significantly higher than near the earlier minimum. It is found that the major source of N2(+) is photoionization, rather than charge exchange with O(+) (2D), and that the major loss process above 300 km is dissociative recombination with electrons. A data sample for which the electron temperature (T sub e) covered the range from 1000 to 3400 K is used to evaluate the rate constant, alpha, for the dissociative-recombination process. The results show good agreement with laboratory measurements given by the expression: alpha = 1.8 x 10 to the -7th (T sub e/300) to the -0.39th cu cm/sec.

Evaluation of a recombinant yeast cell estrogen screening assay.

PubMed Central

Coldham, N G; Dave, M; Sivapathasundaram, S; McDonnell, D P; Connor, C; Sauer, M J

1997-01-01

A wide range of chemicals with diverse structures derived from plant and environmental origins are reported to have hormonal activity. The potential for appreciable exposure of humans to such substances prompts the need to develop sensitive screening methods to quantitate and evaluate the risk to the public. Yeast cells transformed with plasmids encoding the human estrogen receptor and an estrogen responsive promoter linked to a reporter gene were evaluated for screening compounds for estrogenic activity. Relative sensitivity to estrogens was evaluated by reference to 17 beta-estradiol (E2) calibration curves derived using the recombinant yeast cells, MCF-7 human breast cancer cells, and a prepubertal mouse uterotrophic bioassay. The recombinant yeast cell bioassay (RCBA) was approximately two and five orders of magnitude more sensitive to E2 than MCF-7 cells and the uterotrophic assay, respectively. The estrogenic potency of 53 chemicals, including steroid hormones, synthetic estrogens, environmental pollutants, and phytoestrogens, was measured using the RCBA. Potency values produced with the RCBA relative to E2 (100) included estrone (9.6), diethylstilbestrol (74.3), tamoxifen (0.0047), alpha-zearalanol (1.3), equol (0.085), 4-nonylphenol (0.005), and butylbenzyl phathalate (0.0004), which were similar to literature values but generally higher than those produced by the uterotrophic assay. Exquisite sensitivity, absence of test compound biotransformation, ease of use, and the possibility of measuring antiestrogenic activity are important attributes that argue for the suitability of the RCBA in screening for potential xenoestrogens to evaluate risk to humans, wildlife, and the environment. Images Figure 1. Figure 2. Figure 3. Figure 4. PMID:9294720

Pattern differences in experimental fevers induced by endotoxin, endogenous pyrogen, and prostaglandins.

PubMed

Morimoto, A; Nakamori, T; Watanabe, T; Ono, T; Murakami, N

1988-04-01

To distinguish pattern differences in experimentally induced fevers, we investigated febrile responses induced by intravenous (IV), intracerebroventricular (ICV), and intra-preoptic/anterior hypothalamic (POA) administration of bacterial endotoxin (lipopolysaccharide, LPS), endogenous pyrogen (EP), human recombinant interleukin-1 alpha (IL-1), and prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha). Intravenous LPS, EP, or IL-1 in high concentrations caused biphasic fever. In low concentrations, they induced only the first phase of fever. Latency to onset and time to first peak of fever induced by IV injection of LPS or EP were almost the same as those after ICV or POA injection of PGE2. Fever induced by ICV or POA administration of LPS, EP, IL-1, or PGF2 alpha had a long latency to onset and a prolonged time course. There were significant differences among the latencies to fever onset exhibited by groups that received ICV or POA injections of LPS, EP, or PGF2 alpha and by groups given IV injections of LPS or EP and ICV or POA injections of PGE2. Present observations indicate different patterns of fever produced by several kinds of pyrogens when given by various routes. These results permit us to consider the possibility that there are several mediators or multiprocesses underlying the pathogenesis of fever.

Isolated lymphoid follicles are not IgA inductive sites for recombinant Salmonella

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

Hashizume, Tomomi; Momoi, Fumiki; Kurita-Ochiai, Tomoko

2007-08-24

In this study, we investigated whether isolated lymphoid follicles (ILF) play a role in the regulation of intestinal IgA antibody (Ab) responses. The transfer of wild type (WT) bone marrow (BM) to lymphotoxin-{alpha}-deficient (LT{alpha}{sup -/-}) mice resulted in the formation of mature ILF containing T cells, B cells, and FDC clusters in the absence of mesenteric lymph nodes and Peyer's patches. Although the ILF restored total IgA Abs in the intestine, antigen (Ag)-specific IgA responses were not induced after oral immunization with recombinant Salmonella expressing fragment C of tetanus toxin. Moreover, Ag-specific cell proliferation was not detected in the ILF.more » Interestingly, no IgA anti-LPS Abs were detected in the fecal extracts of LT{alpha}{sup -/-} mice reconstituted with WT BM. On the basis of these findings, ILF can be presumed to play a role in the production of IgA Abs, but lymphoid nodules are not inductive sites for the regulation of Ag-specific intestinal IgA responses to recombinant Salmonella.« less

Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

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

Bidart, J.M.; Troalen, F.; Salesse, R.

1987-06-25

We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptidesmore » spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex.« less

Infection, fever, and exogenous and endogenous pyrogens: some concepts have changed.

PubMed

Dinarello, Charles A

2004-01-01

For many years, it was thought that bacterial products caused fever via the intermediate production of a host-derived, fever-producing molecule, called endogenous pyrogen (EP). Bacterial products and other fever-producing substances were termed exogenous pyrogens. It was considered highly unlikely that exogenous pyrogens caused fever by acting directly on the hypothalamic thermoregulatory center since there were countless fever-producing microbial products, mostly large molecules, with no common physical structure. In vivo and in vitro, lipopolysaccharides (LPSs) and other microbial products induced EP, subsequently shown to be interleukin-1 (IL-1). The concept of the 'endogenous pyrogen' cause of fever gained considerable support when pure, recombinant IL-1 produced fever in humans and in animals at subnanomolar concentrations. Subsequently, recombinant tumor necrosis factor-alpha (TNF-alpha), IL-6 and other cytokines were also shown to cause fever and EPs are now termed pyrogenic cytokines. However, the concept was challenged when specific blockade of either IL-1 or TNF activity did not diminish the febrile response to LPS, to other microbial products or to natural infections in animals and in humans. During infection, fever could occur independently of IL-1 or TNF activity. The cytokine-like property of Toll-like receptor (TLR) signal transduction provides an explanation by which any microbial product can cause fever by engaging its specific TLR on the vascular network supplying the thermoregulatory center in the anterior hypothalamus. Since fever induced by IL-1, TNF-alpha, IL-6 or TLR ligands requires cyclooxygenase-2, production of prostaglandin E2 (PGE2) and activation of hypothalamic PGE2 receptors provides a unifying mechanism for fever by endogenous and exogenous pyrogens. Thus, fever is the result of either cytokine receptor or TLR triggering; in autoimmune diseases, fever is mostly cytokine mediated whereas both cytokine and TLR account for fever during infection.

Rapid Immunochromatographic Detection of Serum Anti-α-Galactosidase A Antibodies in Fabry Patients after Enzyme Replacement Therapy.

PubMed

Nakano, Sachie; Tsukimura, Takahiro; Togawa, Tadayasu; Ohashi, Toya; Kobayashi, Masahisa; Takayama, Katsuyoshi; Kobayashi, Yukuharu; Abiko, Hiroshi; Satou, Masatsugu; Nakahata, Tohru; Warnock, David G; Sakuraba, Hitoshi; Shibasaki, Futoshi

2015-01-01

We developed an immunochromatography-based assay for detecting antibodies against recombinant α-galactosidase A proteins in serum. The evaluation of 29 serum samples from Fabry patients, who had received enzyme replacement therapy with agalsidase alpha and/or agalsidase beta, was performed by means of this assay method, and the results clearly revealed that the patients exhibited the same level of antibodies against both agalsidase alpha and agalsidase beta, regardless of the species of recombinant α-galactosidase A used for enzyme replacement therapy. A conventional enzyme-linked immunosorbent assay supported the results. Considering these, enzyme replacement therapy with agalsidase alpha or agalsidase beta would generate antibodies against the common epitopes in both agalsidase alpha and agalsidase beta. Most of the patients who showed immunopositive reaction exhibited classic Fabry phenotype and harbored gene mutations affecting biosynthesis of α-galactosidase A. As immunochromatography is a handy and simple assay system which can be available at bedside, this assay method would be extremely useful for quick evaluation or first screening of serum antibodies against agalsidase alpha or agalsidase beta in Fabry disease with enzyme replacement therapy.

Dose-ranging study of the novel recombinant plasminogen activator BM 06.022 in healthy volunteers.

PubMed

Martin, U; von Möllendorff, E; Akpan, W; Kientsch-Engel, R; Kaufmann, B; Neugebauer, G

1991-10-01

The novel recombinant plasminogen activator BM 06.022 consists of the kringle 2 and protease domains of human tissue-type plasminogen activator and is unglycosylated because of its expression in Escherichia coli cells. Pharmacokinetics for activity and hemostatic effects of BM 06.022 were studied in 18 healthy male volunteers after an intravenous bolus injection over 2 minutes. BM 06.022 was administered successively at doses of 0.1125, 0.55, 2.2, 3.3, 4.4, and 5.5 MU to three volunteers. Plasma fibrinogen was unchanged; effects of BM 06.022 were observed on plasminogen only at higher doses, and dose-dependent effects were seen on alpha 2-antiplasmin and fibrin D-dimers. The concentration of plasminogen and alpha 2-antiplasmin was 87% +/- 3% and 79% +/- 3%, respectively, of baseline 2 hours after injection of 5.5 MU of BM 06.022. Fibrin D-dimers were highest with 1147 +/- 380 ng/ml at 5.5 MU of BM 06.022. The area under the activity concentration-time curve (AUC) increased dose-dependently and linearly. At 5.5 MU of BM 06.022, the AUC was 313 +/- 47 IU.hr.ml-1, the total plasma clearance was 306 +/- 40 ml/min, and the half-life was 14.4 +/- 1.1 minutes.

Perioperative recombinant human granulocyte colony-stimulating factor (Filgrastim) treatment prevents immunoinflammatory dysfunction associated with major surgery.

PubMed

Schneider, Christian; von Aulock, Sonja; Zedler, Siegfried; Schinkel, Christian; Hartung, Thomas; Faist, Eugen

2004-01-01

To examine the effects of perioperative rhG-CSF administration on immune function in patients subjected to major surgery. Severe trauma, such as major surgery, initiates acute immunodysfunction which predisposes the patient towards infectious complications. Sixty patients undergoing elective surgery received either recombinant human granulocyte colony-stimulating factor/rh G-CSF (Filgrastim) or a placebo perioperatively. At several time points before and after the surgical intervention immunofunctional parameters were assessed. RESULTS Leukocyte counts and serum levels of anti-inflammatory mediators (IL-1ra and TNF-R) were increased in Filgrastim-treated patients, while the post-operative acute phase response was attenuated. Monocyte deactivation (reduced TNF-alpha release and HLA-DR expression) and lymphocyte anergy (impaired mitogenic proliferation and reduced TH1 lymphokine release) were blunted and the incidence and severity of infectious complications were reduced. These results suggest that Filgrastim treatment reinforces innate immunity, enabling better prevention of infection. Thus, this unique combination of hematopoietic, anti-inflammatory and anti-infectious effects on the innate immune system warrants further study of clinical efficacy and sepsis prophylaxis.

Prostaglandin ethanolamides (prostamides): in vitro pharmacology and metabolism.

PubMed

Matias, I; Chen, J; De Petrocellis, L; Bisogno, T; Ligresti, A; Fezza, F; Krauss, A H-P; Shi, L; Protzman, C E; Li, C; Liang, Y; Nieves, A L; Kedzie, K M; Burk, R M; Di Marzo, V; Woodward, D F

2004-05-01

We investigated whether prostaglandin ethanolamides (prostamides) E(2), F(2alpha), and D(2) exert some of their effects by 1) activating prostanoid receptors either per se or after conversion into the corresponding prostaglandins; 2) interacting with proteins for the inactivation of the endocannabinoid N-arachidonoylethanolamide (AEA), for example fatty acid amide hydrolase (FAAH), thereby enhancing AEA endogenous levels; or 3) activating the vanilloid receptor type-1 (TRPV1). Prostamides potently stimulated cat iris contraction with potency approaching that of the corresponding prostaglandins. However, prostamides D(2), E(2), and F(2alpha) exhibited no meaningful interaction with the cat recombinant FP receptor, nor with human recombinant DP, EP(1-4), FP, IP, and TP prostanoid receptors. Prostamide F(2alpha) was also very weak or inactive in a panel of bioassays specific for the various prostanoid receptors. None of the prostamides inhibited AEA enzymatic hydrolysis by FAAH in cell homogenates, or AEA cellular uptake in intact cells. Furthermore, less than 3% of the compounds were hydrolyzed to the corresponding prostaglandins when incubated for 4 h with homogenates of rat brain, lung, or liver, and cat iris or ciliary body. Very little temperature-dependent uptake of prostamides was observed after incubation with rat brain synaptosomes or RBL-2H3 cells. We suggest that prostamides' most prominent pharmacological actions are not due to transformation into prostaglandins, activation of prostanoid receptors, enhancement of AEA levels, or gating of TRPV1 receptors, but possibly to interaction with novel receptors that seem to be functional in the cat iris.

5 Year Expression and Neutrophil Defect Repair after Gene Therapy in Alpha-1 Antitrypsin Deficiency.

PubMed

Mueller, Christian; Gernoux, Gwladys; Gruntman, Alisha M; Borel, Florie; Reeves, Emer P; Calcedo, Roberto; Rouhani, Farshid N; Yachnis, Anthony; Humphries, Margaret; Campbell-Thompson, Martha; Messina, Louis; Chulay, Jeffrey D; Trapnell, Bruce; Wilson, James M; McElvaney, Noel G; Flotte, Terence R

2017-06-07

Alpha-1 antitrypsin deficiency is a monogenic disorder resulting in emphysema due principally to the unopposed effects of neutrophil elastase. We previously reported achieving plasma wild-type alpha-1 antitrypsin concentrations at 2.5%-3.8% of the purported therapeutic level at 1 year after a single intramuscular administration of recombinant adeno-associated virus serotype 1 alpha-1 antitrypsin vector in alpha-1 antitrypsin deficient patients. We analyzed blood and muscle for alpha-1 antitrypsin expression and immune cell response. We also assayed previously reported markers of neutrophil function known to be altered in alpha-1 antitrypsin deficient patients. Here, we report sustained expression at 2.0%-2.5% of the target level from years 1-5 in these same patients without any additional recombinant adeno-associated virus serotype-1 alpha-1 antitrypsin vector administration. In addition, we observed partial correction of disease-associated neutrophil defects, including neutrophil elastase inhibition, markers of degranulation, and membrane-bound anti-neutrophil antibodies. There was also evidence of an active T regulatory cell response (similar to the 1 year data) and an exhausted cytotoxic T cell response to adeno-associated virus serotype-1 capsid. These findings suggest that muscle-based alpha-1 antitrypsin gene replacement is tolerogenic and that stable levels of M-AAT may exert beneficial neutrophil effects at lower concentrations than previously anticipated. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

In vitro monoamine oxidase inhibition potential of alpha-methyltryptamine analog new psychoactive substances for assessing possible toxic risks.

PubMed

Wagmann, Lea; Brandt, Simon D; Kavanagh, Pierce V; Maurer, Hans H; Meyer, Markus R

2017-04-15

Tryptamines have emerged as new psychoactive substances (NPS), which are distributed and consumed recreationally without preclinical studies or safety tests. Within the alpha-methylated tryptamines, some of the psychoactive effects of the prototypical alpha-methyltryptamine (AMT) have been described decades ago and a contributing factor of its acute toxicity appears to involve the inhibition of monoamine oxidase (MAO). However, detailed information about analogs is scarce. Therefore, thirteen AMT analogs were investigated for their potential to inhibit MAO. An in vitro assay analyzed using hydrophilic interaction liquid chromatography-high resolution-tandem mass spectrometry was developed and validated. The AMT analogs were incubated with recombinant human MAO-A or B and kynuramine, a non-selective MAO substrate to determine the IC 50 values. The known MAO-A inhibitors 5-(2-aminopropyl)indole (5-IT), harmine, harmaline, yohimbine, and the MAO-B inhibitor selegiline were tested for comparison. AMT and all analogs showed MAO-A inhibition properties with IC 50 values between 0.049 and 166μM, whereas four analogs inhibited also MAO-B with IC 50 values between 82 and 376μM. 7-Me-AMT provided the lowest IC 50 value against MAO-A comparable to harmine and harmaline and was identified as a competitive MAO-A inhibitor. Furthermore, AMT, 7-Me-AMT, and nine further analogs inhibited MAO activity in human hepatic S9 fraction used as model for the human liver which expresses both isoforms. The obtained results suggested that MAO inhibition induced by alpha-methylated tryptamines might be clinically relevant concerning possible serotonergic and adrenergic effects and interactions with drugs (of abuse) particularly acting as monoamine reuptake inhibitors. However, as in vitro assays have only limited conclusiveness, further studies are needed. Copyright © 2017 Elsevier B.V. All rights reserved.

Purification, crystallization and preliminary X-ray diffraction analysis of the human mismatch repair protein MutS[beta

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

Tseng, Quincy; Orans, Jillian; Hast, Michael A.

2012-03-16

MutS{beta} is a eukaryotic mismatch repair protein that preferentially targets extrahelical unpaired nucleotides and shares partial functional redundancy with MutS{alpha} (MSH2-MSH6). Although mismatch recognition by MutS{alpha} has been shown to involve a conserved Phe-X-Glu motif, little is known about the lesion-binding mechanism of MutS{beta}. Combined MSH3/MSH6 deficiency triggers a strong predisposition to cancer in mice and defects in msh2 and msh6 account for roughly half of hereditary nonpolyposis colorectal cancer mutations. These three MutS homologs are also believed to play a role in trinucleotide repeat instability, which is a hallmark of many neurodegenerative disorders. The baculovirus overexpression and purification ofmore » recombinant human MutS{beta} and three truncation mutants are presented here. Binding assays with heteroduplex DNA were carried out for biochemical characterization. Crystallization and preliminary X-ray diffraction analysis of the protein bound to a heteroduplex DNA substrate are also reported.« less

A Combined NMR-Computational Study of the Interaction between Influenza Virus Hemagglutinin and Sialic Derivatives from Human and Avian Receptors on the Surface of Transfected Cells.

PubMed

Vasile, Francesca; Panigada, Maddalena; Siccardi, Antonio; Potenza, Donatella; Tiana, Guido

2018-04-24

The development of small-molecule inhibitors of influenza virus Hemagglutinin could be relevant to the opposition of the diffusion of new pandemic viruses. In this work, we made use of Nuclear Magnetic Resonance (NMR) spectroscopy to study the interaction between two derivatives of sialic acid, Neu5Ac-α-(2,6)-Gal-β-(1⁻4)-GlcNAc and Neu5Ac-α-(2,3)-Gal-β-(1⁻4)-GlcNAc, and hemagglutinin directly expressed on the surface of recombinant human cells. We analyzed the interaction of these trisaccharides with 293T cells transfected with the H5 and H1 variants of hemagglutinin, which thus retain their native trimeric conformation in such a realistic environment. By exploiting the magnetization transfer between the protein and the ligand, we obtained evidence of the binding event, and identified the epitope. We analyzed the conformational features of the glycans with an approach combining NMR spectroscopy and data-driven molecular dynamics simulations, thus obtaining useful information for an efficient drug design.

Specific Genetic Immunotherapy Induced by Recombinant Vaccine Alpha-Fetoprotein-Heat Shock Protein 70 Complex

NASA Astrophysics Data System (ADS)

Wang, Xiaoping; Lin, Huanping; Wang, Qiaoxia

Purposes: To construct a recombinant vaccine alpha-fetoprotein (AFP)-heat shock protein (HSP70) complex, and study its ability to induce specific CTL response and its protective effect against AFP-producing tumor. Material/Methods: A recombinant vaccine was constructed by conjugating mouse alpha-fetoprotein to heat shock protein 70. By way of intracutaneous injection, mice were primed and boosted with recombinant vaccine mAFP/HSP70, whereas single mAFP or HSP70 injection as controls. The ELISPOT and ELISA were used to measure the frequency of cells producing the cytokine IFN-γ in splenocytes and the level of anti-AFP antibody of serum from immunized mice respectively. In vivo tumor challenge were carried out to assess the immune effect of the recombinant vaccine. Results: By recombinant mAFP/HSP70 vaccine immunization, the results of ELISPOT and ELISA showed that the number of splenic cells producing IFN-γ and the level of anti-AFP antibody of serum were significantly higher in mAFP/HSP70 group than those in mAFP and HSP70 groups (108.50±11.70 IFN-γ spots/106 cells vs 41.60±10.40 IFN-γ spots/106 cells, 7.32±3.14 IFN-γ spots/106 cells, P<0.01; 156.32±10.42 μg/mL vs 66.52±7.35 μg/mL, 5.73±2.89 μg/mL, P<0.01). The tumor volume in mAFP/HSP70 group was significantly smaller than that in mAFP and HSP70 groups (42.44±7.14 mm3 vs 392.23±12.46 mm3, 838.63±13.84 mm3, P<0.01). Conclusions: The study further confirmed the function of heat shock protein 70's immune adjuvant. Sequential immunization with recombinant mAFP/HSP70 vaccine could generate effective antitumor immunity on AFP-producing tumor. The recombined mAFP/HSP70 vaccine may be suitable for serving as an immunotherapy for hepatocellular carcinoma.

A BioDesign Approach to Obtain High Yields of Biosimilars by Anti-apoptotic Cell Engineering: a Case Study to Increase the Production Yield of Anti-TNF Alpha Producing Recombinant CHO Cells.

PubMed

Gulce Iz, Sultan; Inevi, Muge Anil; Metiner, Pelin Saglam; Tamis, Duygu Ayyildiz; Kisbet, Nazli

2018-01-01

Recent developments in medical biotechnology have facilitated to enhance the production of monoclonal antibodies (mAbs) and recombinant proteins in mammalian cells. Human mAbs for clinical applications have focused on three areas, particularly cancer, immunological disorders, and infectious diseases. Tumor necrosis factor alpha (TNF-α), which has both proinflammatory and immunoregulatory functions, is an important target in biopharmaceutical industry. In this study, a humanized anti-TNF-α mAb producing stable CHO cell line which produces a biosimilar of Humira (adalimumab) was used. Adalimumab is a fully human anti-TNF mAb among the top-selling mAb products in recent years as a biosimilar. Products from mammalian cell bioprocesses are a derivative of cell viability and metabolism, which is mainly disrupted by cell death in bioreactors. Thus, different strategies are used to increase the product yield. Suppression of apoptosis, also called anti-apoptotic cell engineering, is the most remarkable strategy to enhance lifetime of cells for a longer production period. In fact, using anti-apoptotic cell engineering as a BioDesign approach was inspired by nature; nature gives prolonged life span to some cells like stem cells, tumor cells, and memory B and T cells, and researchers have been using this strategy for different purposes. In this study, as a biomimicry approach, anti-apoptotic cell engineering was used to increase the anti-TNF-α mAb production from the humanized anti-TNF-α mAb producing stable CHO cell line by Bcl-xL anti-apoptotic protein. It was shown that transient transfection of CHO cells by the Bcl-xL anti-apoptotic protein expressing plasmid prolonged the cell survival rate and protected cells from apoptosis. The transient expression of Bcl-xL using CHO cells enhanced the anti-TNF-α production. The production of anti-TNF-α in CHO cells was increased up to 215 mg/L with an increase of 160% after cells were transfected with Bcl-xL expressing plasmid with polyethylenimine (PEI) reagent at the ratio of 1:6 (DNA:PEI). In conclusion, the anti-apoptotic efficacy of the Bcl-xL expressing plasmid in humanized anti-TNF-α MAb producing stable CHO cells is compatible with curative effect for high efficiency recombinant protein production. Thus, this model can be used for large-scale production of biosimilars through transient Bcl-xL gene expression as a cost-effective method.

Purification and characterization of a bioactive alpha-fetoprotein produced by HEK-293 cells.

PubMed

Lin, Bo; Peng, Guoqing; Feng, Haipeng; Li, Wei; Dong, Xu; Chen, Yi; Lu, Yan; Wang, Qiaoyun; Xie, Xieju; Zhu, Mingyue; Li, Mengsen

2017-08-01

Alpha-fetoprotein (AFP) is a biomarker that is used to diagnose hepatocellular carcinoma (HCC) and can promote malignancy in HCC. AFP is an important target in the treatment of liver cancer. To obtain enough AFP to screen for AFP inhibitors, we expressed and purified AFP in HEK-293 cells. In the present study, we produced AFP in the cells and harvested highly pure rAFP (or recombinant expression AFP in HEK-293 cells). We also analysed the bioactivity of rAFP and found that rAFP promoted growth of the human HCC cells, antagonize paclitaxel inhibition of HCC cell proliferation, suppress expression of active caspase-3, and promote expression of Ras and survivin. This study provides a method to produce significant amounts of AFP for use in biochemical assays and functional studies and to screen AFP inhibitors for use in HCC therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

Specific starch digestion of maize alpha-limit dextrins by recombinant mucosal glucosidase enzymes

USDA-ARS?s Scientific Manuscript database

Starch digestion requires two luminal enzymes, salivary and pancreatic alpha-amylase (AMY), and four small intestinal mucosal enzyme activities from the N- and C-terminals of maltase-glucoamylase (MGAM) and sucrose-isomaltase (SI) complexes. AMY is not a requirement for starch digestion to glucose b...

Low-Dose Irradiation Enhances Gene Targeting in Human Pluripotent Stem Cells.

PubMed

Hatada, Seigo; Subramanian, Aparna; Mandefro, Berhan; Ren, Songyang; Kim, Ho Won; Tang, Jie; Funari, Vincent; Baloh, Robert H; Sareen, Dhruv; Arumugaswami, Vaithilingaraja; Svendsen, Clive N

2015-09-01

Human pluripotent stem cells (hPSCs) are now being used for both disease modeling and cell therapy; however, efficient homologous recombination (HR) is often crucial to develop isogenic control or reporter lines. We showed that limited low-dose irradiation (LDI) using either γ-ray or x-ray exposure (0.4 Gy) significantly enhanced HR frequency, possibly through induction of DNA repair/recombination machinery including ataxia-telangiectasia mutated, histone H2A.X and RAD51 proteins. LDI could also increase HR efficiency by more than 30-fold when combined with the targeting tools zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats. Whole-exome sequencing confirmed that the LDI administered to hPSCs did not induce gross genomic alterations or affect cellular viability. Irradiated and targeted lines were karyotypically normal and made all differentiated lineages that continued to express green fluorescent protein targeted at the AAVS1 locus. This simple method allows higher throughput of new, targeted hPSC lines that are crucial to expand the use of disease modeling and to develop novel avenues of cell therapy. The simple and relevant technique described in this report uses a low level of radiation to increase desired gene modifications in human pluripotent stem cells by an order of magnitude. This higher efficiency permits greater throughput with reduced time and cost. The low level of radiation also greatly increased the recombination frequency when combined with developed engineered nucleases. Critically, the radiation did not lead to increases in DNA mutations or to reductions in overall cellular viability. This novel technique enables not only the rapid production of disease models using human stem cells but also the possibility of treating genetically based diseases by correcting patient-derived cells. ©AlphaMed Press.

A novel recombinantly produced banana lectin isoform is a valuable tool for glycoproteomics and a potent modulator of the proliferation response in CD3+, CD4+, and CD8+ populations of human PBMCs.

PubMed

Gavrovic-Jankulovic, Marija; Poulsen, Knud; Brckalo, Tamara; Bobic, Sonja; Lindner, Buko; Petersen, Arnd

2008-01-01

Lectins as carbohydrate-binding proteins have been employed in various biological assays for the detection and characterization of glycan structures on glycoproteins, including clinical biomarkers in disease states. A mannose-specific banana lectin (BanLec) is unique in its specificity for internal alpha1,3 linkages as well as beta1,3 linkages at the reducing termini. The immunomodulatory potential of natural BanLec was recognized by a strong immunoglobulin G4 antibody response and T cell mitogen activity in humans. To explore its applicability in glycoproteomics and its modulatory potential, the gene of banana lectin was cloned, sequenced and a recombinant protein was produced in Escherichia coli. The obtained cDNA revealed a novel banana lectin isoform, with an open reading frame of 426 nucleotides, encoding a cytoplasmatic protein of 141 amino acids. The molecular mass of rBanLec determined by ESI FT-MS and N-terminal sequencing confirmed the cDNA at the protein level. The specificity of rBanLec for detection glycan structures was the same as for natural BanLec as examined with five protein extracts rich in glycoprotein content, as well as with horseradish peroxidase glycoprotein. Besides, the immunomodulatory potential of rBanLec and nBanLec were comparable as assessed by an inhibition assay and a human T cell proliferation assay where they induced a strong proliferation response in CD3+, CD4+, and CD8+ populations of human PBMCs. This recombinant BanLec is a useful reagent for glycoproteomics and lectin microarrays, with a potential for modulation of the immune response.

Rapid Immunochromatographic Detection of Serum Anti-α-Galactosidase A Antibodies in Fabry Patients after Enzyme Replacement Therapy

PubMed Central

Nakano, Sachie; Tsukimura, Takahiro; Togawa, Tadayasu; Ohashi, Toya; Kobayashi, Masahisa; Takayama, Katsuyoshi; Kobayashi, Yukuharu; Abiko, Hiroshi; Satou, Masatsugu; Nakahata, Tohru; Warnock, David G.; Sakuraba, Hitoshi; Shibasaki, Futoshi

2015-01-01

We developed an immunochromatography-based assay for detecting antibodies against recombinant α-galactosidase A proteins in serum. The evaluation of 29 serum samples from Fabry patients, who had received enzyme replacement therapy with agalsidase alpha and/or agalsidase beta, was performed by means of this assay method, and the results clearly revealed that the patients exhibited the same level of antibodies against both agalsidase alpha and agalsidase beta, regardless of the species of recombinant α-galactosidase A used for enzyme replacement therapy. A conventional enzyme-linked immunosorbent assay supported the results. Considering these, enzyme replacement therapy with agalsidase alpha or agalsidase beta would generate antibodies against the common epitopes in both agalsidase alpha and agalsidase beta. Most of the patients who showed immunopositive reaction exhibited classic Fabry phenotype and harbored gene mutations affecting biosynthesis of α-galactosidase A. As immunochromatography is a handy and simple assay system which can be available at bedside, this assay method would be extremely useful for quick evaluation or first screening of serum antibodies against agalsidase alpha or agalsidase beta in Fabry disease with enzyme replacement therapy. PMID:26083343

Treatment of three patients with systemic mastocytosis with interferon alpha-2b.

PubMed

Worobec, A S; Kirshenbaum, A S; Schwartz, L B; Metcalfe, D D

1996-08-01

It has been reported that the administration of interferon alpha-2b is of potential benefit in the treatment of mastocytosis based on a single patient study (NEJM, Feb 27, 1992, 326(9):619-623). Following this report, we administered interferon alpha-2b at a dose of 4 to 5 million units per square meter of body surface area for at least 12 months to one patient with mastocytosis with an associated hematologic disorder (patient 1), one patient with aggressive systemic mastocytosis (patient 2), and one patient with indolent mastocytosis (patient 3). Patients were monitored with the following clinical and laboratory parameters: serial bone marrow biopsies and aspirates, patient log of histamine release attacks, medication dependency, plasma tryptase levels, serum lactate dehydrogenase (LDH) levels, white blood cell counts and differentials, extent of urticaria pigmentosa lesions, bony involvement, and extent of gastrointestinal involvement and hepatomegaly. We also examined the ability of interferon alpha-2b to inhibit recombinant human stem cell factor (rhSCF)-dependent mast cell proliferation from CD34+ bone marrow-derived cells. All patients demonstrated continued progression of disease in one or more clinical criteria at one year of therapy. Similarly, interferon alpha-2b did not inhibit the culture of mast cells from CD34+ bone marrow-derived cells in the presence of SCF. Thus, in our study of three patients with systemic mastocytosis, treatment with interferon alpha-2b was found to be ineffective in controlling progression of disease.

Preparation and screening of an arrayed human genomic library generated with the P1 cloning system.

PubMed Central

Shepherd, N S; Pfrogner, B D; Coulby, J N; Ackerman, S L; Vaidyanathan, G; Sauer, R H; Balkenhol, T C; Sternberg, N

1994-01-01

We describe here the construction and initial characterization of a 3-fold coverage genomic library of the human haploid genome that was prepared using the bacteriophage P1 cloning system. The cloned DNA inserts were produced by size fractionation of a Sau3AI partial digest of high molecular weight genomic DNA isolated from primary cells of human foreskin fibroblasts. The inserts were cloned into the pAd10sacBII vector and packaged in vitro into P1 phage. These were used to generate recombinant bacterial clones, each of which was picked robotically from an agar plate into a well of a 96-well microtiter dish, grown overnight, and stored at -70 degrees C. The resulting library, designated DMPC-HFF#1 series A, consists of approximately 130,000-140,000 recombinant clones that were stored in 1500 microtiter dishes. To screen the library, clones were combined in a pooling strategy and specific loci were identified by PCR analysis. On average, the library contains two or three different clones for each locus screened. To date we have identified a total of 17 clones containing the hypoxanthine-guanine phosphoribosyltransferase, human serum albumin-human alpha-fetoprotein, p53, cyclooxygenase I, human apurinic endonuclease, beta-polymerase, and DNA ligase I genes. The cloned inserts average 80 kb in size and range from 70 to 95 kb, with one 49-kb insert and one 62-kb insert. Images PMID:8146166

Maintaining HNF6 expression prevents AdHNF3beta-mediated decrease in hepatic levels of Glut-2 and glycogen.

PubMed

Tan, Yongjun; Adami, Guy; Costa, Robert H

2002-04-01

The hepatocyte nuclear factor 3 (HNF-3) proteins are members of the Forkhead Box (Fox) family of transcription factors that play important roles in regulating expression of genes involved in cellular proliferation, differentiation, and metabolic homeostasis. In previous studies we increased liver expression of HNF-3beta by using either transgenic mice (transthyretin HNF-3beta) or recombinant adenovirus infection (AdHNF3beta), and observed diminished hepatic levels of glycogen, and glucose transporter 2 (Glut-2), as well as the HNF-6, HNF-3, HNF-1alpha, HNF-4alpha, and C/EBPalpha transcription factors. We conducted the present study to determine whether maintaining HNF-6 protein expression during AdHNF3beta infection prevents reduction of hepatic levels of glycogen and the earlier-mentioned genes. Here, we show that AdHNF3beta- and AdHNF6-infected mouse liver displayed increased hepatic levels of glycogen, Glut-2, HNF-3gamma, HNF-1alpha, and HNF-4alpha at 2 and 3 days postinfection (PI). Furthermore, restoration of hepatic glycogen levels after AdHNF3beta and AdHNF6 coinfection was associated with increased Glut-2 expression. AdHNF6 infection alone caused a 2-fold increase in hepatic Glut-2 levels, suggesting that HNF 6 stimulates in vivo transcription of the Glut-2 gene. DNA binding assays showed that only recombinant HNF-6 protein, but not the HNF-3 proteins, binds to the mouse -185 to -144 bp Glut-2 promoter sequences. Cotransfection assays in human hepatoma (HepG2) cells with either HNF-3 or HNF-6 expression vectors show that only HNF-6 provided significant transcriptional activation of the Glut-2 promoter. In conclusion, these studies show that the hepatic Glut-2 promoter is a direct target for HNF-6 transcriptional activation.

Interleukin 1 alpha-induced expression of manganous superoxide dismutase reduces myocardial reperfusion injury in the rat.

PubMed

Nogae, C; Makino, N; Hata, T; Nogae, I; Takahashi, S; Suzuki, K; Taniguchi, N; Yanaga, T

1995-10-01

We investigated the effects of pretreatment with interleukin (IL)-1 alpha on the expression of manganous (Mn) superoxide dismutase (SOD) mRNA and reperfusion-induced arrhythmias and the size of myocardial infarct in rats. Male Wistar rats received 10 mg intraperitoneal injections of human recombinant IL-1 alpha. Their hearts were thereafter isolated at 6, 12, 24, 36 h. A Northern analysis showed that Mn-SOD mRNA was mainly expressed in the heart and slightly in kidney, but not in any other organs. The expression of Mn-SOD mRNA peaked at 6 h after the injection of IL-1 alpha. The Mn-SOD protein content was most increased 12 h after injection. In the isolated heart model, the rats were pretreated with IL-1 alpha 24 h earlier and their hearts were perfused by the Langendorff method. After 20 min of ischemia which was induced by a ligation of a coronary artery, reperfusion-induced arrhythmias were observed. There were no significant differences in the incidence of ventricular arrhythmias between the IL-1 alpha pretreated and the untreated hearts. IL-1 alpha pretreatment significantly reduced the mean duration of the ventricular arrhythmias and also delayed the onset of arrhythmias. The effect of IL-1 alpha pretreatment was also investigated in a 30-min model of ischemia followed by a 3-min reperfusion in anesthetized rats. The infarct size expressed as a percentage of the area at risk was significantly reduced in the IL-1 alpha pretreated hearts compared with the untreated hearts. The left ventricular systolic pressure increased significantly in rat hearts pretreated with IL-1 alpha. Our results therefore showed that the pretreatment with IL-1 alpha induced the overexpression of Mn-SOD mRNA in the rat hearts and also suggested that pretreatment with IL-1 alpha 24 h before ischemia reduced the risk of ischemia-reperfusion injury.

Intracarotid tumor necrosis factor-alpha administration increases the blood-brain barrier permeability in cerebral cortex of the newborn pig: quantitative aspects of double-labelling studies and confocal laser scanning analysis.

PubMed

Abraham, C S; Deli, M A; Joo, F; Megyeri, P; Torpier, G

1996-04-19

Tumor necrosis factor-alpha (TNF-alpha) plays a crucial role in the pathogenesis of the central nervous system infections. The aim of the present study was to analyze quantitatively the changes in the blood-brain barrier (BBB) permeability after the intracarotid injection of TNF-alpha. Recombinant human TNF-alpha was injected into the left internal carotid artery of anesthetized newborn pigs (n = 48) in the doses of 0, 1000, 10 000 and 100 000 IU, respectively. Before, as well as 1, 2, 4, 8, and 16 h after the challenge, the extravasation of a small (sodium fluorescein (SF), mw 376), and a large (Evan's blue-albumin (EBA), mw 67 000) tracer was determined concomitantly by spectrophotometry in the cerebral cortex of the animals. There was a time- and dose-dependent increase in BBB permeability both for SF and EBA; however, significant (P < 0.05) BBB opening for albumin only developed 2 h after the challenge. In the morphological study the same excitable tracers, identical experimental protocol and groups were used. Cryostat sections of brain tissue were viewed for optical sectioning with a confocal laser scanning microscope equipped with an argon/krypton ion laser. A diffuse BBB opening for SF and a moderate perivascular extravasation for EBA were found in the cortices of TNF-alpha-treated animals. We conclude that significant increases in intravascular TNF-alpha-concentration during neonatal infections may result in vasogenic brain edema formation.

Restoration of central nervous system alpha-N-acetylglucosaminidase activity and therapeutic benefits in mucopolysaccharidosis IIIB mice by a single intracisternal recombinant adeno-associated viral type 2 vector delivery.

PubMed

Fu, Haiyan; DiRosario, Julianne; Kang, Lu; Muenzer, Joseph; McCarty, Douglas M

2010-07-01

Finding efficient central nervous system (CNS) delivery approaches has been the major challenge facing therapeutic development for treating diseases with global neurological manifestation, such as mucopolysaccharidosis (MPS) IIIB, a lysosomal storage disease, caused by autosomal recessive defect of alpha-N-acetylglucosaminidase (NaGlu). Previously, we developed an approach, intracisternal (i.c.) injection, to deliver recombinant adeno-associated viral (rAAV) vector to the CNS of mice, leading to a widespread periventricular distribution of transduction. In the present study, we delivered rAAV2 vector expressing human NaGlu into the CNS of MPS IIIB mice by an i.c. injection approach, to test its therapeutic efficacy and feasibility for treating the neurological manifestation of the disease. We demonstrated significant functional neurological benefits of a single i.c. vector infusion in adult MPS IIIB mice. The treatment slowed the disease progression by mediating widespread recombinant NaGlu expression in the CNS, resulting in the reduction of brain lysosomal storage pathology, significantly improved cognitive function and prolonged survival. However, persisting motor function deficits suggested that pathology in areas outside the CNS contributes to the MPS IIIB behavioral phenotype. The therapeutic benefit of i.c. rAAV2 delivery was dose-dependent and could be attribute solely to the CNS transduction because the procedure did not lead to detectable transduction in somatic tissues. A single IC rAAV2 gene delivery is functionally beneficial for treating the CNS disease of MPS IIIB in mice. It is immediately clinically translatable, with the potential of improving the quality of life for patients with MPS IIIB.

Successful ovulation induction, conception, and normal delivery after chronic therapy with etanercept: a recombinant fusion anti-cytokine treatment for rheumatoid arthritis.

PubMed

Sills, E S; Perloe, M; Tucker, M J; Kaplan, C R; Palermo, G D

2001-11-01

Etanercept (Enbrel; Wyeth-Ayerst/Immunex Inc, Seattle, WA, USA) is a subcutaneously administered novel fusion protein consisting of the extracellular ligand-binding domain of the 75 kD receptor for tumor necrosis factor-alpha (anti-TNFalpha) and the Fc portion of human IgG1. The agent is synthesized by plasmid transfection of a Chinese hamster ovary cell line, utilizing recombinant DNA technology. Etanercept was approved by the US FDA for treatment of multi-drug resistant rheumatoid arthritis in 1998, but no human data exist regarding the impact of anti-TNFalpha therapy on human reproductive function or its use before ovulation induction. As TNFalpha potentiates collagenolysis via matrix metalloproteinase gene expression (thereby facilitating ovulation), there exists a theoretical risk that TNFalpha-inhibition could exert an undesirable effect on ovulation and pregnancy. In this report, we describe the first case of ovulation induction, intrauterine insemination, normal pregnancy and singleton delivery of a healthy infant following chronic ( > 1 year) pre-ovulatory TNFalpha-inhibitor therapy for rheumatoid arthritis. Reproductive endocrinologists and obstetrician-gynecologists should be familiar with etanercept therapy in the context of severe rheumatic disease, and offer appropriate reassurance regarding its safe use for infertility patients planning ovulation induction.

Analysis of the CDR3 length repertoire and the diversity of TCR alpha chain in human peripheral blood T lymphocytes.

PubMed

Yao, Xin Sheng; Diao, Ying; Sun, Wan Bang; Luo, Jun Min; Qin, Ming; Tang, Xian Ying

2007-06-01

Analysis of complementarity determining region 3 (CDR3) length of T lymphocyte receptors (TCRs) by immunoscope spectratyping technique has been used successfully to investigate the diversity of TCR in autoimmune diseases and infection diseases. In this study, we investigated the patterns of CDR3 length distribution for all 32 TCR AV gene families in human peripheral blood lymphocytes of four normal volunteers by the immunoscope spectratyping technique. It was found that PCR products exhibited an obscure band on 1.5% agarose gel electrophoresis. Each TCR AV family exhibited more than 8 bands on 6% sequencing gel electrophoresis. The CDR3 spectratyping of all TCR AV families showed a standard Gaussian distribution with different CDR3 length, and the expression frequency of CDR3 was similar among the gene families. Most of CDR3 in TCR AV family recombine in frame. However, some of the CDR3 showed out-of frame gene rearrangement. Additionally, we found that in some of TCR AV families there were 18 amino acid discrepancies between the longest CDR3 and shortest CDR3. These results may be helpful to further study the recombination mechanism of human TCR genes, the TCR CDR3 gene repertoire, and the repertoire drift in health people and disease state.

Tumor necrosis factor (cachectin) is an endogenous pyrogen and induces production of interleukin 1.

PubMed

Dinarello, C A; Cannon, J G; Wolff, S M; Bernheim, H A; Beutler, B; Cerami, A; Figari, I S; Palladino, M A; O'Connor, J V

1986-06-01

Recombinant human tumor necrosis factor (rTNF alpha) injected intravenously into rabbits produces a rapid-onset, monophasic fever indistinguishable from the fever produced by rIL-1. On a weight basis (1 microgram/kg) rTNF alpha and rIL-1 produce the same amount of fever and induce comparable levels of PGE2 in rabbit hypothalamic cells in vitro; like IL-1, TNF fever is blocked by drugs that inhibit cyclooxygenase. At higher doses (10 micrograms/kg) rTNF alpha produces biphasic fevers. The first fever reaches peak elevation 45-55 min after bolus injection and likely represents a direct action on the thermoregulatory center. During the second fever peak (3 h later), a circulating endogenous pyrogen can be shown present using passive transfer of plasma into fresh rabbits. This likely represents the in vivo induction of IL-1. In vitro, rTNF alpha induces the release of IL-1 activity from human mononuclear cells with maximal production observed at 50-100 ng/ml of rTNF alpha. In addition, rTNF alpha and rIFN-gamma have a synergistic effect on IL-1 production. The biological activity of rTNF alpha could be distinguished from IL-1 in three ways: the monophasic pyrogenic activity of rIL-1 was destroyed at 70 degrees C, whereas rTNF alpha remained active; anti-IL-1 neutralized IL-1 but did recognize rTNF alpha or natural cachectin nor neutralize its cytotoxic effect; and unlike IL-1, rTNF alpha was not active in the mitogen-stimulated T cell proliferation assay. The possibility that endotoxin was responsible for rTNF alpha fever and/or the induction of IL-1 was ruled-out in several studies: rTNF alpha produced fever in the endotoxin-resistant C3H/HeJ mice; the IL-1-inducing property of rTNF alpha was destroyed either by heat (70 degrees C) or trypsinization, and was unaffected by polymyxin B; pyrogenic tolerance to daily injections of rTNF alpha did not occur; levels of endotoxin, as determined in the Limulus amebocyte lysate, were below the minimum rabbit pyrogen dose; and these levels of endotoxin were confirmed by gas chromatography/mass spectrometry analysis for the presence of beta-hydroxymyristic acid. Although rTNF alpha is not active in T cell proliferation assays, it may mimic IL-1 in a T cell assay, since high concentrations of rTNF alpha induced IL-1 from epithelial or macrophagic cells in the thymocyte preparations. These studies show that TNF (cachectin) is another endogenous pyrogen which, like IL-1 and IFN-alpha, directly stimulate hypothalamic PGE2 synthesis. In addition, rTNF alpha is an endogenous inducer of IL-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Progranulin expression in advanced human atherosclerotic plaque.

PubMed

Kojima, Yoji; Ono, Koh; Inoue, Katsumi; Takagi, Yasushi; Kikuta, Ken-ichiro; Nishimura, Masaki; Yoshida, Yoshinori; Nakashima, Yasuhiro; Matsumae, Hironobu; Furukawa, Yutaka; Mikuni, Nobuhiro; Nobuyoshi, Masakiyo; Kimura, Takeshi; Kita, Toru; Tanaka, Makoto

2009-09-01

Progranulin (PGRN) is a unique growth factor that plays an important role in cutaneous wound healing. It has an anti-inflammatory effect and promotes cell proliferation. However, when it is degraded to granulin peptides (GRNs) by neutrophil proteases, a pro-inflammatory reaction occurs. Since injury, inflammation and repair are common features in the progression of atherosclerosis, it is conceivable that PGRN plays a role in atherogenesis. Immunohistochemical analysis of human carotid endoatherectomy specimens indicated that vascular smooth muscle cells (vSMCs) in the intima expressed PGRN. Some macrophages in the plaque also expressed PGRN. We assessed the effect of PGRN on a human monocytic leukemia cell line (THP-1) and human aortic smooth muscle cells (HASMCs). PGRN alone had no effect on HASMC or THP-1 proliferation or migration. However, when THP-1 cells were stimulated with MCP-1, the number of migrated cells decreased in a PGRN-dose-dependent manner. TNF-alpha-induced HASMC migration was enhanced only at 10nM of PGRN. Interleukin-8 (IL-8) secretion from HASMCs was reduced by forced expression of PGRN and increased by RNAi-mediated knockdown of PGRN. While exogenous treatment with recombinant PGRN decreased IL-8 secretion, degraded recombinant GRNs increased IL-8 secretion from HASMCs. The expression of PGRN mainly reduces inflammation and its degradation into GRNs enhances inflammation in atherosclerotic plaque and may contribute to the progression of atherosclerosis.

Alpha-Tocopherol alters transcription activities that modulate tumor necrosis factor alpha (TNF-¿)-induced inflammatory response in bovine cells

USDA-ARS?s Scientific Manuscript database

To further investigate the potential role of '-tocopherol in maintaining immuno-homeostasis in bovine cells (Madin-Darby bovine kidney epithelial cell line), we undertook in vitro experiments using recombinant TNF-a as an immuno-stimulant to simulate inflammation response in cells with and without '...

The highly obscured nucleus of 3C 219

NASA Technical Reports Server (NTRS)

Fabbiano, G.; Willner, S. P.; Carleton, N. P; Elvis, M.

1986-01-01

The detection of a strong, and possibly broad, Paschen-alpha line from the narrow-line radio galaxy 3C 219 is reported. The detected flux is larger than predicted from the H-alpha line and the case B recombination. This implies the presence of a highly reddened line-emitting region in the nucleus.

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.

Immunogenicity of a Trivalent Recombinant Vaccine Against Clostridium perfringens Alpha, Beta, and Epsilon Toxins in Farm Ruminants.

PubMed

Moreira, Gustavo Marçal Schmidt Garcia; Salvarani, Felipe Masiero; da Cunha, Carlos Eduardo Pouey; Mendonça, Marcelo; Moreira, Ângela Nunes; Gonçalves, Luciana Aramuni; Pires, Prhiscylla Sadanã; Lobato, Francisco Carlos Faria; Conceição, Fabricio Rochedo

2016-03-23

Clostridium perfringens is an anaerobic bacterium that produces several toxins. Of these, the alpha, beta, and epsilon toxins are responsible for causing the most severe C. perfringens-related diseases in farm animals. The best way to control these diseases is through vaccination. However, commercially available vaccines are based on inactivated toxins and have many production drawbacks, which can be overcome through the use of recombinant antigens. In this study, we produced recombinant alpha, beta, and epsilon toxins in Escherichia coli to formulate a trivalent vaccine. Its effectiveness was evaluated through a potency test in rabbits, in which the vaccine generated 9.6, 24.4, and 25.0 IU/mL of neutralizing antibodies against the respective toxins. Following this, cattle, sheep, and goats received the same formulation, generating, respectively, 5.19 ± 0.48, 4.34 ± 0.43, and 4.70 ± 0.58 IU/mL against alpha toxin, 13.71 ± 1.17 IU/mL (for all three species) against beta toxin, and 12.74 ± 1.70, 7.66 ± 1.69, and 8.91 ± 2.14 IU/mL against epsilon toxin. These levels were above the minimum recommended by international protocols. As such, our vaccine was effective in generating protective antibodies and, thus, may represent an interesting alternative for the prevention of C. perfringens-related intoxications in farm animals.

Tip-alpha (hp0596 gene product) is a highly immunogenic Helicobacter pylori protein involved in colonization of mouse gastric mucosa.

PubMed

Godlewska, Renata; Pawlowski, Marcin; Dzwonek, Artur; Mikula, Michal; Ostrowski, Jerzy; Drela, Nadzieja; Jagusztyn-Krynicka, Elzbieta K

2008-03-01

A product of the Helicobacter pylori hp0596 gene (Tip-alpha) is a highly immunogenic homodimeric protein, unique for this bacterium. Cell fractionation experiments indicate that Tip-alpha is anchored to the inner membrane. In contrast, the three-dimensional model of the protein suggests that Tip-alpha is soluble or, at least, largely exposed to the solvent. hp0596 gene knockout resulted in a significant decrease in the level of H. pylori colonization as measured by real-time PCR assay. In addition, the Tip-alpha recombinant protein was determined to stimulate macrophage to produce IL-1alpha and TNF-alpha. Both results imply that Tip-alpha is rather loosely connected to the inner membrane and potentially released during infection.

The dissociative recombination of O2/+/ in the ionosphere

NASA Technical Reports Server (NTRS)

Torr, M. R.; Torr, D. G.

1981-01-01

Aeronomical determinations of the dissociative recombination reaction rate coefficient for O2(+) and alpha depend directly on a knowledge of the rate coefficient for the charge exchange of O(+) with O2 and k. The aeronomical determination of alpha is reevaluated using Atmosphere Explorer satellite data in light of a subsequent laboratory measurement of k (Chen et al., 1978). The results are found to be in good agreement with laboratory determinations of the coefficient for night-time conditions. For data obtained under sunlit conditions, however, the results differed significantly with those of the laboratory measurements. These results imply that the state of the O2(+) molecule major thermospheric processes needs to be examined in greater detail.

Biobusiness in the pharmaceutical industry.

PubMed

Werner, R G

1987-09-01

Although conventional biotechnology used for the synthesis of antibiotics, vitamins, amino acids, nucleotides, enzyme inhibitors and immunomodulating compounds has still a major impact in the production of pharmaceutical compounds, the importance of the new biotechnology is increasing. Whereas in conventional biotechnology naturally occurring strains are screened for production of pharmacologically active compounds, in new biotechnology known organisms are programmed by genetic engineering to produce a distinct protein or glycoprotein of human origin for substitution therapy. Such complex compounds from new biotechnology can be divided into products which might replace compounds which are already on the market by safer recombinant products such as human insulin, human growth hormone, urokinase, factor VIII and products which are new on the market such as interferons, lymphokines, tissue plasminogen activator, oligonucleotide probes, monoclonal antibodies and subunit vaccines. However, only a few of these recombinant products have reached the market such as human insulin, interferon alpha, interferon beta, human growth hormone and recombivax HB. In most cases, depending on the difficulties in demonstrating clinical efficacy, the investigated drugs have reached the marketing phase much faster than conventional chemical drugs. Return on investment of biotechnical produced pharmaceutics mainly depends on the issues of whether the product has to compete with chemically synthesized drugs, whether it is totally new but competes with other bioproducts, whether it is exceptional but the proof of clinical efficacy is difficult, or whether it is totally new and clinical studies are promising.(ABSTRACT TRUNCATED AT 250 WORDS)

Bayesian Inference of Shared Recombination Hotspots Between Humans and Chimpanzees

PubMed Central

Wang, Ying; Rannala, Bruce

2014-01-01

Recombination generates variation and facilitates evolution. Recombination (or lack thereof) also contributes to human genetic disease. Methods for mapping genes influencing complex genetic diseases via association rely on linkage disequilibrium (LD) in human populations, which is influenced by rates of recombination across the genome. Comparative population genomic analyses of recombination using related primate species can identify factors influencing rates of recombination in humans. Such studies can indicate how variable hotspots for recombination may be both among individuals (or populations) and over evolutionary timescales. Previous studies have suggested that locations of recombination hotspots are not conserved between humans and chimpanzees. We made use of the data sets from recent resequencing projects and applied a Bayesian method for identifying hotspots and estimating recombination rates. We also reanalyzed SNP data sets for regions with known hotspots in humans using samples from the human and chimpanzee. The Bayes factors (BF) of shared recombination hotspots between human and chimpanzee across regions were obtained. Based on the analysis of the aligned regions of human chromosome 21, locations where the two species show evidence of shared recombination hotspots (with high BFs) were identified. Interestingly, previous comparative studies of human and chimpanzee that focused on the known human recombination hotspots within the β-globin and HLA regions did not find overlapping of hotspots. Our results show high BFs of shared hotspots at locations within both regions, and the estimated locations of shared hotspots overlap with the locations of human recombination hotspots obtained from sperm-typing studies. PMID:25261696

Regulation by interferon alpha of immunoglobulin isotype selection and lymphokine production in mice

PubMed Central

1991-01-01

Antigens and infectious agents that stimulate interferon alpha(IFN- alpha) production in mice induce antibody responses that are predominantly of the immunoglobulin (Ig)G2a isotype and contain little or no IgE. This suggested the possibility that IFN-alpha might have a role in directing Ig isotype selection. Consistent with this possibility, we have found that injection of mice with recombinant mouse IFN-alpha suppresses IgE secretion, enhances IgG2a secretion, and has no independent effect on IgG1 secretion in mice stimulated with a foreign anti-IgD antibody. Injection of mice with polyinosinic acid.polycytidylic acid (poly I.C), an inducer of macrophage IFN-alpha production, also suppresses the anti-IgD antibody-induced IgE response and stimulates the IgG2a response; these effects are blocked by a sheep antibody that neutralizes mouse IFN-alpha/beta. Both recombinant IFN- alpha and poly I.C have maximum IgE suppressive and IgG2a stimulatory effects when injected early in the anti-IgD antibody-induced immune response. Addition of IFN-alpha to mouse B cells cultured with lipopolysaccharide (LPS) + interleukin 4 (IL-4) suppresses both IgG1 and IgE production, but much less potently than IFN-gamma. IFN-alpha suppresses anti-IgD antibody-induced increases in the level of splenic IL-4 mRNA, but enhances the anti-IgD antibody-induced increase in the splenic level of IFN-gamma mRNA. These results are consistent with the effect of IFN-alpha on Ig isotype expression in mice, as IL-4 stimulates IgE and suppresses IgG2a secretion while IFN-gamma exerts opposite effects. These observations suggest that antigen presenting cells, by secreting IFN-alpha early in the course of an immune response, can influence the nature of that response both through direct effects on B cells and by influencing the differentiation of T cells. PMID:1940796

The Novel, Nicotinic Alpha7 Receptor Partial Agonist, BMS-933043, Improves Cognition and Sensory Processing in Preclinical Models of Schizophrenia

PubMed Central

Bristow, Linda J.; Easton, Amy E.; Li, Yu-Wen; Sivarao, Digavalli V.; Lidge, Regina; Jones, Kelli M.; Post-Munson, Debra; Daly, Christopher; Lodge, Nicholas J.; Gallagher, Lizbeth; Molski, Thaddeus; Pieschl, Richard; Chen, Ping; Hendricson, Adam; Westphal, Ryan; Cook, James; Iwuagwu, Christiana; Morgan, Daniel; Benitex, Yulia; King, Dalton; Macor, John E.; Zaczek, Robert; Olson, Richard

2016-01-01

The development of alpha7 nicotinic acetylcholine receptor agonists is considered a promising approach for the treatment of cognitive symptoms in schizophrenia patients. In the present studies we characterized the novel agent, (2R)-N-(6-(1H-imidazol-1-yl)-4-pyrimidinyl)-4'H-spiro[4-azabicyclo[2.2.2]octane-2,5'-[1,3]oxazol]-2'-amine (BMS-933043), in vitro and in rodent models of schizophrenia-like deficits in cognition and sensory processing. BMS-933043 showed potent binding affinity to native rat (Ki = 3.3 nM) and recombinant human alpha7 nicotinic acetylcholine receptors (Ki = 8.1 nM) and agonist activity in a calcium fluorescence assay (EC50 = 23.4 nM) and whole cell voltage clamp electrophysiology (EC50 = 0.14 micromolar (rat) and 0.29 micromolar (human)). BMS-933043 exhibited a partial agonist profile relative to acetylcholine; the relative efficacy for net charge crossing the cell membrane was 67% and 78% at rat and human alpha7 nicotinic acetylcholine receptors respectively. BMS-933043 showed no agonist or antagonist activity at other nicotinic acetylcholine receptor subtypes and was at least 300 fold weaker at binding to and antagonizing human 5-HT3A receptors (Ki = 2,451 nM; IC50 = 8,066 nM). BMS-933043 treatment i) improved 24 hour novel object recognition memory in mice (0.1–10 mg/kg, sc), ii) reversed MK-801-induced deficits in Y maze performance in mice (1–10 mg/kg, sc) and set shift performance in rats (1–10 mg/kg, po) and iii) reduced the number of trials required to complete the extradimensional shift discrimination in neonatal PCP treated rats performing the intra-dimensional/extradimensional set shifting task (0.1–3 mg/kg, po). BMS-933043 also improved auditory gating (0.56–3 mg/kg, sc) and mismatch negativity (0.03–3 mg/kg, sc) in rats treated with S(+)ketamine or neonatal phencyclidine respectively. Given this favorable preclinical profile BMS-933043 was selected for further development to support clinical evaluation in humans. PMID:27467081

The pharmacology and toxicology of three new biologic agents used in pulmonary medicine.

PubMed

Albertson, T E; Walby, W F; Allen, R P; Tharratt, R S

1995-01-01

Biological agents have played an important role in the evolution of modern medical therapeutics. Recent advances in biologicals have in part been stimulated by the biotechnology revolution seen over the last several years. Toxicologists need to be aware of the proposed mechanisms and approved and experimental uses of these new biologic agents. Further, controversies about their use, efficacy, cost issues and potential toxicities should be known. Often these drugs are designed for small patient populations thus limiting the availability of human toxicological data bases. This paper reviews the pharmacology and toxicology of three new biologics (recombinant human DNase I, alpha 1-protease inhibitor, and nitric oxide). These agents appear to have important roles in treating specific diseases or disease states seen in pulmonary medicine.

Flowing afterglow studies of the electron recombination of protonated cyanides (RCN)H+ and their proton-bound dimer ions (RCN)2H+ where R is H, CH3, and CH3CH2

NASA Astrophysics Data System (ADS)

McLain, J. L.; Molek, C. D.; , D. Osborne, Jr.; Adams, N. G.

2009-05-01

A study has been made of the electron-ion dissociative recombination of the protonated cyanides (RCNH+, R = H, CH3, C2H5) and their proton-bound dimers (RCN)2H+ at 300 K. This has been accomplished with the flowing afterglow technique using an electrostatic Langmuir probe to determine the electron density decay along the flow tube. For the protonated species, the recombination coefficients, [alpha]e(cm3 s-1), are (3.6 +/- 0.5) × 10-7, (3.4 +/- 0.5) × 10-7, (4.6 +/- 0.7) × 10-7 for R = H, CH3, C2H5, respectively. For the proton-bound dimers, the [alpha]e are substantially greater being (2.4 +/- 0.4) × 10-6, (2.8 +/- 0.4) × 10-6, (2.3 +/- 0.3) × 10-6 for R = H, CH3, C2H5, respectively. Fitting of the electron density decay data to a simple model has shown that the rate coefficients for the three-body association of RCNH+ with RCN are very large being (2.0 +/- 0.5) × 10-26 cm6 s-1. The significance of these data to the Titan ionosphere is discussed.

Mucosal C-terminal maltase-glucoamylase hydrolyzes large size starch digestion products that may contribute to rapid postprandial glucose generation

USDA-ARS?s Scientific Manuscript database

The four mucosal alpha-glucosidases, which differ in their digestive roles, generate glucose from glycemic carbohydrates and accordingly can be viewed as a control point for rate of glucose delivery to the body. In this study, individual recombinant enzymes were used to understand how alpha-glucan o...

Actions of alpha2 adrenoceptor ligands at alpha2A and 5-HT1A receptors: the antagonist, atipamezole, and the agonist, dexmedetomidine, are highly selective for alpha2A adrenoceptors.

PubMed

Newman-Tancredi, A; Nicolas, J P; Audinot, V; Gavaudan, S; Verrièle, L; Touzard, M; Chaput, C; Richard, N; Millan, M J

1998-08-01

This study examined the activity of chemically diverse alpha2 adrenoceptor ligands at recombinant human (h) and native rat (r) alpha2A adrenoceptors compared with 5-HT1A receptors. First, in competition binding experiments at h alpha2A and h5-HT1A receptors expressed in CHO cells, several compounds, including the antagonists 1-(2-pyrimidinyl)piperazine (1-PP), (+/-)-idazoxan, benalfocin (SKF 86466), yohimbine and RX 821,002, displayed preference for h alpha2A versus h5-HT1A receptors of only 1.4-, 3.6-, 4-, 10- and 11-fold, respectively (based on differences in pKi values). Clonidine, brimonidine (UK 14304), the benzopyrrolidine fluparoxan and the guanidines guanfacine and guanabenz exhibited intermediate selectivity (22- to 31-fold) for h alpha2A receptors. Only the antagonist atipamezole and the agonist dexmedetomidine (DMT) displayed high preference for alpha2 adrenoceptors (1290- and 91-fold, respectively). Second, the compounds were tested for their ability to induce h5-HT1A receptor-mediated G-protein activation, as indicated by the stimulation of [35S]GTPgammaS binding. All except atipamezole and RX 821,002 exhibited agonist activity, with potencies which correlated with their affinity for h5-HT1A receptors. Relative efficacies (Emax values) were 25-35% for guanabenz, guanfacine, WB 4101 and benalfocin, 50-65% for 1-PP, (+/-)-idazoxan and clonidine, and over 70% for fluparoxan, oxymetazoline and yohimbine (relative to 5-HT = 100%). Yohimbine-induced [35S]GTPgammaS binding was inhibited by the selective 5-HT1A receptor antagonist WAY 100,635. In contrast, RX 821,002 was the only ligand which exhibited antagonist activity at h5-HT1A receptors, inhibiting 5-HT-stimulated [35S]GTPgammaS binding. Atipamezole, which exhibited negligeable affinity for 5-HT1A receptors, was inactive. Third, the affinities for r alpha2A differed considerably from the affinities for h alpha2A receptors whereas the affinities for r5-HT1A differed much less from the affinities for h5-HT1A receptors. This affected markedly the affinity ratios of certain compounds. For example, (+/-)-idazoxan was only 3.6-fold selective for h alpha2A versus h5-HT1A but 51-fold selective for r alpha2A versus r5-HT1A receptors. Conversely, yohimbine was tenfold selective for h alpha2A versus h5-HT1A adrenoceptors but 4.2-fold selective for r alpha2A versus r5-HT1A receptors. Nevertheless, both atipamezole and DMT were highly selective for both rat and human alpha2A versus rat or human 5-HT1A receptors. In conclusion, these data indicate that: (1) the agonist DMT and the antagonist atipamezole are the ligands of choice to distinguish alpha2-mediated from 5-HT1A-mediated actions, whilst several of the other compounds show only low or modest selectivity for alpha2A over 5-HT1A receptors; (2) caution should be exercised in experimental and clinical interpretation of the actions of traditionally employed alpha2 ligands, such as clonidine, yohimbine and (+/-)-idazoxan, which exhibit marked agonist activity at 5-HT1A receptors.

Effect of novel negative allosteric modulators of neuronal nicotinic receptors on cells expressing native and recombinant nicotinic receptors: implications for drug discovery.

PubMed

González-Cestari, Tatiana F; Henderson, Brandon J; Pavlovicz, Ryan E; McKay, Susan B; El-Hajj, Raed A; Pulipaka, Aravinda B; Orac, Crina M; Reed, Damon D; Boyd, R Thomas; Zhu, Michael X; Li, Chenglong; Bergmeier, Stephen C; McKay, Dennis B

2009-02-01

Allosteric modulation of nAChRs is considered to be one of the most promising approaches for drug design targeting nicotinic acetylcholine receptors (nAChRs). We have reported previously on the pharmacological activity of several compounds that seem to act noncompetitively to inhibit the activation of alpha3beta4(*) nAChRs. In this study, the effects of 51 structurally similar molecules on native and recombinant alpha3beta4 nAChRs are characterized. These 51 molecules inhibited adrenal neurosecretion activated via stimulation of native alpha3beta4(*) nAChR, with IC(50) values ranging from 0.4 to 13.0 microM. Using cells expressing recombinant alpha3beta4 nAChRs, these molecules inhibited calcium accumulation (a more direct assay to establish nAChR activity), with IC(50) values ranging from 0.7 to 38.2 microM. Radiolabeled nAChR binding studies to orthosteric sites showed no inhibitory activity on either native or recombinant nAChRs. Correlation analyses of the data from both functional assays suggested additional, non-nAChR activity of the molecules. To test this hypothesis, the effects of the drugs on neurosecretion stimulated through non-nAChR mechanisms were investigated; inhibitory effects ranged from no inhibition to 95% inhibition at concentrations of 10 microM. Correlation analyses of the functional data confirmed this hypothesis. Several of the molecules (24/51) increased agonist binding to native nAChRs, supporting allosteric interactions with nAChRs. Computational modeling and blind docking identified a binding site for our negative allosteric modulators near the orthosteric binding site of the receptor. In summary, this study identified several molecules for potential development as negative allosteric modulators and documented the importance of multiple screening assays for nAChR drug discovery.

Profile of follitropin alpha/lutropin alpha combination for the stimulation of follicular development in women with severe luteinizing hormone and follicle-stimulating hormone deficiency

PubMed Central

Rinaldi, Leonardo; Selman, Helmy

2016-01-01

A severe gonadotropin deficiency together with chronic estradiol deficiency leading to amenorrhea characterizes patients suffering from hypogonadotropic hypogonadism. Administration of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to these patients has been shown to be essential in achieving successful stimulation of follicular development, ovulation, and rescue of fertility. In recent years, the availability of both recombinant FSH (rFSH) and recombinant LH (rLH) has provided a new therapeutic option for the stimulation of follicular growth in hypopituitary–hypogonadotropic women (World Health Organization Group I). In this article, we review the data reported in the literature to highlight the role and the efficacy of using recombinant gonadotropins, rFSH and rLH, in the treatment of women with severe LH/FSH deficiency. Although the studies on this issue are limited and the experiences available in the literature are few due to the small number of such patients, it is clearly evident that the recombinant gonadotropins rFSH and rLH are efficient in treating patients affected by hypogonadotropic hypogonadism. The results observed in the studies reported in this review suggest that recombinant gonadotropins are able to induce proper follicular growth, oocyte maturation, and eventually pregnancy in this group of women. Moreover, the clinical use of recombinant gonadotropins in this type of patients has given more insight into some endocrinological aspects of ovarian function that have not yet been fully understood. PMID:27307766

Expression of feline interferon-alpha subtypes in Esherichia coli, and their antiviral activity and animal species specificity.

PubMed

Taira, Osamu; Suzuki, Makoto; Takeuchi, Yuko; Aramaki, Yoshitaka; Sakurai, Itsuki; Watanabe, Takao; Motokawa, Kenji; Arai, Setsuo; Sato, Hisaaki; Maehara, Nobutoshi

2005-05-01

Two kinds of FeIFN-alpha consisting of 166 amino acids (aa) and 171 aa were expressed in Escherichia coli, and the purified proteins were tested for antiviral activity on homologous and heterologous animal cells. Crude FeIFN induced in feline cells revealed antiviral activity on both homologous and heterologous animal cells. In contrast, both types of recombinant FeIFN-alpha revealed antiviral activity only on the feline cells. All of the FeIFN-alpha subtypes showed high activity to vesicular stomatitis virus, and the three species of feline viruses belonging to different families.

Bayesian inference of shared recombination hotspots between humans and chimpanzees.

PubMed

Wang, Ying; Rannala, Bruce

2014-12-01

Recombination generates variation and facilitates evolution. Recombination (or lack thereof) also contributes to human genetic disease. Methods for mapping genes influencing complex genetic diseases via association rely on linkage disequilibrium (LD) in human populations, which is influenced by rates of recombination across the genome. Comparative population genomic analyses of recombination using related primate species can identify factors influencing rates of recombination in humans. Such studies can indicate how variable hotspots for recombination may be both among individuals (or populations) and over evolutionary timescales. Previous studies have suggested that locations of recombination hotspots are not conserved between humans and chimpanzees. We made use of the data sets from recent resequencing projects and applied a Bayesian method for identifying hotspots and estimating recombination rates. We also reanalyzed SNP data sets for regions with known hotspots in humans using samples from the human and chimpanzee. The Bayes factors (BF) of shared recombination hotspots between human and chimpanzee across regions were obtained. Based on the analysis of the aligned regions of human chromosome 21, locations where the two species show evidence of shared recombination hotspots (with high BFs) were identified. Interestingly, previous comparative studies of human and chimpanzee that focused on the known human recombination hotspots within the β-globin and HLA regions did not find overlapping of hotspots. Our results show high BFs of shared hotspots at locations within both regions, and the estimated locations of shared hotspots overlap with the locations of human recombination hotspots obtained from sperm-typing studies. Copyright © 2014 by the Genetics Society of America.

Unsaturated free fatty acids increase benzodiazepine receptor agonist binding depending on the subunit composition of the GABAA receptor complex.

PubMed

Witt, M R; Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nielsen, M

1996-11-01

It has been shown previously that unsaturated free fatty acids (FFAs) strongly enhance the binding of agonist benzodiazepine receptor ligands and GABAA receptor ligands in the CNS in vitro. To investigate the selectivity of this effect, recombinant human GABAA/benzodiazepine receptor complexes formed by different subunit compositions (alpha x beta y gamma 2, x = 1, 2, 3, and 5; y = 1, 2, and 3) were expressed using the baculovirus-transfected Sf9 insect cell system. At 10(-4) M, unsaturated FFAs, particularly arachidonic (20:4) and docosahexaenoic (22:6) acids, strongly stimulated (> 200% of control values) the binding of [3H]flunitrazepam ([3H]FNM) to the alpha 3 beta 2 gamma 2 receptor combination in whole cell preparations. No effect or small increases in levels of unsaturated FFAs on [3H]FNM binding to alpha 1 beta x gamma 2 and alpha 2 beta x gamma 2 receptor combinations were observed, and weak effects (130% of control values) were detected using the alpha 5 beta 2 gamma 2 receptor combination. The saturated FFAs, stearic and palmitic acids, were without effect on [3H]FNM binding to any combination of receptor complexes. The hydroxylated unsaturated FFAs, ricinoleic and ricinelaidic acids, were shown to decrease the binding of [3H]FNM only if an alpha 1 beta 2 gamma 2 receptor combination was used. Given the heterogeneity of the GABAA/ benzodiazepine receptor subunit distribution in the CNS, the effects of FFAs on the benzodiazepine receptor can be assumed to vary at both cellular and regional levels.

The Pneumococcal Alpha-Glycerophosphate Oxidase Enhances Nasopharyngeal Colonization through Binding to Host Glycoconjugates.

PubMed

Mahdi, Layla K; Higgins, Melanie A; Day, Christopher J; Tiralongo, Joe; Hartley-Tassell, Lauren E; Jennings, Michael P; Gordon, David L; Paton, Adrienne W; Paton, James C; Ogunniyi, Abiodun D

2017-04-01

Streptococcus pneumoniae (the pneumococcus) is a major human pathogen, causing a broad spectrum of diseases including otitis media, pneumonia, bacteraemia and meningitis. Here we examined the role of a potential pneumococcal meningitis vaccine antigen, alpha-glycerophosphate oxidase (SpGlpO), in nasopharyngeal colonization. We found that serotype 4 and serotype 6A strains deficient in SpGlpO have significantly reduced capacity to colonize the nasopharynx of mice, and were significantly defective in adherence to human nasopharyngeal carcinoma cells in vitro. We also demonstrate that intranasal immunization with recombinant SpGlpO significantly protects mice against subsequent nasal colonization by wild type serotype 4 and serotype 6A strains. Furthermore, we show that SpGlpO binds strongly to lacto/neolacto/ganglio host glycan structures containing the GlcNAcβ1-3Galβ disaccharide, suggesting that SpGlpO enhances colonization of the nasopharynx through its binding to host glycoconjugates. We propose that SpGlpO is a promising vaccine candidate against pneumococcal carriage, and warrants inclusion in a multi-component protein vaccine formulation that can provide robust, serotype-independent protection against all forms of pneumococcal disease. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Aldose reductase inhibitors from the leaves of Myrciaria dubia (H. B. & K.) McVaugh.

PubMed

Ueda, H; Kuroiwa, E; Tachibana, Y; Kawanishi, K; Ayala, F; Moriyasu, M

2004-11-01

Ellagic acid (1) and its two derivatives, 4-O-methylellagic acid (2) and 4-(alpha-rhamnopyranosyl)ellagic acid (3) were isolated as inhibitors of aldose reductase (AR) from Myrciaria dubia (H. B. & K.) McVaugh. Compound 2 was the first isolated from the nature. Compound 3 showed the strongest inhibition against human recombinant AR (HRAR) and rat lens AR (RLAR). Inhibitory activity of compound 3 against HRAR (IC50 value = 4.1 x 10(-8) M) was 60 times more than that of quercetin (2.5 x 10(-6) M). The type of inhibition against HRAR was uncompetitive.

Can microbes compete with cows for sustainable protein production - A feasibility study on high quality protein

NASA Astrophysics Data System (ADS)

Vestergaard, Mike; Chan, Siu Hung Joshua; Jensen, Peter Ruhdal

2016-11-01

An increasing population and their increased demand for high-protein diets will require dramatic changes in the food industry, as limited resources and environmental issues will make animal derived foods and proteins, gradually more unsustainable to produce. To explore alternatives to animal derived proteins, an economic model was built around the genome-scale metabolic network of E. coli to study the feasibility of recombinant protein production as a food source. Using a novel model, we predicted which microbial production strategies are optimal for economic return, by capturing the tradeoff between the market prices of substrates, product output and the efficiency of microbial production. A case study with the food protein, Bovine Alpha Lactalbumin was made to evaluate the upstream economic feasibilities. Simulations with different substrate profiles at maximum productivity were used to explore the feasibility of recombinant Bovine Alpha Lactalbumin production coupled with market prices of utilized materials. We found that recombinant protein production could be a feasible food source and an alternative to traditional sources.

Assessment of a recombinant androgen receptor binding assay: initial steps towards validation.

PubMed

Freyberger, Alexius; Weimer, Marc; Tran, Hoai-Son; Ahr, Hans-Jürgen

2010-08-01

Despite more than a decade of research in the field of endocrine active compounds with affinity for the androgen receptor (AR), still no validated recombinant AR binding assay is available, although recombinant AR can be obtained from several sources. With funding from the European Union (EU)-sponsored 6th framework project, ReProTect, we developed a model protocol for such an assay based on a simple AR binding assay recently developed at our institution. Important features of the protocol were the use of a rat recombinant fusion protein to thioredoxin containing both the hinge region and ligand binding domain (LBD) of the rat AR (which is identical to the human AR-LBD) and performance in a 96-well plate format. Besides two reference compounds [dihydrotestosterone (DHT), androstenedione] ten test compounds with different affinities for the AR [levonorgestrel, progesterone, prochloraz, 17alpha-methyltestosterone, flutamide, norethynodrel, o,p'-DDT, dibutylphthalate, vinclozolin, linuron] were used to explore the performance of the assay. At least three independent experiments per compound were performed. The AR binding properties of reference and test compounds were well detected, in terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using recombinant AR preparations. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.6. Our data demonstrate that the assay reliably ranked compounds with strong, weak, and no/marginal affinity for the AR with high accuracy. It avoids the manipulation and use of animals, as a recombinant protein is used and thus contributes to the 3R concept. On the whole, this assay is a promising candidate for further validation. Copyright 2009 Elsevier Inc. All rights reserved.

Effect of Recombinant Human Growth Hormone and Rosiglitazone for HIV-Associated Abdominal Fat Accumulation on Adiponectin and other Markers of Inflammation

PubMed Central

Leung, Vivien; Chiu, Ya-Lin; Kotler, Donald P.; Albu, Jeanine; Zhu, Yuan-Shan; Ham, Kirsis; Engelson, Ellen S.; Hammad, Hoda; Christos, Paul; Donovan, Daniel S.; Ginsberg, Henry N.; Glesby, Marshall J.

2016-01-01

Background/Objective In a previous report of HIV-infected patients with fat redistribution, we found that recombinant human growth hormone (rhGH) therapy reduced visceral adipose tissue (VAT) but increased insulin resistance, and that the addition of rosiglitazone reversed the negative effects of rhGH on insulin sensitivity. In this study, we sought to determine the effects of recombinant human growth hormone (rhGH) and rosiglitazone therapy on an array of inflammatory and fibrinolytic markers. Methods 72 patients with HIV-associated abdominal obesity and insulin resistance were randomized to treatment with rhGH, rosiglitazone, the combination of rhGH and rosiglitazone, or placebo for 12 weeks. Subjects with plasma and serum samples available at weeks 0 (n = 63) and 12 (n = 46-48) were assessed for adiponectin, C-reactive protein (CRP), homocysteine, interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), fibrinogen, plasminogen activator inhibitor-1 (PAI-1) antigen, and tissue plasminogen activator (tPA) antigen. Results Treatment with both rosiglitazone alone and the combination of rosiglitazone and rhGH for 12 weeks resulted in significant increases in adiponectin levels from baseline. Adiponectin levels did not change significantly in the rhGH alone arm. There were no significant changes in the other biomarkers amongst the different treatment groups. Discussion In this study of HIV-infected patients with altered fat distribution, treatment with rosiglitazone had beneficial effects on adiponectin concentrations, an effect that was also seen with combination rosiglitazone and rhGH. RhGH administration alone, however, did not demonstrate any significant impact on adiponectin levels despite reductions in VAT. PMID:27077672

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

Parvinen, M.; Soeder, O.M.; Mali, P.

Levels of rat testicular interleukin-1-like factor (tIL-1) have been shown to correlate with DNA synthetic activity during the cycle of the rat seminiferous epithelium, suggesting its role as a spermatogonial or meiotic growth factor. To explore this further, a new in vitro model system was developed. Rat seminiferous tubule segments from stages I, V, VIIa, and VIII-IX of the cycle were isolated by transillumination-assisted microdissection, cultured in chemically defined serum-free medium supplemented with human recombinant IL-1 {alpha}, and labeled with (3H)thymidine. During incubation, spontaneous progression of spermatogenesis was noted. Inactive stage VIIa tubule segments differentiated to stage VIII and initiatedmore » DNA synthesis, and concomitantly started to secrete IL-1-like factor. DNA synthesis of stages VIII-IX ceased through differentiation of spermatocytes to leptotene-zygotene (stages XII-XIII of the cycle). IL-1 {alpha} stimulated DNA synthesis significantly in spermatogonia of stage I. Meiotic DNA synthesis at stage VIIa was stimulated (48 h/34 C) and maintained at stages VIII-IX (48 h/34 C). IL-1 {alpha} seems to act as a regulator of spermatogenic DNA synthesis in both mitotic and meiotic phases. It has mainly stimulating and maintaining effects, but it may also be inhibitory under certain conditions.« less

A peptide affinity column for the identification of integrin alpha IIb-binding proteins.

PubMed

Daxecker, Heide; Raab, Markus; Bernard, Elise; Devocelle, Marc; Treumann, Achim; Moran, Niamh

2008-03-01

To understand the regulation of integrin alpha(IIb)beta(3), a critical platelet adhesion molecule, we have developed a peptide affinity chromatography method using the known integrin regulatory motif, LAMWKVGFFKR. Using standard Fmoc chemistry, this peptide was synthesized onto a Toyopearl AF-Amino-650 M resin on a 6-aminohexanoic acid (Ahx) linker. Peptide density was controlled by acetylation of 83% of the Ahx amino groups. Four recombinant human proteins (CIB1, PP1, ICln and RN181), previously identified as binding to this integrin regulatory motif, were specifically retained by the column containing the integrin peptide but not by a column presenting an irrelevant peptide. Hemoglobin, creatine kinase, bovine serum albumin, fibrinogen and alpha-tubulin failed to bind under the chosen conditions. Immunodetection methods confirmed the binding of endogenous platelet proteins, including CIB1, PP1, ICln RN181, AUP-1 and beta3-integrin, from a detergent-free platelet lysate. Thus, we describe a reproducible method that facilitates the reliable extraction of specific integrin-binding proteins from complex biological matrices. This methodology may enable the sensitive and specific identification of proteins that interact with linear, membrane-proximal peptide motifs such as the integrin regulatory motif LAMWKVGFFKR.

Immunogenicity of a Trivalent Recombinant Vaccine Against Clostridium perfringens Alpha, Beta, and Epsilon Toxins in Farm Ruminants

PubMed Central

Moreira, Gustavo Marçal Schmidt Garcia; Salvarani, Felipe Masiero; da Cunha, Carlos Eduardo Pouey; Mendonça, Marcelo; Moreira, Ângela Nunes; Gonçalves, Luciana Aramuni; Pires, Prhiscylla Sadanã; Lobato, Francisco Carlos Faria; Conceição, Fabricio Rochedo

2016-01-01

Clostridium perfringens is an anaerobic bacterium that produces several toxins. Of these, the alpha, beta, and epsilon toxins are responsible for causing the most severe C. perfringens-related diseases in farm animals. The best way to control these diseases is through vaccination. However, commercially available vaccines are based on inactivated toxins and have many production drawbacks, which can be overcome through the use of recombinant antigens. In this study, we produced recombinant alpha, beta, and epsilon toxins in Escherichia coli to formulate a trivalent vaccine. Its effectiveness was evaluated through a potency test in rabbits, in which the vaccine generated 9.6, 24.4, and 25.0 IU/mL of neutralizing antibodies against the respective toxins. Following this, cattle, sheep, and goats received the same formulation, generating, respectively, 5.19 ± 0.48, 4.34 ± 0.43, and 4.70 ± 0.58 IU/mL against alpha toxin, 13.71 ± 1.17 IU/mL (for all three species) against beta toxin, and 12.74 ± 1.70, 7.66 ± 1.69, and 8.91 ± 2.14 IU/mL against epsilon toxin. These levels were above the minimum recommended by international protocols. As such, our vaccine was effective in generating protective antibodies and, thus, may represent an interesting alternative for the prevention of C. perfringens-related intoxications in farm animals. PMID:27004612

Recombinant methionyl human leptin administration activates signal transducer and activator of transcription 3 signaling in peripheral blood mononuclear cells in vivo and regulates soluble tumor necrosis factor-alpha receptor levels in humans with relative leptin deficiency.

PubMed

Chan, Jean L; Moschos, Stergios J; Bullen, John; Heist, Kathleen; Li, Xian; Kim, Young-Bum; Kahn, Barbara B; Mantzoros, Christos S

2005-03-01

Studies of congenital complete leptin deficiency in animals and humans support a role for leptin in regulating immune function. Whether acquired relative leptin deficiency affects immunological parameters in healthy humans remains unknown. We thus used experimental models of relative leptin deficiency and recombinant methionyl human leptin (r-metHuLeptin) administration in humans to investigate whether r-metHuLeptin would activate signaling pathways in peripheral blood mononuclear cells (PBMCs) and whether acquired relative leptin deficiency and/or increasing circulating leptin levels into the physiologic range would change PBMC subpopulations and cytokines important in the T-helper cell and systemic immune responses. We found that r-metHuLeptin administration to healthy humans activates signal transducer and activator of transcription-3 signaling in PBMCs in vivo. Neither short-term leptin deficiency, induced by 3-d complete fasting, nor physiologic r-metHuLeptin replacement for the same period of time had a major effect on PBMC subpopulations or serum cytokines in healthy men. In contrast, normalizing serum leptin levels over 8 wk in lean women with relative leptin deficiency for 5.1 +/- 1.4 yr (mean +/- se) due to chronic energy deficit increased soluble TNFalpha receptor levels, indicating activation of the TNFalpha system. These findings suggest that relative leptin deficiency due to more long-term energy deprivation is associated with defects in immunological parameters that may be corrected with exogenous r-metHuLeptin administration. Further studies are warranted to assess the implications of acquired relative hypoleptinemia and/or r-metHuLeptin administration on the immunosuppression associated with energy- and leptin-deficient states in humans.

Conjugation of gold nanoparticles and recombinant human endostatin modulates vascular normalization via interruption of anterior gradient 2-mediated angiogenesis.

PubMed

Pan, Fan; Yang, Wende; Li, Wei; Yang, Xiao-Yan; Liu, Shuhao; Li, Xin; Zhao, Xiaoxu; Ding, Hui; Qin, Li; Pan, Yunlong

2017-07-01

Several studies have revealed the potential of normalizing tumor vessels in anti-angiogenic treatment. Recombinant human endostatin is an anti-angiogenic agent which has been applied in clinical tumor treatment. Our previous research indicated that gold nanoparticles could be a nanoparticle carrier for recombinant human endostatin delivery. The recombinant human endostatin-gold nanoparticle conjugates normalized vessels, which improved chemotherapy. However, the mechanism of recombinant human endostatin-gold nanoparticle-induced vascular normalization has not been explored. Anterior gradient 2 has been reported to be over-expressed in many malignant tumors and involved in tumor angiogenesis. To date, the precise efficacy of recombinant human endostatin-gold nanoparticles on anterior gradient 2-mediated angiogenesis or anterior gradient 2-related signaling cohort remained unknown. In this study, we aimed to explore whether recombinant human endostatin-gold nanoparticles could normalize vessels in metastatic colorectal cancer xenografts, and we further elucidated whether recombinant human endostatin-gold nanoparticles could interrupt anterior gradient 2-induced angiogenesis. In vivo, it was indicated that recombinant human endostatin-gold nanoparticles increased pericyte expression while inhibit vascular endothelial growth factor receptor 2 and anterior gradient 2 expression in metastatic colorectal cancer xenografts. In vitro, we uncovered that recombinant human endostatin-gold nanoparticles reduced cell migration and tube formation induced by anterior gradient 2 in human umbilical vein endothelial cells. Treatment with recombinant human endostatin-gold nanoparticles attenuated anterior gradient 2-mediated activation of MMP2, cMyc, VE-cadherin, phosphorylation of p38, and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in human umbilical vein endothelial cells. Our findings demonstrated recombinant human endostatin-gold nanoparticles might normalize vessels by interfering anterior gradient 2-mediated angiogenesis in metastatic colorectal cancer.

A bipolar outflow of ionized gas in K3-50A: H76 alpha radio recombination line and continuum observations of K3-50

NASA Technical Reports Server (NTRS)

Depree, C. G.; Goss, W. M.; Palmer, Patrick; Rubin, Robert H.

1994-01-01

The H II regions near K3-50 (G70.3 + 1.6) have been imaged at high angular resolution (approximately 1 sec .3) in the continuum and the recombination lines H76(sub alpha and He76(sub alpha) using the Very Large Array (VLA). The helium line is detected in only the brightest component K3-50A while the hydrogen line is detected in three components (K3-50A, B and C1). K3-50A shows a pronounced velocity gradient of approximately 150 km/sec/pc along its major axis (P.A. = 160 deg); in addition a wide range of line widths are observed, from 20 to 65 km/sec. Kinematics from the line data and the morphology of the continuum emission suggest that the ionized material associated with K3-50A is undergoing a high-velocity bipolar outflow.

Design and synthesis of novel sulfonamide-containing bradykinin hB2 receptor antagonists. 1. Synthesis and SAR of alpha,alpha-dimethylglycine sulfonamides.

PubMed

Fattori, Daniela; Rossi, Cristina; Fincham, Christopher I; Berettoni, Marco; Calvani, Federico; Catrambone, Fernando; Felicetti, Patrizia; Gensini, Martina; Terracciano, Rosa; Altamura, Maria; Bressan, Alessandro; Giuliani, Sandro; Maggi, Carlo A; Meini, Stefania; Valenti, Claudio; Quartara, Laura

2006-06-15

We recently published the extensive in vivo pharmacological characterization of MEN 16132 (J. Pharmacol. Exp. Ther. 2005, 616-623; Eur. J. Pharmacol. 2005, 528, 7), a member of the sulfonamide-containing human B(2) receptor (hB(2)R) antagonists. Here we report, in detail, how this family of compounds was designed, synthesized, and optimized to provide a group of products with subnanomolar affinity for the hB(2)R and high in vivo potency after topical administration to the respiratory tract. The series was designed on the basis of indications from the X-ray structures of the key structural motifs A and B present in known antagonists and is characterized by the presence of an alpha,alpha-dialkyl amino acid. The first lead (17) of the series was submitted to extensive chemical work to elucidate the structural requirements to increase hB(2) receptor affinity and antagonist potency in bioassays expressing the human B(2) receptor (hB(2)R). The following structural features were selected: a 2,4-dimethylquinoline moiety and a piperazine linker acylated with a basic amino acid. The representative lead compound 68 inhibited the specific binding of [(3)H]BK to hB(2)R with a pKi of 9.4 and antagonized the BK-induced inositolphosphate (IP) accumulation in recombinant cell systems expressing the hB(2)R with a pA(2) of 9.1. Moreover, compound 68 when administered (300 nmol/kg) intratracheally in the anesthetized guinea pig, was able to significantly inhibit BK-induced bronchoconstriction for up to 120 min after its administration, while having a lower and shorter lasting effect on hypotension.

Functional characterization of recombinant prefoldin complexes from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1.

PubMed

Iizuka, Ryo; Sugano, Yuri; Ide, Naoki; Ohtaki, Akashi; Yoshida, Takao; Fujiwara, Shinsuke; Imanaka, Tadayuki; Yohda, Masafumi

2008-03-28

Prefoldin is a heterohexameric molecular chaperone complex that is found in the eukaryotic cytosol and also in archaea. It captures a nonnative protein and subsequently delivers it to a group II chaperonin for proper folding. Archaeal prefoldin is a heterocomplex containing two alpha subunits and four beta subunits with the structure of a double beta-barrel assembly, with six long coiled coils protruding from it like a jellyfish with six tentacles. We have studied the protein folding mechanism of group II chaperonin using those of Thermococcus sp. strain KS-1 (T. KS-1) because they exhibit high protein folding activity in vitro. We have also demonstrated functional cooperation between T. KS-1 chaperonins and prefoldin from Pyrococcus horikoshii OT3. Recent genome analysis has shown that Thermococcus kodakaraensis KOD1 contains two pairs of prefoldin subunit genes, correlating with the existence of two different chaperonin subunits. In this study, we characterized four different recombinant prefoldin complexes composed of two pairs of prefoldin subunits (alpha1, alpha2, beta1, and beta2) from T. KS-1. All of them (alpha1-beta1, alpha2-beta1, alpha1-beta2, and alpha2-beta2) exist as alpha(2)beta(4) heterohexamers and can protect several proteins from forming aggregates with different activities. We have also compared the collaborative activity between the prefoldin complexes and the cognate chaperonins. Prefoldin complexes containing the beta1 subunit interacted with the chaperonins more strongly than those with the beta2 subunit. The results suggest that Thermococcus spp. express different prefoldins for different substrates or conditions as chaperonins.

Structural characterization by NMR of the natively unfolded extracellular domain of beta-dystroglycan: toward the identification of the binding epitope for alpha-dystroglycan.

PubMed

Bozzi, Manuela; Bianchi, Marzia; Sciandra, Francesca; Paci, Maurizio; Giardina, Bruno; Brancaccio, Andrea; Cicero, Daniel O

2003-11-25

Dystroglycan (DG) is an adhesion molecule playing a crucial role for tissue stability during both early embriogenesis and adulthood and is composed by two tightly interacting subunits: alpha-DG, membrane-associated and highly glycosylated, and the transmembrane beta-DG. Recently, by solid-phase binding assays and NMR experiments, we have shown that the C-terminal domain of alpha-DG interacts with a recombinant extracellular fragment of beta-DG (positions 654-750) independently from glycosylation and that the linear binding epitope is located between residues 550 and 565 of alpha-DG. In order to elucidate which moieties of beta-DG are specifically involved in the complex with alpha-DG, the ectodomain has been recombinantly expressed and purified in a labeled ((13)C,(15)N) form and studied by multidimensional NMR. Although it represents a natively unfolded protein domain, we obtained an almost complete backbone assignment. Chemical shift index, (1)H-(15)N heteronuclear single-quantum coherence and nuclear Overhauser effect (HSQC-NOESY) spectra and (3)J(HN,H)(alpha) coupling constant values confirm that this protein is highly disordered, but (1)H-(15)N steady-state NOE experiments indicate that the protein presents two regions of different mobility. The first one, between residues 659 and 722, is characterized by a limited degree of mobility, whereas the C-terminal portion, containing about 30 amino acids, is highly flexible. The binding of beta-DG(654-750) to the C-terminal region of the alpha subunit, alpha-DG(485-620), has been investigated, showing that the region of beta-DG(654-750) between residues 691 and 719 is involved in the interaction.

Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme.

PubMed Central

Dong, G; Vieille, C; Zeikus, J G

1997-01-01

The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide. The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the alpha-amylase family. In particular, none of the consensus regions present in the alpha-amylase family could be identified. P. furiosus APU showed similarity to three proteins, including the P. furiosus intracellular alpha-amylase and Dictyoglomus thermophilum alpha-amylase A. The mature protein had a molecular weight of 89,000. The recombinant P. furiosus APU remained folded after denaturation at temperatures of < or = 70 degrees C and showed an apparent molecular weight of 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Denaturating temperatures of above 100 degrees C were required for complete unfolding. The enzyme was extremely thermostable, with an optimal activity at 105 degrees C and pH 5.5. Ca2+ increased the enzyme activity, thermostability, and substrate affinity. The enzyme was highly resistant to chemical denaturing reagents, and its activity increased up to twofold in the presence of surfactants. PMID:9293009

A Proposed Mechanism for the Thermal Denaturation of a Recombinant Bacillus Halmapalus Alpha-amylase - the Effect of Calcium Ions

NASA Technical Reports Server (NTRS)

Nielsen, Anders D.; Pusey, Marc L.; Fuglsang, Claus C.; Westh, Peter

2003-01-01

The thermal stability of a recombinant alpha-amylase from Bacillus halmapalus alpha-amylase (BHA) has been investigated using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). This alpha-amylase is homologous to other Bacillus alpha-amylases where previous crystallographic studies have identified the existence of 3 calcium binding sites in the structure. Denaturation of BHA is irreversible with a Tm of approximately 89 C, and DSC thermograms can be described using a one-step irreversible model. A 5 C increase in T(sub m) in the presence of 10 fold excess CaCl2 was observed. However, a concomitant increase in the tendency to aggregate was also observed. The presence of 30-40 fold excess calcium chelator (EDTA or EGTA) results in a large destabilization of BHA corresponding to about 40 C lower T(sub m), as determined by both CD and DSC. Ten fold excess EGTA reveals complex DSC thermograms corresponding to both reversible and irreversible transitions, which possibly originate from different populations of BHA:calcium complexes. The observations in the present study have, in combination with structural information of homologous alpha-amylases, provided the basis for the proposal of a simple denaturation mechanism of BHA. The proposed mechanism describes the irreversible thermal denaturation of different BHA:calcium complexes and the calcium binding equilibrium involved. Furthermore, the model accounts for a temperature induced reversible structural change associated with calcium binding.

Phospholipase C produced by Clostridium botulinum types C and D: comparison of gene, enzymatic, and biological activities with those of Clostridium perfringens alpha-toxin.

PubMed

Fatmawati, Ni Nengah Dwi; Sakaguchi, Yoshihiko; Suzuki, Tomonori; Oda, Masataka; Shimizu, Kenta; Yamamoto, Yumiko; Sakurai, Jun; Matsushita, Osamu; Oguma, Keiji

2013-01-01

Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival.

Influence of recombinant interferon alpha on nutritional status and growth pattern in children with chronic viral hepatitis.

PubMed

Gottrand, F; Michaud, L; Guimber, D; Ategbo, S; Dubar, G; Turck, D; Farriaux, J P

1996-12-01

Anorexia and weight loss are frequently reported as adverse effects during recombinant interferon alpha (rIFN-alpha) treatment. The aim of the present study was to assess both nutritional status and growth of children and adolescents treated with rIFN-alpha for chronic viral hepatitis. Eleven patients aged 4-16 years with histologically proven chronic active hepatitis (hepatitis B, n = 9; hepatitis C, n = 2) receiving rIFN-alpha subcutaneously thrice a week for 6 months were studied. Weight and height increments were assessed during the 6 months before starting rIFN-alpha. Weight and height were measured every 3 months (M0, M3, M6) during the 6 months of rIFN-alpha treatment, then every 6 months during the follow up period (6-36 months). Weight decreased in every child during rIFN-alpha treatment (weight loss varies from 0.5 to 2.6 kg after 3 months of treatment). Weight/age Z-score decreased from 0.12 at M0 to -0.69 at M3 (P < 0.01), then increased between M3 and M6 (-0.33) (P < 0.01), but normalized (0.02) only 6 months after completion of treatment. Nutritional status was significantly impaired during treatment (Z-score for weight/height decreased from 0.18 at M0 to -0.74 at M3, P < 0.01) and recovered progressively thereafter. Height and height velocity were not modified by rIFN-alpha treatment. A reduction of the caloric intake observed between M0 and M3 might explain these features. Significant but transient abnormalities of the nutritional status are encountered constantly at the beginning of rIFN-alpha therapy without any deleterious effect on growth. Information of the families and nutritional intervention during treatment should be required, in order to limit the importance of weight loss.

Effects of various vitamins and coenzymes Q on reactions involving alpha-hydroxyl-containing radicals.

PubMed

Shadyro, Oleg I; Sosnovskaya, Anna A; Edimecheva, Irina P; Grintsevich, Ivan B; Lagutin, Petr Yu; Alekseev, Aleksei V; Kazem, Kamel

2005-07-01

Effects of vitamins B, C, E, K and P, as well as coenzymes Q, on formation of final products of radiation-induced free-radical transformations of ethanol, ethylene glycol, alpha-methylglycoside and glucose in aqueous solutions were studied. Based on the obtained results, it can be concluded that there are substances among vitamins and coenzymes that effectively interact with alpha-hydroxyl-containing radicals. In the presence of these substances, recombination reactions of alpha-hydroxyalkyl radicals and fragmentation of alpha-hydroxy-beta-substituted organic radicals are suppressed. It has been established that the observed effects are due to the ability of the vitamins and coenzymes under study to either oxidize alpha-hydroxyl-containing radicals yielding the respective carbonyl compounds or reduce them into the initial molecules.

Group I but not group II NPV induces antiviral effects in mammalian cells.

PubMed

Liang, Changyong; Song, Jianhua; Hu, Zhihong; Chen, Xinwen

2006-10-01

Nucleopolyhedrovirus (NPV) is divided into Group I and Group II based on the phylogenetic analysis. It has been reported that Group I NPVs such as Autographa californica multiple NPV (AcMNPV) can transduce mammalian cells, while Group II NPVs such as Helicoverpa armigera single NPV (HaSNPV) cannot. Here we report that AcMNPV was capable of stimulating antiviral activity in human hepatoma cells (SMMC-7721) manifested by inhibition of Vesicular Stomatitis virus (VSV) replication. In contrast, the HaSNPV and the Spodoptera exigua multiple NPV (SeMNPV) of group II had no inhibitory effect on VSV. Recombinant AcMNPV was shown to induce interferons alpha/beta even in the absence of transgene expression in human SMMC-7721 cells, while it mediated transgene expression in BHK and L929 mammalian cells without an ensuing antiviral activity.

An Aspergillus oryzae acetyl xylan esterase: molecular cloning and characteristics of recombinant enzyme expressed in Pichia pastoris.

PubMed

Koseki, Takuya; Miwa, Yozo; Akao, Takeshi; Akita, Osamu; Hashizume, Katsumi

2006-02-10

We screened 20,000 clones of an expressed sequence tag (EST) library from Aspergillus oryzae (http://www.nrib.go.jp/ken/EST/db/index.html) and obtained one cDNA clone encoding a protein with similarity to fungal acetyl xylan esterase. We also cloned the corresponding gene, designated as Aoaxe, from the genomic DNA. The deduced amino acid sequence consisted of a putative signal peptide of 31-amino acids and a mature protein of 276-amino acids. We engineered Aoaxe for heterologous expression in P. pastoris. Recombinant AoAXE (rAoAXE) was secreted by the aid of fused alpha-factor secretion signal peptide and accumulated as an active enzyme in the culture medium to a final level of 190 mg/l after 5 days. Purified rAoAXEA before and after treatment with endoglycosidase H migrated by SDS-PAGE with a molecular mass of 31 and 30 kDa, respectively. Purified rAoAXE displayed the greatest hydrolytic activity toward alpha-naphthylacetate (C2), lower activity toward alpha-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. The recombinant enzyme catalyzed the release of acetic acid from birchwood xylan. No activity was detectable using methyl esters of ferulic, caffeic or sinapic acids. rAoAXE was thermolabile in comparison to other AXEs from Aspergillus.

Recombinant Human Acidic Fibroblast Growth Factor (aFGF) Expressed in Nicotiana benthamiana Potentially Inhibits Skin Photoaging.

PubMed

Ha, Jang-Ho; Kim, Ha-Neul; Moon, Ki-Beom; Jeon, Jae-Heung; Jung, Dai-Hyun; Kim, Su-Jung; Mason, Hugh S; Shin, Seo-Yeon; Kim, Hyun-Soon; Park, Kyung-Mok

2017-07-01

Responding to the need for recombinant acidic fibroblast growth factor in the pharmaceutical and cosmetic industries, we established a scalable expression system for recombinant human aFGF using transient and a DNA replicon vector expression in Nicotiana benthamiana . Recombinant human-acidic fibroblast growth factor was recovered following Agrobacterium infiltration of N. benthamiana . The optimal time point at which to harvest recombinant human acidic fibroblast growth factor expressing leaves was found to be 4 days post-infiltration, before necrosis was evident. Commassie-stained SDS-PAGE gels of His-tag column eluates, concentrated using a 10 000 molecular weight cut-off column, showed an intense band at the expected molecular weight for recombinant human acidic fibroblast growth factor. An immunoblot confirmed that this band was recombinant human acidic fibroblast growth factor. Up to 10 µg recombinant human-acidic fibroblast growth factor/g of fresh leaves were achieved by a simple affinity purification protocol using protein extract from the leaves of agroinfiltrated N. benthamiana . The purified recombinant human acidic fibroblast growth factor improved the survival rate of UVB-irradiated HaCaT and CCD-986sk cells approximately 89 and 81 %, respectively. N. benthamiana -derived recombinant human acidic fibroblast growth factor showed similar effects on skin cell proliferation and UVB protection compared to those of Escherichia coli -derived recombinant human acidic fibroblast growth factor. Additionally, N. benthamiana- derived recombinant human acidic fibroblast growth factor increased type 1 procollagen synthesis up to 30 % as well as reduced UVB-induced intracellular reactive oxygen species generation in fibroblast (CCD-986sk) cells.UVB is a well-known factor that causes various types of skin damage and premature aging. Therefore, the present study demonstrated that N. benthamiana -derived recombinant human acidic fibroblast growth factor effectively protects skin cell from UVB, suggesting its potential use as a cosmetic or therapeutic agent against skin photoaging. Georg Thieme Verlag KG Stuttgart · New York.

[Preventive and therapeutic effect of genetic vaccine based on recombinant alpha virus against mouse mastocytoma P815].

PubMed

Ni, Bing; Yang, Ri-gao; Li, Yan-qiu; Wu, Yu-zhang

2004-01-01

To explore the immunological effect of genetic vaccine based on alpha-virus and to seek out better forms of gene vaccines. Expression plasmid P1A/pSMART2a and packaging plasmid helper were cotransfected into mammalian 293 cells by calcium phosphate precipitation method and high level of recombinant alpha-virus P1A/SFV was prepared. Following identification of rSFV and its expression, BALB/c mice were inoculated with rSFV, and the production of antigen-specific antibody and the cytotoxic effect of CTLs were determined. In the preventive and therapeutic experiments, the percents of tumor-free and of survival mice immunized with rSFV were observed. The recombinant SFV could express correctly in cultured cells. After being inoculated into the mice, rSFV could prime stronger CTL response than that in control mice. When the ratio of E/T cells was 100:1, the (51)Cr release rate reached 75%. No antibody could be detected in mice from all groups. The immunological effect of P1A/SFV among all groups was the best in both preventive and therapeutic experiment within experimental deadline. On 60th day in preventive experiment, the percent of tumor-free animal in P1A/SFV group reached 60%, whereas that was only 20% in P1A/pCI-neogroup. On 60th day in therapeutic experiment, survival rate of mice in P1A/SFV group reached 50%, but only 10% mice could survive in all control groups. Compared with common gene vaccines, the genetic vaccine based on recombinant SFV has the best immunological effect, which provides some new strategies for clinical genetic therapy of tumors.

NF546 [4,4'-(carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)-carbonylimino))-bis(1,3-xylene-alpha,alpha'-diphosphonic acid) tetrasodium salt] is a non-nucleotide P2Y11 agonist and stimulates release of interleukin-8 from human monocyte-derived dendritic cells.

PubMed

Meis, Sabine; Hamacher, Alexandra; Hongwiset, Darunee; Marzian, Claudia; Wiese, Michael; Eckstein, Niels; Royer, Hans-Dieter; Communi, Didier; Boeynaems, Jean-Marie; Hausmann, Ralf; Schmalzing, Günther; Kassack, Matthias U

2010-01-01

The G protein-coupled P2Y(11) receptor is involved in immune system modulation. In-depth physiological evaluation is hampered, however, by a lack of selective and potent ligands. By screening a library of sulfonic and phosphonic acid derivatives at P2Y(11) receptors recombinantly expressed in human 1321N1 astrocytoma cells (calcium and cAMP assays), the selective non-nucleotide P2Y(11) agonist NF546 [4,4'-(carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)carbonylimino))-bis(1,3-xylene-alpha,alpha'-diphosphonic acid) tetrasodium salt] was identified. NF546 had a pEC(50) of 6.27 and is relatively selective for P2Y(11) over P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(12), P2X(1), P2X(2), and P2X(2)-X(3). Adenosine-5'-O-(3-thio)triphosphate (ATPgammaS), a nonhydrolyzable analog of the physiological P2Y(11) agonist ATP, and NF546 use a common binding site as suggested by molecular modeling studies and their competitive behavior toward the nanomolar potency antagonist NF340 [4,4'-(carbonylbis(imino-3,1-(4-methyl-phenylene)carbonylimino))bis(naphthalene-2,6-disulfonic acid) tetrasodium salt] in Schild analysis. The pA(2) of NF340 was 8.02 against ATPgammaS and 8.04 against NF546 (calcium assays). NF546 was further tested for P2Y(11)-mediated effects in monocyte-derived dendritic cells. Similarly to ATPgammaS, NF546 led to thrombospondin-1 secretion and inhibition of lipopolysaccharide-stimulated interleukin-12 release, whereas NF340 inhibited these effects. Further, for the first time, it was shown that ATPgammaS or NF546 stimulation promotes interleukin 8 (IL-8) release from dendritic cells, which could be inhibited by NF340. In conclusion, we have described the first selective, non-nucleotide agonist NF546 for P2Y(11) receptors in both recombinant and physiological expression systems and could show a P2Y(11)-stimulated IL-8 release, further supporting the immunomodulatory role of P2Y(11) receptors.

A randomized phase II trial comparing two different sequence combinations of autologous vaccine and human recombinant interferon gamma and human recombinant interferon alpha2B therapy in patients with metastatic renal cell carcinoma: clinical outcome and analysis of immunological parameters.

PubMed

Schwaab, T; Heaney, J A; Schned, A R; Harris, R D; Cole, B F; Noelle, R J; Phillips, D M; Stempkowski, L; Ernstoff, M S

2000-04-01

The clinical observation of spontaneous regression in patients with renal cell carcinoma (RCC) and the response to various immunotherapeutic therapies strongly suggest a role for the host immune system in this disease. Prior studies showed that sequential administration of interferon (IFN) gamma and IFN alpha to RCC patients was safe. Clinical responses as well as immune changes in the peripheral blood mononuclear cell compartment were observed. Autologous tumor cell vaccines (AV) have also demonstrated activity in renal cell carcinoma. We hypothesize that the addition of AV to sequential IFN gamma and a therapy might improve the tumor-specific immune response by providing an appropriate source of antigen in the appropriate cytokine environment. To our knowledge, this is the first trial using AV combined with IFN alpha and IFN gamma. The purpose of this study was to evaluate the feasibility of manufacturing and administering (AV) from resected tumor samples, and administration of AV with combination IFN gamma and IFN alpha therapy. Finally, the impact on immunological parameters of these treatment options was assessed. Patients with metastatic RCC were randomly assigned to receive AV plus bCG along with a sequential administration of IFN gamma and a either together or after initiation of vaccine. Toxicity and clinical responses were evaluated. Modulations of the immune system were investigated by analyzing phenotype, cytokine mRNA expression, T cell proliferation and cytotoxicity in the peripheral blood mononuclear cell compartment. Fourteen patients with metastatic renal cell carcinoma were enrolled in this study; 9 were available for response evaluation. In a 70 day period, 3 (33%) showed mixed responses, 5 (56%) stable disease and 1 (11%) progression of disease. Toxicities were consistent with previous clinical reports. In the flow-cytometry phenotype analysis, stimulation of distinct subsets of circulating T-lymphocytes and a decrease of CD8+ T cell subsets was demonstrated. T-cell proliferation to allogeneic tumor cell stimulation improved following treatment. IL-4 and IL-5 mRNA levels were reduced in all patients after treatment. Patients who responded to treatment did not produce any IL-4 mRNA at all, before or after treatment. AV with IFNgamma and IFNalpha therapy might induce a MHC class-mediated cytotoxic T lymphocyte (CTL) response. We suggest that adequate therapy might direct T cell response toward a Th1 type response. We hypothesize a state of improved immune readiness in patients who might eventually respond to immunotherapy.

Successful reinstitution of agalsidase beta therapy in Fabry disease patients with previous IgE-antibody or skin-test reactivity to the recombinant enzyme.

PubMed

Bodensteiner, David; Scott, C Ronald; Sims, Katherine B; Shepherd, Gillian M; Cintron, Rebecca D; Germain, Dominique P

2008-05-01

To determine if enzyme replacement therapy, involving intravenous infusions of recombinant human alpha-galactosidase A (agalsidase beta; Fabrazyme), could be safely continued in patients with Fabry disease who had been withdrawn from a previous clinical trial as a precautionary, protocol-specified measure due to detection of serum IgE antibodies or skin-test reactivity to agalsidase beta. The rechallenge infusion protocol specified strict patient monitoring conditions and graded dosing and infusion-rate schemes that were adjusted according to each patient's tolerance to the infusion. Six males (age: 26-66 years) were enrolled. During rechallenge, five patients received between 4 and 27 infusions; one patient voluntarily withdrew after one infusion because of recurrence of infusion-associated reactions. No anaphylactic reactions occurred. All adverse events, including four serious adverse events, were mild or moderate in intensity. Most treatment-related adverse events occurred during infusions (most commonly urticaria, vomiting, nausea, chills, pruritus, hypertension) and were resolved by infusion rate reductions and/or medication. After participation in the study, all patients, including the one who withdrew after one infusion, transitioned to commercial drug. Agalsidase beta therapy can be successfully reinstated in patients with Fabry disease who have developed IgE antibodies or skin test reactivity to the recombinant enzyme.

Protective specific immunity induced by doxorubicin plus TNF-alpha combination treatment of EL4 lymphoma-bearing C57BL/6 mice.

PubMed

Ehrke, M J; Verstovsek, S; Maccubbin, D L; Ujházy, P; Zaleskis, G; Berleth, E; Mihich, E

2000-07-01

The therapeutic efficacy of a single (day 8), moderate dose (4 mg/kg, i.v.) of doxorubicin (DOX, Adriamycin) combined with recombinant human TNF-alpha (3 different doses and 5 different schedules, i.v.) was evaluated in C57BL/6 mice bearing an implant (s.c.) of the DOX-sensitive, TNF-alpha-resistant EL4 lymphoma. In parallel to monitoring survival, the levels of several host anti-tumor cytolytic effector functions of splenocytes and thymocytes were evaluated throughout the treatment period and in long-term survivors (LTS). DOX treatment alone resulted in a moderate (approx. 20%) increase in life span but no cures. TNF-alpha alone, at any tested dose or schedule, had little or no positive effect on survival. The combinations of DOX and TNF-alpha were only slightly better than DOX alone with respect to the time to death of mice that died (approx. 29% increase); however, each of the combinations involving 1,000 U TNF-alpha/injection produced a fraction (20% to 80%) of LTS. The host defense activities examined included those of splenic and thymic cytolytic T lymphocytes (CTL) and lymphokine-activated killer cells as well as splenic tumoricidal macrophages. Although most activities were modulated by tumor growth and/or treatment, only CTL responsiveness appeared to correlate with survival. CTL activity in the treated groups with LTS was significantly higher than in control groups late in the treatment period. Finally, ex vivo analyses of splenocytes and thymocytes together with the rejection of implanted tumor at 17 months established that LTS displayed specific long-term immune memory. Copyright 2000 Wiley-Liss, Inc.

Human HOXA5 homeodomain enhances protein transduction and its application to vascular inflammation

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

Lee, Ji Young; Park, Kyoung sook; Cho, Eun Jung

2011-07-01

Highlights: {yields} We have developed an E. coli protein expression vector including human specific gene sequences for protein cellular delivery. {yields} The plasmid was generated by ligation the nucleotides 770-817 of the homeobox A5 mRNA sequence. {yields} HOXA5-APE1/Ref-1 inhibited TNF-alpha-induced monocyte adhesion to endothelial cells. {yields} Human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins. -- Abstract: Cellular protein delivery is an emerging technique by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an Escherichia coli expression vector including humanmore » specific gene sequences for protein cellular delivery. The plasmid was generated by ligation the nucleotides 770-817 of the homeobox A5 mRNA sequence which was matched with protein transduction domain (PTD) of homeodomain protein A5 (HOXA5) into pET expression vector. The cellular uptake of HOXA5-PTD-EGFP was detected in 1 min and its transduction reached a maximum at 1 h within cell lysates. The cellular uptake of HOXA5-EGFP at 37 {sup o}C was greater than in 4 {sup o}C. For study for the functional role of human HOXA5-PTD, we purified HOXA5-APE1/Ref-1 and applied it on monocyte adhesion. Pretreatment with HOXA5-APE1/Ref-1 (100 nM) inhibited TNF-{alpha}-induced monocyte adhesion to endothelial cells, compared with HOXA5-EGFP. Taken together, our data suggested that human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins.« less

Distribution, metabolism and excretion of a synthetic androgen 7alpha-methyl-19-nortestosterone, a potential male-contraceptive.

PubMed

Prasad, Pramod Vishwanath; Arumugam, Ramamani; Willman, Mark; Ge, Ren-Shan; Sitruk-Ware, Regine; Kumar, Narender

2009-01-01

A synthetic androgen 7alpha-Methyl-19-nortestosterone (MENT) has a potential for therapeutic use in 'androgen replacement therapy' for hypogonadal men or as a hormonal male-contraceptive in normal men. Its tissue distribution, excretion and metabolic enzyme(s) have not been reported. Therefore, the present study tested the distribution and excretion of MENT in Sprague-Dawley rats castrated 24h prior to the injection of tritium-labeled MENT ((3)H-MENT). Rats were euthanized at different time intervals after dosing, and the amount of radioactivity in various tissues/organs was measured following combustion in a Packard oxidizer. The radioactivity (% injected dose) was highest in the duodenal contents in the first 30min of injection. Specific uptake of the steroid was observed in target tissues such as ventral prostate and seminal vesicles at 6h, while in other tissues radioactivity equilibrated with blood. Liver and duodenum maintained high radioactivity throughout, as these organs were actively involved in the metabolism and excretion of most drugs. The excretion of (3)H-MENT was investigated after subcutaneous injection of (3)H-MENT into male rats housed in metabolic cages. Urine and feces were collected at different time intervals (up to 72h) following injection. Results showed that the radioactivity was excreted via feces and urine in equal amounts by 30h. Aiming to identify enzyme(s) involved in the MENT metabolism, we performed in vitro metabolism of (3)H-MENT using rat and human liver microsomes, cytosol and recombinant cytochrome P(450) (CYP) isozymes. The metabolites were separated by thin-layer chromatography (TLC). Three putative metabolites (in accordance with the report of Agarwal and Monder [Agarwal AK, Monder C. In vitro metabolism of 7alpha-methyl-19-nortestosterone by rat liver, prostate, and epididymis. Endocrinology 1988;123:2187-93]), [i] 3-hydroxylated MENT by both rat and human liver cytosol; [ii] 16alpha-hydroxylated MENT (a polar metabolite) by both rat and human hepatic microsomes; and [iii] 7alpha-methyl-19-norandrostenedione (a non-polar metabolite) by human hepatic microsomes, were obtained. By employing chemical inhibitors and specific anti-CYP antibodies, (3)H-MENT was found to be metabolized specifically by rat CYP 2C11 and 3-hydroxysteroid dehydrogenase (3-HSD) enzymes whereas in humans it was accomplished by CYP 3A4, 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and 3-HSD enzymes.

Evaluation of the immuno-stimulatory potential of stopper extractables and leachables by using dendritic cells as readout.

PubMed

Mueller, Robert; Karle, Anette; Vogt, Anne; Kropshofer, Harald; Ross, Alfred; Maeder, Karsten; Mahler, Hanns-Christian

2009-10-01

Recombinant protein pharmaceuticals may bear some risks and undesirable side effects, such as the appearance of immunogenic reactions. The increased incidence of antibody-mediated pure red cell aplasia (PRCA) outside the United States after administration of a human serum albumin (HSA)-free EPREX (recombinant human erythropoietin alpha) formulation was explained with the generation of rubber stopper related leachables, possibly acting as immunogenic adjuvants. In our study, we have investigated the potential of extractable and leachable preparations of three different pharmaceutical relevant stoppers to generate a "danger signal" in a dendritic cell assay. Furthermore, the investigated extractable and leachable preparations were characterized by NMR and a micelle-based polysorbate quantification method. In summary, we could demonstrate that stopper extractables, either generated by extraction or by leaching conditions, were not acting as danger signals for dendritic cells. Instead we identified degradation products of polysorbate 80, oleic acid and follow-up products, occur only under very accelerated conditions (100 degrees C for 4 days) as a potential stimulator for these immune cells. As this degradation did not occur at real-time, the authors however do not consider their finding to be linked to any direct safety implications of polysorbate-containing formulations in clinical practice.

Live attenuated yellow fever 17D infects human DCs and allows for presentation of endogenous and recombinant T cell epitopes.

PubMed

Barba-Spaeth, Giovanna; Longman, Randy S; Albert, Matthew L; Rice, Charles M

2005-11-07

The yellow fever (YF) 17D vaccine is one of the most successful live attenuated vaccines available. A single immunization induces both long-lasting neutralizing antibody and YF-specific T cell responses. Surprisingly, the mechanism for this robust immunity has not been addressed. In light of several recent reports suggesting flavivirus interaction with dendritic cells (DCs), we investigated the mechanism of YF17D interaction with DCs and the importance of this interaction in generating T cell immunity. Our results show that YF17D can infect immature and mature human DCs. Viral entry is Ca(2+) dependent, but it is independent of DC-SIGN as well as multiple integrins expressed on the DC surface. Similar to infection of cell lines, YF infection of immature DCs is cytopathic. Although infection itself does not induce DC maturation in vitro, TNF-alpha-induced maturation protects DCs from YF-induced cytopathogenicity. Furthermore, we show that DCs infected with YF17D or YF17D carrying a recombinant epitope can process and present antigens for CD8(+) T cell stimulation. These findings offer insight into the immunologic mechanisms associated with the highly capable YF17D vaccine that may guide effective vaccine design.

Antibody-based enzyme-linked lectin assay (ABELLA) for the sialylated recombinant human erythropoietin present in culture supernatant.

PubMed

Kim, Hyoung Jin; Lee, Seung Jae; Kim, Hong-Jin

2008-11-04

The terminal sialic acid of human erythropoietin (hEPO) is essential for in vivo activity. The current resorcinol and HPLC methods for analyzing alpha2,3-linked sialic acid require more than a microgram of purified rhEPO, and purification takes a great deal of time and labor. In this study, we assessed the use of an antibody-based enzyme-linked lectin assay (ABELLA) for analyzing non-purified recombinant hEPO (rhEPO). The major problem of this method was the high background due to terminal sialylation of components of the assay (antibody and bovine serum albumin) other than rhEPO. To solve this problem, we used a monoclonal antibody (Mab 287) to capture the rhEPO, and oxidized the bovine serum albumin used for blocking with meta-periodate. The sialic acid content of non-purified rhEPO measured by ABELLA was similar to that obtained by the resorcinol method on purified rhEPO. ABELLA has advantages such as adaptability and need for minimal amounts of rhEPO (40 ng/ml). Our observations suggest that ABELLA should reduce the time and labor needed to improve culture conditions so as to increase protein sialylation, and also facilitate the study of sialylation mechanisms.

Characterization of the translation products of the major mRNA species from rabbit lactating mammary glands and construction of bacterial recombinants containing casein and alpha-lactalbumin complementary DNA.

PubMed Central

Suard, Y M; Tosi, M; Kraehenbuhl, J P

1982-01-01

Total cytoplasmic polyadenylated RNA from lactating rabbit mammary glands was analysed on methylmercury hydroxide-agarose gels. The size of the most abundant mRNA species ranged between 0.5 and 5.0 kb (kilobases), with major bands at 0.55, 0.84, 0.92, 1.18 and 2.4 kb and discrete minor bands of 1.5, 1.7, 3.0 and 3.9 kb. Translation in vitro of total mRNA with [3H]leucine or [35S]methionine as precursor yielded four major bands with apparent Mr values of 16 000, 25 000, 26 000 and 29 000. The four protein bands were identified by immunoprecipitation by using specific antisera as alpha-lactalbumin and x-, kappa- and alpha-caseins, respectively. Labelling with (35S]cysteine followed by immunoprecipitation with anti-transferrin or anti-alpha-lactalbumin sera allowed the identification of two whey proteins. Translated transferrin was resolved as an 80 000-dalton band and alpha-lactalbumin appeared as a 16 000-dalton protein. A library of recombinant plasmids containing cDNA (complementary DNA) sequences representing cytoplasmic polyadenylated RNA was used to isolate clones for the major rabbit caseins and alpha-lactalbumin. A preliminary characterization of these cDNA clones was achieved by colony hybridization with enriched RNA fractions as probes. Positive clones were identified by use of hybrid-promoted translation in vitro and immunoprecipitation of the translation products. The corresponding mRNA species were further identified by hybridizing RNA blots with radioactively labelled cDNA clones. We present the restriction map of alpha-casein and kappa-casein cDNA clones. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:6123313

Comparison of nitric oxide-induced oxidation of recombinant oxyhemoglobin subunits using a competition experiment.

PubMed

Lin, Yen-Lin; Huang, Kuang-Tse

2009-08-01

A low reaction rate with nitric oxide (NO) is one of the important characteristics of hemoglobin (Hb)-based oxygen carriers. The reaction rate between oxyHb and NO is usually measured by stopped-flow spectrophotometry. However, the reported rates vary due to the difficulty of accurately determining the NO concentration and the limit of the instrument dead time. To circumvent these problems, we developed an experiment using oxymyoglobin (oxyMb) to compete with oxyHb for NO that is released from an NO donor. Determination of the rate constants in the competition experiment no longer depends on accurate measurement of time or NO concentration, since this approach instead measures the ratio of rate constants for the reaction of oxyHb and oxyMb with NO. For recombinant mutant Hb alpha(L29F)beta the rates for alpha(L29F) and beta are approximately 15- and 1.6-fold smaller than for wild-type Hb. In conclusion, the competition experiment provides an alternative method for determination of relative reaction rates of recombinant Hb subunits with NO.

Ovarian response markers lead to appropriate and effective use of corifollitropin alpha in assisted reproduction.

PubMed

La Marca, Antonio; D'Ippolito, Giovanni

2014-02-01

Corifollitropin alpha is a highly effective gonadotrophin, which maintains multifollicular growth for a week. The advantages of its administration include ease of use of the drug, making the treatment more patient friendly, resulting in a lower level of distress for the patient. At the same time, the pregnancy rate resulting from its use in IVF/intracytoplasmic sperm injection cycles is similar to that found when daily recombinant FSH is administered. The ovarian response to corifollitropin alpha is dependent on clinically established predictors such as baseline FSH, antral follicle count (AFC) and age. There is a general trend towards a higher ovarian response with an increasing AFC and the number of oocytes per attempt decreased with increasing baseline FSH and age. Even if the risk of ovarian hyperstimulation syndrome following corifollitropin alpha is very similar to the rate reported in literature for young women undergoing IVF, the risk of overstimulation may be reduced by avoiding maximal ovarian stimulation in women anticipated to be hyperresponders. High basal anti-Müllerian hormone and/or AFC can identify women with enhanced functional ovarian reserve at risk of overstimulation, and the risk is even higher if maximally stimulated with corifollitropin alpha or high dose of daily recombinant FSH. Corifollitropin alpha is a highly effective gonadotrophin which maintains multifollicular growth for a week. The ovarian response to corifollitropin was demonstrated to be dependent on clinically established predictors such as baseline FSH, antral follicle count (AFC) and age. There was a general trend toward a higher ovarian response with an increasing AFC and the mean number of oocytes per attempt decreased with increasing baseline FSH and age. Even if the risk of ovarian hyperstimulation syndrome (OHSS) following corifollitropin alpha is very similar to the rate of OHSS reported in literature for young women undergoing IVF, the risk of overstimulation may be reduced by avoiding maximal ovarian stimulation in women anticipated to be hyperresponders. Increasing evidence demonstrates that anti-Müllerian hormone and AFC exhibit a very good diagnostic performance in the prediction of hyperresponse. High basal anti-Müllerian hormone and/or AFC can identify women with enhanced functional ovarian reserve who are at risk of overstimulation if stimulated in IVF cycles and the risk is even higher if maximally stimulated with corifollitropin alpha or high dose of daily recombinant FSH. Copyright © 2013 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Age-Dependent Recombination Rates in Human Pedigrees

PubMed Central

Hussin, Julie; Roy-Gagnon, Marie-Hélène; Gendron, Roxanne; Andelfinger, Gregor; Awadalla, Philip

2011-01-01

In humans, chromosome-number abnormalities have been associated with altered recombination and increased maternal age. Therefore, age-related effects on recombination are of major importance, especially in relation to the mechanisms involved in human trisomies. Here, we examine the relationship between maternal age and recombination rate in humans. We localized crossovers at high resolution by using over 600,000 markers genotyped in a panel of 69 French-Canadian pedigrees, revealing recombination events in 195 maternal meioses. Overall, we observed the general patterns of variation in fine-scale recombination rates previously reported in humans. However, we make the first observation of a significant decrease in recombination rates with advancing maternal age in humans, likely driven by chromosome-specific effects. The effect appears to be localized in the middle section of chromosomal arms and near subtelomeric regions. We postulate that, for some chromosomes, protection against non-disjunction provided by recombination becomes less efficient with advancing maternal age, which can be partly responsible for the higher rates of aneuploidy in older women. We propose a model that reconciles our findings with reported associations between maternal age and recombination in cases of trisomies. PMID:21912527

Immunogenicity of HSP-70, KMP-11 and PFR-2 leishmanial antigens in the experimental model of canine visceral leishmaniasis.

PubMed

Carrillo, Eugenia; Crusat, Martín; Nieto, Javier; Chicharro, Carmen; Thomas, Maria del Carmen; Martínez, Enrique; Valladares, Basilio; Cañavate, Carmen; Requena, Jose María; López, Manuel Carlos; Alvar, Jorge; Moreno, Javier

2008-03-28

Zoonotic visceral leishmaniasis (ZVL) is a parasitic disease caused by Leishmania infantum/L. chagasi that is emerging as an important medical and veterinary problem. Dogs are the domestic reservoir for this parasite and, therefore, the main target for controlling the transmission to humans. In the present work, we have evaluated the immunogenicity of the Leishmania infantum heat shock protein (HSP)-70, paraflagellar rod protein (PFR)-2 and kinetoplastida membrane protein (KMP)-11 recombinant proteins in dogs experimentally infected with the parasite. We have shown that peripheral blood mononuclear cells (PBMC) from experimentally infected dogs proliferated in response to these recombinant antigens and against the soluble leishmanial antigen (SLA). We have also quantified the mRNA expression level of the cytokines induced in PBMC upon stimulation with the HSP-70, PFR-2 and KMP-11 proteins. These recombinant proteins induced an up-regulation of IFN-gamma. HSP-70 and PFR-2 also produced an increase of the TNF-alpha transcripts abundance. No measurable induction of IL-10 was observed and low levels of IL-4 mRNA were produced in response to the three mentioned recombinant antigens. Serum levels of specific antibodies against HSP-70, PFR-2 and KMP-11 recombinant proteins were also determined in these animals. Our study showed that HSP-70, KMP-11 and PFR-2 proteins are recognized by infected canines. Furthermore, these antigens produce a Th1-type immune response, suggesting that they may be involved in protection. The identification as vaccine candidates of Leishmania antigens that elicit appropriate immune responses in the canine model is a key step in the rational approach to generate a vaccine for canine visceral leishmaniasis.

Can indirect tests detect a known recombination event in human mtDNA?

PubMed

White, Daniel James; Gemmell, Neil John

2009-07-01

Whether human mitochondrial DNA (mtDNA) recombines sufficiently to influence its evolution, evolutionary analysis, and disease etiology, remains equivocal. Overall, evidence from indirect studies of population genetic data suggests that recombination is not occurring at detectable levels. This may be explained by no, or low, recombination or, alternatively, current indirect tests may be incapable of detecting recombination in human mtDNA. To investigate the latter, we have tested whether six well-established indirect tests of recombination could detect recombination in a human mtDNA data set, in which its occurrence had been empirically confirmed. Three showed statistical evidence for recombination (r(2) vs. distance, the Homoplasy test, Neighborhood Similarity Score), and three did not (D' vs. distance, Max Chi Squared, Pairwise Homoplasy Index). Possible reasons for detection failure are discussed. Further, evidence from earlier studies suggesting a lack of recombination in mtDNA in humans is reconsidered, taking into account the appropriateness of the tests used, based on our new findings.

Thermodynamics of Aryl-Dihydroxyphenyl-Thiadiazole Binding to Human Hsp90

PubMed Central

Kazlauskas, Egidijus; Petrikaitė, Vilma; Michailovienė, Vilma; Revuckienė, Jurgita; Matulienė, Jurgita; Grinius, Leonas; Matulis, Daumantas

2012-01-01

The design of specific inhibitors against the Hsp90 chaperone and other enzyme relies on the detailed and correct understanding of both the thermodynamics of inhibitor binding and the structural features of the protein-inhibitor complex. Here we present a detailed thermodynamic study of binding of aryl-dihydroxyphenyl-thiadiazole inhibitor series to recombinant human Hsp90 alpha isozyme. The inhibitors are highly potent, with the intrinsic Kd approximately equal to 1 nM as determined by isothermal titration calorimetry (ITC) and thermal shift assay (TSA). Dissection of protonation contributions yielded the intrinsic thermodynamic parameters of binding, such as enthalpy, entropy, Gibbs free energy, and the heat capacity. The differences in binding thermodynamic parameters between the series of inhibitors revealed contributions of the functional groups, thus providing insight into molecular reasons for improved or diminished binding efficiency. The inhibitor binding to Hsp90 alpha primarily depended on a large favorable enthalpic contribution combined with the smaller favorable entropic contribution, thus suggesting that their binding was both enthalpically and entropically optimized. The enthalpy-entropy compensation phenomenon was highly evident when comparing the inhibitor binding enthalpies and entropies. This study illustrates how detailed thermodynamic analysis helps to understand energetic reasons for the binding efficiency and develop more potent inhibitors that could be applied for therapeutic use as Hsp90 inhibitors. PMID:22655030

Immunochemical characterization of Glycine max L. Merr. var Raiden, as a possible hypoallergenic substitute for cow's milk-allergic patients.

PubMed

Curciarello, R; Lareu, J F; Fossati, C A; Docena, G H; Petruccelli, S

2008-09-01

Cows' milk allergy (CMA) is the most common cause of food allergy in infancy. The only proven treatment is the complete elimination of cows' milk proteins (CMPs) from the diet by means of hypoallergenic formulas. Soybean-based formulae are widely used although intolerance to soy has been reported to occur in 15-40% of infants with CMA. The aim of this work was to analyse the in vitro reactivity of the soybean cultivar Raiden, which naturally lacks glycinin A(4)A(5)B(3), to evaluate whether this genotype could be a safe CMP substitute for CMA patients. The reactivity of conventional soybean (CS) and Raiden soybean (RS) genotypes and also recombinant glycinin A(4)A(5)B(3) and alphabeta-conglycinin with casein-specific monoclonal antibodies and CMP-specific polyclonal serum was evaluated by immunoblotting and ELISA. A sequential competitive ELISA with the polyclonal antiserum and different soluble inhibitors was performed. In addition, an indirect ELISA with sera of atopic children with CMA was carried out to analyse the IgE-binding capacity of the different soybean components. We have shown that CS contains four components that cross-react with CMP, while RS has only one. The remaining cross-reactive component in RS was identified as alpha-subunit beta-conglycinin. By means of inhibitory ELISA, we demonstrated that CS, RS and the alpha-subunit beta-conglycinin extracts inhibited the binding of CMP-specific antibodies to the CMP-coated solid phase. Finally, we showed that CS, RS and the recombinant proteins were recognized by human CMP-specific IgE antibodies. This work shows that although Raiden has fewer cross-reactive components than conventional soybean, it still has a residual cross-reactive component: the alpha-subunit beta-conglycinin. This reactivity might make this genotype unsuitable to treat CMA and also explains adverse reactions to soybean in CMA infants.

The paradox of MHC-DRB exon/intron evolution: alpha-helix and beta-sheet encoding regions diverge while hypervariable intronic simple repeats coevolve with beta-sheet codons.

PubMed

Schwaiger, F W; Weyers, E; Epplen, C; Brün, J; Ruff, G; Crawford, A; Epplen, J T

1993-09-01

Twenty-one different caprine and 13 ovine MHC-DRB exon 2 sequences were determined including part of the adjacent introns containing simple repetitive (gt)n(ga)m elements. The positions for highly polymorphic DRB amino acids vary slightly among ungulates and other mammals. From man and mouse to ungulates the basic (gt)n(ga)m structure is fixed in evolution for 7 x 10(7) years whereas ample variations exist in the tandem (gt)n and (ga)m dinucleotides and especially their "degenerated" derivatives. Phylogenetic trees for the alpha-helices and beta-pleated sheets of the ungulate DRB sequences suggest different evolutionary histories. In hoofed animals as well as in humans DRB beta-sheet encoding sequences and adjacent intronic repeats can be assembled into virtually identical groups suggesting coevolution of noncoding as well as coding DNA. In contrast alpha-helices and C-terminal parts of the first DRB domain evolve distinctly. In the absence of a defined mechanism causing specific, site-directed mutations, double-recombination or gene-conversion-like events would readily explain this fact. The role of the intronic simple (gt)n(ga)m repeat is discussed with respect to these genetic exchange mechanisms during evolution.

Mapping of the Pim-1 oncogene in mouse t-haplotypes and its use to define the relative map positions of the tcl loci t0(t6) and tw12 and the marker tf (tufted).

PubMed

Ark, B; Gummere, G; Bennett, D; Artzt, K

1991-06-01

Pim-1 is an oncogene activated in mouse T-cell lymphomas induced by Moloney and AKR mink cell focus (MCF) viruses. Pim-1 was previously mapped to chromosome 17 by somatic cell hybrids, and subsequently to the region between the hemoglobin alpha-chain pseudogene 4 (Hba-4ps) and the alpha-crystalline gene (Crya-1) by Southern blot analysis of DNA obtained from panels of recombinant inbred strains. We have now mapped Pim-1 more accurately in t-haplotypes by analysis of recombinant t-chromosomes. The recombinants were derived from Tts6tf/t12 parents backcrossed to + tf/ + tf, and scored for recombination between the loci of T and tf. For simplicity all t-complex lethal genes properly named tcl-tx are shortened to tx. The Pim-1 gene was localized 0.6 cM proximal to the tw12 lethal gene, thus placing the Pim-1 gene 5.2 cM distal to the H-2 region in t-haplotypes. Once mapped, the Pim-1 gene was used as a marker for further genetic analysis of t-haplotypes. tw12 is so close to tf that even with a large number of recombinants it was not possible to determine whether it is proximal or distal to tf. Southern blot analysis of DNA from T-tf recombinants with a separation of tw12 and tf indicated that tw12 is proximal to tf. The mapping of two allelic t-lethals, t0 and t6 with respect to tw12 and tf has also been a problem.(ABSTRACT TRUNCATED AT 250 WORDS)

Four rice seed cDNA clones belonging to the alpha-amylase/trypsin inhibitor gene family encode potential rice allergens.

PubMed

Alvarez, A M; Fukuhara, E; Nakase, M; Adachi, T; Aoki, N; Nakamura, R; Matsuda, T

1995-07-01

Four rice seed proteins encoded by cDNAs belonging to the alpha-amylase/trypsin inhibitor gene family were overexpressed as TrpE-fusion proteins in E. coli. The expressed rice proteins were detected by SDS-PAGE as major proteins in bacterial cell lysates. Western blot analyses showed that all the recombinant proteins were immunologically reactive to rabbit polyclonal antibodies and to a mouse monoclonal antibody (25B9) specific for a previously isolated rice allergen of 16 kDa. Some truncated proteins from deletion mutants of the cDNAs retained their reactivity to the specific antibodies. These results suggest that the cDNAs encode potential rice allergens and that some epitopes of the recombinant proteins are still immunoreactive when they are expressed as their fragments.

Engineered Modular Recombinant Transporters: Application of New Platform for Targeted Radiotherapeutic Agents to {alpha}-Particle Emitting {sup 211}At

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

Rosenkranz, Andrey A.; Department of Biophysics, Biological Faculty, Moscow State University, Moscow; Vaidyanathan, Ganesan

2008-09-01

Purpose: To generate and evaluate a modular recombinant transporter (MRT) for targeting {sup 211}At to cancer cells overexpressing the epidermal growth factor receptor (EGFR). Methods and Materials: The MRT was produced with four functional modules: (1) human epidermal growth factor as the internalizable ligand, (2) the optimized nuclear localization sequence of simian vacuolating virus 40 (SV40) large T-antigen, (3) a translocation domain of diphtheria toxin as an endosomolytic module, and (4) the Escherichia coli hemoglobin-like protein (HMP) as a carrier module. MRT was labeled using N-succinimidyl 3-[{sup 211}At]astato-5-guanidinomethylbenzoate (SAGMB), its {sup 125}I analogue SGMIB, or with {sup 131}I using Iodogen.more » Binding, internalization, and clonogenic assays were performed with EGFR-expressing A431, D247 MG, and U87MG.wtEGFR human cancer cell lines. Results: The affinity of SGMIB-MRT binding to A431 cells, determined by Scatchard analysis, was 22 nM, comparable to that measured before labeling. The binding of SGMIB-MRT and its internalization by A431 cancer cells was 96% and 99% EGFR specific, respectively. Paired label assays demonstrated that compared with Iodogen-labeled MRT, SGMIB-MRT and SAGMB-MRT exhibited more than threefold greater peak levels and durations of intracellular retention of activity. SAGMB-MRT was 10-20 times more cytotoxic than [{sup 211}At]astatide for all three cell lines. Conclusion: The results of this study have demonstrated the initial proof of principle for the MRT approach for designing targeted {alpha}-particle emitting radiotherapeutic agents. The high cytotoxicity of SAGMB-MRT for cancer cells overexpressing EGFR suggests that this {sup 211}At-labeled conjugate has promise for the treatment of malignancies, such as glioma, which overexpress this receptor.« less

Enzyme replacement therapy with velmanase alfa (human recombinant alpha-mannosidase): Novel global treatment response model and outcomes in patients with alpha-mannosidosis.

PubMed

Harmatz, Paul; Cattaneo, Federica; Ardigò, Diego; Geraci, Silvia; Hennermann, Julia B; Guffon, Nathalie; Lund, Allan; Hendriksz, Christian J; Borgwardt, Line

2018-06-01

Alpha-mannosidosis is an ultra-rare monogenic disorder resulting from a deficiency in the lysosomal enzyme alpha-mannosidase, with a prevalence estimated to be as low as 1:1,000,000 live births. The resulting accumulation of mannose-rich oligosaccharides in all tissues leads to a very heterogeneous disorder with a continuum of clinical manifestations with no distinctive phenotypes. Long-term enzyme replacement therapy (ERT) with velmanase alfa is approved in Europe for the treatment of non-neurological manifestations in patients with mild to moderate alpha-mannosidosis. The clinical heterogeneity and rarity of the disease limit the sensitivity of single parameters to detect clinically relevant treatment effects. Thus, we propose a novel multiple variable responder analysis to evaluate the efficacy of ERT for alpha-mannosidosis and present efficacy analyses for velmanase alfa using this method. Global treatment response to velmanase alfa (defined by response to ≥2 domains comprising pharmacodynamic, functional, and quality of life outcomes) was applied post hoc to data from the pivotal placebo-controlled rhLAMAN-05 study and to the longer-term integrated data from all patients in the clinical development program (rhLAMAN-10). After 12 months of treatment, a global treatment response was achieved by 87% of patients receiving velmanase alfa (n = 15) compared with 30% of patients receiving placebo (n = 10). Longer-term data from all patients in the clinical program (n = 33) showed 88% of patients were global responders, including all (100%) pediatric patients (n = 19) and the majority (71%) of adult patients (n = 14). The responder analysis model demonstrates a clinically meaningful treatment effect with velmanase alfa and supports the early initiation and continued benefit of longer-term treatment of all patients with alpha-mannosidosis with this ERT. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Alpha-tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II.

PubMed

Dong, L-F; Low, P; Dyason, J C; Wang, X-F; Prochazka, L; Witting, P K; Freeman, R; Swettenham, E; Valis, K; Liu, J; Zobalova, R; Turanek, J; Spitz, D R; Domann, F E; Scheffler, I E; Ralph, S J; Neuzil, J

2008-07-17

Alpha-tocopheryl succinate (alpha-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of alpha-TOS has not been identified. Here, we show that alpha-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ)-binding site (Q(P) and Q(D), respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of alpha-TOS compared to that of UbQ for the Q(P) and Q(D) sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and underwent apoptosis in the presence of alpha-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to alpha-TOS. We propose that alpha-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy.

Molecular cloning and heterologous expression of the isopullulanase gene from Aspergillus niger A.T.C.C. 9642.

PubMed Central

Aoki, H; Yopi; Sakano, Y

1997-01-01

Isopullulanase (IPU) from Aspergillus niger A.T.C.C. (American Type Culture Collection) 9642 hydrolyses pullulan to isopanose. IPU is important for the production of isopanose and is used in the structural analysis of oligosaccharides with alpha-1,4 and alpha-1,6 glucosidic linkages. We have isolated the ipuA gene encoding IPU from the filamentous fungi A. niger A.T.C.C. 9642. The ipuA gene encodes an open reading frame of 1695 bp (564 amino acids). IPU contained a signal sequence of 19 amino acids, and the molecular mass of the mature form was calculated to be 59 kDa. IPU has no amino-acid-sequence similarity with the other pullulan-hydrolysing enzymes, which are pullulanase, neopullulanase and glucoamylase. However, IPU showed a high amino-acid-sequence similarity with dextranases from Penicillium minioluteum (61%) and Arthrobacter sp. (56%). When the ipuA gene was expressed in Aspergillus oryzae, the expressed protein (recombinant IPU) had IPU activity and was immunologically reactive with antibodies raised against native IPU. The substrate specificity, thermostability and pH profile of recombinant IPU were identical with those of the native enzyme, but recombinant IPU (90 kDa) was larger than the native enzyme (69-71 kDa). After deglycosylation with peptide-N-glycosidase F, the deglycosylated recombinant IPU had the same molecular mass as deglycosylated native enzyme (59 kDa). This result suggests that the carbohydrate chain of recombinant IPU differed from that of the native enzyme. PMID:9169610

Enzyme replacement therapy of Fabry disease.

PubMed

Clarke, Joe T R; Iwanochko, R Mark

2005-08-01

Fabry disease is an X-linked lysosomal storage disease caused by deficiency of the enzyme alpha-galactosidase A and results in pain, progressive renal impairment, cardiomyopathy, and cerebrovascular disease. The results of two major randomized, double-blind, placebo-controlled clinical trials and open-label extensions have shown that replacement of the deficient enzyme with either of two preparations of recombinant human alpha-galactosidase A, agalsidase-alfa, and agalsidase-beta is safe. Biweekly i.v. infusions of 0.2 mg/kg of agalsidase-alfa were associated with a significant decrease in pain and stabilization of renal function. Biweekly infusions of 1 mg/kg of agalsidase-beta were associated with virtually complete clearing of accumulated glycolipid substrate from renal and cutaneous capillary endothelial cells. Several smaller, open-label studies, along with observations made in the course of monitoring large numbers of patients on enzyme replacement therapy, indicated that treatment stabilizes renal function and produces significant improvements in myocardial mass and function. Treatment of Fabry disease by enzyme replacement has a significant impact on at least some serious complications of the disease.

Recombinant human B7.2 IgV-like domain expressed in bacteria maintains its co-stimulatory activity in vitro.

PubMed

Yan, Xiaocai; Ma, Jun; Zheng, Jin; Lai, Baochang; Geng, Yiping; Wang, Yili; Si, Lüsheng

2002-07-01

To investigate which of the two immunoglobulin (Ig)-like domains, the immunoglobulin variable region homologous domain IgV (hB7.2 IgV) and the immunoglobulin constant region homologous domain IgC (hB7.2 IgC) on the human B7.2 molecule contains receptor binding sites, and to evaluate whether the B7.2 protein expressed in bacteria has biological activity in vitro. Three fragments of hB7.2 IgV,hB7.2 IgC and the complete extracellular region of human B7.2 containing both the IgV and IgC domains,hB7.2 Ig (V+C), were amplified by PCR and subcloned into pGEM-Teasy. Three recombinants,pGEX-4T-3-hB7.2 IgV,pGEX-4T-3-hB7.2 IgC and pGEX-4T-3-hB7.2 Ig (V+C), were generated by cloning the fragments into a prokaryote expression plasmid (pGEX-4T-3) and transformed into the host strain E. coli DH5alpha. The relevant target fusion proteins consisting of GST and hB7.2 IgV,hB7.2 IgC and hB7.2 Ig (V+C), were identified by SDS-PAGE and Western blotting. With the presence of the first signal imitated by anti-CD3 antibody, T cell activation was observed by exposing purified T lymphocytes to each soluble form of the three bacterially-produced human B7.2 fusion proteins by [(3)H]-TdR incorporation. Three recombinant fusion proteins of human B7.2, GST-hB7.2 IgV, GST-hB7.2 IgC and GST-hB7.2 Ig (V+C) were produced and detected in inclusion body form from engineered bacteria. With the first signal present,T lymphocytes proliferated when co-stimulated by bacterially-produced either GST-hB7.2 Ig (V+C) or GST-hB7.2 IgV fusion proteins, but not by GST-hB7.2 IgC. Functional human B7.2 fusion protein can be produced in bacteria. The IgV-like domain of human B7.2 is sufficient for B7.2 to interact with its counter-receptors and co-stimulate T lymphocytes.

Hydrogen line ratios in Seyfert galaxies and low redshift quasars

NASA Technical Reports Server (NTRS)

Kriss, G. R.

1984-01-01

New observations of the Lymal alpha radiation/hydrogen alpha radiation ratio in a set of X-ray selected active galactic nuclei and an archival study of International Ultraviolet Explorer (IUE) observations of Lymal alpha low redshift quasars and Seyfert galaxies have been used to form a large sample for studying the influence of soft X-rays on the enhancement of Balmer emission in the broad line region. In common models of broad line clouds, the Balmer lines are formed deep in the interior, largely by collisional excitation. Heating within the clouds is provided by soft X-ray radiation, while Lymal alpha is formed mainly by recombination after photoionization. The ratio Lymal alpha/Halpha is expected to depend weakly on the ratio of ionizing ultraviolet luminosity to X-ray luminosity (L sub UV/l sub x). If the Lymal alpha luminosity is used as a measure of L sub UV' a weak dependence of Lymal/H alpha on the X-ray luminosity is found similar to previous results.

Alpha-Mannosidosis: Therapeutic Strategies.

PubMed

Ceccarini, Maria Rachele; Codini, Michela; Conte, Carmela; Patria, Federica; Cataldi, Samuela; Bertelli, Matteo; Albi, Elisabetta; Beccari, Tommaso

2018-05-17

Alpha-mannosidosis (α-mannosidosis) is a rare lysosomal storage disorder with an autosomal recessive inheritance caused by mutations in the gene encoding for the lysosomal α-d-mannosidase. So far, 155 variants from 191 patients have been identified and in part characterized at the biochemical level. Similarly to other lysosomal storage diseases, there is no relationship between genotype and phenotype in alpha-mannosidosis. Enzyme replacement therapy is at the moment the most effective therapy for lysosomal storage disease, including alpha-mannosidosis. In this review, the genetic of alpha-mannosidosis has been described together with the results so far obtained by two different therapeutic strategies: bone marrow transplantation and enzyme replacement therapy. The primary indication to offer hematopoietic stem cell transplantation in patients affected by alpha-mannosidosis is preservation of neurocognitive function and prevention of early death. The results obtained from a Phase I⁻II study and a Phase III study provide evidence of the positive clinical effect of the recombinant enzyme on patients with alpha-mannosidosis.

High-level expression of human stem cell factor fused with erythropoietin mimetic peptide in Escherichia coli.

PubMed

Su, Lin; Chen, Song-Sen; Yang, Ke-Gong; Liu, Chang-Zheng; Zhang, Yan-Li; Liang, Zhi-Quan

2006-06-01

Stem cell factor (SCF) and erythropoietin are essential for normal erythropoiesis and induce proliferation and differentiation synergistically for erythroid progenitor cells. Here, we report our work on construction of SCF/erythropoietin mimetic peptide (EMP) fusion protein gene, in which human SCF cDNA (1-165aa) and EMP sequence (20aa) were connected using a short (GGGGS) or long (GGGGSGGGGGS) linker sequence. The SCF/EMP gene was cloned into the pBV220 vector and expressed in the Escherichia coli DH5alpha strain. The expression level of the fusion protein was about 30% of total cell protein. The resulting inclusion bodies were solubilized with 8 M urea, followed by dilution refolding. The renatured protein was subsequently purified by Q-Sepharose FF column. The final product was >95% pure by SDS-PAGE and the yield of fusion protein was about 40 mg/L of culture. UT-7 cell proliferation and human cord blood cell colony-forming assays showed that the fusion proteins exhibited more potent activity than recombinant human SCF, suggesting a new strategy to enhance biological activities of growth factors.

Localization of the panhypopituitary dwarf mutation (df) on mouse chromosome 11 in an intersubspecific backcross.

PubMed

Buckwalter, M S; Katz, R W; Camper, S A

1991-07-01

Ames dwarf (df) is an autosomal recessive mutation characterized by severe dwarfism and infertility. This mutation provides a mouse model for panhypopituitarism. The dwarf phenotype results from failure in the differentiation of the cells which produce growth hormone, prolactin, and thyroid stimulating hormone. Using the backcross (DF/B-df/df X CASA/Rk) X DF/B-df/df, we confirmed the assignment of df to mouse chromosome 11 and demonstrated recombination between df and the growth hormone gene. This backcross is an invaluable resource for screening candidate genes for the df mutation. The df locus maps to less than 1 cM distal to Pad-1 (0.85 +/- 0.85 cM). Two new genes localized on mouse chromosome 11, Rpo2-1, and Edp-1, map to a region of conserved synteny with human chromosome 17. The localization of the alpha 1 adrenergic receptor, Adra-1, extends a known region of synteny conservation between mouse chromosome 11 and human chromosome 5, and suggests that a human counterpart to df would map to human chromosome 5.

Identification of endogenous surrogate ligands for human P2Y receptors through an in silico search.

PubMed

Hiramoto, Takeshi; Nonaka, Yosuke; Inoue, Kazuko; Yamamoto, Takefumi; Omatsu-Kanbe, Mariko; Matsuura, Hiroshi; Gohda, Keigo; Fujita, Norihisa

2004-05-01

G protein-coupled receptors (GPCRs) are distributed widely throughout the human body, and nearly 50% of current medicines act on a GPCR. GPCRs are considered to consist of seven transmembrane alpha-helices that form an alpha-helical bundle in which agonists and antagonists bind. A 3D structure of the target GPCR is indispensable for designing novel medicines acting on a GPCR. We have previously constructed the 3D structure of human P2Y(1) (hP2Y(1)) receptor, a GPCR, by homology modeling with the 3D structure of bovine rhodopsin as a template. In the present study, we have employed an in silico screening for compounds that could bind to the hP2Y(1)-receptor model using AutoDock 3.0. We selected 21 of the 30 top-ranked compounds, and by measuring intracellular Ca(2+) concentration, we identified 12 compounds that activated or blocked the hP2Y(1) receptor stably expressed in recombinant CHO cells. 5-Phosphoribosyl-1-pyrophosphate (PRPP) was found to activate the hP2Y(1) receptor with a low ED(50) value of 15 nM. The Ca(2+) assays showed it had no significant effect on P2Y(2), P2Y(6), or P2X(2) receptors, but acted as a weak agonist on the P2Y(12) receptor. This is the first study to rationally identify surrogate ligands for the P2Y-receptor family.

Cytokine-like factor-1, a novel soluble protein, shares homology with members of the cytokine type I receptor family.

PubMed

Elson, G C; Graber, P; Losberger, C; Herren, S; Gretener, D; Menoud, L N; Wells, T N; Kosco-Vilbois, M H; Gauchat, J F

1998-08-01

In this report we describe the identification, cloning, and expression pattern of human cytokine-like factor 1 (hCLF-1) and the identification and cloning of its murine homologue. They were identified from expressed sequence tags using amino acid sequences from conserved regions of the cytokine type I receptor family. Human CLF-1 and murine CLF-1 shared 96% amino acid identity and significant homology with many cytokine type I receptors. CLF-1 is a secreted protein, suggesting that it is either a soluble subunit within a cytokine receptor complex, like the soluble form of the IL-6R alpha-chain, or a subunit of a multimeric cytokine, e.g., IL-12 p40. The highest levels of hCLF-1 mRNA were observed in lymph node, spleen, thymus, appendix, placenta, stomach, bone marrow, and fetal lung, with constitutive expression of CLF-1 mRNA detected in a human kidney fibroblastic cell line. In fibroblast primary cell cultures, CLF-1 mRNA was up-regulated by TNF-alpha, IL-6, and IFN-gamma. Western blot analysis of recombinant forms of hCLF-1 showed that the protein has the tendency to form covalently linked di- and tetramers. These results suggest that CLF-1 is a novel soluble cytokine receptor subunit or part of a novel cytokine complex, possibly playing a regulatory role in the immune system and during fetal development.

Crystallization and preliminary X-ray diffraction studies of delta-toxin from Clostridium perfringens.

PubMed

Huyet, Jessica; Gilbert, Maryse; Popoff, Michel R; Basak, Ajit

2011-03-01

Clostridium perfringens is a Gram-positive anaerobic bacterium that is responsible for a wide range of diseases in humans and both wild and domesticated animals, including birds. C. perfringens is notable for its ability to produce a plethora of toxins, e.g. phospholipases C (alpha-toxin), pore-forming toxins (epsilon-toxin, beta-toxin and enterotoxin) and binary toxins (iota-toxin). Based on alpha-, beta-, epsilon- and iota-toxin production, the bacterium is classified into five different toxinotypes (A-E). Delta-toxin, which is a 32.6 kDa protein with 290 amino acids, is one of three haemolysins released by type C and possibly by type B strains of C. perfringens. This toxin is immunogenic and lytic to erythrocytes from the even-toed ungulates sheep, goats and pigs, and is cytotoxic to other cell types such as rabbit macrophages, human monocytes and blood platelets from goats, rabbits, guinea pigs and humans. The recombinant delta-toxin has been cloned, expressed, purified and crystallized in two different crystal forms by the hanging-drop vapour-diffusion method. Of these two different crystal forms, only the form II crystal diffracted to atomic resolution (dmin=2.4 Å), while the form I crystal diffracted to only 15 Å resolution. The form II crystals belonged to space group P2(1)2(1)2, with one molecule in the crystallographic asymmetric unit and unit-cell parameters a=49.66, b=58.48, c=112.93 Å.

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.

How good are indirect tests at detecting recombination in human mtDNA?

PubMed

White, Daniel James; Bryant, David; Gemmell, Neil John

2013-07-08

Empirical proof of human mitochondrial DNA (mtDNA) recombination in somatic tissues was obtained in 2004; however, a lack of irrefutable evidence exists for recombination in human mtDNA at the population level. Our inability to demonstrate convincingly a signal of recombination in population data sets of human mtDNA sequence may be due, in part, to the ineffectiveness of current indirect tests. Previously, we tested some well-established indirect tests of recombination (linkage disequilibrium vs. distance using D' and r(2), Homoplasy Test, Pairwise Homoplasy Index, Neighborhood Similarity Score, and Max χ(2)) on sequence data derived from the only empirically confirmed case of human mtDNA recombination thus far and demonstrated that some methods were unable to detect recombination. Here, we assess the performance of these six well-established tests and explore what characteristics specific to human mtDNA sequence may affect their efficacy by simulating sequence under various parameters with levels of recombination (ρ) that vary around an empirically derived estimate for human mtDNA (population parameter ρ = 5.492). No test performed infallibly under any of our scenarios, and error rates varied across tests, whereas detection rates increased substantially with ρ values > 5.492. Under a model of evolution that incorporates parameters specific to human mtDNA, including rate heterogeneity, population expansion, and ρ = 5.492, successful detection rates are limited to a range of 7-70% across tests with an acceptable level of false-positive results: the neighborhood similarity score incompatibility test performed best overall under these parameters. Population growth seems to have the greatest impact on recombination detection probabilities across all models tested, likely due to its impact on sequence diversity. The implications of our findings on our current understanding of mtDNA recombination in humans are discussed.

How Good Are Indirect Tests at Detecting Recombination in Human mtDNA?

PubMed Central

White, Daniel James; Bryant, David; Gemmell, Neil John

2013-01-01

Empirical proof of human mitochondrial DNA (mtDNA) recombination in somatic tissues was obtained in 2004; however, a lack of irrefutable evidence exists for recombination in human mtDNA at the population level. Our inability to demonstrate convincingly a signal of recombination in population data sets of human mtDNA sequence may be due, in part, to the ineffectiveness of current indirect tests. Previously, we tested some well-established indirect tests of recombination (linkage disequilibrium vs. distance using D′ and r2, Homoplasy Test, Pairwise Homoplasy Index, Neighborhood Similarity Score, and Max χ2) on sequence data derived from the only empirically confirmed case of human mtDNA recombination thus far and demonstrated that some methods were unable to detect recombination. Here, we assess the performance of these six well-established tests and explore what characteristics specific to human mtDNA sequence may affect their efficacy by simulating sequence under various parameters with levels of recombination (ρ) that vary around an empirically derived estimate for human mtDNA (population parameter ρ = 5.492). No test performed infallibly under any of our scenarios, and error rates varied across tests, whereas detection rates increased substantially with ρ values > 5.492. Under a model of evolution that incorporates parameters specific to human mtDNA, including rate heterogeneity, population expansion, and ρ = 5.492, successful detection rates are limited to a range of 7−70% across tests with an acceptable level of false-positive results: the neighborhood similarity score incompatibility test performed best overall under these parameters. Population growth seems to have the greatest impact on recombination detection probabilities across all models tested, likely due to its impact on sequence diversity. The implications of our findings on our current understanding of mtDNA recombination in humans are discussed. PMID:23665874

Recombinant human alpha fetoprotein synergistically potentiates the anti-cancer effects of 1'-S-1'-acetoxychavicol acetate when used as a complex against human tumours harbouring AFP-receptors.

PubMed

Arshad, Norhafiza M; In, Lionel L A; Soh, Tchen Lin; Azmi, Mohamad Nurul; Ibrahim, Halijah; Awang, Khalijah; Dudich, Elena; Tatulov, Eduard; Nagoor, Noor Hasima

2015-06-30

Previous in vitro and in vivo studies have reported that 1'-S-1'-acetoxychavicol acetate (ACA) isolated from rhizomes of the Malaysian ethno-medicinal plant Alpinia conchigera Griff (Zingiberaceae) induces apoptosis-mediated cell death in tumour cells via dysregulation of the NF-κB pathway. However there were some clinical development drawbacks such as poor in vivo solubility, depreciation of biological activity upon exposure to an aqueous environment and non-specific targeting of tumour cells. In the present study, all the problems above were addressed using the novel drug complex formulation involving recombinant human alpha fetoprotein (rhAFP) and ACA. To study the synergistic effect of both agents on human cancer xenografts, athymic nude (Nu/Nu) mice were used and treated with various combination regimes intraperitoneally. Serum levels of tumour markers for carcinoembryonic antigen (CEA) and prostate specific antigen (PSA) were assessed using sandwich ELISA. IHC and Western blotting were also conducted on in vivo tumour biopsies to investigate the involvement of NF-κB regulated genes and inflammatory biomarkers. Quantification and correlation between drug efficacies and AFP-receptors were done using IF-IC and Pearson's correlation analysis. Mice exposed to combined treatments displayed higher reductions in tumour volume compared to stand alone agents, consistent with in vitro cytotoxicity assays. Milder signs of systemic toxicity, such as loss in body weight and inflammation of vital organs were also demonstrated compared to stand alone treatments. Tumour marker levels were consistent within all rhAFP/ACA treatment groups where levels of CEA and PSA were initially elevated upon commencement of treatment, and consecutively reduced corresponding to a decrease in tumour bulk volume. Both IHC and Western blotting results indicated that the combined action of rhAFP/ACA was not only able to down-regulate NF-κB activation, but also reduce the expression of NF-κB regulated genes and inflammatory biomarkers. The efficacy of rhAFP/ACA complex was also found to be weakly negatively correlated to the level of surface AFP-receptors between tumour types. This drug complex formulation shows great therapeutic potential against AFP-receptor positive tumours, and serves as a basis to overcome insoluble and non-specific anti-neoplastic molecules.

Recombinant human alpha fetoprotein synergistically potentiates the anti-cancer effects of 1′-S-1′-acetoxychavicol acetate when used as a complex against human tumours harbouring AFP-receptors

PubMed Central

Arshad, Norhafiza M.; In, Lionel L.A.; Soh, Tchen Lin; Azmi, Mohamad Nurul; Ibrahim, Halijah; Awang, Khalijah; Dudich, Elena; Tatulov, Eduard; Nagoor, Noor Hasima

2015-01-01

Purpose Previous in vitro and in vivo studies have reported that 1′-S-1′-acetoxychavicol acetate (ACA) isolated from rhizomes of the Malaysian ethno-medicinal plant Alpinia conchigera Griff (Zingiberaceae) induces apoptosis-mediated cell death in tumour cells via dysregulation of the NF-κB pathway. However there were some clinical development drawbacks such as poor in vivo solubility, depreciation of biological activity upon exposure to an aqueous environment and non-specific targeting of tumour cells. In the present study, all the problems above were addressed using the novel drug complex formulation involving recombinant human alpha fetoprotein (rhAFP) and ACA. Experimental Design To study the synergistic effect of both agents on human cancer xenografts, athymic nude (Nu/Nu) mice were used and treated with various combination regimes intraperitoneally. Serum levels of tumour markers for carcinoembryonic antigen (CEA) and prostate specific antigen (PSA) were assessed using sandwich ELISA. IHC and Western blotting were also conducted on in vivo tumour biopsies to investigate the involvement of NF-κB regulated genes and inflammatory biomarkers. Quantification and correlation between drug efficacies and AFP-receptors were done using IF-IC and Pearson's correlation analysis. Results Mice exposed to combined treatments displayed higher reductions in tumour volume compared to stand alone agents, consistent with in vitro cytotoxicity assays. Milder signs of systemic toxicity, such as loss in body weight and inflammation of vital organs were also demonstrated compared to stand alone treatments. Tumour marker levels were consistent within all rhAFP/ACA treatment groups where levels of CEA and PSA were initially elevated upon commencement of treatment, and consecutively reduced corresponding to a decrease in tumour bulk volume. Both IHC and Western blotting results indicated that the combined action of rhAFP/ACA was not only able to down-regulate NF-κB activation, but also reduce the expression of NF-κB regulated genes and inflammatory biomarkers. The efficacy of rhAFP/ACA complex was also found to be weakly negatively correlated to the level of surface AFP-receptors between tumour types. Conclusions This drug complex formulation shows great therapeutic potential against AFP-receptor positive tumours, and serves as a basis to overcome insoluble and non-specific anti-neoplastic molecules. PMID:26158863

alpha(2)-adrenoceptor antagonist properties of OPC-28326, a novel selective peripheral vasodilator.

PubMed

Orito, K; Kishi, M; Imaizumi, T; Nakazawa, T; Hashimoto, A; Mori, T; Kambe, T

2001-10-01

1. Antagonistic properties of OPC-28326 ([4-(N-methyl-2-phenylethylamino)-1-(3,5-dimethyl-4-propionyl-aminobenzoyl)] piperidine hydrochloride monohydrate), a selective peripheral vasodilator, were investigated by analysing the data from functional studies in various tissues from the rat and binding studies of the drug to alpha(2)-adrenoceptor subtypes. 2. Using a human recombinant receptor and rat kidney cortex, we found that OPC-28326 displays affinities to alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptors with K(i) values of 2040, 285, and 55 nM, respectively. The K(i) values of yohimbine for alpha(2A)-, alpha(2B)-, and alpha(2C)-adrenoceptors were 3.0, 2.0 and 11.0 nM, respectively. 3. B-HT 920, an alpha(2)-adrenoceptor agonist, produced a pressor response via peripheral postsynaptic alpha(2)-adrenoceptor stimulation (thought to be an alpha(2B)-subtype) in a reserpine-pretreated pithed rat preparation. OPC-28326 (3 - 30 mg kg(-1), i.v.) and yohimbine (0.3 - 3 mg kg(-1), i.v.) caused dose-dependent rightward shift in the pressor dose-response curve induced by B-HT 920. The apparent pA(2) values were 1.55 (0.87 - 2.75, 95% confidence interval) and 0.11 (0.06 - 0.21) mg kg(-1), respectively. The potency of OPC-28326 was about 14 times less than that of yohimbine. 4. Clonidine inhibited the tension developed by electrical stimulation, of the rat vas deferens, by its peripheral presynaptic alpha(2A/D)-adrenoceptor action. OPC-28326 (1 - 100 microM) and yohimbine (10 - 1000 nM) caused a rightward shift in the concentration-response curve of clonidine. The pA(2) values were 5.73 (5.54 - 5.91) and 7.92 (7.84 - 8.01), respectively, providing evidence for a potency of OPC-28326 of about 155 times less than that of yohimbine. 5. Mydriasis was induced by brimonidine via stimulation of central alpha(2A/D)-adrenoceptors in anaesthetized rats. Intravenous OPC-28326 had no effect on this action, even at a very high dose of 10 mg kg(-1) i.v., while yohimbine (0.1 - 0.3 mg kg(-1) i.v.) inhibited mydriasis in a dose-dependent manner, indicating that OPC-28326 was at least 100 times less potent than yohimbine in regard to the anti-mydriatic effect. 6. These data suggest that OPC-28326 preferentially exerts peripheral and postsynaptic antagonistic actions on the alpha(2B)- and alpha(2C)-adrenoceptor subtypes.

A 4'-phosphopantetheinyl transferase mediates non-ribosomal peptide synthetase activation in Aspergillus fumigatus.

PubMed

Neville, Claire; Murphy, Alan; Kavanagh, Kevin; Doyle, Sean

2005-04-01

Aspergillus fumigatus is a significant human pathogen. Non-ribosomal peptide (NRP) synthesis is thought to be responsible for a significant proportion of toxin and siderophore production in the organism. Furthermore, it has been shown that 4'-phosphopantetheinylation is required for the activation of key enzymes involved in non-ribosomal peptide synthesis in other species. Here we report the cloning, recombinant expression and functional characterisation of a 4'-phosphopantetheinyl transferase from A. fumigatus and the identification of an atypical NRP synthetase (Afpes1), spanning 14.3 kb. Phylogenetic analysis has shown that the NRP synthetase exhibits greatest identity to NRP synthetases from Metarhizium anisolpiae (PesA) and Alternaria brassicae (AbrePsy1). Northern hybridisation and RT-PCR analysis have confirmed that both genes are expressed in A. fumigatus. A 120 kDa fragment of the A. fumigatus NRP synthetase, containing a putative thiolation domain, was cloned and expressed in the baculovirus expression system. Detection of a 4'-phosphopantetheinylated peptide (SFSAMK) from this protein, by MALDI-TOF mass spectrometric analysis after coincubation of the 4'-phosphopantetheinyl transferase with the recombinant NRP synthetase fragment and acetyl CoA, confirms that it is competent to play a role in NRP synthetase activation in A. fumigatus. The 4'-phosphopantetheinyl transferase also activates, by 4'-phosphopantetheinylation, recombinant alpha-aminoadipate reductase (Lys2p) from Candida albicans, a key enzyme involved in lysine biosynthesis.

Long-term safety and efficacy of enzyme replacement therapy for Fabry disease.

PubMed

Wilcox, William R; Banikazemi, Maryam; Guffon, Nathalie; Waldek, Stephen; Lee, Philip; Linthorst, Gabor E; Desnick, Robert J; Germain, Dominique P

2004-07-01

Elsewhere, we reported the safety and efficacy results of a multicenter phase 3 trial of recombinant human alpha -galactosidase A (rh-alpha GalA) replacement in patients with Fabry disease. All 58 patients who were enrolled in the 20-wk phase 3 double-blind, randomized, and placebo-controlled study received subsequently 1 mg/kg of rh-alpha GalA (agalsidase beta, Fabrazyme, Genzyme Corporation) biweekly in an ongoing open-label extension study. Evidence of long-term efficacy, even in patients who developed IgG antibodies against rh- alpha GalA, included the continuously normal mean plasma globotriaosylceramide (GL-3) levels during 30 mo of the extension study and the sustained capillary endothelial GL-3 clearance in 98% (39/40) of patients who had a skin biopsy taken after treatment for 30 mo (original placebo group) or 36 mo (original enzyme-treated group). The mean serum creatinine level and estimated glomerular filtration rate also remained stable after 30-36 mo of treatment. Infusion-associated reactions decreased over time, as did anti-rh- alpha GalA IgG antibody titers. Among seroconverted patients, after 30-36 mo of treatment, seven patients tolerized (no detectable IgG antibody), and 59% had > or =4-fold reductions in antibody titers. As of 30 mo into the extension trial, three patients were withdrawn from the study because of positive serum IgE or skin tests; however, all have been rechallenged successfully at the time of this report. Thus, enzyme replacement therapy for 30-36 mo with agalsidase beta resulted in continuously decreased plasma GL-3 levels, sustained endothelial GL-3 clearance, stable kidney function, and a favorable safety profile.

THE LYMAN ALPHA REFERENCE SAMPLE: EXTENDED LYMAN ALPHA HALOS PRODUCED AT LOW DUST CONTENT

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

Hayes, Matthew; Oestlin, Goeran; Duval, Florent

2013-03-10

We report on new imaging observations of the Lyman alpha emission line (Ly{alpha}), performed with the Hubble Space Telescope, that comprise the backbone of the Lyman alpha Reference Sample. We present images of 14 starburst galaxies at redshifts 0.028 < z < 0.18 in continuum-subtracted Ly{alpha}, H{alpha}, and the far ultraviolet continuum. We show that Ly{alpha} is emitted on scales that systematically exceed those of the massive stellar population and recombination nebulae: as measured by the Petrosian 20% radius, R{sub P20}, Ly{alpha} radii are larger than those of H{alpha} by factors ranging from 1 to 3.6, with an average ofmore » 2.4. The average ratio of Ly{alpha}-to-FUV radii is 2.9. This suggests that much of the Ly{alpha} light is pushed to large radii by resonance scattering. Defining the Relative Petrosian Extension of Ly{alpha} compared to H{alpha}, {xi}{sub Ly{alpha}} = R {sup Ly{alpha}}{sub P20}/R {sup H{alpha}}{sub P20}, we find {xi}{sub Ly{alpha}} to be uncorrelated with total Ly{alpha} luminosity. However, {xi}{sub Ly{alpha}} is strongly correlated with quantities that scale with dust content, in the sense that a low dust abundance is a necessary requirement (although not the only one) in order to spread Ly{alpha} photons throughout the interstellar medium and drive a large extended Ly{alpha} halo.« less

Theoretical evidence of the observed kinetic order dependence on temperature during the N(2)O decomposition over Fe-ZSM-5.

PubMed

Guesmi, Hazar; Berthomieu, Dorothee; Bromley, Bryan; Coq, Bernard; Kiwi-Minsker, Lioubov

2010-03-28

The characterization of Fe/ZSM5 zeolite materials, the nature of Fe-sites active in N(2)O direct decomposition, as well as the rate limiting step are still a matter of debate. The mechanism of N(2)O decomposition on the binuclear oxo-hydroxo bridged extraframework iron core site [Fe(II)(mu-O)(mu-OH)Fe(II)](+) inside the ZSM-5 zeolite has been studied by combining theoretical and experimental approaches. The overall calculated path of N(2)O decomposition involves the oxidation of binuclear Fe(II) core sites by N(2)O (atomic alpha-oxygen formation) and the recombination of two surface alpha-oxygen atoms leading to the formation of molecular oxygen. Rate parameters computed using standard statistical mechanics and transition state theory reveal that elementary catalytic steps involved into N(2)O decomposition are strongly dependent on the temperature. This theoretical result was compared to the experimentally observed steady state kinetics of the N(2)O decomposition and temperature-programmed desorption (TPD) experiments. A switch of the reaction order with respect to N(2)O pressure from zero to one occurs at around 800 K suggesting a change of the rate determining step from the alpha-oxygen recombination to alpha-oxygen formation. The TPD results on the molecular oxygen desorption confirmed the mechanism proposed.

Pyramiding of insecticidal compounds for control of the cowpea bruchid (Callosobruchus maculatus F.).

PubMed

Tarver, Matthew R; Shade, Richard E; Shukle, Richard H; Moar, William J; Muir, William M; Murdock, Larry M; Pittendrigh, Barry R

2007-05-01

The cowpea bruchid (Callosobruchus maculatus F.) (Chrysomelidae: Bruchini) is a major pest of stored cowpea grain. With limited available technologies for controlling the bruchid, transgenic cowpeas with bruchid resistance genes engineered into them could become the next management tools. An investigation was made of two different sets of potential transgenic insecticidal compounds using an artificial seed system: (i) CIP-PH-BT-J and recombinant egg white avidin, and (ii) avidin and wheat alpha-amylase inhibitor. CIP-PH-BT-J (0.1%; 1000 mg kg(-1)) and recombinant egg white avidin (0.006%; 60 mg kg(-1)) incorporated separately into artificial seeds caused 98.2 and 99% larval mortality rates respectively. Combining CIP-PH-BT-J and avidin in the same artificial seed provided additional mortality compared with each factor incorporated singly; no insects survived in seeds with the combined toxins. Similarly, when avidin and wheat alpha-amylase inhibitor (alphaAI) (1%; 10 g kg(-1)) were incorporated separately into artificial seeds, this caused 99.8 and 98% mortality respectively. However, in combination, avidin and alphaAI did not increase mortality, but they did cause a significant increase in developmental time of the cowpea bruchids. These results emphasize that the joint action of potential insecticidal compounds cannot be predicted from results obtained separately for each compound, and they suggest potential transgenes for further consideration.

Establishment of a robust dengue virus NS3-NS5 binding assay for identification of protein-protein interaction inhibitors.

PubMed

Takahashi, Hirotaka; Takahashi, Chikako; Moreland, Nicole J; Chang, Young-Tae; Sawasaki, Tatsuya; Ryo, Akihide; Vasudevan, Subhash G; Suzuki, Youichi; Yamamoto, Naoki

2012-12-01

Whereas the dengue virus (DENV) non-structural (NS) proteins NS3 and NS5 have been shown to interact in vitro and in vivo, the biological relevance of this interaction in viral replication has not been fully clarified. Here, we first applied a simple and robust in vitro assay based on AlphaScreen technology in combination with the wheat-germ cell-free protein production system to detect the DENV-2 NS3-NS5 interaction in a 384-well plate. The cell-free-synthesized NS3 and NS5 recombinant proteins were soluble and in possession of their respective enzymatic activities in vitro. In addition, AlphaScreen assays using the recombinant proteins detected a specific interaction between NS3 and NS5 with a robust Z' factor of 0.71. By employing the AlphaScreen assay, we found that both the N-terminal protease and C-terminal helicase domains of NS3 are required for its association with NS5. Furthermore, a competition assay revealed that the binding of full-length NS3 to NS5 was significantly inhibited by the addition of an excess of NS3 protease or helicase domains. Our results demonstrate that the AlphaScreen assay can be used to discover novel antiviral agents targeting the interactions between DENV NS proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

IgE enhances Fc epsilon RI expression and IgE-dependent TNF-alpha release from canine skin mast cells.

PubMed

Brazís, P; De Mora, F; Ferrer, L; Puigdemont, A

2002-03-01

The role of IgE on mast cell (MC) activation is well known. Recent studies have demonstrated that IgE also has the ability to up-regulate the high affinity IgE receptor (Fc epsilon RI) on the surface of human and murine MC, leading to an increased production of cytokines and chemokines. In the present study, we have examined the influence of IgE levels on Fc epsilon RI expression, and its consequences on TNF-alpha production from canine skin MC. Mature MC were enzymatically dispersed from the skin biopsies of 6-8 dogs and were cultured for up to 5 days in medium supplemented with recombinant canine stem cell factor (SCF) (6 ng/ml), in the presence of increasing serum IgE concentrations (ranging from 0 to 80 microg/ml). Subsequently, skin MC were activated with anti-IgE, and TNF-alpha concentration was assessed 5h post-activation by a cytotoxic bioassay. Fc epsilon RI receptors were identified in MC surface by flow cytometry. MC cultured for up to 5 days in the presence of high serum IgE concentration (8 microg/ml) produced twice the quantity of TNF-alpha than MC cultured in the absence of serum IgE, in response to stimulation with anti-IgE. Moreover, the percentage of Fc epsilon RI-positive skin cells was found to be approximately double in cells cultured with serum IgE compared to that cultured in the absence of IgE, following saturation of IgE receptors. These results suggest that, as found in human and murine MC, IgE may induce an up-regulation of the Fc epsilon RI density and an enhancement in the secretory activity of canine skin MC. This study could be of great interest in designing new therapeutic strategies for controlling MC activation in inflammatory and allergic processes.

A 3,387 bp 5'-flanking sequence of the goat alpha-S1-casein gene provides correct tissue-specific expression of human granulocyte colony-stimulating factor (hG-CSF) in the mammary gland of transgenic mice.

PubMed

Serova, Irina A; Dvoryanchikov, Gennady A; Andreeva, Ludmila E; Burkov, Ivan A; Dias, Luciene P B; Battulin, Nariman R; Smirnov, Alexander V; Serov, Oleg L

2012-06-01

A new expression vector containing the 1,944 bp 5'-flanking regulatory region together with exon 1 and intron 1 of the goat alpha-S1-casein gene (CSN1S1), the full-sized human granulocyte colony-stimulating factor gene (hGCSF) and the 3'-flanking sequence of the bovine CSN1S1, was created. The vector DNA was used for generation of four mouse transgenic lines. The transgene was integrated into chromosomes 8 and 12 of two founders as 2 and 5 copies, respectively. Tissue-specific secretion of hG-CSF into the milk of transgenic mice was in the range of 19-40 μg/ml. RT-PCR analysis of various tissues of the transgenic mice demonstrated that expression of hGCSF was detected in only the mammary gland in the progeny of all founders. Moreover, cells were shown to be positive for hG-CSF by immunofluorescent analysis in the mammary glands but not in any other tissues. There were no signs of mosaic expression in the mammary gland. Trace amounts of hG-CSF were detected in the serum of females of two transgenic lines during lactation only. However, no transgenic mice showed any changes in hematopoiesis based on the number of granulocytes in blood. Immunoblotting of hG-CSF in the milk of transgenic mice revealed two forms, presumably the glycosylated and non-glycosylated forms. The hematopoietic activity of hG-CSF in the milk of transgenic females is comparable to that of recombinant G-CSF. In general, the data obtained in this study show that the new expression vector is able to provide correct tissue-specific expression of hG-CSF with high biological activity in transgenic mice.

Endotoxin-induced lethality in neonatal mice is counteracted by interleukin-10 (IL-10) and exacerbated by anti-IL-10.

PubMed Central

Nicoletti, F; Mancuso, G; Ciliberti, F A; Beninati, C; Carbone, M; Franco, S; Cusumano, V

1997-01-01

The lethal effects occurring in neonatal (<24-h-old) BALB/c mice after challenge with 25 mg of lipopolysaccharide (LPS) per kg of body weight were significantly counteracted by pretreatment with recombinant interleukin-10 (rIL-10; 25 or 50 ng/mouse). Concordantly, blockage of endogenous IL-10 with the SXC1 monoclonal antibody increased LPS-induced mortality. Both IL-10 and SXC1 modulated the release of tumor necrosis factor alpha (TNF-alpha) so that, relative to controls, peak TNF-alpha values after LPS challenge were decreased by rIL-10 and increased by anti-IL-10. PMID:9302214

Investigation of atomic oxygen-surface interactions related to measurements with dual air density explorer satellites

NASA Technical Reports Server (NTRS)

Wood, B. J.; Ablow, C. M.; Wise, H.

1973-01-01

For a number of candidate materials of construction for the dual air density explorer satellites the rate of oxygen atom loss by adsorption, surface reaction, and recombination was determined as a function of surface and temperature. Plain aluminum and anodized aluminum surfaces exhibit a collisional atom loss probability alpha .01 in the temperature range 140 - 360 K, and an initial sticking probability. For SiO coated aluminum in the same temperature range, alpha .001 and So .001. Atom-loss on gold is relatively rapid alpha .01. The So for gold varies between 0.25 and unity in the temperature range 360 - 140 K.

ONR Far East Scientific Bulletin. Volume 10, Number 3, July-September 1985,

DTIC Science & Technology

1985-09-01

to alpha and beta interferon. " Vaccines Using recombinant DNA technology, researchers at Osaka University have developed a vaccine against the Chicken ... Pox virus. Using recombinant DNA technology, researchers at Kyushu University have developed a vaccine against the Herpes simplex virus. " Drugs...Germany 9 Norway 8 Holland 7 U.K. 6 France 4 Denmark 4 Austria 4 U.S.S.R. 3 Australia 2 Singapore 2 Spain 2 Poland I Egypt I Israel I Mexico I 20

Pharmacokinetic and hemostatic properties of the recombinant plasminogen activator bm 06.022 in healthy volunteers.

PubMed

Martin, U; von Möllendorff, E; Akpan, W; Kientsch-Engel, R; Kaufmann, B; Neugebauer, G

1991-11-01

In a randomized, single-blind, placebo-controlled, cross-over Phase-I study pharmacokinetic and hemostatic properties of BM 06.022 were investigated in seven healthy, male human volunteers. The novel recombinant plasminogen activator BM 06.022 consists of the kringle 2 domain and the protease domain of human t-PA and is unglycosylated due to its expression in Escherichia coli cells. Vehicle or 6 MU (= 10.4 mg) BM 06.022 was administered as a single i.v. bolus injection of 10 ml over 2 min. BM 06.022 was well tolerated. Fibrinogen levels and clotting times remained unchanged at baseline levels after 6 MU BM 06.022; plasminogen and alpha 2-antiplasmin (collected on chloromethylketone) decreased maximally to 83 +/- 1% and 64 +/- 3%, respectively, of baseline. D-dimers and fibrinogen degradation products increased to 1,006 +/- 234 ng/ml and 555 +/- 155 ng/ml, respectively, after BM 06.022. Half-life of BM 06.022-activity was 11.2 +/- 0.4 min and of antigen was 13.9 +/- 0.7 min, followed by a terminal half-life only for antigen of 173 +/- 33 min. Plasma clearance of BM 06.022 was 371 +/- 13 ml/min for activity and 183 +/- 15 ml/min for antigen. Thus, BM 06.022 is not fibrinogenolytic at 6 MU and is a fibrinolytic agent with a longer half-life than t-PA.

Sequential promotion of normal and leukemic hemopoiesis by recombinant human granulocyte colony-stimulating factor during the course of myelodysplastic syndrome.

PubMed

Ueda, T; Kawai, Y; Sugiyama, T; Takeuchi, N; Yoshida, A; Iwasaki, H; Wano, Y; Tsutani, H; Kamada, N; Nakamura, T

1993-12-01

A 48-year-old man developed refractory anemia with excess of blasts in transformation. Complete response was achieved by low-dose ara-C therapy, but he relapsed 15 months later, with pancytopenia and 13.0% myeloblasts in normocellular marrow. He was treated unsuccessfully with prednisolone, metenolone, and 1-alpha-hydroxyvitamin D3 for 8 weeks. He then developed life-threatening pneumonia and was treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF Filgrastim; 125 micrograms/day s.c.). The pneumonia resolved and, interestingly, he achieved a partial response, with normal blood cell counts and only a few dysmyelopoietic cells in the marrow. However, thrombocytopenia progressed when rhG-CSF administration was tapered. When the dose was increased again, leukemic blasts were found to proliferate. When rhG-CSF was discontinued, blasts rapidly decreased in the peripheral blood. Chromosomal analysis revealed a complex abnormality during the first relapse, a normal 46,XY karyotype during the partial response, and recurrence of the same complex abnormality during leukemic transformation. The stimulation index of marrow mononuclear cells cultured with rhG-CSF increased with disease progression. These findings suggest that rhG-CSF initially stimulated the selective proliferation of normal hemopoietic cells, but the evolution or selection of a leukemic clone responsive to rhG-CSF appears to have occurred subsequently.

Mortality Benefit of Recombinant Human Interleukin-1 Receptor Antagonist for Sepsis Varies by Initial Interleukin-1 Receptor Antagonist Plasma Concentration.

PubMed

Meyer, Nuala J; Reilly, John P; Anderson, Brian J; Palakshappa, Jessica A; Jones, Tiffanie K; Dunn, Thomas G; Shashaty, Michael G S; Feng, Rui; Christie, Jason D; Opal, Steven M

2018-01-01

Plasma interleukin-1 beta may influence sepsis mortality, yet recombinant human interleukin-1 receptor antagonist did not reduce mortality in randomized trials. We tested for heterogeneity in the treatment effect of recombinant human interleukin-1 receptor antagonist by baseline plasma interleukin-1 beta or interleukin-1 receptor antagonist concentration. Retrospective subgroup analysis of randomized controlled trial. Multicenter North American and European clinical trial. Five hundred twenty-nine subjects with sepsis and hypotension or hypoperfusion, representing 59% of the original trial population. Random assignment of placebo or recombinant human interleukin-1 receptor antagonist × 72 hours. We measured prerandomization plasma interleukin-1 beta and interleukin-1 receptor antagonist and tested for statistical interaction between recombinant human interleukin-1 receptor antagonist treatment and baseline plasma interleukin-1 receptor antagonist or interleukin-1 beta concentration on 28-day mortality. There was significant heterogeneity in the effect of recombinant human interleukin-1 receptor antagonist treatment by plasma interleukin-1 receptor antagonist concentration whether plasma interleukin-1 receptor antagonist was divided into deciles (interaction p = 0.046) or dichotomized (interaction p = 0.028). Interaction remained present across different predicted mortality levels. Among subjects with baseline plasma interleukin-1 receptor antagonist above 2,071 pg/mL (n = 283), recombinant human interleukin-1 receptor antagonist therapy reduced adjusted mortality from 45.4% to 34.3% (adjusted risk difference, -0.12; 95% CI, -0.23 to -0.01), p = 0.044. Mortality in subjects with plasma interleukin-1 receptor antagonist below 2,071 pg/mL was not reduced by recombinant human interleukin-1 receptor antagonist (adjusted risk difference, +0.07; 95% CI, -0.04 to +0.17), p = 0.230. Interaction between plasma interleukin-1 beta concentration and recombinant human interleukin-1 receptor antagonist treatment was not statistically significant. We report a heterogeneous effect of recombinant human interleukin-1 receptor antagonist on 28-day sepsis mortality that is potentially predictable by plasma interleukin-1 receptor antagonist in one trial. A precision clinical trial of recombinant human interleukin-1 receptor antagonist targeted to septic patients with high plasma interleukin-1 receptor antagonist may be worthy of consideration.

Modulation of the immunogenicity of the Trypanosoma congolense cysteine protease, congopain, through complexation with alpha(2)-macroglobulin.

PubMed

Huson, Laura Elizabeth Joan; Authié, Edith; Boulangé, Alain Francçois; Goldring, James Phillip Dean; Coetzer, Theresa Helen Taillefer

2009-01-01

The protozoan parasite Trypanosoma congolense is the main causative agent of livestock trypanosomosis. Congopain, the major lysosomal cysteine proteinase of T. congolense, contributes to disease pathogenesis, and antibody-mediated inhibition of this enzyme may contribute to mechanisms of trypanotolerance. The potential of different adjuvants to facilitate the production of antibodies that would inhibit congopain activity was evaluated in the present study. Rabbits were immunised with the recombinant catalytic domain of congopain (C2), either without adjuvant, with Freund's adjuvant or complexed with bovine or rabbit alpha(2)-macroglobulin (alpha(2)M). The antibodies were assessed for inhibition of congopain activity. Rabbits immunised with C2 alone produced barely detectable anti-C2 antibody levels and these antibodies had no effect on recombinant C2 or native congopain activity. Rabbits immunised with C2 and Freund's adjuvant produced the highest levels of anti-C2 antibodies. These antibodies either inhibited C2 and native congopain activity to a small degree, or enhanced their activity, depending on time of production after initial immunisation. Rabbits receiving C2-alpha(2)M complexes produced moderate levels of anti-C2 antibodies and these antibodies consistently showed the best inhibition of C2 and native congopain activity of all the antibodies, with maximum inhibition of 65%. Results of this study suggest that antibodies inhibiting congopain activity could be raised in livestock with a congopain catalytic domain-alpha(2)M complex. This approach improves the effectiveness of the antigen as an anti-disease vaccine candidate for African trypanosomosis.

Cloning and Characterization of the Gene Encoding Alpha-Pinene Oxide Lyase Enzyme (Prα-POL) from Pseudomonas rhodesiae CIP 107491 and Production of the Recombinant Protein in Escherichia coli.

PubMed

Dubessay, Pascal; Larroche, Christian; Fontanille, Pierre

2017-12-28

The alpha-pinene oxide lyase (Prα-POL) from Pseudomonas rhodesiae CIP107491 belongs to catabolic alpha-pinene degradation pathway. In this study, the gene encoding Prα-POL has been identified using mapping approach combined to inverse PCR (iPCR) strategy. The Prα-POL gene included a 609-bp open reading frame encoding 202 amino acids and giving rise to a 23.7 kDa protein, with a theoretical isoelectric point (pI) of 5.23. The amino acids sequence analysis showed homologies with those of proteins with unknown function from GammaProteobacteria group. Identification of a conserved domain in amino acid in positions 18 to 190 permitted to classify Prα-POL among the nuclear transport factor 2 (NTF2) protein superfamily. Heterologous expression of Prα-POL, both under its native form and with a histidin tag, was successfully performed in Escherichia coli, and enzymatic kinetics were analyzed. Bioconversion assay using recombinant E. coli strain allowed to reach a rate of isonovalal production per gramme of biomass about 40-fold higher than the rate obtained with P. rhodesiae.

Human alpha 7 acetylcholine receptor: cloning of the alpha 7 subunit from the SH-SY5Y cell line and determination of pharmacological properties of native receptors and functional alpha 7 homomers expressed in Xenopus oocytes.

PubMed

Peng, X; Katz, M; Gerzanich, V; Anand, R; Lindstrom, J

1994-03-01

The alpha-bungarotoxin-binding acetylcholine receptors from the human neuroblastoma cell line SH-SY5Y were found to cross-react with some monoclonal antibodies to alpha 7 subunits of nicotinic acetylcholine receptors from chicken brain. The human alpha 7 subunit cDNA from SH-SY5Y was cloned, revealing 94% amino acid sequence identity to rat alpha 7 subunits and 92% identity to chicken alpha 7 subunits. Native human alpha 7 receptors showed affinities for some ligands similar to those previously observed with native chicken alpha 7 receptors, but for other ligands there were large species-specific differences in binding affinity. These results paralleled properties of alpha 7 homomers expressed in Xenopus oocytes. Human alpha 7 homomers exhibited rapidly desensitizing, inwardly rectifying, agonist-induced, cation currents that triggered Ca(2+)-sensitive Cl- channels in the oocytes. A change in efficacy from partial agonist for chicken alpha 7 homomers to full agonist for human alpha 7 homomers was exhibited by 1,1-dimethyl-4-phenylpiperazinium. This result reveals a large species-specific pharmacological difference, despite small differences in alpha 7 sequences. This is important for understanding the effects of these drugs in humans and for identifying amino acids that may contribute to the acetylcholine binding site, for analysis by in vitro mutagenesis. These results also characterize properties of native alpha 7 receptors and alpha 7 homomers that will provide criteria for functional properties expected of structural subunits, when these can be identified, cloned, and coexpressed with alpha 7 subunits.

Construction of amylolytic industrial brewing yeast strain with high glutathione content for manufacturing beer with improved anti-staling capability and flavor.

PubMed

Wang, Jinjing; Wang, Zhao-Yue; He, Xiu-Ping; Zhang, Bo-Run

2010-11-01

Glutathione in beer works as the main antioxidant compounds which correlates with beer flavor stability. High residual sugars in beer contribute to major non-volatile components which correlate to high caloric content. In this work, Saccharomyces cerevisiae GSH1 gene encoding glutamylcysteine synthetase and Scharomycopsis fibuligera ALP1 gene encoding alpha-amylase were co-expressed in industrial brewing yeast strain Y31 targeting at alpha-acetolactate synthase (AHAS) gene (ILV2) and alcohol dehydrogenase gene (ADH2), and new recombinant strain TY3 was constructed. The glutathione content from the fermentation broth of TY3 increased to 43.83 mg/l compared to 33.34 mg/l from Y31. The recombinant strain showed high alpha-amylase activity and utilized more than 46% of starch after 5 days growing on starch as sole carbon source. European Brewery Convention tube fermentation tests comparing the fermentation broth of TY3 and Y31 showed that the flavor stability index increased to 1.3 fold and residual sugar concentration were reduced by 76.8%, respectively. Due to the interruption of ILV2 gene and ADH2 gene, the amounts of off-flavor compounds diacetyl and acetaldehyde were reduced by 56.93% and 31.25%, comparing with the amounts of these from Y31 fermentation broth. In addition, as no drug-resistance genes were introduced to new recombinant strain, consequently, it should be more suitable for use in beer industry because of its better flavor stability and other beneficial characteristics.

rhEGF-containing thermosensitive and mucoadhesive polymeric sol-gel for endoscopic treatment of gastric ulcer and bleeding.

PubMed

Maeng, Jin Hee; So, Jung Won; Kim, Jungju; Kim, In Ae; Jung, Ji Hoon; Min, Kyunghyun; Lee, Don Haeng; Yang, Su-Geun

2014-03-01

Gastrointestinal endoscopy is a standard diagnostic tool for gastrointestinal ulcers and cancer. In this study, we have developed recombinant human epidermal growth factor-containing ulcer-coating polymeric sol-gel for endoscopic application. Chitosan and pluronic F127 were employed for their thermoresponsive and bioadhesive properties. At temperatures below 21, polymeric sol-gel remains liquid during endoscopic application and transforms to gel at body temperature after application on ulcers. In an in vitro cellular wounding assay, recombinant human epidermal growth factor sol-gel significantly enhanced the cell migration and decreased the wounding area (68%) compared to nontreated, recombinant human epidermal growth factor solution, and sol-gel without recombinant human epidermal growth factor (42, 49, and 32 % decreased at day 1). The in vivo ulcer-healing study was performed in an acetic acid-induced gastric ulcer rat model and proved that our recombinant human epidermal growth factor endoscopic sol-gel facilitated the ulcer-healing process more efficiently than the other treatments. Ulcer sizes in the recombinant human epidermal growth factor sol-gel group were decreased 2.9- and 2.1-fold compared with those in the nontreated group on days 1 and 3 after ulceration, respectively. The mucosal thickness in the recombinant human epidermal growth factor sol-gel group was significantly increased compared to that in the nontreated group (3.2- and 6.9-fold on days 1 and 3 after ulceration, respectively). In a gastric retention study, recombinant human epidermal growth factor sol-gel stayed on the gastric mucosa more than 2 h after application. The present study suggests that recombinant human epidermal growth factor sol-gel is a prospective candidate for treating gastric ulcers via endoscopic application.

Far-Infrared Hydrogen Lasers in the Peculiar Star MWC 349A

NASA Technical Reports Server (NTRS)

Strelnitski, Vladimir; Haas, Michael R.; Smith, Howard A.; Erickson, Edwin F.; Colgan, Sean W. J.; Hollenbach, David J.

1996-01-01

Far-infrared hydrogen recombination lines H15(alpha)(169.4 micrometers), H12(alpha)(88.8 micrometers), and H10(alpha)(52.5 micrometers) were detected in the peculiar luminous star MWC 349A from the Kuiper Airborne Observatory. Here it is shown that at least H15(alpha) is strongly amplified, with the probable amplification factor being greater than or about equal to 10(exp 3) and a brightness temperature that is greater than or about equal to 10(exp 7) kelvin. The other two lines also show signs of amplification, although to a lesser degree. Beyond H10(alpha) the amplification apparently vanishes. The newly detected amplified lines fall into the laser wavelength domain. These lasers, as well as the previously detected hydrogen masers may originate in the photoionized circumstellar disk of MWC 349A and constrain the disk's physics and structure.

Activation of peroxisome proliferator-activated receptor-{alpha} enhances fatty acid oxidation in human adipocytes

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

Lee, Joo-Young; Hashizaki, Hikari; Goto, Tsuyoshi

2011-04-22

Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of adipocyte differentiation marker genes and GPDH activity in human adipocytes. {yields} PPAR{alpha} activation also increased insulin-dependent glucose uptake in human adipocytes. {yields} PPAR{alpha} activation did not affect lipid accumulation in human adipocytes. {yields} PPAR{alpha} activation increased fatty acid oxidation through induction of fatty acid oxidation-related genes in human adipocytes. -- Abstract: Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPAR{alpha} in adipocytes have been unclarified. We examined the functions of PPAR{alpha} using human multipotent adipose tissue-derived stem cells as a humanmore » adipocyte model. Activation of PPAR{alpha} by GW7647, a potent PPAR{alpha} agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPAR{gamma}, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPAR{alpha} activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPAR{gamma} is activated. On the other hand, PPAR{alpha} activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPAR{alpha}-dependent manner. Moreover, PPAR{alpha} activation increased the production of CO{sub 2} and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPAR{alpha} stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPAR{alpha} agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected effects of PPAR{alpha} activation are very valuable for managing diabetic conditions accompanied by obesity, because PPAR{gamma} agonists, usually used as antidiabetic drugs, induce excessive lipid accumulation in adipocytes in addition to improvement of insulin resistance.« less

Crystal structure of human complement protein C8{gamma} at 1.2 {Angstrom} resolution reveals a lipocalin fold and a distinct ligand binding site.

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

Ortlund, E.; Parker, C. L.; Schreck, A. F.

2002-01-01

C8gamma is a 22-kDa subunit of human C8, which is one of five components of the cytolytic membrane attack complex of complement (MAC). C8gamma is disulfide-linked to a C8alpha subunit that is noncovalently associated with a C8beta chain. In the present study, the three-dimensional structure of recombinant C8gamma was determined by X-ray diffraction to 1.2 A resolution. The structure displays a typical lipocalin fold forming a calyx with a distinct binding pocket that is indicative of a ligand-binding function for C8gamma. When compared to other lipocalins, the overall structure is most similar to neutrophil gelatinase associated lipocalin (NGAL), a proteinmore » released from granules of activated neutrophils. Notable differences include a much deeper binding pocket in C8gamma as well as variation in the identity and position of residues lining the pocket. In C8gamma, these residues allow ligand access to a large hydrophobic cavity at the base of the calyx, whereas corresponding residues in NGAL restrict access. This suggests the natural ligands for C8gamma and NGAL are significantly different in size. Cys40 in C8gamma, which forms the disulfide bond to C8alpha, is located in a partially disordered loop (loop 1, residues 38-52) near the opening of the calyx. Access to the calyx may be regulated by movement of this loop in response to conformational changes in C8alpha during MAC formation.« less

Novel Multiplexed Assay for Identifying SH2 Domain Antagonists of STAT Family Proteins

PubMed Central

Takakuma, Kazuyuki; Ogo, Naohisa; Uehara, Yutaka; Takahashi, Susumu; Miyoshi, Nao; Asai, Akira

2013-01-01

Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z’ values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors. PMID:23977103

Novel multiplexed assay for identifying SH2 domain antagonists of STAT family proteins.

PubMed

Takakuma, Kazuyuki; Ogo, Naohisa; Uehara, Yutaka; Takahashi, Susumu; Miyoshi, Nao; Asai, Akira

2013-01-01

Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z' values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.

Generation of an approximately 2.4 Mb human X centromere-based minichromosome by targeted telomere-associated chromosome fragmentation in DT40.

PubMed

Mills, W; Critcher, R; Lee, C; Farr, C J

1999-05-01

A linear mammalian artificial chromosome (MAC) will require at least three types of functional element: a centromere, two telomeres and origins of replication. As yet, our understanding of these elements, as well as many other aspects of structure and organization which may be critical for a fully functional mammalian chromosome, remains poor. As a way of defining these various requirements, minichromosome reagents are being developed and analysed. Approaches for minichromosome generation fall into two broad categories: de novo assembly from candidate DNA sequences, or the fragmentation of an existing chromosome to reduce it to a minimal size. Here we describe the generation of a human minichromosome using the latter, top-down, approach. A human X chromosome, present in a DT40-human microcell hybrid, has been manipulated using homologous recombination and the targeted seeding of a de novo telomere. This strategy has generated a linear approximately 2.4 Mb human X centromere-based minichromosome capped by two artificially seeded telomeres: one immediately flanking the centromeric alpha-satellite DNA and the other targeted to the zinc finger gene ZXDA in Xp11.21. The chromosome retains an alpha-satellite domain of approximately 1. 8 Mb, a small array of gamma-satellite repeat ( approximately 40 kb) and approximately 400 kb of Xp proximal DNA sequence. The mitotic stability of this minichromosome has been examined, both in DT40 and following transfer into hamster and human cell lines. In all three backgrounds, the minichromosome is retained efficiently, but in the human and hamster microcell hybrids its copy number is poorly regulated. This approach of engineering well-defined chromosome reagents will allow key questions in MAC development (such as whether a lower size limit exists) to be addressed. In addition, the 2.4 Mb minichromosome described here has potential to be developed as a vector for gene delivery.

[Construction of the eukaryotic recombinant vector and expression of the outer membrane protein LipL32 gene from Leptospira serovar Lai].

PubMed

Huang, Bi; Bao, Lang; Zhong, Qi; Shang, Zheng-ling; Zhang, Hui-dong; Zhang, Ying

2008-02-01

To construct the eukaryotic experssion vector of LipL32 gene from Leptospira serovar Lai and express the recombinant plasmid in COS-7 cell. The LipL32 gene was amplified from Leptospira strain 017 genomic DNA by PCR and cloned into pcDNA3.1, through restriction nuclease enzyme digestion. Then the recombinant plasmid was transformed into E.coli DH5alpha. After identified by nuclease digestion, PCR and sequencing analysis, the recombinant vector was transfected into COS-7 cell with lipsome. The expression of the target gene was detected by RT-PCR and Western blot. The eukaryotic experssion vector pcDNA3.1-LipL32 was successfully constructed and stably expressed in COS-7 cell. The eukaryotic recombinant vector of outer membrane protein LipL32 gene from Leptospira serovar Lai can be expressed in mammalian cell, which provides an experimental basis for the application of the Leptospira DNA vaccine.

Human and mouse homo-oligomeric meprin A metalloendopeptidase: substrate and inhibitor specificities.

PubMed

Bylander, John E; Bertenshaw, Greg P; Matters, Gail L; Hubbard, Simon J; Bond, Judith S

2007-11-01

Meprin metalloproteinases have been implicated in the susceptibility to and progression of diabetic nephropathy and inflammatory bowel diseases. Our studies with experimental models of these diseases in mice are congruent with the conclusion that meprins modulate the inflammatory responses and tissue damage. To determine whether the mouse and human enzymes differ, recombinant forms of meprin A from the two species were compared with respect to structure, substrates and inhibitors. Human homo-oligomeric meprin A formed oligomers ranging from 950,000 to 1,500,000 Da vs. 900,000 Da for mouse meprin A. Human and mouse meprin A exhibited similar activity against azocasein, fibronectin, collagen IV, and peptides such as parathyroid hormone, ghrelin, and gastrin-releasing peptide. The human enzyme had lower activity against gelatin, bradykinin, alpha-melanocyte-stimulating hormone and neurotensin, and higher activity against secretin and orcokinin. Human meprin A showed a preference for acidic residues in the P1' position of the substrate, unlike mouse meprin A. Several metalloproteinase inhibitors had IC(50) values in the nanomolar range, but potency ranged from similar values to a difference of several orders of magnitude for meprins from the two species. This work provides valuable data to improve predictability for human systems based on meprin functions in mouse models.

Role of tolerogen conformation in induction of oral tolerance in experimental autoimmune myasthenia gravis.

PubMed

Im, S H; Barchan, D; Souroujon, M C; Fuchs, S

2000-10-01

We recently demonstrated that oral or nasal administration of recombinant fragments of the acetylcholine receptor (AChR) prevents the induction of experimental autoimmune myasthenia gravis (EAMG) and suppresses ongoing EAMG in rats. We have now studied the role of spatial conformation of these recombinant fragments in determining their tolerogenicity. Two fragments corresponding to the extracellular domain of the human AChR alpha-subunit and differing in conformation were tested: Halpha1-205 expressed with no fusion partner and Halpha1-210 fused to thioredoxin (Trx), and designated Trx-Halpha1-210. The conformational similarity of the fragments to intact AChR was assessed by their reactivity with alpha-bungarotoxin and with anti-AChR mAbs, specific for conformation-dependent epitopes. Oral administration of the more native fragment, Trx-Halpha1-210, at the acute phase of disease led to exacerbation of EAMG, accompanied by an elevation of AChR-specific humoral and cellular reactivity, increased levels of Th1-type cytokines (IL-2, IL-12), decreased levels of Th2 (IL-10)- or Th3 (TGF-beta)-type cytokines, and higher expression of costimulatory factors (CD28, CTLA4, B7-1, B7-2, CD40L, and CD40). On the other hand, oral administration of the less native fragments Halpha1-205 or denatured Trx-Halpha1-210 suppressed ongoing EAMG and led to opposite changes in the immunological parameters. It thus seems that native conformation of AChR-derived fragments renders them immunogenic and immunopathogenic and therefore not suitable for treatment of myasthenia gravis. Conformation of tolerogens should therefore be given careful attention when considering oral tolerance for treatment of autoimmune diseases.

Interactome analysis of the lymphocytic choriomeningitis virus nucleoprotein in infected cells reveals ATPase Na+/K+ transporting subunit Alpha 1 and prohibitin as host-cell factors involved in the life cycle of mammarenaviruses

PubMed Central

Iwasaki, Masaharu; Caì, Yíngyún; de la Torre, Juan C.

2018-01-01

Several mammalian arenaviruses (mammarenaviruses) cause hemorrhagic fevers in humans and pose serious public health concerns in their endemic regions. Additionally, mounting evidence indicates that the worldwide-distributed, prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), is a neglected human pathogen of clinical significance. Concerns about human-pathogenic mammarenaviruses are exacerbated by of the lack of licensed vaccines, and current anti-mammarenavirus therapy is limited to off-label use of ribavirin that is only partially effective. Detailed understanding of virus/host-cell interactions may facilitate the development of novel anti-mammarenavirus strategies by targeting components of the host-cell machinery that are required for efficient virus multiplication. Here we document the generation of a recombinant LCMV encoding a nucleoprotein (NP) containing an affinity tag (rLCMV/Strep-NP) and its use to capture the NP-interactome in infected cells. Our proteomic approach combined with genetics and pharmacological validation assays identified ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1) and prohibitin (PHB) as pro-viral factors. Cell-based assays revealed that ATP1A1 and PHB are involved in different steps of the virus life cycle. Accordingly, we observed a synergistic inhibitory effect on LCMV multiplication with a combination of ATP1A1 and PHB inhibitors. We show that ATP1A1 inhibitors suppress multiplication of Lassa virus and Candid#1, a live-attenuated vaccine strain of Junín virus, suggesting that the requirement of ATP1A1 in virus multiplication is conserved among genetically distantly related mammarenaviruses. Our findings suggest that clinically approved inhibitors of ATP1A1, like digoxin, could be repurposed to treat infections by mammarenaviruses pathogenic for humans. PMID:29462184

High level expression, purification and physico- and immunochemical characterisation of recombinant Pen a 1: a major allergen of shrimp.

PubMed

Albrecht, Melanie; Alessandri, Stefano; Conti, Amedeo; Reuter, Andreas; Lauer, Iris; Vieths, Stefan; Reese, Gerald

2008-11-01

Well-characterised and immunologically active recombinant allergens are of eminent importance for improvement of diagnostic tools and immunotherapy of allergic diseases. The use of recombinant allergens has several advantages such as the more precise quantification of the active substance compared to allergen extracts and the reduced risk of contamination with other allergenic proteins compared to purified natural allergens. Optimised standard protocols for expression and purification and a detailed physico-chemical characterisation of such recombinant allergens are necessary to ensure consistent quality and comparability of results obtained with recombinant material. In this study the major allergen Pen a 1 of brown shrimp (Penaeus aztecus) was expressed in E. coli and purified in two steps by immobilised metal chelate-affinity chromatography (IMAC) and size-exclusion chromatography. Identity and purity were verified with N-terminal sequencing and peptide mass fingerprinting. Circular dichroism and NMR-spectroscopy indicated an alpha-helical flexible structure of rPen a 1 which is in accordance with the known structure of tropomyosins. Finally, the recombinant allergen proved to be immunologically reactive in IgE Western blot analysis and ELISA. This study provides a protocol for the preparation of recombinant shrimp tropomyosin in standardised quality.

Heterologous mitochondrial DNA recombination in human cells.

PubMed

D'Aurelio, Marilena; Gajewski, Carl D; Lin, Michael T; Mauck, William M; Shao, Leon Z; Lenaz, Giorgio; Moraes, Carlos T; Manfredi, Giovanni

2004-12-15

Inter-molecular heterologous mitochondrial DNA (mtDNA) recombination is known to occur in yeast and plants. Nevertheless, its occurrence in human cells is still controversial. To address this issue we have fused two human cytoplasmic hybrid cell lines, each containing a distinct pathogenic mtDNA mutation and specific sets of genetic markers. In this hybrid model, we found direct evidence of recombination between these two mtDNA haplotypes. Recombinant mtDNA molecules in the hybrid cells were identified using three independent experimental approaches. First, recombinant molecules containing genetic markers from both parental alleles were demonstrated with restriction fragment length polymorphism of polymerase chain reaction products, by measuring the relative frequencies of each marker. Second, fragments of recombinant mtDNA were cloned and sequenced to identify the regions involved in the recombination events. Finally, recombinant molecules were demonstrated directly by Southern blot using appropriate combinations of polymorphic restriction sites and probes. This combined approach confirmed the existence of heterogeneous species of recombinant mtDNA molecules in the hybrid cells. These findings have important implications for mtDNA-related diseases, the interpretation of human evolution and population genetics and forensic analyses based on mtDNA genotyping.

Essential components for ex vivo proliferation of mesenchymal stromal cells.

PubMed

Fekete, Natalie; Rojewski, Markus Thomas; Lotfi, Ramin; Schrezenmeier, Hubert

2014-02-01

Mesenchymal stromal cells (MSCs) are highly interesting candidates for clinical applications in regenerative medicine. Due to their low occurrence in human tissues, extensive in vitro expansion is necessary to obtain sufficient cell numbers applicable as a clinical dose in the context of cellular therapy. Current cell culture media formulations for the isolation and expansion of MSCs include fetal calf serum (FCS), human AB serum (ABS), or human platelet lysate (PL) as a supplement. However, these established supplements are inherently ill-defined formulations that contain a variety of bioactive molecules in varying batch-to-batch compositions and the risk of transmitting pathogens that escape routine screening procedures. In this study, we have comparatively characterized the capacity of commonly used basal media, such as the Minimum Essential Medium alpha (αMEM), Dulbecco's modified Eagle's medium (DMEM), Iscove's Modified Dulbecco's Medium (IMDM), and RPMI 1640 as well as human- and animal-derived supplements, that is, PL, ABS, and FCS to stimulate cell proliferation. MSC proliferation was observed to be optimal in the PL-supplemented αMEM. Using a combinatorial approach, we then assessed a library of soluble factors, including mitogens (TGF-β1, Activin A, bFGF, EGF, IGF-I, PDGF-BB, and VEGF), chemokines (CCL21, CCL25, CXCL12, and RANTES), proteins (human serum albumin), lipids (e.g., oleic acid, linoleic acid, and arachidonic acid), and hormones (dexamethasone, insulin, and TSH), to create a defined medium as well as coating of cell culture surfaces to promote robust MSC proliferation in vitro. A combination of recombinant human factors partially met the nutritional requirements of bone marrow-derived MSCs, and was able to promote cell proliferation comparable to about 5% PL if supplemented with auxiliary 0.6%-1.2% PL. Maximal MSC proliferation was achieved by combining 5% PL with a cocktail of recombinant factors and did not depend on coating of cell culture surfaces.

Inhibition of human mast cell growth and differentiation by interferon gamma-1b.

PubMed

Kirshenbaum, A S; Worobec, A S; Davis, T A; Goff, J P; Semere, T; Metcalfe, D D

1998-03-01

In an effort to identify cytokines that inhibit human mast cell growth, we cultured HMC-1 cells and recombinant human stem cell factor (rhSCF)-dependent human bone marrow-derived mast cells (HBMCs) in the presence of interferon gamma (IFNgamma)-1b and interferon alpha (IFNalpha)-2b. HMC-1 cell numbers decreased in the presence of 1000 U/mL IFNgamma-1b but were unaffected by 1000 U/mL of IFNalpha-2b. HBMCs were then cultured for 0 to 7 days with 100 ng/mL rhSCF and 10 ng/mL recombinant human IL-3 (rhIL-3), followed by culture in rhSCF and administration of either 1000 U/mL IFNalpha-2b or 1000 U/mL IFNgamma-1b. HBMCs appearing in cultures with rhSCF alone or in combination with IFNalpha-2b were virtually identical in number through 8 weeks of culture. In cultures supplemented with IFNgamma-1b, HBMCs significantly decreased in number and incidence of granular metachromasia by 4 to 5 weeks (p<0.001). Similar results were obtained when human marrow was cultured from day 0 with rhSCF and IFNgamma-1b. Mature rhSCF-dependent HBMCs were also cultured at 5 weeks with rhSCF alone or in combination with IFNgamma-1b. Compared with cells cultured in rhSCF, mature 5-week HBMC cultures treated with rhSCF plus IFNgamma-1b revealed a decrease in mast cells, and those mast cells that remained had fewer toluidine blue- and tryptase-positive granules after 5 to 8 weeks. FACS analysis of rhSCF plus IFNgamma-1b-treated mature HBMCs revealed increased c-kit and Fc(epsilon)RI expression. Mast cell releasibility was not increased. IFNgamma-lb was thus able to suppress mast cell growth from CD34+ cells, suggesting that this agent should be considered as a candidate cytokine for the treatment of disorders of mast cell proliferation.

High-Resolution Patterns of Meiotic Recombination across the Human Major Histocompatibility Complex

PubMed Central

Cullen, Michael; Perfetto, Stephen P.; Klitz, William; Nelson, George; Carrington, Mary

2002-01-01

Definitive characteristics of meiotic recombination events over large (i.e., >1 Mb) segments of the human genome remain obscure, yet they are essential for establishing the haplotypic structure of the genome and for efficient mapping of complex traits. We present a high-resolution map of recombination at the kilobase level across a 3.3-Mb interval encompassing the major histocompatibility complex (MHC). Genotyping of 20,031 single sperm from 12 individuals resulted in the identification and fine mapping of 325 recombinant chromosomes within genomic intervals as small as 7 kb. Several principal characteristics of recombination in this region were observed: (1) rates of recombination can differ significantly between individuals; (2) intense hot spots of recombination occur at least every 0.8 Mb but are not necessarily evenly spaced; (3) distribution in the location of recombination events can differ significantly among individuals; (4) between hot spots, low levels of recombination occur fairly evenly across 100-kb segments, suggesting the presence of warm spots of recombination; and (5) specific sequence motifs associate significantly with recombination distribution. These data provide a plausible model for recombination patterns of the human genome overall. PMID:12297984

Brucella β 1,2 Cyclic Glucan Is an Activator of Human and Mouse Dendritic Cells

PubMed Central

Martirosyan, Anna; Pérez-Gutierrez, Camino; Banchereau, Romain; Dutartre, Hélène; Lecine, Patrick; Dullaers, Melissa; Mello, Marielle; Pinto Salcedo, Suzana; Muller, Alexandre; Leserman, Lee; Levy, Yves; Zurawski, Gerard; Zurawski, Sandy; Moreno, Edgardo; Moriyón, Ignacio; Klechevsky, Eynav; Banchereau, Jacques; Oh, SangKon; Gorvel, Jean-Pierre

2012-01-01

Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella β 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella β 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8+ T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4+ and CD8+ T cell responses including cross-presentation by different human DC subsets. Brucella β 1,2 cyclic glucans increased the memory CD4+ T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies. PMID:23166489

SAM domain-dependent activity of PfTKL3, an essential tyrosine kinase-like kinase of the human malaria parasite Plasmodium falciparum.

PubMed

Abdi, Abdirahman; Eschenlauer, Sylvain; Reininger, Luc; Doerig, Christian

2010-10-01

Over the last decade, several protein kinases inhibitors have reached the market for cancer chemotherapy. The kinomes of pathogens represent potentially attractive targets in infectious diseases. The functions of the majority of protein kinases of Plasmodium falciparum, the parasitic protist responsible for the most virulent form of human malaria, remain unknown. Here we present a thorough characterisation of PfTKL3 (PF13_0258), an enzyme that belongs to the tyrosine kinase-like kinase (TKL) group. We demonstrate by reverse genetics that PfTKL3 is essential for asexual parasite proliferation in human erythrocytes. PfTKL3 is expressed in both asexual and gametocytes stages, and in the latter the protein co-localises with cytoskeleton microtubules. Recombinant PfTKL3 displays in vitro autophosphorylation activity and is able to phosphorylate exogenous substrates, and both activities are dramatically dependent on the presence of an N-terminal "sterile alpha-motif" domain. This study identifies PfTKL3 as a validated drug target amenable to high-throughput screening.

Recombination of mitochondrial DNA in skeletal muscle of individuals with multiple mitochondrial DNA heteroplasmy.

PubMed

Zsurka, Gábor; Kraytsberg, Yevgenia; Kudina, Tatiana; Kornblum, Cornelia; Elger, Christian E; Khrapko, Konstantin; Kunz, Wolfram S

2005-08-01

Experimental evidence for human mitochondrial DNA (mtDNA) recombination was recently obtained in an individual with paternal inheritance of mtDNA and in an in vitro cell culture system. Whether mtDNA recombination is a common event in humans remained to be determined. To detect mtDNA recombination in human skeletal muscle, we analyzed the distribution of alleles in individuals with multiple mtDNA heteroplasmy using single-cell PCR and allele-specific PCR. In all ten individuals who carried a heteroplasmic D-loop mutation and a distantly located tRNA point mutation or a large deletion, we observed a mixture of four allelic combinations (tetraplasmy), a hallmark of recombination. Twelve of 14 individuals with closely located heteroplasmic D-loop mutation pairs contained a mixture of only three types of mitochondrial genomes (triplasmy), consistent with the absence of recombination between adjacent markers. These findings indicate that mtDNA recombination is common in human skeletal muscle.

The role of DNA repair in herpesvirus pathogenesis.

PubMed

Brown, Jay C

2014-10-01

In cells latently infected with a herpesvirus, the viral DNA is present in the cell nucleus, but it is not extensively replicated or transcribed. In this suppressed state the virus DNA is vulnerable to mutagenic events that affect the host cell and have the potential to destroy the virus' genetic integrity. Despite the potential for genetic damage, however, herpesvirus sequences are well conserved after reactivation from latency. To account for this apparent paradox, I have tested the idea that host cell-encoded mechanisms of DNA repair are able to control genetic damage to latent herpesviruses. Studies were focused on homologous recombination-dependent DNA repair (HR). Methods of DNA sequence analysis were employed to scan herpesvirus genomes for DNA features able to activate HR. Analyses were carried out with a total of 39 herpesvirus DNA sequences, a group that included viruses from the alpha-, beta- and gamma-subfamilies. The results showed that all 39 genome sequences were enriched in two or more of the eight recombination-initiating features examined. The results were interpreted to indicate that HR can stabilize latent herpesvirus genomes. The results also showed, unexpectedly, that repair-initiating DNA features differed in alpha- compared to gamma-herpesviruses. Whereas inverted and tandem repeats predominated in alpha-herpesviruses, gamma-herpesviruses were enriched in short, GC-rich initiation sequences such as CCCAG and depleted in repeats. In alpha-herpesviruses, repair-initiating repeat sequences were found to be concentrated in a specific region (the S segment) of the genome while repair-initiating short sequences were distributed more uniformly in gamma-herpesviruses. The results suggest that repair pathways are activated differently in alpha- compared to gamma-herpesviruses. Copyright © 2014. Published by Elsevier Inc.

Recombinant guinea pig CCL5 (RANTES) differentially modulates cytokine production in alveolar and peritoneal macrophages.

PubMed

Skwor, Troy A; Cho, Hyosun; Cassidy, Craig; Yoshimura, Teizo; McMurray, David N

2004-12-01

The CC chemokine ligand 5 (CCL5; regulated on activation, normal T expressed and secreted) is known to recruit and activate leukocytes; however, its role in altering the responses of host cells to a subsequent encounter with a microbial pathogen has rarely been studied. Recombinant guinea pig (rgp)CCL5 was prepared, and its influence on peritoneal and alveolar macrophage activation was examined by measuring cytokine and chemokine mRNA expression in cells stimulated with rgpCCL5 alone or exposed to rgpCCL5 prior to lipopolysaccharide (LPS) stimulation. Levels of mRNA for guinea pig tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, CCL2 (monocyte chemoattractant protein-1), and CXC chemokine ligand 8 (IL-8) were analyzed by reverse transcription followed by real-time polymerase chain reaction analysis using SYBR Green. Bioactive TNF-alpha protein concentration was measured using the L929 bioassay. Both macrophage populations displayed significant enhancement of all the genes and TNF-alpha protein levels when stimulated with rgpCCL5, except for CCL2 in alveolar macrophages. When peritoneal or alveolar macrophages were pretreated with rgpCCL5 for 2 h and then exposed to low concentrations of LPS, diminished cytokine and chemokine mRNA levels were apparent at 6 h compared with LPS alone. At the protein level, there was a reduction in TNF-alpha protein at 6 h in the CCL5-pretreated cells compared with LPS alone. These results further support a role for CCL5 in macrophage activation in addition to chemotactic properties and suggest a role in regulating the inflammatory response to LPS in the guinea pig by modulating the production of proinflammatory cytokines by macrophages.

The time course of follicle-stimulating hormone suppression by recombinant human inhibin A in the adult male rhesus monkey (Macaca mulatta).

PubMed

Ramaswamy, S; Pohl, C R; McNeilly, A S; Winters, S J; Plant, T M

1998-08-01

In higher primates, FSH secretion appears to be regulated by a control system consistent with that described by the classical inhibin hypothesis. The purpose of the present experiment was to examine the time course of inhibin's action to suppress FSH secretion in the intact adult male rhesus monkey. To this end, five adult males implanted with indwelling venous catheters and exhibiting typical episodic patterns of LH and testosterone (T) secretion received a 4-day i.v. infusion of recombinant human (rh) inhibin A (832 ng/h x kg) followed, after a 4-week interval, by vehicle infusion of similar duration. Changes in circulating FSH concentrations during the inhibin and vehicle infusions were determined using a sensitive homologous macaque RIA, whereas enzyme-linked immunosorbent assays were employed to track inhibin A, inhibin B, and inhibin pro-alpha-C levels during the experiment. Normal pulsatile activity in the hypothalamic-pituitary-Leydig cell axis was confirmed by monitoring changes in circulating concentrations of LH and T in 12-h windows of sequential blood collection (1200-2400 h; every 20 min) before, during, and after the rh inhibin A and vehicle infusions. Although infusion of rh inhibin A, which led to a 12 ng/ml square wave increment in circulating levels of this inhibin dimer, produced a marked decline in circulating FSH concentrations, significant suppression of the secretion of this gonadotropin was not manifest until 54 h after initiation of the infusion. Despite the marked decline in FSH secretion during the last 24 h of the 4-day infusion of recombinant hormone, circulating inhibin B and pro-alpha-C concentrations were maintained at preinfusion control levels (1 ng/ml). The finding that imposition of an exaggerated circulating inhibin signal led to suppression of FSH secretion in the male monkey only after 2 days of exposure to the hormone indicates that in this species the feedback action of testicular inhibin on FSH secretion is heavily lagged. Moreover, as the decrease in FSH did not lead to changes in native inhibin secretion, it seems reasonable to propose that the FSH-inhibin feedback loop that governs testicular function in higher primates operates with considerable hysteresis at both the pituitary and gonadal levels. The failure of dramatically elevated inhibin A levels to influence the pulsatile secretion of LH in the monkey reinforces the idea that in this species the pituitary action of testicular inhibin is specific for FSH and does not involve modulation of GnRH receptor levels.

The effects of SB 216469, an antagonist which discriminates between the alpha 1A-adrenoceptor and the human prostatic alpha 1-adrenoceptor.

PubMed Central

Chess-Williams, R.; Chapple, C. R.; Verfurth, F.; Noble, A. J.; Couldwell, C. J.; Michel, M. C.

1996-01-01

1. The affinity of the alpha 1-adrenoceptor antagonist SB 216469 (also known as REC 15/2739) has been determined at native and cloned alpha 1-adrenoceptor subtypes by radioligand binding and at functional alpha 1-adrenoceptor subtypes in isolated tissues. 2. In radioligand binding studies with [3H]-prazosin, SB 216469 had a high affinity at the alpha 1A-adrenoceptors of the rat cerebral cortex and kidney (9.5-9.8) but a lower affinity at the alpha 1B-adrenoceptors of the rat spleen and liver (7.7-8.2). 3. At cloned rat alpha 1-adrenoceptor subtypes transiently expressed in COS-1 cells and also at cloned human alpha 1-adrenoceptor subtypes stably transfected in Rat-1 cells, SB 216469 exhibited a high affinity at the alpha 1a-adrenoceptors (9.6-10.4) with a significantly lower affinity at the alpha 1b-adrenoceptor (8.0-8.4) and an intermediate affinity at the alpha 1d-adrenoceptor (8.7-9.2). 4. At functional alpha 1-adrenoceptors, SB 216469 had a similar pharmacological profile, with a high affinity at the alpha 1A-adrenoceptors of the rat vas deferens and anococcygeus muscle (pA2 = 9.5-10.0), a low affinity at the alpha 1B-adrenoceptors of the rat spleen (6.7) and guinea-pig aorta (8.0), and an intermediate affinity at the alpha 1D-adrenoceptors of the rat aorta (8.8). 5. Several recent studies have concluded that the alpha 1-adrenoceptor present in the human prostate has the pharmacological characteristics of the alpha 1A-adrenoceptor subtype. However, the affinity of SB 216469 at human prostatic alpha 1-adrenoceptors (pA2 = 8.1) determined in isolated tissue strips, was significantly lower than the values obtained at either the cloned alpha 1a-adrenoceptors (human, rat, bovine) or the native alpha 1A-adrenoceptors in radioligand binding and functional studies in the rat. 6. Our results with SB 216469, therefore, suggest that the alpha 1-adrenoceptor mediating contractile responses of the human prostate has properties which distinguish it from the cloned alpha 1a-adrenoceptor or native alpha 1A-adrenoceptor. Since it has previously been shown that the receptor is not the alpha 1B- or alpha 1D-adrenoceptor, the functional alpha 1-adrenoceptor of the human prostate may represent a novel receptor with properties which differ from any of the alpha 1-adrenoceptors currently defined by pharmacological means. PMID:8937710

Neuroendocrine mediators up-regulate alpha1b- and alpha1d-adrenergic receptor subtypes in human monocytes.

PubMed

Rouppe van der Voort, C; Kavelaars, A; van de Pol, M; Heijnen, C J

1999-03-01

Beta2- and alpha2-adrenergic receptors (AR) are thought to be the main AR subtypes to exert the effects of catecholamines on the immune system. However, in the present study, we demonstrate that another subtype of AR can be induced in human monocytes. Expression of alpha1b- and alpha1d-AR mRNA can be obtained by culturing freshly isolated human peripheral blood monocytes with the neuroendocrine mediators dexamethasone or the beta2-AR agonist terbutaline. Using the human monocytic cell line THP-1, we demonstrate that increased levels of alpha1b- and alpha1d-mRNA are accompanied by increased levels of receptor protein as determined by Western blot analysis and radioligand binding assays. This study describes for the first time regulated expression of alpha1-AR subtypes in human monocytes.

[Cloning and sequence analysis of recombinant fusion gene of Escherichia coli heat-liable enterotoxin B subunit and Actinobacillus actinomycetemcomitans fimbria associative protein].

PubMed

Li, Yi; Sun, Hong-chen; Guo, Xue-jun; Feng, Shu-zhang

2005-02-01

To clone the recombinant fusion gene of Escherichia coli heat-liable enterotoxin B subunit (Ltb) and Actinobacillus actinomycetemcomitans fimbria associative protein (Fap). Two couples of primers were designed for PCR according to the known sequence of ltb and fap. The ltb and fap gene were obtained by amplification PCR technique from plasmid EWD299 of Escherichia coli and Actinobacillus actinomycetemcomitans 310 DNA respectively, and fused them by PCR. The fusion gene ltb-fap were cloning into plasmid pET28a(+). The recombined plasmid pET28a ltb-fap was transformed into Escherichia coli DH5alpha. The recombinant was screened and identified by restriction enzyme and PCR. The cloned gene was sequenced. The ltb-fap about 531bp in size was obtained successfully, and identified by PCR, restrictive enzyme and sequence analysis. The vector of pET28a ltb-fap was obtained.

Activation of orphan nuclear constitutive androstane receptor requires subnuclear targeting by peroxisome proliferator-activated receptor gamma coactivator-1 alpha. A possible link between xenobiotic response and nutritional state.

PubMed

Shiraki, Takuma; Sakai, Noriko; Kanaya, Eiko; Jingami, Hisato

2003-03-28

In contrast to the classical nuclear receptors, the constitutive androstane receptor (CAR) is transcriptionally active in the absence of ligand. In the course of searching for the mediator of CAR activation, we found that ligand-independent activation of CAR was achieved in cooperation with the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha). PGC-1 beta, a PGC-1 alpha homologue, also activated CAR to less of an extent than PGC-1 alpha. Coexpression of the ligand-binding domain of a heterodimerization partner, retinoid X receptor alpha, enhanced the PGC-1 alpha-mediated activation of CAR, although it had a weak effect on the basal activity of CAR in the absence of PGC-1 alpha. Both the N-terminal region, with the LXXLL motif, and the C-terminal region, with a serine/arginine-rich domain (RS domain), in PGC-1 alpha were required for full activation of CAR. Pull-down experiments using recombinant proteins revealed that CAR directly interacted with both the LXXLL motif and the RS domain. Furthermore, we demonstrated that the RS domain of PGC-1 alpha was required for CAR localization at nuclear speckles. These results indicate that PGC-1 alpha mediates the ligand-independent activation of CAR by means of subnuclear targeting through the RS domain of PGC-1 alpha.

Regulated expression of the rat recombinant P2X(3) receptor in stably transfected CHO-K1 tTA cells.

PubMed

Lachnit, W G; Oglesby, I B; Gever, J R; Gever, M; Huang, C; Li, X C; Jin, H; McGivern, J G; Ford, A P

2000-07-03

In this report, the regulatable expression by tetracycline of the rat recombinant P2X(3) receptor in stably transfected Chinese hamster ovary (CHO-K1) expressing the tetracycline-controlled transactivator (tTA) is described. cDNA encoding the rat P2X(3)-receptor was subcloned into pTRE (a tetracycline-repressible expression vector) which was used to transfect stably CHO-K1 tTA cells. Using whole cell patch clamp techniques, 100 microM ATP evoked inward currents of 2.9+/-1.6 nA in transfected cells grown in the absence of tetracycline (tet-). The P2X(3) receptor protein was detectable by immunoblot as early as 24 h and protein expression levels continued to increase as much as 192 h following activation of tTA by the removal of the antibiotic. Saturation binding isotherms using [35S]ATP gamma S yielded a pK(d) of 8.2+/-0.1 and a B(max) of 31.9+/-3.5 pmol/mg protein in tet- cell membranes and a pK(d) of 8.1+/-0.1 and a B(max) of 5.8+/-0.8 pmol/mg protein in tet+ cell membranes. The agonist ligands 2MeSATP and alpha beta MeATP displaced the binding of [35S]ATP gamma S in tet- cell membranes with very high affinity, yielding pIC(50) values of 9.4+/-0.2 and 7.5+/-0. 2, respectively. In tet+ cell membrane, displacement of [35S]ATP gamma S by 2MeSATP and alpha beta MeATP was of much lower affinity (pIC(50) values of 7.8 and 6.2, respectively). ATP, ADP and UTP showed similar displacement of [35S]ATP gamma S binding in tet- and tet+ cell membranes. In other experiments, cytosolic Ca(2+) was monitored using the fluorescent indicator, fluo-3. Increases in cytosolic Ca(2+) were elicited by 100 nM alpha beta MeATP in tet- cells while no increases in cytosolic Ca(2+) were detected below 100 microM alpha beta MeATP in either tet+ cells or untransfected cells. These calcium responses to alpha beta MeATP had a pEC(50) of 6.7 and were transient, returning to baseline within 120 s. Suramin produced concentration-dependent, parallel, dextral shifts of E/[A] curves to alpha beta MeATP yielding a pK(B) of 5.6. PPADS produced non-parallel, dextral shifts of E/[A] curves to alpha beta MeATP which were insurmountable. These results show for the first time, expression of a functional, homomeric recombinant rat P2X(3) receptor which is under regulated expression in a stably transfected mammalian cell line.

Producing recombinant human milk proteins in the milk of livestock species.

PubMed

Bösze, Zsuzsanna; Baranyi, Mária; Whitelaw, C Bruce A

2008-01-01

Recombinant human proteins produced by the mammary glands of genetically modified transgenic livestock mammals represent a special aspect of milk bioactive components. For therapeutic applications, the often complex posttranslational modifications of human proteins should be recapitulated in the recombinant products. Compared to alternative production methods, mammary gland production is a viable option, underlined by a number of transgenic livestock animal models producing abundant biologically active foreign proteins in their milk. Recombinant proteins isolated from milk have reached different phases of clinical trials, with the first marketing approval for human therapeutic applications from the EMEA achieved in 2006.

Ultrastructural studies of human and rabbit alpha-M-globulins.

PubMed

Bloth, B; Chesebro, B; Svehag, S E

1968-04-01

Electron micrographs of isolated human alpha(2)M-molecules, obtained by the negative contrast technique, revealed morphologically homogenous structures resembling a graceful monogram of the two letters H and I. The modal values for the length and width of the alpha(2)M particles were 170 A and 100 A, respectively. Purified rabbit alphamacroglobulins contained about 80% alpha(1)M- and 20% alpha(2)M-globulins. The isolated rabbit alpha(1)M- and alpha(2)M-molecules were morphologically indistinguishable from one another and from human alpha(2)M-molecules. Preliminary immunoprecipitation studies demonstrated that the two rabbit alphaM-globulins were antigenically different. Sedimentation constant determinations gave s(20, w) values of 18.8 and 18.2 for rabbit alpha(1)M and alpha(2)M, respectively.

Effectiveness of Recombinant Human Growth Hormone for Pharyngocutaneous Fistula Closure.

PubMed

Kucuk, Nurten; Sari, Murat; Midi, Ahmet; Yumusakhuylu, Ali Cemal; Findik, Ozan; Binnetoglu, Adem

2015-12-01

In laryngeal cancer, which comprises 25% of head and neck cancer, chemotherapy has come into prominence with the increase in organ-protective treatments. With such treatment, salvage surgery has increased following recurrence; the incidence of pharyngocutaneous fistula has also increased in both respiratory and digestive system surgery. We investigated the effects of recombinant human growth hormone on pharyngocutaneous fistula closure in Sprague-Dawley rats, based on an increase in amino acid uptake and protein synthesis for wound healing, an increase in mitogenesis, and enhancement of collagen formation by recombinant human growth hormone. This study was experimental animal study. Forty Sprague-Dawley rats were separated into two groups, and pharyngoesophagotomy was performed. The pharyngoesophagotomy was sutured with vicryl in both groups. Rats in group 1 (control group) received no treatment, while those in group 2 were administered a subcutaneous injection of recombinant human growth hormone daily. On day 14, the pharynx, larynx, and upper oesophagus were excised and examined microscopically. Pharyngocutaneous fistula exhibited better closure macroscopically in the recombinant human growth hormone group. There was a significant difference in collagen formation and epithelisation in the recombinant human growth hormone group compared to the control group. This study is believed to be the first in which the effect of recombinant human growth hormone on pharyngocutaneous fistula closure was evaluated, and the findings suggest the potential of use of growth hormone for treatment of pharyngocutaneous fistula.

Specific Modifications of Histone Tails, but Not DNA Methylation, Mirror the Temporal Variation of Mammalian Recombination Hotspots

PubMed Central

Zeng, Jia; Yi, Soojin V.

2014-01-01

Recombination clusters nonuniformly across mammalian genomes at discrete genomic loci referred to as recombination hotspots. Despite their ubiquitous presence, individual hotspots rapidly lose their activities, and the molecular and evolutionary mechanisms underlying such frequent hotspot turnovers (the so-called “recombination hotspot paradox”) remain unresolved. Even though some sequence motifs are significantly associated with hotspots, multiple lines of evidence indicate that factors other than underlying sequences, such as epigenetic modifications, may affect the evolution of recombination hotspots. Thus, identifying epigenetic factors that covary with recombination at fine-scale is a promising step for this important research area. It was previously reported that recombination rates correlate with indirect measures of DNA methylation in the human genome. Here, we analyze experimentally determined DNA methylation and histone modification of human sperms, and show that the correlation between DNA methylation and recombination in long-range windows does not hold with respect to the spatial and temporal variation of recombination at hotspots. On the other hand, two histone modifications (H3K4me3 and H3K27me3) overlap extensively with recombination hotspots. Similar trends were observed in mice. These results indicate that specific histone modifications rather than DNA methylation are associated with the rapid evolution of recombination hotspots. Furthermore, many human recombination hotspots occupy “bivalent” chromatin regions that harbor both active (H3K4me3) and repressive (H3K27me3) marks. This may explain why human recombination hotspots tend to occur in nongenic regions, in contrast to yeast and Arabidopsis hotspots that are characterized by generally active chromatins. Our results highlight the dynamic epigenetic underpinnings of recombination hotspot evolution. PMID:25326136

Specific modifications of histone tails, but not DNA methylation, mirror the temporal variation of mammalian recombination hotspots.

PubMed

Zeng, Jia; Yi, Soojin V

2014-10-16

Recombination clusters nonuniformly across mammalian genomes at discrete genomic loci referred to as recombination hotspots. Despite their ubiquitous presence, individual hotspots rapidly lose their activities, and the molecular and evolutionary mechanisms underlying such frequent hotspot turnovers (the so-called "recombination hotspot paradox") remain unresolved. Even though some sequence motifs are significantly associated with hotspots, multiple lines of evidence indicate that factors other than underlying sequences, such as epigenetic modifications, may affect the evolution of recombination hotspots. Thus, identifying epigenetic factors that covary with recombination at fine-scale is a promising step for this important research area. It was previously reported that recombination rates correlate with indirect measures of DNA methylation in the human genome. Here, we analyze experimentally determined DNA methylation and histone modification of human sperms, and show that the correlation between DNA methylation and recombination in long-range windows does not hold with respect to the spatial and temporal variation of recombination at hotspots. On the other hand, two histone modifications (H3K4me3 and H3K27me3) overlap extensively with recombination hotspots. Similar trends were observed in mice. These results indicate that specific histone modifications rather than DNA methylation are associated with the rapid evolution of recombination hotspots. Furthermore, many human recombination hotspots occupy "bivalent" chromatin regions that harbor both active (H3K4me3) and repressive (H3K27me3) marks. This may explain why human recombination hotspots tend to occur in nongenic regions, in contrast to yeast and Arabidopsis hotspots that are characterized by generally active chromatins. Our results highlight the dynamic epigenetic underpinnings of recombination hotspot evolution. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Homologous ELISA for detection of oligomeric human TNF: properties of the assay.

PubMed

Petyovka, N; Lyach, L; Voitenok, N N

1995-10-26

In order to quantify oligomeric human tumor necrosis factor-alpha (TNF), we have developed a sensitive homologous enzyme-linked immunosorbent assay (Hm-ELISA) using the same monoclonal antibody (MoAb) for both solid and liquid phase. Different anti-TNF MoAb have been compared in terms of their efficacy in the Hm-ELISA, affinity, neutralization capacity and epitope specificity. The data suggest, that effectiveness in the Hm-ELISA may represent a novel characteristic of MoAb. Of the MoAbs tested, 5 N was capable of recognizing oligomeric TNF in the Hm-ELISA with a detection limit of 15 pg/ml. Furthermore, using Hm-ELISA against human TNF, interleukin-8 (IL-8) and lymphotoxin, we have demonstrated that these cytokines are oligomeric in physiological solutions, but are converted into monomeric forms in the presence of the non-ionic detergent Tween 20. High salt buffer was employed to abrogate a nonspecific false positive reaction in the Hm-ELISA found in nearly half of the plasma samples obtained from healthy subjects. Finally, a good correlation between the Hm-ELISA and the L929 bioassay was observed for natural and recombinant TNF measured in human plasma.

Activation of triggering receptor expressed on myeloid cells-1 on human neutrophils by marburg and ebola viruses.

PubMed

Mohamadzadeh, Mansour; Coberley, Sadie S; Olinger, Gene G; Kalina, Warren V; Ruthel, Gordon; Fuller, Claudette L; Swenson, Dana L; Pratt, William D; Kuhns, Douglas B; Schmaljohn, Alan L

2006-07-01

Marburg virus (MARV) and Ebola virus (EBOV), members of the viral family Filoviridae, cause fatal hemorrhagic fevers in humans and nonhuman primates. High viral burden is coincident with inadequate adaptive immune responses and robust inflammatory responses, and virus-mediated dysregulation of early host defenses has been proposed. Recently, a novel class of innate receptors called the triggering receptors expressed in myeloid cells (TREM) has been discovered and shown to play an important role in innate inflammatory responses and sepsis. Here, we report that MARV and EBOV activate TREM-1 on human neutrophils, resulting in DAP12 phosphorylation, TREM-1 shedding, mobilization of intracellular calcium, secretion of proinflammatory cytokines, and phenotypic changes. A peptide specific to TREM-1 diminished the release of tumor necrosis factor alpha by filovirus-activated human neutrophils in vitro, and a soluble recombinant TREM-1 competitively inhibited the loss of cell surface TREM-1 that otherwise occurred on neutrophils exposed to filoviruses. These data imply direct activation of TREM-1 by filoviruses and also indicate that neutrophils may play a prominent role in the immune and inflammatory responses to filovirus infections.

Sero-detection of Toxocara canis infection in human with T.canis recombinant arginine kinase, cathepsin L-1 and TES-26 antigens.

PubMed

Varghese, Anju; Raina, Opinder K; Chandra, Dinesh; Mirdha, Bijay R; Kelawala, Naresh H; Solanki, Jayesh B; Kumar, Niranjan; Ravindran, Reghu; Arun, Anandanarayanan; Rialch, Ajayta; Lalrinkima, Hniang; Kelawala, Rohan N; Samanta, Subhamoy

2017-12-20

Three recombinant antigens viz. arginine kinase, cathepsin L-1 and TES-26 of Toxocara canis were expressed in Escherichia coli and evaluated for their potential in the detection of T. canis larval infection in human in immunoglobulin G-enzyme linked immunosorbent assay (IgG-ELISA). Results of the IgG-ELISA with the above recombinant antigens were confirmed with commercially available IgG detection kit for T. canis infection used as a standard test. All three recombinant antigens were 100% sensitive in the detection of positive cases (n = 6) of T. canis infection in human and were screened for their cross-reactivity in human patients with history of Toxoplasma gondii, Plasmodium vivax, Entamoeba histolytica, hydatid and hookworm infections. The recombinant TES-26 antigen showed higher specificity and cross-reacted with T. gondii infection sera only. However, arginine kinase and cathepsin L-1 recombinant antigens showed cross-reactions with sera of patients infected with T. gondii, P. vivax and E. histolytica but not with the patient sera infected with hydatid and hookworm. These results show that recombinant TES-26 is a potential diagnostic candidate antigen for human toxocarosis caused by migrating T. canis larvae.

Aldo-keto reductase family 1 B10 protein detoxifies dietary and lipid-derived alpha, beta-unsaturated carbonyls at physiological levels.

PubMed

Zhong, Linlin; Liu, Ziwen; Yan, Ruilan; Johnson, Stephen; Zhao, Yupei; Fang, Xiubin; Cao, Deliang

2009-09-18

Alpha, beta-unsaturated carbonyls are highly reactive mutagens and carcinogens to which humans are exposed on a daily basis. This study demonstrates that aldo-keto reductase family 1 member B10 (AKR1B10) is a critical protein in detoxifying dietary and lipid-derived unsaturated carbonyls. Purified AKR1B10 recombinant protein efficiently catalyzed the reduction to less toxic alcohol forms of crotonaldehyde at 0.90 microM, 4-hydroxynonenal (HNE) at 0.10 microM, trans-2-hexanal at 0.10 microM, and trans-2,4-hexadienal at 0.05 microM, the concentrations at or lower than physiological exposures. Ectopically expressed AKR1B10 in 293T cells eliminated immediately HNE at 1 (subtoxic) or 5 microM (toxic) by converting to 1,4-dihydroxynonene, protecting the cells from HNE toxicity. AKR1B10 protein also showed strong enzymatic activity toward glutathione-conjugated carbonyls. Taken together, our study results suggest that AKR1B10 specifically expressed in the intestine is physiologically important in protecting the host cell against dietary and lipid-derived cytotoxic carbonyls.

Aldo-keto reductase family 1 B10 protein detoxifies dietary and lipid-derived alpha, beta-unsaturated carbonyls at physiological levels

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

Zhong, Linlin; Department of Neurobiology and Anatomy, China Medical University, Shenyang 110001; Liu, Ziwen

2009-09-18

Alpha, beta-unsaturated carbonyls are highly reactive mutagens and carcinogens to which humans are exposed on a daily basis. This study demonstrates that aldo-keto reductase family 1 member B10 (AKR1B10) is a critical protein in detoxifying dietary and lipid-derived unsaturated carbonyls. Purified AKR1B10 recombinant protein efficiently catalyzed the reduction to less toxic alcohol forms of crotonaldehyde at 0.90 {mu}M, 4-hydroxynonenal (HNE) at 0.10 {mu}M, trans-2-hexanal at 0.10 {mu}M, and trans-2,4-hexadienal at 0.05 {mu}M, the concentrations at or lower than physiological exposures. Ectopically expressed AKR1B10 in 293T cells eliminated immediately HNE at 1 (subtoxic) or 5 {mu}M (toxic) by converting to 1,4-dihydroxynonene,more » protecting the cells from HNE toxicity. AKR1B10 protein also showed strong enzymatic activity toward glutathione-conjugated carbonyls. Taken together, our study results suggest that AKR1B10 specifically expressed in the intestine is physiologically important in protecting the host cell against dietary and lipid-derived cytotoxic carbonyls.« less

The inhibition of postinfarct ventricle remodeling without polycythaemia following local sustained intramyocardial delivery of erythropoietin within a supramolecular hydrogel.

PubMed

Wang, Tao; Jiang, Xue-Jun; Lin, Tao; Ren, Shan; Li, Xiao-Yan; Zhang, Xian-Zheng; Tang, Qi-zhu

2009-09-01

Erythropoietin (EPO) can protect myocardium from ischemic injury, but it also plays an important role in promoting polycythaemia, the potential for thrombo-embolic complications. Local sustained delivery of bioactive agents directly to impaired tissues using biomaterials is an approach to limit systemic toxicity and improve the efficacy of therapies. The present study was performed to investigate whether local intramyocardial injection of EPO with hydrogel could enhance cardioprotective effect without causing polycythaemia after myocardial infarction (MI). To test the hypothesis, phosphate buffered solution (PBS), alpha-cyclodextrin/MPEG-PCL-MPEG hydrogel, recombined human erythropoietin (rhEPO) in PBS, or rhEPO in hydrogel were injected into the infarcted area immediately after MI in rats. The hydrogel allowed a sustained release of EPO, which inhibited cell apoptosis and increased neovasculature formation, and subsequently reduced infarct size and improved cardiac function compared with other groups. Notably, there was no evidence of polycythaemia from this therapy, with no differences in erythrocyte count and hematocrit compared with the animals received PBS or hydrogel blank injection. In conclusion, intramyocardial delivery of rhEPO with alpha-cyclodextrin/MPEG-PCL-MPEG hydrogel may lead to cardiac performance improvement after MI without apparent adverse effect.

Transgenic mice overexpressing tyrosine-to-cysteine mutant human alpha-synuclein: a progressive neurodegenerative model of diffuse Lewy body disease.

PubMed

Zhou, Wenbo; Milder, Julie B; Freed, Curt R

2008-04-11

Abnormal aggregation of human alpha-synuclein in Lewy bodies and Lewy neurites is a pathological hallmark of Parkinson disease and dementia with Lewy bodies. Studies have shown that oxidation and nitration of alpha-synuclein lead to the formation of stable dimers and oligomers through dityrosine cross-linking. Previously we have reported that tyrosine-to-cysteine mutations, particularly at the tyrosine 39 residue (Y39C), significantly enhanced alpha-synuclein fibril formation and neurotoxicity. In the current study, we have generated transgenic mice expressing the Y39C mutant human alpha-synuclein gene controlled by the mouse Thy1 promoter. Mutant human alpha-synuclein was widely expressed in transgenic mouse brain, resulting in 150% overexpression relative to endogenous mouse alpha-synuclein. At age 9-12 months, transgenic mice began to display motor dysfunction in rotarod testing. Older animals aged 15-18 months showed progressive accumulation of human alpha-synuclein oligomers, associated with worse motor function and cognitive impairment in the Morris water maze. By age 21-24 months, alpha-synuclein aggregates were further increased, accompanied by severe behavioral deficits. At this age, transgenic mice developed neuropathology, such as Lewy body-like alpha-synuclein and ubiquitin-positive inclusions, phosphorylation at Ser(129) of human alpha-synuclein, and increased apoptotic cell death. In summary, Y39C human alpha-synuclein transgenic mice show age-dependent, progressive neuronal degeneration with motor and cognitive deficits similar to diffuse Lewy body disease. The time course of alpha-synuclein oligomer accumulation coincided with behavioral and pathological changes, indicating that these oligomers may initiate protein aggregation, disrupt cellular function, and eventually lead to neuronal death.

Effects of B group vitamins on reactions of various alpha-hydroxyl-containing organic radicals.

PubMed

Lagutin, P Yu; Shadyro, O I

2005-08-15

Effects of vitamins B1, B2, B6, and pyridoxal phosphate (PPh) on final product formation in radiolysis of aqueous solutions of ethanol, ethylene glycol, alpha-methylglycoside, and maltose were studied. It has been found that vitamin B2 and PPh effectively oxidize R*CHOH species, while suppressing their recombination and fragmentation reactions, thereby increasing the yields of the respective oxidation products. Vitamins B1 and B2 are capable of reducing alcohol radicals to the respective initial molecules, decreasing the yields of the radical transformation products.

Immunoassay for Visualization of Protein-Protein Interactions on Ni-Nitrilotriacetate Support: Example of a Laboratory Exercise with Recombinant Heterotrimeric G[alpha][subscript i2][beta][subscript 1[gamma]2] Tagged by Hexahistidine from sf9 Cells

ERIC Educational Resources Information Center

Bavec, Aljosa

2004-01-01

We have developed an "in vitro assay" for following the interaction between the [alpha][subscript i2] subunit and [beta][subscript 1[gamma]2] dimer from sf9 cells. This method is suitable for education purposes because it is easy, reliable, nonexpensive, can be applied for a big class of 20 students, and avoid the commonly used kinetic approach,…

Frequency and genetic characterization of V(DD)J recombinants in the human peripheral blood antibody repertoire.

PubMed

Briney, Bryan S; Willis, Jordan R; Hicar, Mark D; Thomas, James W; Crowe, James E

2012-09-01

Antibody heavy-chain recombination that results in the incorporation of multiple diversity (D) genes, although uncommon, contributes substantially to the diversity of the human antibody repertoire. Such recombination allows the generation of heavy chain complementarity determining region 3 (HCDR3) regions of extreme length and enables junctional regions that, because of the nucleotide bias of N-addition regions, are difficult to produce through normal V(D)J recombination. Although this non-classical recombination process has been observed infrequently, comprehensive analysis of the frequency and genetic characteristics of such events in the human peripheral blood antibody repertoire has not been possible because of the rarity of such recombinants and the limitations of traditional sequencing technologies. Here, through the use of high-throughput sequencing of the normal human peripheral blood antibody repertoire, we analysed the frequency and genetic characteristics of V(DD)J recombinants. We found that these recombinations were present in approximately 1 in 800 circulating B cells, and that the frequency was severely reduced in memory cell subsets. We also found that V(DD)J recombination can occur across the spectrum of diversity genes, indicating that virtually all recombination signal sequences that flank diversity genes are amenable to V(DD)J recombination. Finally, we observed a repertoire bias in the diversity gene repertoire at the upstream (5') position, and discovered that this bias was primarily attributable to the order of diversity genes in the genomic locus. © 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.

The Development of Novel Recombinant Human Gelatins as Replacements for Animal-Derived Gelatin in Pharmaceutical Applications

NASA Astrophysics Data System (ADS)

Olsen, David; Chang, Robert; Williams, Kim E.; Polarek, James W.

We have developed a recombinant expression system to produce a series of novel recombinant human gelatins that can substitute for animal sourced gelatin preparations currently used in pharmaceutical and nutraceutical applications. This system allows the production of human sequence gelatins, or, if desired, gelatins from any other species depending on the availability of the cloned gene. The gelatins produced with this recombinant system are of defined molecular weight, unlike the animal-sourced gelatins, which consist of numerous polypeptides of varying size. The fermentation and purification process used to prepare these recombinant gelatins does not use any human- or animal-derived components and thus this recombinant material should be free from viruses and agents that cause transmissible spongiform encephalopathies. The recombinant gelatins exhibit lot-to-lot reproducibility and we have performed extensive analytical testing on them. We have demonstrated the utility of these novel gelatins as biological stabilizers and plasma expanders, and we have shown they possess qualities that are important in applications where gel formation is critical. Finally, we provide examples of how our system allows the engineering of these recombinant gelatins to optimize the production process.

Yersinia pestis Yop secretion protein F: purification, characterization, and protective efficacy against bubonic plague.

PubMed

Swietnicki, Wieslaw; Powell, Bradford S; Goodin, Jeremy

2005-07-01

Yersinia pestis is a gram-negative human pathogen that uses a type III secretion system to deliver virulence factors into human hosts. The delivery is contact-dependent and it has been proposed that polymerization of Yop secretion protein F (YscF) is used to puncture mammalian cell membranes to facilitate delivery of Yersinia outer protein effectors into host cells. To evaluate the potential immunogenicity and protective efficacy of YscF against Y. pestis, we used a purified recombinant YscF protein as a potential vaccine candidate in a mouse subcutaneous infection model. YscF was expressed and purified from Escherichia coli by immobilized metal-ion affinity chromatography and protein identity was confirmed by ion trap mass spectrometry. The recombinant protein was highly alpha-helical and formed relatively stable aggregates under physiological conditions. The properties were consistent with behavior expected for the native YscF, suggesting that the antigen was properly folded. Ten mice were inoculated subcutaneously, administered booster injections after one month, and challenged with 130 LD(50) of wild type Y. pestis CO92. Six animals in the vaccinated group but none in the control group survived the challenge. The vaccinated animals produced high levels of specific antibodies against YscF as determined by Western blot. The data were statistically significant (P = 0.053 by two-tailed Fisher's test), suggesting that the YscF protein can provide a protective immune response against lethal plague challenge during subcutaneous plague infection.

Mixed nicotinic-muscarinic properties of the alpha9 nicotinic cholinergic receptor.

PubMed

Verbitsky, M; Rothlin, C V; Katz, E; Elgoyhen, A B

2000-10-01

The rat alpha9 nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus laevis oocytes and tested for its sensitivity to a wide variety of cholinergic compounds. Acetylcholine (ACh), carbachol, choline and methylcarbachol elicited agonist-evoked currents, giving maximal or near maximal responses. Both the nicotinic agonist suberyldicholine as well as the muscarinic agonists McN-A-343 and methylfurtrethonium behaved as weak partial agonists of the receptor. Most classical cholinergic compounds tested, being either nicotinic (nicotine, epibatidine, cytisine, methyllycaconitine, mecamylamine, dihydro-beta-erythroidine), or muscarinic (muscarine, atropine, gallamine, pilocarpine, bethanechol) agonists and antagonists, blocked the recombinant alpha9 receptor. Block by nicotine, epibatidine, cytisine, methyllycaconitine and atropine was overcome at high ACh concentrations, suggesting a competitive type of block. The present results indicate that alpha9 displays mixed nicotinic-muscarinic features that resemble the ones described for the cholinergic receptor of cochlear outer hair cells (OHCs). We suggest that alpha9 contains the structural determinants responsible for the pharmacological properties of the native receptor.

EEG alpha power and creative ideation☆

PubMed Central

Fink, Andreas; Benedek, Mathias

2014-01-01

Neuroscientific studies revealed first insights into neural mechanisms underlying creativity, but existing findings are highly variegated and often inconsistent. Despite the disappointing picture on the neuroscience of creativity drawn in recent reviews, there appears to be robust evidence that EEG alpha power is particularly sensitive to various creativity-related demands involved in creative ideation. Alpha power varies as a function of creativity-related task demands and the originality of ideas, is positively related to an individuals’ creativity level, and has been observed to increase as a result of creativity interventions. Alpha increases during creative ideation could reflect more internally oriented attention that is characterized by the absence of external bottom-up stimulation and, thus, a form of top-down activity. Moreover, they could indicate the involvement of specific memory processes such as the efficient (re-)combination of unrelated semantic information. We conclude that increased alpha power during creative ideation is among the most consistent findings in neuroscientific research on creativity and discuss possible future directions to better understand the manifold brain mechanisms involved in creativity. PMID:23246442

LDSplitDB: a database for studies of meiotic recombination hotspots in MHC using human genomic data.

PubMed

Guo, Jing; Chen, Hao; Yang, Peng; Lee, Yew Ti; Wu, Min; Przytycka, Teresa M; Kwoh, Chee Keong; Zheng, Jie

2018-04-20

Meiotic recombination happens during the process of meiosis when chromosomes inherited from two parents exchange genetic materials to generate chromosomes in the gamete cells. The recombination events tend to occur in narrow genomic regions called recombination hotspots. Its dysregulation could lead to serious human diseases such as birth defects. Although the regulatory mechanism of recombination events is still unclear, DNA sequence polymorphisms have been found to play crucial roles in the regulation of recombination hotspots. To facilitate the studies of the underlying mechanism, we developed a database named LDSplitDB which provides an integrative and interactive data mining and visualization platform for the genome-wide association studies of recombination hotspots. It contains the pre-computed association maps of the major histocompatibility complex (MHC) region in the 1000 Genomes Project and the HapMap Phase III datasets, and a genome-scale study of the European population from the HapMap Phase II dataset. Besides the recombination profiles, related data of genes, SNPs and different types of epigenetic modifications, which could be associated with meiotic recombination, are provided for comprehensive analysis. To meet the computational requirement of the rapidly increasing population genomics data, we prepared a lookup table of 400 haplotypes for recombination rate estimation using the well-known LDhat algorithm which includes all possible two-locus haplotype configurations. To the best of our knowledge, LDSplitDB is the first large-scale database for the association analysis of human recombination hotspots with DNA sequence polymorphisms. It provides valuable resources for the discovery of the mechanism of meiotic recombination hotspots. The information about MHC in this database could help understand the roles of recombination in human immune system. DATABASE URL: http://histone.scse.ntu.edu.sg/LDSplitDB.

Strong gender differences in reproductive success variance, and the times to the most recent common ancestors.

PubMed

Favre, Maroussia; Sornette, Didier

2012-10-07

The Time to the Most Recent Common Ancestor (TMRCA) based on human mitochondrial DNA (mtDNA) is estimated to be twice that based on the non-recombining part of the Y chromosome (NRY). These TMRCAs have special demographic implications because mtDNA is transmitted only from mother to child, while NRY is passed along from father to son. Therefore, the former locus reflects female history, and the latter, male history. To investigate what caused the two-to-one female-male TMRCA ratio r(F/M)=T(F)/T(M) in humans, we develop a forward-looking agent-based model (ABM) with overlapping generations. Our ABM simulates agents with individual life cycles, including life events such as reaching maturity or menopause. We implemented two main mating systems: polygynandry and polygyny with different degrees in between. In each mating system, the male population can be either homogeneous or heterogeneous. In the latter case, some males are 'alphas' and others are 'betas', which reflects the extent to which they are favored by female mates. A heterogeneous male population implies a competition among males with the purpose of signaling as alpha males. The introduction of a heterogeneous male population is found to reduce by a factor 2 the probability of finding equal female and male TMRCAs and shifts the distribution of r(F/M) to higher values. In order to account for the empirical observation of the factor 2, a high level of heterogeneity in the male population is needed: less than half the males can be alphas and betas can have at most half the fitness of alphas for the TMRCA ratio to depart significantly from 1. In addition, we find that, in the modes that maximize the probability of having 1.5

alpha-Lactalbumin species variation, HAMLET formation, and tumor cell death.

PubMed

Pettersson, Jenny; Mossberg, Ann-Kristin; Svanborg, Catharina

2006-06-23

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex of apo alpha-lactalbumin and oleic acid, formed in casein after low pH treatment of human milk. This study examined if HAMLET-like complexes are present in casein from different species and if isolated alpha-lactalbumin from those species can form such complexes with oleic acid. Casein from human, bovine, equine, and porcine milk was separated by ion exchange chromatography and active complexes were only found in human casein. This was not explained by alpha-lactalbumin sequence variation, as purified bovine, equine, porcine, and caprine alpha-lactalbumins formed complexes with oleic acid with biological activity similar to HAMLET. We conclude that structural variation of alpha-lactalbumins does not preclude the formation of HAMLET-like complexes and that natural HAMLET formation in casein was unique to human milk, which also showed the highest oleic acid content.

Population Demographic History Can Cause the Appearance of Recombination Hotspots

PubMed Central

Johnston, Henry R.; Cutler, David J.

2012-01-01

Although the prevailing view among geneticists suggests that recombination hotspots exist ubiquitously across the human genome, there is only limited experimental evidence from a few genomic regions to support the generality of this claim. A small number of true recombination hotspots are well supported experimentally, but the vast majority of hotspots have been identified on the basis of population genetic inferences from the patterns of linkage disequilibrium (LD) seen in the human population. These inferences are made assuming a particular model of human history, and one of the assumptions of that model is that the effective population size of humans has remained constant throughout our history. Our results show that relaxation of the constant population size assumption can create LD and variation patterns that are qualitatively and quantitatively similar to human populations without any need to invoke localized hotspots of recombination. In other words, apparent recombination hotspots could be an artifact of variable population size over time. Several lines of evidence suggest that the vast majority of hotspots identified on the basis of LD information are unlikely to have elevated recombination rates. PMID:22560089

Chaperokine function of recombinant Hsp72 produced in insect cells using a baculovirus expression system is retained.

PubMed

Zheng, Hongying; Nagaraja, Ganachari M; Kaur, Punit; Asea, Edwina E; Asea, Alexzander

2010-01-01

Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72(bv) (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72(bv) enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72(bv) in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72(bv) can now be used to unlock the important role Hsp72 plays in modulating immune function.

Chaperokine Function of Recombinant Hsp72 Produced in Insect Cells Using a Baculovirus Expression System Is Retained*

PubMed Central

Zheng, Hongying; Nagaraja, Ganachari M.; Kaur, Punit; Asea, Edwina E.; Asea, Alexzander

2010-01-01

Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72bv (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72bv enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72bv in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72bv can now be used to unlock the important role Hsp72 plays in modulating immune function. PMID:19861412

Effectiveness of Recombinant Human Growth Hormone for Pharyngocutaneous Fistula Closure

PubMed Central

Sari, Murat; Midi, Ahmet; Yumusakhuylu, Ali Cemal; Findik, Ozan; Binnetoglu, Adem

2015-01-01

Objectives In laryngeal cancer, which comprises 25% of head and neck cancer, chemotherapy has come into prominence with the increase in organ-protective treatments. With such treatment, salvage surgery has increased following recurrence; the incidence of pharyngocutaneous fistula has also increased in both respiratory and digestive system surgery. We investigated the effects of recombinant human growth hormone on pharyngocutaneous fistula closure in Sprague-Dawley rats, based on an increase in amino acid uptake and protein synthesis for wound healing, an increase in mitogenesis, and enhancement of collagen formation by recombinant human growth hormone. Methods This study was experimental animal study. Forty Sprague-Dawley rats were separated into two groups, and pharyngoesophagotomy was performed. The pharyngoesophagotomy was sutured with vicryl in both groups. Rats in group 1 (control group) received no treatment, while those in group 2 were administered a subcutaneous injection of recombinant human growth hormone daily. On day 14, the pharynx, larynx, and upper oesophagus were excised and examined microscopically. Results Pharyngocutaneous fistula exhibited better closure macroscopically in the recombinant human growth hormone group. There was a significant difference in collagen formation and epithelisation in the recombinant human growth hormone group compared to the control group. Conclusion This study is believed to be the first in which the effect of recombinant human growth hormone on pharyngocutaneous fistula closure was evaluated, and the findings suggest the potential of use of growth hormone for treatment of pharyngocutaneous fistula. PMID:26622960

Regulation of the human SLC25A20 expression by peroxisome proliferator-activated receptor alpha in human hepatoblastoma cells

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

Tachibana, Keisuke, E-mail: nya@phs.osaka-u.ac.jp; Takeuchi, Kentaro; Inada, Hirohiko

2009-11-20

Solute carrier family 25, member 20 (SLC25A20) is a key molecule that transfers acylcarnitine esters in exchange for free carnitine across the mitochondrial membrane in the mitochondrial {beta}-oxidation. The peroxisome proliferator-activated receptor alpha (PPAR{alpha}) is a ligand-activated transcription factor that plays an important role in the regulation of {beta}-oxidation. We previously established tetracycline-regulated human cell line that can be induced to express PPAR{alpha} and found that PPAR{alpha} induces the SLC25A20 expression. In this study, we analyzed the promoter region of the human slc25a20 gene and showed that PPAR{alpha} regulates the expression of human SLC25A20 via the peroxisome proliferator responsive element.

Cloning and expression of Clostridium perfringens type D vaccine strain epsilon toxin gene in E. coli as a recombinant vaccine candidate.

PubMed

Aziminia, Parastoo; Pilehchian-Langroudi, Reza; Esmaeilnia, Kasra

2016-08-01

Clostridium perfringens, a Gram-positive obligate anaerobic bacterium, is able to form resistant spores which are widely distributed in the environment. C. perfringens is subdivided into five types A to E based on its four major alpha, beta, epsilon and iota toxins. The aim of the present study was cloning and expression of C. perfringens type D vaccine strain epsilon toxin gene. Genomic DNA was extracted and the epsilon toxin gene was amplified using Pfu DNA polymerase. The PCR product was cloned into pJET1.2/blunt cloning vector. The recombinant vector (pJETε) was sequenced using universal primers. At the next step epsilon toxin gene was subcloned into pET22b(+) expression vector and transformed into E. coli Rosetta (DE3) host strain. The recombinant protein has been expressed in E. coli Rosetta (DE3) cells after subcloning of C. perfringens etx gene (1008 bp) into the expression vector. We concluded that E. coli Rosetta strain was suitable for the expression of recombinant C. perfringens epsilon toxin protein from pET22ε expression vector. This recombinant cell can be used for further research on recombinant vaccine development.

V(D)J recombination on minichromosomes is not affected by transcription.

PubMed

Hsieh, C L; McCloskey, R P; Lieber, M R

1992-08-05

It has been shown previously by others that transcription is temporally correlated with the onset of V(D)J recombination at the endogenous antigen receptor loci. We have been interested in determining whether this temporal correlation indicates a causal connection between these two processes. We have compared V(D)J recombination minichromosome substrates that have transcripts running through the recombination zone with substrates that do not in a transient transfection assay. In this system, the substrates acquire a minichromosome conformation within the first several hours after transfection. We find that the substrates recombine equally well over a 100-fold range in transcriptional variation. In additional studies, we have taken substrates that have low levels of transcription and inhibited transcription further by methylating the substrate DNA or by treating the cells with a general transcription inhibitor (alpha-amanitin). Although these treatments decrease the level of expression an additional 10-100-fold, there is still no observable effect on V(D)J recombination. Based on these results, we conclude that transcription is not necessary for the V(D)J reaction mechanism and does not alter substrate structure at the DNA level or at the simplest levels of chromatin structure in a way that affects the reaction.

a/alpha-specific effect on the mms3 mutation on ultraviolet mutagenesis in Saccharomyces cerevisiae.

PubMed

Martin, P; Prakash, L; Prakash, S

1981-05-01

A new gene involved in error-prone repair of ultraviolet (UV) damage has been identified in Saccharomyces cerevisiae by the mms3-1 mutation. UV-induced reversion is reduced in diploids that are homozygous for mms3-1, only if they are also heterozygous (MATa/MAT alpha) at the mating type locus. The mms3-1 mutation has no effect on UV-induced reversion either in haploids or MATa/MATa or MAT alpha/MAT alpha diploids. The mutation confers sensitivity to UV and methyl methane sulfonate in both haploids and diploids. Even though mutation induction by UV is restored to wild-type levels in MATa/MATa mms3-1/mms3-1 or MAT alpha/MAT alpha mms3-1/mms3-1 diploids, such strains still retain sensitivity to the lethal effects of UV. Survival after UV irradiation in mms3-1 rad double mutant combinations indicates that mms3-1 is epistatic to rad6-1 whereas non-epistatic interactions are observed with rad3 and rad52 mutants. When present in the homozygous state in MATa/MAT alpha his1-1/his1-315 heteroallelic diploids, mms3-1 was found to lower UV-induced mitotic recombination.

A Novel High-Throughput 1536-well Notch1 γ-Secretase AlphaLISA Assay

PubMed Central

Chau, De-ming; Shum, David; Radu, Constantin; Bhinder, Bhavneet; Gin, David; Gilchrist, M. Lane; Djaballah, Hakim; Li, Yue-Ming

2013-01-01

The Notch pathway plays a crucial role in cell fate decisions through controlling various cellular processes. Overactive Notch signal contributes to cancer development from leukemias to solid tumors. γ-Secretase is an intramembrane protease responsible for the final proteolytic step of Notch that releases the membrane-tethered Notch fragment for signaling. Therefore, γ-secretase is an attractive drug target in treating Notch-mediated cancers. However, the absence of high-throughput γ-secretase assay using Notch substrate has limited the identification and development of γ-secretase inhibitors that specifically target the Notch signaling pathway. Here, we report on the development of a 1536-well γ-secretase assay using a biotinylated recombinant Notch1 substrate. We effectively assimilated and miniaturized this newly developed Notch1 substrate with the AlphaLISA detection technology and demonstrated its robustness with a calculated Z’ score of 0.66. We further validated this optimized assay by performing a pilot screening against a chemical library consisting of ~5,600 chemicals and identified known γ-secretase inhibitors e.g. DAPT, and Calpeptin; as well as a novel γ-secretase inhibitor referred to as KD-I-085. This assay is the first reported 1536-well AlphaLISA format and represents a novel high-throughput Notch1-γ-secretase assay, which provides an unprecedented opportunity to discover Notch-selective γ-secretase inhibitors that can be potentially used for the treatment of cancer and other human disorders. PMID:23448293

Transcriptional activation of transforming growth factor alpha by estradiol: requirement for both a GC-rich site and an estrogen response element half-site.

PubMed

Vyhlidal, C; Samudio, I; Kladde, M P; Safe, S

2000-06-01

17beta-Estradiol (E2) induces transforming growth factor alpha (TGFalpha) gene expression in MCF-7 cells and previous studies have identified a 53 bp (-252 to -200) sequence containing two imperfect estrogen responsive elements (EREs) that contribute to E2 responsiveness. Deletion analysis of the TGFalpha gene promoter in this study identified a second upstream region of the promoter (-623 to -549) that is also E2 responsive. This sequence contains three GC-rich sites and an imperfect ERE half-site, and the specific cis-elements and trans-acting factors were determined by promoter analysis in transient transfection experiments, gel mobility shift assays and in vitro DNA footprinting. The results are consistent with an estrogen receptor alpha (ERalpha)/Sp1 complex interacting with an Sp1(N)(30) ERE half-site ((1/2)) motif in which both ERalpha and Sp1 bind promoter DNA. The ER/Sp1-DNA complex is formed using nuclear extracts from MCF-7 cells but not with recombinant human ERalpha or Sp1 proteins, suggesting that other nuclear factor(s) are required for complex stabilization. The E2-responsive Sp1(N)(x)ERE(1/2) motif identified in the TGFalpha gene promoter has also been characterized in the cathepsin D and heat shock protein 27 gene promoters; however, in the latter two promoters the numbers of intervening nucleotides are 23 and 10 respectively.

Interleukin 1 amplifies receptor-mediated activation of phospholipase A2 in 3T3 fibroblasts.

PubMed Central

Burch, R M; Connor, J R; Axelrod, J

1988-01-01

Human recombinant interleukin 1 alpha (IL-1 alpha) and IL-1 beta stimulated prostaglandin E2 synthesis in 3T3 fibroblasts in a time- and concentration-dependent manner. Enhanced prostaglandin E2 synthesis after IL-1 treatment was apparent by 1 hr and continued to increase for at least 2 days. Half-maximal stimulation occurred at 0.5 pM IL-1 alpha or IL-1 beta, and both interleukins were equally effective, with maximal stimulation occurring in response to 5-10 pM IL-1. In contrast to IL-1, bradykinin stimulation of prostaglandin E2 synthesis is rapid; its effect is maximal by 5 min. In cells that had been pretreated with IL-1 for 24 hr, prostaglandin E2 synthesis in response to bradykinin was amplified more than 10-fold. IL-1 also amplified the receptor-mediated formation of prostaglandin E2 by bombesin and thrombin. The lymphokine did not affect bradykinin receptor number or affinity. IL-1 treatment induced phospholipase A2 and cyclooxygenase but not phospholipase C or prostaglandin E isomerase. It also enhanced bradykinin-stimulated GTPase activity, suggesting possible induction of the GTP-binding regulatory protein coupled to the bradykinin receptor. Thus, IL-1 enhanced receptor-mediated release of prostaglandin E2 in response to bradykinin, bombesin, and thrombin by increasing the cellular levels of phospholipase A2, cyclooxygenase, and GTP-binding regulatory protein(s). PMID:2901097

Mouse alpha1(I)-collagen promoter is the best known promoter to drive efficient Cre recombinase expression in osteoblast.

PubMed

Dacquin, Romain; Starbuck, Michael; Schinke, Thorsten; Karsenty, Gérard

2002-06-01

Cell- and time-specific gene inactivation should enhance our knowledge of bone biology. Implementation of this technique requires construction of transgenic mouse lines expressing Cre recombinase in osteoblasts, the bone forming cell. We tested several promoter fragments for their ability to drive efficient Cre expression in osteoblasts. In the first mouse transgenic line, the Cre gene was placed under the control of the 2.3-kb proximal fragment of the alpha1(I)-collagen promoter, which is expressed at high levels in osteoblasts throughout their differentiation. Transgenic mice expressing this transgene in bone were bred with the ROSA26 reporter (R26R) strain in which the ROSA26 locus is targeted with a conditional LacZ reporter cassette. In R26R mice, Cre expression and subsequent Cre-mediated recombination lead to expression of the LacZ reporter gene, an event that can be monitored by LacZ staining. LacZ staining was detected in virtually all osteoblasts of alpha1(I)-Cre;R26R mice indicating that homologous recombination occurred in these cells. No other cell type stained blue. In the second line studied, the 1.3-kb fragment of osteocalcin gene 2 (OG2) promoter, which is active in differentiated osteoblasts, was used to drive Cre expression. OG2-Cre mice expressed Cre specifically in bone. However, cross of OG2-Cre mice with R26R mice did not lead to any detectable LacZ staining in osteoblasts. Lastly, we tested a more active artificial promoter derived from the OG2 promoter. The artificial OG2-Cre transgene was expressed by reverse transcriptase-polymerase chain reaction in cartilage and bone samples. After cross of the artificial OG2-Cre mice with R26R mice, we detected a LacZ staining in articular chondrocytes but not in osteoblasts. Our data suggest that the only promoter able to drive Cre expression at a level sufficient to induce recombination in osteoblasts is the alpha1(I)-collagen promoter. Copyright 2002 Wiley-Liss, Inc.

Natural protection from zoonosis by alpha-gal epitopes on virus particles in xenotransmission.

PubMed

Kim, Na Young; Jung, Woon-Won; Oh, Yu-Kyung; Chun, Taehoon; Park, Hong-Yang; Lee, Hoon-Taek; Han, In-Kwon; Yang, Jai Myung; Kim, Young Bong

2007-03-01

Clinical transplantation has become one of the preferred treatments for end-stage organ failure, and one of the novel approaches being pursued to overcome the limited supply of human organs involves the use of organs from other species. The pig appears to be a near ideal animal due to proximity to humans, domestication, and ability to procreate. The presence of Gal-alpha1,3-Gal residues on the surfaces of pig cells is a major immunological obstacle to xenotransplantation. Alpha1,3galactosyltransferase (alpha1,3GT) catalyzes the synthesis of Gal alpha 1-3Gal beta 1-4GlcNAc-R (alpha-gal epitope) on the glycoproteins and glycolipids of non-primate mammals, but this does not occur in humans. Moreover, the alpha-gal epitope causes hyperacute rejection of pig organs in humans, and thus, the elimination of this antigen from pig tissues is highly desirable. Recently, concerns have been raised that the risk of virus transmission from such pigs may be increased due to the absence of alpha-gal on their viral particles. In this study, transgenic cells expressing alpha1,3GT were selected using 1.25 mg/ml neomycin. The development of HeLa cells expressing alpha1,3GT now allows accurate studies to be conducted on the function of the alpha-gal epitope in xenotransmission. The expressions of alpha-gal epitopes on HeLa/alpha-gal cells were demonstrated by flow cytometry and confocal microscopy using cells stained with IB4-fluorescein isothiocyanate lectin. Vaccinia viruses propagated in HeLa/alpha-gal cells also expressed alpha-gal on their viral envelopes and were more sensitive to inactivation by human sera than vaccinia virus propagated in HeLa cells. Moreover, neutralization of vaccinia virus was inhibited in human serum by 10 mm ethylene glycol bis(beta-aminoethylether)tetraacetic acid (EDTA) treatment. Our data indicated that alpha-gal epitopes are one of the major barriers to zoonosis via xenotransmission.

Modulation of Phosphorylation of Tocopherol and Phosphatidylinositol by hTAP1/SEC14L2-Mediated Lipid Exchange

PubMed Central

Zingg, Jean-Marc; Libinaki, Roksan; Meydani, Mohsen; Azzi, Angelo

2014-01-01

The vitamin E derivative, alpha-tocopheryl phosphate (αTP), is detectable in cultured cells, plasma and tissues in small amounts, suggesting the existence of enzyme(s) with α-tocopherol (αT) kinase activity. Here, we characterize the production of αTP from αT and [γ-32P]-ATP in primary human coronary artery smooth muscle cells (HCA-SMC) using separation by thin layer chromatography (TLC) and subsequent analysis by Ultra Performance Liquid Chromatography (UPLC). In addition to αT, although to a lower amount, also γT is phosphorylated. In THP-1 monocytes, γTP inhibits cell proliferation and reduces CD36 scavenger receptor expression more potently than αTP. Both αTP and γTP activate the promoter of the human vascular endothelial growth factor (VEGF) gene with similar potency, whereas αT and γT had no significant effect. The recombinant human tocopherol associated protein 1 (hTAP1, hSEC14L2) binds both αT and αTP and stimulates phosphorylation of αT possibly by facilitating its transport and presentation to a putative αT kinase. Recombinant hTAP1 reduces the in vitro activity of the phosphatidylinositol-3-kinase gamma (PI3Kγ) indicating the formation of a stalled/inactive hTAP1/PI3Kγ heterodimer. The addition of αT, βT, γT, δT or αTP differentially stimulates PI3Kγ, suggesting facilitated egress of sequestered PI from hTAP1 to the enzyme. It is suggested that the continuous competitive exchange of different lipophilic ligands in hTAPs with cell enzymes and membranes may be a way to make these lipophiles more accessible as substrates for enzymes and as components of specific membrane domains. PMID:24983950

CD1 molecule expression on human monocytes induced by granulocyte-macrophage colony-stimulating factor.

PubMed

Kasinrerk, W; Baumruker, T; Majdic, O; Knapp, W; Stockinger, H

1993-01-15

In this paper we demonstrate that granulocyte-macrophage CSF (GM-CSF) specifically induces the expression of CD1 molecules, CD1a, CD1b and CD1c, upon human monocytes. CD1 molecules appeared upon monocytes on day 1 of stimulation with rGM-CSF, and expression was up-regulated until day 3. Monocytes cultured in the presence of LPS, FMLP, PMA, recombinant granulocyte-CSF, rIFN-gamma, rTNF-alpha, rIL-1 alpha, rIL-1 beta, and rIL-6 remained negative. The induction of CD1 molecules by rGM-CSF was restricted to monocytes, since no such effect was observed upon peripheral blood granulocytes, PBL, and the myeloid cell lines Monomac1, Monomac6, MV4/11, HL60, U937, THP1, KG1, and KG1A. CD1a mRNA was detectable in rGM-CSF-induced monocytes but not in those freshly isolated. SDS-PAGE and immunoblotting analyses of CD1a mAb VIT6 immunoprecipitate from lysate of rGM-CSF-activated monocytes revealed an appropriate CD1a polypeptide band of 49 kDa associated with beta 2-microglobulin. Expression of CD1 molecules on monocytes complements the distribution of these structures on accessory cells, and their specific induction by GM-CSF strengthens the suggestion that CD1 is a family of crucial structures required for interaction between accessory cells and T cells.

Tissue distribution and developmental expression of type XVI collagen in the mouse.

PubMed

Lai, C H; Chu, M L

1996-04-01

The expression of a recently identified collagen, alpha 1 (XVI), in adult mouse tissue and developing mouse embryo was examined by immunohistochemistry and in situ hybridization. A polyclonal antiserum was raised against a recombinant fusion protein, which contained a segment of 161 amino acids in the N-terminal noncollagenous domain of the human alpha 1 (XVI) collagen. Immunoprecipitation of metabolically labelled human or mouse fibroblast cell lysates with this antibody revealed a major, bacterial collagenase sensitive polypeptide of approximately 210 kDa. The size agrees with the prediction from the full-length cDNA. Immunofluorescence examination of adult mouse tissues using the affinity purified antibody revealed a rather broad distribution of the protein. The heart, kidney, intestine, ovary, testis, eye, arterial walls and smooth muscles all exhibited significant levels of expression, while the skeletal muscle, lung and brain showed very restricted and low signals. During development, no significant expression of the mRNA or protein was observed in embryo of day 8 of gestation, but strong signals was detected in placental trophoblasts. Expression in embryos was detectable first after day 11 of gestation with weak positive signals appearing in the heart. In later stages of development, stronger RNA hybridizations were observed in a variety of tissues, particularly in atrial and ventricular walls of the developing heart, spinal root neural fibers and skin. These data demonstrate that type XVI collagen represents another collagenous component widely distributed in the extracellular matrix and may contribute to the structural integrity of various tissues.

Dehydroepiandrosterone (DHEA) metabolism in Saccharomyces cerevisiae expressing mammalian steroid hydroxylase CYP7B: Ayr1p and Fox2p display 17beta-hydroxysteroid dehydrogenase activity.

PubMed

Vico, Pedro; Cauet, Gilles; Rose, Ken; Lathe, Richard; Degryse, Eric

2002-07-01

We have engineered recombinant yeast to perform stereospecific hydroxylation of dehydroepiandrosterone (DHEA). This mammalian pro-hormone promotes brain and immune function; hydroxylation at the 7alpha position by P450 CYP7B is the major pathway of metabolic activation. We have sought to activate DHEA via yeast expression of rat CYP7B enzyme. Saccharomyces cerevisiae was found to metabolize DHEA by 3beta-acetylation; this was abolished by mutation at atf2. DHEA was also toxic, blocking tryptophan (trp) uptake: prototrophic strains were DHEA-resistant. In TRP(+) atf2 strains DHEA was then converted to androstene-3beta,17beta-diol (A/enediol) by an endogenous 17beta-hydroxysteroid dehydrogenase (17betaHSD). Seven yeast polypeptides similar to human 17betaHSDs were identified: when expressed in yeast, only AYR1 (1-acyl dihydroxyacetone phosphate reductase) increased A/enediol accumulation, while the hydroxyacyl-CoA dehydrogenase Fox2p, highly homologous to human 17betaHSD4, oxidized A/enediol to DHEA. The presence of endogenous yeast enzymes metabolizing steroids may relate to fungal pathogenesis. Disruption of AYR1 eliminated reductive 17betaHSD activity, and expression of CYP7B on the combination background (atf2, ayr1, TRP(+)) permitted efficient (>98%) bioconversion of DHEA to 7alpha-hydroxyDHEA, a product of potential medical utility. Copyright 2002 John Wiley & Sons, Ltd.

Partial mapping and sequencing of a fish iridovirus genome reveals genes homologous to the frog virus 3 p31, p40 and human eIF2alpha.

PubMed

Yu, Y X; Béarzotti, M; Vende, P; Ahne, W; Brémont, M

1999-09-01

Iridovirus-like pathogens have been recognized as a cause of serious systemic diseases among feral, cultured and ornamental fish in the recent years. Mortalities of fish due to systemic iridovirus infection reaching 30-100% were observed in Europe, Australia, Japan and Thailand. Up to now, the molecular biology of these important pathogens has been poorly documented. To get better insights on the genomic organization of these piscine iridoviruses, we have constructed a cosmid viral DNA library from the epizootic hematopoietic necrosis virus (EHNV). Two recombinant cosmids (Cos7 and Cos12) have been selected for systematic sequencing. Cos7 and 12 are localized side by side along the genome and cover the 2/3 part of the total EHNV genome which has been estimated to be approximately 101.47 kb in length. Thirty five kilobase pairs (kbps) from Cos7 and 10 kbps from Cos12 have been determined. Sequence analysis revealed open reading frames (ORF) sharing homologies with sequences from the Frog virus 3 such as the p31 and p40 proteins. Among the others identified ORFs, some of them presented homologies with known protein sequences, such as the human eIF2alpha protein, and some did not show any significant homologies with sequences available in the databases. But, none were related to Lymphocystis virus, a member of the Iridoviridae family, for which the full genome nucleotide sequence has been determined.

Genetic recombination pathways and their application for genome modification of human embryonic stem cells.

PubMed

Nieminen, Mikko; Tuuri, Timo; Savilahti, Harri

2010-10-01

Human embryonic stem cells are pluripotent cells derived from early human embryo and retain a potential to differentiate into all adult cell types. They provide vast opportunities in cell replacement therapies and are expected to become significant tools in drug discovery as well as in the studies of cellular and developmental functions of human genes. The progress in applying different types of DNA recombination reactions for genome modification in a variety of eukaryotic cell types has provided means to utilize recombination-based strategies also in human embryonic stem cells. Homologous recombination-based methods, particularly those utilizing extended homologous regions and those employing zinc finger nucleases to boost genomic integration, have shown their usefulness in efficient genome modification. Site-specific recombination systems are potent genome modifiers, and they can be used to integrate DNA into loci that contain an appropriate recombination signal sequence, either naturally occurring or suitably pre-engineered. Non-homologous recombination can be used to generate random integrations in genomes relatively effortlessly, albeit with a moderate efficiency and precision. DNA transposition-based strategies offer substantially more efficient random strategies and provide means to generate single-copy insertions, thus potentiating the generation of genome-wide insertion libraries applicable in genetic screens. 2010 Elsevier Inc. All rights reserved.

Loss of tumorigenic potential by human lung tumor cells in the presence of antisense RNA specific to the ectopically synthesized alpha subunit of human chorionic gonadotropin.

PubMed

Rivera, R T; Pasion, S G; Wong, D T; Fei, Y B; Biswas, D K

1989-06-01

A clonal strain of human lung tumor cells in culture (ChaGo), derived from a bronchogenic carcinoma, synthesizes and secretes large amounts of alpha (alpha) and a comparatively lower level of beta (beta) subunit of the glycoprotein hormone, human chorionic gonadotropin (HCG). ChaGo cells lost their characteristic anchorage-independent growth phenotype in the presence of anti-alpha-HCG antibody. The effect of the antibody was partially reversed by addition of alpha-HCG to the culture medium. ChaGo cells were transfected with an expression vector (pRSV-anti-alpha-HCG), that directs synthesis of RNA complementary to alpha-HCG mRNA. The transfectants produced alpha-HCG antisense RNA which was associated with the reduced level of alpha-HCG. Transfectants also displayed several altered phenotypic properties, including altered morphology, less mitosis, reduced growth rate, loss of anchorage-independent growth, and loss of tumorigenicity in nude mice. Treatment of transfectants with 8,bromo-cAMP resulted in increased accumulation of alpha-HCG mRNA, no change in the level of alpha-HCG antisense RNA, release of the inhibition of [3H]thymidine incorporation, and restoration of anchorage-independent growth phenotype. The overexpression of c-myc, observed in ChaGo cells, was unaffected by the reduced level of alpha-HCG. These results suggest that ectopic synthesis of the alpha subunit of HCG plays a functional role in the transformation of these human lung cells.

Escherichia coli ArgR mutants defective in cer/Xer recombination, but not in DNA binding.

PubMed

Sénéchal, Hélène; Delesques, Jérémy; Szatmari, George

2010-04-01

The Escherichia coli arginine repressor (ArgR) is an L-arginine-dependent DNA-binding protein that controls the expression of the arginine biosynthetic genes and is required as an accessory factor for Xer site-specific recombination at cer and related recombination sites in plasmids. We used the technique of pentapeptide scanning mutagenesis to isolate a series of ArgR mutants that were considerably reduced in cer recombination, but were still able to repress an argA::lacZ fusion. DNA sequence analysis showed that all of the mutants mapped to the same nucleotide, resulting in a five amino acid insertion between residues 149 and 150 of ArgR, corresponding to the end of the alpha6 helix. A truncated ArgR containing a stop codon at residue 150 displayed the same phenotype as the protein with the five amino acid insertion, and both mutants displayed sequence-specific DNA-binding activity that was L-arginine dependent. These results show that the C-terminus of ArgR is more important in cer/Xer site-specific recombination than in DNA binding.

Inhibitory effects of hepatocyte growth factor and interleukin-6 on transforming growth factor-beta1 mediated vocal fold fibroblast-myofibroblast differentiation.

PubMed

Vyas, Bimal; Ishikawa, Keiko; Duflo, Suzy; Chen, Xia; Thibeault, Susan L

2010-05-01

The role of myofibroblasts in vocal fold scarring has not been extensively studied, partly because of the lack of a robust in vitro model. The objective of this investigation was to develop and characterize a myofibroblast in vitro model that could be utilized to investigate the molecular mechanism of myofibroblast differentiation and function in injured vocal fold tissue. Differentiation of human primary vocal fold fibroblasts (hVFFs) to myofibroblasts was stimulated with 5, 10, or 20 ng/mL of recombinant transforming growth factor-beta1 (TGF-beta1). Cultures were analyzed by immunofluorescence and Western blotting, with an alpha-smooth muscle actin (alpha-SMA) antibody used as a myofibroblast marker. Normal rabbit vocal folds were treated with 10 ng/mL of TGF-beta1 for 7 days for in vivo corroboration. The effects of interleukin-6 (IL-6) and hepatocyte growth factor (HGF) on myofibroblast differentiation were studied with Western blots. The hVFFs demonstrated positive alpha-SMA labeling in cells stimulated by 10 and 20 ng/mL TGF-beta1, indicating that hVFFs were capable of differentiation to myofibroblasts. Transforming growth factor-beta1 induced the largest increase in alpha-SMA at 10 ng/mL on day 5 of treatment. Both HGF and IL-6 suppressed the expression of TGF-beta1-induced alpha-SMA. Our work characterizes a useful in vitro model of TGF-beta1-mediated vocal fold fibroblast-myofibroblast differentiation. The extent of differentiation appears to be attenuated by HGF, suggesting a potential mechanism to support prior work indicating that HGF plays a protective role in reducing scar formation in vocal fold injuries. Paradoxically, IL-6, which has been shown to play a profibrotic role in dermal studies, also attenuated the TGF-beta1 response.

Low-Frequency Carbon Recombination Lines in the Orion Molecular Cloud Complex

NASA Astrophysics Data System (ADS)

Tremblay, Chenoa D.; Jordan, Christopher H.; Cunningham, Maria; Jones, Paul A.; Hurley-Walker, Natasha

2018-05-01

We detail tentative detections of low-frequency carbon radio recombination lines from within the Orion molecular cloud complex observed at 99-129 MHz. These tentative detections include one alpha transition and one beta transition over three locations and are located within the diffuse regions of dust observed in the infrared at 100 μm, the Hα emission detected in the optical, and the synchrotron radiation observed in the radio. With these observations, we are able to study the radiation mechanism transition from collisionally pumped to radiatively pumped within the H ii regions within the Orion molecular cloud complex.

Nonhomologous recombination between defective poliovirus and coxsackievirus genomes suggests a new model of genetic plasticity for picornaviruses.

PubMed

Holmblat, Barbara; Jégouic, Sophie; Muslin, Claire; Blondel, Bruno; Joffret, Marie-Line; Delpeyroux, Francis

2014-08-05

Most of the circulating vaccine-derived polioviruses (cVDPVs) implicated in poliomyelitis outbreaks in Madagascar have been shown to be recombinants between the type 2 poliovirus (PV) strain of the oral polio vaccine (Sabin 2) and another species C human enterovirus (HEV-C), such as type 17 coxsackie A virus (CA17) in particular. We studied intertypic genetic exchanges between PV and non-PV HEV-C by developing a recombination model, making it possible to rescue defective type 2 PV RNA genomes with a short deletion at the 3' end by the cotransfection of cells with defective or infectious CA17 RNAs. We isolated over 200 different PV/CA17 recombinants, using murine cells expressing the human PV receptor (PVR) and selecting viruses with PV capsids. We found some homologous (H) recombinants and, mostly, nonhomologous (NH) recombinants presenting duplications of parental sequences preferentially located in the regions encoding proteins 2A, 2B, and 3A. Short duplications appeared to be stable, whereas longer duplications were excised during passaging in cultured cells or after multiplication in PVR-transgenic mice, generating H recombinants with diverse sites of recombination. This suggests that NH recombination events may be a transient, intermediate step in the generation and selection of the fittest H recombinants. In addition to the classical copy-choice mechanism of recombination thought to generate mostly H recombinants, there may also be a modular mechanism of recombination, involving NH recombinant precursors, shaping the genomes of recombinant enteroviruses and other picornaviruses. Importance: The multiplication of circulating vaccine-derived polioviruses (cVDPVs) in poorly immunized human populations can render these viruses pathogenic, causing poliomyelitis outbreaks. Most cVDPVs are intertypic recombinants between a poliovirus (PV) strain and another human enterovirus, such as type 17 coxsackie A viruses (CA17). For further studies of the genetic exchanges between PV and CA17, we have developed a model of recombination, making it possible to rescue defective PV RNA genomes with a short deletion by cotransfecting cells with the defective PV genome and CA17 genomic RNA. Numerous recombinants were found, including homologous PV/CA17 recombinants, but mostly nonhomologous recombinants presenting duplications of parental sequences preferentially located in particular regions. Long duplications were excised by passages in cultured cells or in mice, generating diverse homologous recombinants. Recombination leading to nonhomologous recombinants, which evolve into homologous recombinants, may therefore be seen as a model of genetic plasticity in enteroviruses and, possibly, in other RNA viruses. Copyright © 2014 Holmblat et al.

Population patch-clamp electrophysiology analysis of recombinant GABAA alpha1beta3gamma2 channels expressed in HEK-293 cells.

PubMed

Hollands, Emma C; Dale, Tim J; Baxter, Andrew W; Meadows, Helen J; Powell, Andrew J; Clare, Jeff J; Trezise, Derek J

2009-08-01

Gamma-amino butyric acid (GABA)-activated Cl- channels are critical mediators of inhibitory postsynaptic potentials in the CNS. To date, rational design efforts to identify potent and selective GABA(A) subtype ligands have been hampered by the absence of suitable high-throughput screening approaches. The authors describe 384-well population patch-clamp (PPC) planar array electrophysiology methods for the study of GABA(A) receptor pharmacology. In HEK293 cells stably expressing human alpha1beta3gamma2 GABA(A) channels, GABA evoked outward currents at 0 mV of 1.05 +/- 0.08 nA, measured 8 s post GABA addition. The I(GABA) was linear and reversed close to the theoretical E(Cl) (-56 mV). Concentration-response curve analysis yielded a mean pEC(50) value of 5.4 and Hill slope of 1.5, and for a series of agonists, the rank order of potency was muscimol > GABA > isoguvacine. A range of known positive modulators, including diazepam and pentobarbital, produced concentration-dependent augmentation of the GABA EC( 20) response (1 microM). The competitive antagonists bicuculline and gabazine produced concentration-dependent, parallel, rightward displacement of GABA curves with pA(2) and slope values of 5.7 and 1.0 and 6.7 and 1.0, respectively. In contrast, picrotoxin (0.2-150 microM) depressed the maximal GABA response, implying a non-competitive antagonism. Overall, the pharmacology of human alpha1beta3gamma2 GABA(A) determined by PPC was highly similar to that obtained by conventional patch-clamp methods. In small-scale single-shot screens, Z' values of >0.5 were obtained in agonist, modulator, and antagonist formats with hit rates of 0% to 3%. The authors conclude that despite the inability of the method to resolve the peak agonist responses, PPC can rapidly and usefully quantify pharmacology for the alpha1beta3gamma2 GABA(A) isoform. These data suggest that PPC may be a valuable approach for a focused set and secondary screening of GABA(A) receptors and other slow ligand-gated ion channels.

Application of four anti-human interferon-alpha monoclonal antibodies for immunoassay and comparative analysis of natural interferon-alpha mixtures

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

Andersson, G.; Lundgren, E.; Ekre, H.P.

Four different mouse monoclonal antibodies to human interferon-alpha (IFN-alpha) were evaluated for application in quantitative and comparative analysis of natural IFN-alpha mixtures. Binding to IFN-alpha subtypes in solution revealed individual reactivity patterns. These patterns changed if the IFN-alpha molecules were immobilized either passively to a surface or bound by another antibody. Also, substitution of a single amino acid in IFN-alpha 2 affected the binding, apparently by altering the conformation. Isoelectric focusing of three natural IFN-alpha preparations from different sources, followed by immunoblotting, resulted in individual patterns with each of the four mAbs and also demonstrated variation in the composition ofmore » the IFN-alpha preparations. None of the mAbs was subtype specific, but by combining the different mAbs, and also applying polyclonal anti-human IFN-alpha antibodies, it was possible to design sensitive sandwich ELISAs with broad or more limited IFN-alpha subtype specificity.« less

Method of artificial DNA splicing by directed ligation (SDL).

PubMed Central

Lebedenko, E N; Birikh, K R; Plutalov, O V; Berlin YuA

1991-01-01

An approach to directed genetic recombination in vitro has been devised, which allows for joining together, in a predetermined way, a series of DNA segments to give a precisely spliced polynucleotide sequence (DNA splicing by directed ligation, SDL). The approach makes use of amplification, by means of several polymerase chain reactions (PCR), of a chosen set of DNA segments. Primers for the amplifications contain recognition sites of the class IIS restriction endonucleases, which transform blunt ends of the amplification products into protruding ends of unique primary structures, the ends to be used for joining segments together being mutually complementary. Ligation of the mixture of the segments so synthesized gives the desired sequence in an unambiguous way. The suggested approach has been exemplified by the synthesis of a totally processed (intronless) gene encoding human mature interleukin-1 alpha. Images PMID:1662363

STRUCTURAL AND FUNCTIONAL INTERACTION OF FATTY ACIDS WITH HUMAN LIVER FATTY ACID BINDING PROTEIN (L-FABP) T94A VARIANT

PubMed Central

Huang, Huan; McIntosh, Avery L.; Martin, Gregory G.; Landrock, Kerstin K.; Landrock, Danilo; Gupta, Shipra; Atshaves, Barbara P.; Kier, Ann B.; Schroeder, Friedhelm

2014-01-01

The human liver fatty acid binding protein (L-FABP) T94A variant, the most common in the FABP family, has been associated with elevated liver triglyceride (TG) levels. How this amino acid substitution elicits these effects is not known. This issue was addressed with human recombinant wild-type (WT, T94T) and T94A variant L-FABP proteins as well as cultured primary human hepatocytes expressing the respective proteins (genotyped as TT, TC, and CC). T94A substitution did not or only slightly alter L-FABP binding affinities for saturated, monounsaturated, or polyunsaturated long chain fatty acids (LCFA), nor did it change the affinity for intermediates in TG synthesis. Nevertheless, T94A substitution markedly altered the secondary structural response of L-FABP induced by binding LCFA or intermediates of TG synthesis. Finally, T94A substitution markedly diminished polyunsaturated fatty acid, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), induction of peroxisome proliferator-activated receptor alpha (PPARα) - regulated proteins such as L-FABP, fatty acid transport protein 5 (FATP5), and PPARα itself in cultured primary human hepatocytes. Thus, while T94A substitution did not alter the affinity of human L-FABP for LCFAs, it significantly altered human L-FABP structure and stability as well as conformational and functional response to these ligands. PMID:24628888

Tocopherol metabolites 2, 5, 7, 8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC) and 2, 7, 8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman (gamma-CEHC) in human serum after a single dose of natural vitamin E.

PubMed

Radosavac, Dragan; Graf, Peter; Polidori, M Cristina; Sies, Helmut; Stahl, Wilhelm

2002-06-01

alpha- and gamma-Tocopherol are vitamin E compounds in human blood and tissues. alpha-CEHC (2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman) and gamma-CEHC (2,7,8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman) have been identified as water-soluble metabolites which are excreted with the urine in humans. To assess over-time changes of serum levels of alpha- and gamma-CEHC in humans after a single dose of vitamin E from a natural source. Twenty-one healthy subjects ingested a single dose of vitamin E (306 mg of RRR-alpha-tocopherol and 1.77 mg of gamma-tocopherol). Blood was collected before (baseline) and 2, 6, 12, 24, 35, 50, and 74 h after ingestion. Serum was separated and levels of alpha- and gamma-tocopherol and alpha- and gamma-CEHC were determined by HPLC. After vitamin E ingestion, a statistically significant increase was observed for alpha-tocopherol and alpha-CEHC. Maximum serum levels for both compounds were measured 12 h after application (33.3 +/- 11.1 micromol alpha-toco-pherol /L and 42.4 +/- 18.3 nmol alpha-CEHC /L); baseline values were reached again after 72 h. While gamma-tocopherol levels decreased during the study period, an increase in the metabolite gamma-CEHC was observed. The optical isomer formed in the metabolism of RRR-alpha-tocopherol was assigned as S-alpha-CEHC. alpha-CEHC levels increase after administration of a single dose of natural vitamin E in humans. The appearance of the metabolite in blood parallels that of the parent compound. The gamma-tocopherol analog appears to be metabolized more efficiently than alpha-tocopherol.

Distribution of alpha3, alpha5 and alpha(v) integrin subunits in mature and immature human oocytes.

PubMed

Capmany, G; Mart, M; Santaló, J; Bolton, V N

1998-10-01

The distribution of three integrin subunits, alpha3, alpha5 and alpha(v), in immature and mature human oocytes has been examined using immunofluorescence and confocal microscopy. The results demonstrate that both alpha5 and alpha(v) are present at the germinal vesicle stage, while alpha3 was only detected in oocytes after germinal vesicle breakdown, in metaphase I and II stage oocytes. The cortical concentration of integrin subunits alpha3 and alpha5 is consistent with their localization in the oolemma. In contrast, the homogeneous distribution of alpha(v) throughout the oocyte suggests the existence of cytoplasmic reservoirs of this protein in the oocyte.

Use of a purified and functional recombinant calcium-channel beta4 subunit in surface-plasmon resonance studies.

PubMed Central

Geib, Sandrine; Sandoz, Guillaume; Mabrouk, Kamel; Matavel, Alessandra; Marchot, Pascale; Hoshi, Toshinori; Villaz, Michel; Ronjat, Michel; Miquelis, Raymond; Lévêque, Christian; de Waard, Michel

2002-01-01

Native high-voltage-gated calcium channels are multi-subunit complexes comprising a pore-forming subunit Ca(v) and at least two auxiliary subunits alpha(2)delta and beta. The beta subunit facilitates cell-surface expression of the channel and contributes significantly to its biophysical properties. In spite of its importance, detailed structural and functional studies are hampered by the limited availability of native beta subunit. Here, we report the purification of a recombinant calcium-channel beta(4) subunit from bacterial extracts by using a polyhistidine tag. The purified protein is fully functional since it binds on the alpha1 interaction domain, its main Ca(v)-binding site, and regulates the activity of P/Q calcium channel expressed in Xenopus oocytes in a similar way to the beta(4) subunit produced by cRNA injection. We took advantage of the functionality of the purified material to (i) develop an efficient surface-plasmon resonance assay of the interaction between two calcium channel subunits and (ii) measure, for the first time, the affinity of the recombinant His-beta(4) subunit for the full-length Ca(v)2.1 channel. The availability of this purified material and the development of a surface-plasmon resonance assay opens two immediate research perspectives: (i) drug screening programmes applied to the Ca(v)/beta interaction and (ii) crystallographic studies of the calcium-channel beta(4) subunit. PMID:11988102

Characterization of three types of human alpha s1-casein mRNA transcripts.

PubMed Central

Johnsen, L B; Rasmussen, L K; Petersen, T E; Berglund, L

1995-01-01

Here we report the molecular cloning and sequencing of three types of human alpha s1-casein transcripts and present evidence indicating that exon skipping is responsible for deleted mRNA transcripts. The largest transcript comprised 981 bp encoding a signal peptide of 15 amino acids followed by the mature alpha s1-casein sequence of 170 amino acids. Human alpha s1-casein has been reported to exist naturally as a multimer in complex with kappa-casein in mature human milk, thereby being unique among alpha s1-caseins [Rasmussen, Due and Petersen (1995) Comp. Biochem. Physiol., in the press]. The present demonstration of three cysteines in the mature protein provides a molecular explanation of the interactions in this complex. Tissue-specific expression of human alpha s1-casein was indicated by Northern-blot analysis. In addition, two cryptic exons were localized in the bovine alpha s1-casein gene. Images Figure 3 PMID:7619062

Human cells: new platform for recombinant therapeutic protein production.

PubMed

Swiech, Kamilla; Picanço-Castro, Virgínia; Covas, Dimas Tadeu

2012-07-01

The demand for recombinant therapeutic proteins is significantly increasing. There is a constant need to improve the existing expression systems, and also developing novel approaches to face the therapeutic proteins demands. Human cell lines have emerged as a new and powerful alternative for the production of human therapeutic proteins because this expression system is expected to produce recombinant proteins with post translation modifications more similar to their natural counterpart and reduce the potential immunogenic reactions against nonhuman epitopes. Currently, little information about the cultivation of human cells for the production of biopharmaceuticals is available. These cells have shown efficient production in laboratory scale and represent an important tool for the pharmaceutical industry. This review presents the cell lines available for large-scale recombinant proteins production and evaluates critically the advantages of this expression system in comparison with other expression systems for recombinant therapeutic protein production. Copyright © 2012 Elsevier Inc. All rights reserved.

The IL-6/sIL-6R treatment of a malignant melanoma cell line enhances susceptibility to TNF-{alpha}-induced apoptosis

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

Wagley, Yadav; Yoo, Yung-Choon; Seo, Han Geuk

2007-03-23

Melanoma is an intractable tumor that has shown very impressive and promising response to local administration of high dose recombinant TNF-{alpha} in combination with IFN-{gamma} in clinical studies. In this study, we investigated the effect of IL-6/sIL-6R on TNF-{alpha}-resistant B16/F10.9 melanoma cells. A low dose of TNF-{alpha} or IL-6/sIL-6R had minimal affect on the cell growth. However, the highly active fusion protein of sIL-6R and IL-6 (IL6RIL6), covalently linked by a flexible peptide, sensitized TNF-{alpha}-resistant F10.9 melanoma cells to TNF-{alpha}-induced apoptosis. Stimulation of the cells with IL6RIL6 plus TNF-{alpha} resulted in both the activation of caspase-3 and the reduction ofmore » bcl-2 expression. Flow cytometry analysis showed that IL6RIL6-upregulated TNF-R55 and TNF-R75 expression, suggesting an increase in TNF-{alpha} responsiveness by IL6RIL6 resulting from the induction of TNF receptors. Moreover, exposure of F10.9 cells to neutralizing antibody to TNF-R55 significantly inhibited IL6RIL6/TNF-{alpha}-induced cytotoxicity. These results suggest that the IL6/sIL6R/gp130 system, which sensitizes TNF-{alpha}-resistant melanoma cells to TNF-{alpha}-induced apoptosis, may provide a new target for immunotherapy.« less

Genetic battle between Helicobacter pylori and humans. The mechanism underlying homologous recombination in bacteria, which can infect human cells.

PubMed

Hanada, Katsuhiro; Yamaoka, Yoshio

2014-10-01

Helicobacter pylori is a gram-negative pathogenic bacterium that colonises the human stomach. The chronic infection it causes results in peptic ulcers and gastric cancers. H. pylori can easily establish a chronic infection even if the immune system attacks this pathogen with oxidative stress agents and immunoglobulins. This is attributed to bacterial defence mechanisms against these stresses. As a defence mechanism against oxidative stresses, in bacterial genomes, homologous recombination can act as a repair pathway of DNA's double-strand breaks (DSBs). Moreover, homologous recombination is also involved in the antigenic variation in H. pylori. Gene conversion alters genomic structures of babA and babB (encoding outer membrane proteins), resulting in escape from immunoglobulin attacks. Thus, homologous recombination in bacteria plays an important role in the maintenance of a chronic infection. In addition, H. pylori infection causes DSBs in human cells. Homologous recombination is also involved in the repair of DSBs in human cells. In this review, we describe the roles of homologous recombination with an emphasis on the maintenance of a chronic infection. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Effect of TNF{alpha} on activities of different promoters of human apolipoprotein A-I gene

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

Orlov, Sergey V., E-mail: serge@iem.sp.ru; Department of Embryology, St. Petersburg State University, 199034 St. Petersburg; Mogilenko, Denis A.

2010-07-23

Research highlights: {yields} TNF{alpha} stimulates the distal alternative promoter of human apoA-I gene. {yields} TNF{alpha} acts by weakening of promoter competition within apoA-I gene (promoter switching). {yields} MEK1/2 and nuclear receptors PPAR{alpha} and LXRs take part in apoA-I promoter switching. -- Abstract: Human apolipoprotein A-I (ApoA-I) is a major structural and functional protein component of high-density lipoproteins. The expression of the apolipoprotein A-I gene (apoA-I) in hepatocytes is repressed by pro-inflammatory cytokines such as IL-1{beta} and TNF{alpha}. Recently, two novel additional (alternative) promoters for human apoA-I gene have been identified. Nothing is known about the role of alternative promoters inmore » TNF{alpha}-mediated downregulation of apoA-I gene. In this article we report for the first time about the different effects of TNF{alpha} on two alternative promoters of human apoA-I gene. Stimulation of HepG2 cells by TNF{alpha} leads to activation of the distal alternative apoA-I promoter and downregulation of the proximal alternative and the canonical apoA-I promoters. This effect is mediated by weakening of the promoter competition within human apoA-I 5'-regulatory region (apoA-I promoter switching) in the cells treated by TNF{alpha}. The MEK1/2-ERK1/2 cascade and nuclear receptors PPAR{alpha} and LXRs are important for TNF{alpha}-mediated apoA-I promoter switching.« less

Variation in recombination rate may bias human genetic disease mapping studies.

PubMed

Boyle, A Susannah; Noor, Mohamed A F

2004-11-01

The availability of the human genome sequence and variability information (as from the International HapMap project) will enhance our ability to map genetic disorders and choose targets for therapeutic intervention. However, several factors, such as regional variation in recombination rate, can bias conclusions from genetic mapping studies. Here, we examine the impact of regional variation in recombination rate across the human genome. Through computer simulations and literature surveys, we conclude that genetic disorders have been mapped to regions of low recombination more often than expected if such diseases were randomly distributed across the genome. This concentration in low recombination regions may be an artifact, and disorders appearing to be caused by a few genes of large effect may be polygenic. Future genetic mapping studies should be conscious of this potential complication by noting the regional recombination rate of regions implicated in diseases.

GDNF and NGF family members and receptors in human fetal and adult spinal cord and dorsal root ganglia.

PubMed

Josephson, A; Widenfalk, J; Trifunovski, A; Widmer, H R; Olson, L; Spenger, C

2001-11-12

We describe the expression of mRNA encoding ligands and receptors of members of the GDNF family and members of the neurotrophin family in the adult human spinal cord and dorsal root ganglia (DRG). Fetal human spinal cord and ganglia were investigated for the presence of ligands and receptors of the neurotrophin family. Tissues were collected from human organ donors and after routine elective abortions. Messenger RNA was found encoding RET, GFR alpha-1, BDNF, trkB, and trkC in the adult human spinal cord and BDNF, NT-3, p75, trkB, and trkC in the fetal human spinal cord. The percentage of adult human DRG cells expressing p75, trkA, trkB, or trkC was 57, 46, 29, and 24%, respectively, and that of DRG cells expressing RET, GFR alpha-1, GFR alpha-2, or GFR alpha-3 was 79, 20, 51, and 32%, respectively. GFR alpha-2 was expressed selectively in small, GFR alpha-3 principally in small and GFR alpha-1 and RET in both large and small adult human DRG neurons. p75 and trkB were expressed by a wide range of DRG neurons while trkA was expressed in most small diameter and trkC primarily in large DRG neurons. Fetal DRG cells were positive for the same probes as adult DRG cells except for NT-3, which was only found in fetal DRG cells. Messenger RNA species only expressed at detectable levels in fetal but not adult spinal cord tissues included GDNF, GFR alpha-2, NT-3, and p75. Notably, GFR alpha-2, which is expressed in the adult rat spinal cord, was not found in the adult human spinal cord. Copyright 2001 Wiley-Liss, Inc.

Interferometric rotation sensor

NASA Technical Reports Server (NTRS)

Walsh, T. M. (Inventor)

1973-01-01

An interferometric rotation sensor and control system is provided which includes a compound prism interferometer and an associated direction control system. Light entering the interferometer is split into two paths with the light in the respective paths being reflected an unequal number of times, and then being recombined at an exit aperture in phase differing relationships. Incoming light is deviated from the optical axis of the device by an angle, alpha. The angle causes a similar displacement of the two component images at the exit aperture which results in a fringe pattern. Fringe numbers are directly related to angle alpha. Various control systems of the interferometer are given.

21-cm radiation: a new probe of variation in the fine-structure constant.

PubMed

Khatri, Rishi; Wandelt, Benjamin D

2007-03-16

We investigate the effect of variation in the value of the fine-structure constant (alpha) at high redshifts (recombination > z > 30) on the absorption of the cosmic microwave background (CMB) at 21 cm hyperfine transition of the neutral atomic hydrogen. We find that the 21 cm signal is very sensitive to the variations in alpha and it is so far the only probe of the fine-structure constant in this redshift range. A change in the value of alpha by 1% changes the mean brightness temperature decrement of the CMB due to 21 cm absorption by >5% over the redshift range z < 50. There is an effect of similar magnitude on the amplitude of the fluctuations in the brightness temperature. The redshift of maximum absorption also changes by approximately 5%.

Pharmacokinetic and pharmacodynamic comparisons between human granulocyte colony-stimulating factor purified from human bladder carcinoma cell line 5637 culture medium and recombinant human granulocyte colony-stimulating factor produced in Escherichia coli.

PubMed

Tanaka, H; Kaneko, T

1992-07-01

The pharmacokinetics and biological activities of recombinant human granulocyte colony-stimulating factor (hG-CSF) produced in Escherichia coli were compared with those of hG-CSF purified from human bladder carcinoma cell line 5637 culture medium (5637-hG-CSF). Recombinant hG-CSF was biologically active in a bone marrow cell proliferation assay in vitro, with a dose-response curve similar to that of 5637-hG-CSF. The effects of 5637- and recombinant hG-CSF administered via i.v. injection to rats showed similar response patterns of neutrophil counts in peripheral blood. From these results, it is concluded that the O-linked sugar chain of hG-CSF does not contribute to the in vitro and in vivo biological activities. The pharmacokinetics of both forms of hG-CSF in rats were investigated using a sandwich enzyme-linked immunosorbent assay. After i.v. administration, the serum concentration-time curves of 5637- and recombinant hG-CSF declined biexponentially. Total body clearance and steady-state volume of distribution of 5637-hG-CSF were smaller than those for the recombinant form. After s.c. administration, a lower peak serum level, smaller AUC, and lower bioavailability of 5637-hG-CSF were observed compared to recombinant hG-CSF.

[Construction and identification of recombinant human platelet-derived growth factor-B adenoviral vector and transfection into periodontal ligament stem cells].

PubMed

Shang, Shu-huan; Zhang, Yu-feng; Shi, Bin; Cheng, Xiang-rong

2008-10-01

To construct a recombinant human platelet-derived growth factor-B (PDGF-B) adenoviral vector and to transfect it into human periodontal ligament stem cells (PDLSC). The recombinant plasmid pAd-PDGF-B was constructed by homologous recombination and confirmed by restriction endonucleases digestion. Recombinant adenovirus was packaged in HEK293 cells. PDLSC were transfected with recombinant adenovirus and PDGF-B expression was confirmed. Expression of collagen type I gene was determined by quantitative analysis of the products of RT-PCR. The cell proliferation was determined with MTT colorimetric assay. The recombinant plasmid pAd-PDGF-B was confirmed by restriction endonucleases digestion. EGFP expression was observed on the third day after transfecting, and the expression of PDGF-B was detected. Immunohistochemical methods revealed that PDGF-B was expressed in PDLSC. Levels of expression of collagen type I gene were increased significantly by transfer of the exogenous PDGF-B gene to PDLSC. At the same time, findings indicated that Ad-PDGF-B stimulated PDLSC proliferation. MTT assay indicated the absorbance of PDLSC by stimulating with Ad-PDGF-B was (0.68 +/- 0.02), P < 0.01. Using the AdEasy system, the human PDGF-B recombinant adenovirus can be rapidly obtained. These results indicate that recombinant adenoviruses encoding PDGF-B transgenes could modulate proliferative activity of PDLSC, enhance the high expression of collagen type I and lay the foundation for periodontal tissue regeneration and dental implant gene therapy.

The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

PubMed

Jin, Qingwen; Chen, Hong; Wang, Xingxia; Zhao, Liandong; Xu, Qingchen; Wang, Huijuan; Li, Guanyu; Yang, Xiaofan; Ma, Hongming; Wu, Haoquan; Ji, Xiaohui

2015-01-01

Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed. We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects. Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1) infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5. Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

Genetic Recombination between Human and Animal Parasites Creates Novel Strains of Human Pathogen

PubMed Central

Gibson, Wendy; Peacock, Lori; Ferris, Vanessa; Fischer, Katrin; Livingstone, Jennifer; Thomas, James; Bailey, Mick

2015-01-01

Genetic recombination between pathogens derived from humans and livestock has the potential to create novel pathogen strains, highlighted by the influenza pandemic H1N1/09, which was derived from a re-assortment of swine, avian and human influenza A viruses. Here we investigated whether genetic recombination between subspecies of the protozoan parasite, Trypanosoma brucei, from humans and animals can generate new strains of human pathogen, T. b. rhodesiense (Tbr) responsible for sleeping sickness (Human African Trypanosomiasis, HAT) in East Africa. The trait of human infectivity in Tbr is conferred by a single gene, SRA, which is potentially transferable to the animal pathogen Tbb by sexual reproduction. We tracked the inheritance of SRA in crosses of Tbr and Tbb set up by co-transmitting genetically-engineered fluorescent parental trypanosome lines through tsetse flies. SRA was readily transferred into new genetic backgrounds by sexual reproduction between Tbr and Tbb, thus creating new strains of the human pathogen, Tbr. There was no evidence of diminished growth or transmissibility of hybrid trypanosomes carrying SRA. Although expression of SRA is critical to survival of Tbr in the human host, we show that the gene exists as a single copy in a representative collection of Tbr strains. SRA was found on one homologue of chromosome IV in the majority of Tbr isolates examined, but some Ugandan Tbr had SRA on both homologues. The mobility of SRA by genetic recombination readily explains the observed genetic variability of Tbr in East Africa. We conclude that new strains of the human pathogen Tbr are being generated continuously by recombination with the much larger pool of animal-infective trypanosomes. Such novel recombinants present a risk for future outbreaks of HAT. PMID:25816228

Genetic recombination between human and animal parasites creates novel strains of human pathogen.

PubMed

Gibson, Wendy; Peacock, Lori; Ferris, Vanessa; Fischer, Katrin; Livingstone, Jennifer; Thomas, James; Bailey, Mick

2015-03-01

Genetic recombination between pathogens derived from humans and livestock has the potential to create novel pathogen strains, highlighted by the influenza pandemic H1N1/09, which was derived from a re-assortment of swine, avian and human influenza A viruses. Here we investigated whether genetic recombination between subspecies of the protozoan parasite, Trypanosoma brucei, from humans and animals can generate new strains of human pathogen, T. b. rhodesiense (Tbr) responsible for sleeping sickness (Human African Trypanosomiasis, HAT) in East Africa. The trait of human infectivity in Tbr is conferred by a single gene, SRA, which is potentially transferable to the animal pathogen Tbb by sexual reproduction. We tracked the inheritance of SRA in crosses of Tbr and Tbb set up by co-transmitting genetically-engineered fluorescent parental trypanosome lines through tsetse flies. SRA was readily transferred into new genetic backgrounds by sexual reproduction between Tbr and Tbb, thus creating new strains of the human pathogen, Tbr. There was no evidence of diminished growth or transmissibility of hybrid trypanosomes carrying SRA. Although expression of SRA is critical to survival of Tbr in the human host, we show that the gene exists as a single copy in a representative collection of Tbr strains. SRA was found on one homologue of chromosome IV in the majority of Tbr isolates examined, but some Ugandan Tbr had SRA on both homologues. The mobility of SRA by genetic recombination readily explains the observed genetic variability of Tbr in East Africa. We conclude that new strains of the human pathogen Tbr are being generated continuously by recombination with the much larger pool of animal-infective trypanosomes. Such novel recombinants present a risk for future outbreaks of HAT.

The alpha-spectrin gene is on chromosome 1 in mouse and man.

PubMed

Huebner, K; Palumbo, A P; Isobe, M; Kozak, C A; Monaco, S; Rovera, G; Croce, C M; Curtis, P J

1985-06-01

By using alpha-spectrin cDNA clones of murine and human origin and somatic cell hybrids segregating either mouse or human chromosomes, the gene for alpha-spectrin has been mapped to chromosome 1 in both species. This assignment of the mouse alpha-spectrin gene to mouse chromosome 1 by DNA hybridization strengthens the previous identification of the alpha-spectrin locus in mouse with the sph locus, which previously was mapped by linkage analysis to mouse chromosome 1, distal to the Pep-3 locus. By in situ hybridization to human metaphase chromosomes, the human alpha-spectrin gene has been localized to 1q22-1q25; interestingly, the locus for a non-Rh-linked form of elliptocytosis has been provisionally mapped to band 1q2 by family linkage studies.

Computational studies of H5N1 hemagglutinin binding with SA-{alpha}-2, 3-Gal and SA-{alpha}-2, 6-Gal

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

Li Minyong; Wang Binghe

2006-09-01

For influenza H5N1 hemagglutinin, a switch from SA-{alpha}-2, 3-Gal to SA-{alpha}-2, 6-Gal receptor specificity is a critical step leading to the conversion from avian-to-human to human-to-human infection. Therefore, the understanding of the binding modes of SA-{alpha}-2, 3-Gal and SA-{alpha}-2, 6-Gal to H5N1 hemagglutinin will be very important for the examination of possible mutations needed for going from an avian to a human flu virus. Based on the available H5N1 hemagglutinin crystal structure, the binding profiles between H5N1 hemagglutinin and two saccharide ligands, SA-{alpha}-2, 3-Gal and SA-{alpha}-2, 6-Gal, were investigated by ab initio quantum mechanics, molecular docking, molecular mechanics, and molecularmore » dynamics simulations. It was found that SA-{alpha}-2, 3-Gal has strong multiple hydrophobic and hydrogen bond interactions in its trans conformation with H5N1 hemagglutinin, whereas the SA-{alpha}-2, 6-Gal only shows weak interactions in a different conformation (cis type)« less

Mapping of monoclonal antibody- and receptor-binding domains on human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using a surface plasmon resonance-based biosensor.

PubMed

Laricchia-Robbio, L; Liedberg, B; Platou-Vikinge, T; Rovero, P; Beffy, P; Revoltella, R P

1996-10-01

An automated surface plasmon resonance (SPR)-based biosensor system has been used for mapping antibody and receptor-binding regions on the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) molecule. A rabbit antimouse IgG1-Fc antibody (RAM.Fc) was coupled to an extended carboxymethylated-hydrogel matrix attached to a gold surface in order to capture an anti-rhGM-CSF monoclonal antibody (MAb) injected over the sensing layer. rhGM-CSF was subsequently injected and allowed to bind to this antibody. Multisite binding assays were then performed, by flowing sequentially other antibodies and peptides over the surface, and the capacity of the latter to interact with the entrapped rhGM-CSF in a multimolecular complex was monitored in real time with SPR. Eleven MAb (all IgG1K), were analyzed: respectively, four antipeptide MAb raised against three distinct epitopes of the cytokine (two clones against residues 14-24, that includes part of the first alpha-helix toward the N-terminal region; one clone against peptide 30-41, an intrahelical loop; and one clone against residues 79-91, including part of the third alpha-helix) and seven antiprotein MAbs raised against the entire rhGM-CSF, whose target native epitopes are still undetermined. In addition, the binding capacity to rhGM-CSF of a synthetic peptide, corresponding to residues 238-254 of the extracellular human GM-CSF receptor alpha-chain, endowed with rhGM-CSF binding activity, was tested. The results from experiments performed with the biosensor were compared with those obtained by a sandwich enzyme-linked immunosorbent assay (ELISA), using the same reagents. The features of the biosensor technology (fully automated, measure in real time, sharpened yes/no response, less background disturbances, no need for washing step or labeling of the reagent) offered several advantages in these studies of MAb immunoreactivity and epitope mapping, giving a much better resolution and enabling more distinct epitopes to be identified over ELISA.

Carcinoembryonic antigen-stimulated THP-1 macrophages activate endothelial cells and increase cell-cell adhesion of colorectal cancer cells.

PubMed

Aarons, Cary B; Bajenova, Olga; Andrews, Charles; Heydrick, Stanley; Bushell, Kristen N; Reed, Karen L; Thomas, Peter; Becker, James M; Stucchi, Arthur F

2007-01-01

The liver is the most common site for metastasis by colorectal cancer, and numerous studies have shown a relationship between serum carcinoembryonic antigen (CEA) levels and metastasis to this site. CEA activates hepatic macrophages or Kupffer cells via binding to the CEA receptor (CEA-R), which results in the production of cytokines and the up-regulation of endothelial adhesion molecules, both of which are implicated in hepatic metastasis. Since tissue macrophages implicated in the metastatic process can often be difficult to isolate, the aim of this study was to develop an in vitro model system to study the complex mechanisms of CEA-induced macrophage activation and metastasis. Undifferentiated, human monocytic THP-1 (U-THP) cells were differentiated (D-THP) to macrophages by exposure to 200 ng/ml phorbol myristate acetate (PMA) for 18 h. Immunohistochemistry showed two CEA-R isoforms present in both U- and D-THP cells. The receptors were localized primarily to the nucleus in U-THP cells, while a significant cell-surface presence was observed following PMA-differentiation. Incubation of D-THP-1 cells with CEA resulted in a significant increase in tumor necrosis factor-alpha (TNF-alpha) release over 24 h compared to untreated D-THP-1 or U-THP controls confirming the functionality of these cell surface receptors. U-THP cells were unresponsive to CEA. Attachment of HT-29 cells to human umbilical vein endothelial cells significantly increased at 1 h after incubation with both recombinant TNF-alpha and conditioned media from CEA stimulated D-THP cells by six and eightfold, respectively. This study establishes an in vitro system utilizing a human macrophage cell line expressing functional CEA-Rs to study activation and signaling mechanisms of CEA that facilitate tumor cell attachment to activated endothelial cells. Utilization of this in vitro system may lead to a more complete understanding of the expression and function of CEA-R and facilitate the design of anti-CEA-R therapeutic modalities that may significantly diminish the metastatic potential of CEA overexpressing colorectal tumors.

Pulmonary deposition and disappearance of aerosolised secretory leucocyte protease inhibitor.

PubMed Central

Stolk, J.; Camps, J.; Feitsma, H. I.; Hermans, J.; Dijkman, J. H.; Pauwels, E. K.

1995-01-01

BACKGROUND--The neutrophil elastase inhibitor, secretory leucocyte protease inhibitor (SLPI), is a potential therapeutic tool in inflammatory lung diseases such as cystic fibrosis and pulmonary emphysema. The distribution and disappearance in the lung of aerosolised recombinant SLPI (rSLPI) was investigated in healthy humans and in patients with cystic fibrosis or alpha 1-antitrypsin-associated emphysema. METHODS--To distinguish aerosolised rSLPI from endogenous SLPI the recombinant inhibitor was radiolabelled with 99m-technetium (99mTc) pertechnetate. Distribution and disappearance of aerosolised 99mTc-rSLPI in the lungs were studied by gamma radiation imaging. RESULTS--The deposition of 99mTc-rSLPI in normal volunteers was homogeneous in all lung lobes, while in patients with cystic fibrosis or emphysema only well ventilated areas showed deposition of the aerosol. The disappearance rate of 99mTc-rSLPI was biexponential. The half life of the rapid phase was 0.2-2.8 hours, while that of the slow phase was more than 24 hours. CONCLUSIONS--Future aerosol therapy with rSLPI will be most beneficial for well ventilated lung tissue that needs protection against neutrophil derived elastase. It may be more difficult to neutralise the burden of elastase in poorly ventilated, highly inflamed areas as are seen in cystic fibrosis. Images PMID:7638807

Validation of biological activity testing procedure of recombinant human interleukin-7.

PubMed

Lutsenko, T N; Kovalenko, M V; Galkin, O Yu

2017-01-01

Validation procedure for method of monitoring the biological activity of reсombinant human interleukin-7 has been developed and conducted according to the requirements of national and international recommendations. This method is based on the ability of recombinant human interleukin-7 to induce proliferation of T lymphocytes. It has been shown that to control the biological activity of recombinant human interleukin-7 peripheral blood mononuclear cells (PBMCs) derived from blood or cell lines can be used. Validation charac­teristics that should be determined depend on the method, type of product or object test/measurement and biological test systems used in research. The validation procedure for the method of control of biological activity of recombinant human interleukin-7 in peripheral blood mononuclear cells showed satisfactory results on all parameters tested such as specificity, accuracy, precision and linearity.

Skin test performed with highly purified Mycobacterium tuberculosis recombinant protein triggers tuberculin shock in infected guinea pigs.

PubMed

Reece, Stephen T; Stride, Nicole; Ovendale, Pamela; Reed, Steven G; Campos-Neto, Antonio

2005-06-01

Tuberculin shock due to inoculation of Mycobacterium tuberculosis antigens in patients with tuberculosis is a serious syndrome originally described over 100 years ago by Robert Koch. Here, we present experimental evidence that a single M. tuberculosis recombinant protein, CFP-10, triggers this syndrome. Intradermal inoculation of CFP-10 elicits in M. tuberculosis-infected mice high levels of serum tumor necrosis factor alpha and causes tuberculin shock in infected guinea pigs characterized by hypothermia and death within 6 to 48 h after the antigen inoculation. Autopsies of these animals revealed intense polycythemia and hemorrhagic patches in the lung parenchyma, a pathological observation consistent with tuberculin shock. These results point to the possible occurrence of tuberculin shock in sensitive individuals inoculated with highly purified M. tuberculosis recombinant proteins as vaccine candidates or skin test reagents.

Detection of sperm-reactive antibodies in wild sika deer and identification of the sperm antigens.

PubMed

Kawase, Osamu; Jimbo, Mitsuru

2018-03-16

Antisperm antibodies potentially inhibit sperm functions causing the sterility in humans and experimentally treated animals. However, there is no information about antisperm antibodies emerging spontaneously in wildlife. In this study, we searched for the sperm-reactive antibodies, spontaneously produced in wild sika deer (Cervus nippon), and identified the sperm antigens. We collected 529 fecal masses of sika deer in Japanese cities, from which we extracted the mucosal antibodies to test them for reactivities to deer sperm proteins by ELISA. Two of the extracts contained IgAs that were highly reactive to the sperm proteins. The molecular weights of the active IgAs, partially purified by DEAE-sephadex A-50, were estimated at more than 100 kDa, suggesting that the IgAs evaded drastic digestion in the gastrointestinal tract. Two-dimensional electrophoresis and immunoblotting detected three major antigens, and the following LC-MS/MS analysis identified them as alpha-enolase, phosphoglycerate kinase 2 and acrosin-binding protein. The antibodies were cross-reactive to a recombinant human acrosin-binding protein. To our knowledge, this is the first research to find that the sperm-reactive antibodies are produced spontaneously in wildlife and they recognize a common antigen found in humans.

Probing conformational changes in the I-like domain and the cysteine-rich repeat of human beta 3 integrins following disulfide bond disruption by cysteine mutations: identification of cysteine 598 involved in alphaIIbbeta3 activation.

PubMed

Chen, P; Melchior, C; Brons, N H; Schlegel, N; Caen, J; Kieffer, N

2001-10-19

We have investigated receptor function and epitope expression of recombinant alpha(IIb)beta(3) mutated at Cys(177) or Cys(273) in the I-like domain as well as Cys(598), located in the fourth repeat of the membrane-proximal cysteine-rich region and mutated in a Glanzmann's thrombasthenia type II patient. The beta(3) mutants beta(3)C177A, beta(3)C273A, and beta(3)C598Y exhibited a decreased electrophoretic mobility in SDS-polyacrylamide gel electrophoresis under nonreducing conditions, confirming the disruption of the respective disulfide loops. Despite reduced surface expression, the alpha(IIb)beta(3)C177A, alpha(IIb)beta(3)C273A, and alpha(IIb)beta(3)C598Y receptors mediated cell adhesion to immobilized fibrinogen and translocated into focal adhesion plaques. The beta(3)C598Y mutation, but not the beta(3)C177A or beta(3)C273A mutations, induced spontaneous binding of the ligand mimetic monoclonal antibody PAC-1, while the beta(3)C177A and beta(3)C273A mutants exhibited reduced complex stability in the absence of Ca(2+). Epitope mapping of function-blocking monoclonal antibodies (mAbs) allowed the identification of two distinct subgroups; mAbs A2A9, pl2-46, 10E5, and P256 did not interact with alpha(IIb)beta(3)C273A and bound only weakly to alpha(IIb)beta(3)C177A, while mAbs AP2, LM609 and 7E3 bound normally to mutant alpha(IIb)beta(3)C273A, but interacted only weakly with mutant alpha(IIb)beta(3)C177A. Furthermore, a cryptic epitope recognized by mAb 4D10G3 and not exposed on wild type alpha(IIb)beta(3) became accessible only on mutant alpha(IIb)beta(3)C177A and was mapped to the 60-kDa chymotrypsin fragment of beta(3). Finally, the ligand-induced binding site (LIBS) epitopes AP5, D3, LIBS1, and LIBS2 were spontaneously expressed on all three mutants independent of RGDS or dithiothreitol treatment. Our results provide evidence that disruption of a single cysteine disulfide bond in the cysteine-rich repeat domain, but not in the I-like domain, activates integrin alpha(IIb)beta(3). In contrast, disruption of each of the disulfide bonds in the two long insertions of the I-like domain predicted to be in close contact with the alpha subunit beta-propeller domain affect the stability of the alpha(IIb)beta(3) heterodimer and inhibit complex-specific mAb binding without affecting the RGD binding capacity of the metal ion-dependent adhesion site-like domain.

Postjunctional inhibition of contractor responses in the mouse vas deferens by rat and human calcitonin gene-related peptides (CGRP).

PubMed Central

Al-Kazwini, S. J.; Craig, R. K.; Marshall, I.

1986-01-01

The effects of rat and human alpha-calcitonin gene-related peptide (CGRP) were compared in the mouse and rabbit isolated vas deferens preparation contracted by either field stimulation or acetylcholine. The peptides were about equipotent at inhibiting twitch responses of the mouse vas deferens to field stimulation at 0.2 Hz (IC50 12 +/- 4 nM and 15 +/- 3 nM, rat and human alpha-CGRP respectively). Rat alpha-CGRP was less potent at inhibiting responses to 10 Hz than to either 0.2 Hz or 1.0 Hz stimulation. The potency of rat alpha-CGRP at 1.0 Hz was unaltered by halving the calcium concentration of the Krebs solution. The inhibitory effect of human alpha-CGRP was not antagonized by either propranolol (300 nM) or idazoxan (300 nM), although in the same tissues these latter two drugs reduced responses to isoprenaline and clonidine respectively. Rat alpha-CGRP (100 nM) and human alpha-CGRP (1.0 microM) did not alter the uptake of [3H]-noradrenaline (30 nM) into mice isolated vasa deferentia. Rat alpha-CGRP (3-100 nM) did not alter the fractional release per pulse (1.0 Hz, 100 pulses) of tritium from vasa preloaded with [3H]-noradrenaline, although at the same time the peptide inhibited responses of the smooth muscle to field stimulation. Rat and human alpha-CGRP were equipotent at inhibiting contractions of the mouse vas deferens evoked by acetylcholine although the peptides were less potent than against twitch responses. In the rabbit vas deferens neither rat nor human alpha-CGRP (3 nM-1 microM) inhibited either twitch responses or acetylcholine contractions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3486688

Evidence for Alpha Receptors in the Human Ureter

NASA Astrophysics Data System (ADS)

Madeb, Ralph; Knopf, Joy; Golijanin, Dragan; Bourne, Patricia; Erturk, Erdal

2007-04-01

An immunohistochemical and western blot expression analysis of human ureters was performed in order to characterize the alpha-1-adrenergic receptor distribution along the length of the human ureteral wall. Mapping the distribution will assist in understanding the potential role alpha -1-adrenergic receptors and their subtype density might have in the pathophysiology of ureteral colic and stone passage. Patients diagnosed with renal cancer or bladder cancer undergoing nephrectomy, nephroureterectomy, or cystectomy had ureteral specimens taken from the proximal, mid, distal and tunneled ureter. Tissues were processed for fresh frozen examination and fixed in formalin. None of the ureteral specimens were involved with cancer. Serial histologic sections and immunohistochemical studies were performed using antibodies specific for alpha-1-adrenergic receptor subtypes (alpha 1a, alpha 1b, alpha 1d). The sections were examined under a light microscope and scored as positive or negative. In order to validate and quantify the alpha receptor subtypes along the human ureter. Western blotting techniques were applied. Human ureter stained positively for alpha -1-adrenergic receptors. Immunostaining appeared red, with intense reaction in the smooth muscle of the ureter and endothelium of the neighboring blood vessels. There was differential expression between all the receptors with the highest staining for alpha-1D subtype. The highest protein expression for all three subtypes was in the renal pelvis and decreased with advancement along the ureter to the distal ureter. At the distal ureter, there was marked increase in expression as one progressed towards the ureteral orifice. The same pattern of protein expression was exhibited for all three alpha -1-adrenergic receptor subtypes. We provide preliminary evidence for the ability to detect and quantify the alpha-1-receptor subtypes along the human ureter which to the best of our knowledge has never been done with immunohistochemistry and molecular techniques. These findings may lend support to the preliminary studies of the effectiveness of alpha-receptor blockade on ureteral colic and stone passage.

Vaccination with Eimeria tenella Elongation Factor-1alpha Recombinant Protein Induces protective Immunity against E. tenella and E. maxima infections

USDA-ARS?s Scientific Manuscript database

Avian coccidiosis is caused by multiple species of the apicomplexan protozoan, Eimeria, and is one of the most economically devastating enteric diseases for the poultry industry worldwide. Host immunity to Eimeria infection, however, is relatively species-specific. The ability to immunize chickens a...

Cloning and characterization of BmK86, a novel K{sup +}-channel blocker from scorpion venom

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

Mao, Xin; Cao, Zhijian; Yin, Shijin

2007-09-07

Scorpion venom represents a tremendous hitherto unexplored resource for understanding ion channels. BmK86 is a novel K{sup +}-channel toxin gene isolated from a cDNA library of Mesobuthus martensii Karsch, which encodes a signal peptide of 22 amino acid residues and a mature toxin of 35 residues with three disulfide bridges. The genomic sequence of BmK86 consists of two exons disrupted by an intron of 72 bp. Comparison with the other scorpion toxins BmK86 shows low sequence similarity. The GST-BmK86 fusion protein was successfully expressed in Escherichia coli. The fusion protein was cleaved by enterokinase and the recombinant BmK86 was purifiedmore » by HPLC. Using whole-cell patch-clamp recording, the recombinant BmK86 was found to inhibit the potassium current of mKv1.3 channel expressed in COS7 cells. These results indicated that BmK86 belongs to a representative member of a novel subfamily of {alpha}-KTxs. The systematic number assigned to BmK86 is {alpha}-KTx26.1.« less

The construction of recombinant industrial yeasts free of bacterial sequences by directed gene replacement into a nonessential region of the genome.

PubMed

Xiao, W; Rank, G H

1989-03-15

The yeast SMR1 gene was used as a dominant resistance-selectable marker for industrial yeast transformation and for targeting integration of an economically important gene at the homologous ILV2 locus. A MEL1 gene, which codes for alpha-galactosidase, was inserted into a dispensable upstream region of SMR1 in vitro; different treatments of the plasmid (pWX813) prior to transformation resulted in 3' end, 5' end and replacement integrations that exhibited distinct integrant structures. One-step replacement within a nonessential region of the host genome generated a stable integration of MEL1 devoid of bacterial plasmid DNA. Using this method, we have constructed several alpha-galactosidase positive industrial Saccharomyces strains. Our study provides a general method for stable gene transfer in most industrial Saccharomyces yeasts, including those used in the baking, brewing (ale and lager), distilling, wine and sake industries, with solely nucleotide sequences of interest. The absence of bacterial DNA in the integrant structure facilitates the commercial application of recombinant DNA technology in the food and beverage industry.

Molecular cloning of the cDNA encoding laccase from Trametes versicolor and heterologous expression in Pichia methanolica.

PubMed

Guo, Mei; Lu, Fuping; Pu, Jun; Bai, Dongqing; Du, Lianxiang

2005-11-01

A cDNA encoding for laccase was isolated from the ligninolytic fungus Trametes versicolor by RNA-PCR. The cDNA corresponds to the gene Lcc1, which encodes a laccase isoenzyme of 498 amino acid residues preceded by a 22-residue signal peptide. The Lcc1 cDNA was cloned into the vectors pMETA and pMETalphaA and expressed in Pichia methanolica. The laccase activity obtained with the Saccharomyces cerevisiae alpha-factor signal peptide was found to be twofold higher than that obtained with the native secretion signal peptide. The extracellular laccase activity in recombinants with the alpha-factor signal peptide was 9.79 U ml(-1). The presence of 0.2 mM copper was necessary for optimal activity of laccase. The expression level was favoured by lower cultivation temperature. The identity of the recombinant protein was further confirmed by immunodetection using Western blot analysis. As expected, the molecular mass of the mature laccase was 64.0 kDa, similar to that of the native form.

Partial white and grey matter protection with prolonged infusion of recombinant human erythropoietin after asphyxia in preterm fetal sheep.

PubMed

Wassink, Guido; Davidson, Joanne O; Dhillon, Simerdeep K; Fraser, Mhoyra; Galinsky, Robert; Bennet, Laura; Gunn, Alistair J

2017-03-01

Perinatal asphyxia in preterm infants remains a significant contributor to abnormal long-term neurodevelopmental outcomes. Recombinant human erythropoietin has potent non-haematopoietic neuroprotective properties, but there is limited evidence for protection in the preterm brain. Preterm (0.7 gestation) fetal sheep received sham asphyxia (sham occlusion) or asphyxia induced by umbilical cord occlusion for 25 min, followed by an intravenous infusion of vehicle (occlusion-vehicle) or recombinant human erythropoietin (occlusion-Epo, 5000 international units by slow push, then 832.5 IU/h), starting 30 min after asphyxia and continued until 72 h. Recombinant human erythropoietin reduced neuronal loss and numbers of caspase-3-positive cells in the striatal caudate nucleus, CA3 and dentate gyrus of the hippocampus, and thalamic medial nucleus ( P < 0.05 vs. occlusion-vehicle). In the white matter tracts, recombinant human erythropoietin increased total, but not immature/mature oligodendrocytes ( P < 0.05 vs. occlusion-vehicle), with increased cell proliferation and reduced induction of activated caspase-3, microglia and astrocytes ( P < 0.05). Finally, occlusion-Epo reduced seizure burden, with more rapid recovery of electroencephalogram power, spectral edge frequency, and carotid blood flow. In summary, prolonged infusion of recombinant human erythropoietin after severe asphyxia in preterm fetal sheep was partially neuroprotective and improved electrophysiological and cerebrovascular recovery, in association with reduced apoptosis and inflammation.

Partial white and grey matter protection with prolonged infusion of recombinant human erythropoietin after asphyxia in preterm fetal sheep

PubMed Central

Wassink, Guido; Davidson, Joanne O; Dhillon, Simerdeep K; Fraser, Mhoyra; Galinsky, Robert; Bennet, Laura

2016-01-01

Perinatal asphyxia in preterm infants remains a significant contributor to abnormal long-term neurodevelopmental outcomes. Recombinant human erythropoietin has potent non-haematopoietic neuroprotective properties, but there is limited evidence for protection in the preterm brain. Preterm (0.7 gestation) fetal sheep received sham asphyxia (sham occlusion) or asphyxia induced by umbilical cord occlusion for 25 min, followed by an intravenous infusion of vehicle (occlusion-vehicle) or recombinant human erythropoietin (occlusion-Epo, 5000 international units by slow push, then 832.5 IU/h), starting 30 min after asphyxia and continued until 72 h. Recombinant human erythropoietin reduced neuronal loss and numbers of caspase-3-positive cells in the striatal caudate nucleus, CA3 and dentate gyrus of the hippocampus, and thalamic medial nucleus (P < 0.05 vs. occlusion-vehicle). In the white matter tracts, recombinant human erythropoietin increased total, but not immature/mature oligodendrocytes (P < 0.05 vs. occlusion-vehicle), with increased cell proliferation and reduced induction of activated caspase-3, microglia and astrocytes (P < 0.05). Finally, occlusion-Epo reduced seizure burden, with more rapid recovery of electroencephalogram power, spectral edge frequency, and carotid blood flow. In summary, prolonged infusion of recombinant human erythropoietin after severe asphyxia in preterm fetal sheep was partially neuroprotective and improved electrophysiological and cerebrovascular recovery, in association with reduced apoptosis and inflammation. PMID:27207167

Effects of recombinant human growth hormone and nandrolone phenylpropionate on the healing of ischemic colon anastomosis in rats.

PubMed

Yarimkaya, Ali; Apaydin, Berat; Unal, Ethem; Karabicak, Ilhan; Aydogan, Fatih; Uslu, Ezel; Erginoz, Ethem; Artis, Tarik; Eyuboglu, Erhun

2003-12-01

Recombinant human growth hormone and nandrolone phenylpropionate are two different anabolic agents. This study was designed to investigate the effects of these anabolic agents on the healing of ischemic colon anastomosis in rats. Seventy adult male Wistar rats were divided into five groups (n = 14). Group I was the sham laparotomy group. In the other groups, surgical procedures consisting of transsection and anastomosis were made at a distance 3 cm from the peritoneal reflection. Group II was the nonischemic control group. Ischemic colon model was produced in the remaining groups. Group III was the untreated control group. Groups IV and V received recombinant human growth hormone and nandrolone phenylpropionate, respectively. Bursting pressure and hydroxyproline levels were measured on the third and seventh postoperative days to evaluate anastomotic healing. Recombinant human growth hormone increased both collagen deposition and bursting pressure significantly at postoperative Days 3 and 7 compared with the sham and untreated control groups (P < 0.005). When compared with the untreated control, nandrolone phenylpropionate significantly increased collagen deposition at postoperative Days 3 and 7 (P < 0.005) and bursting pressure only at postoperative Day 3 (P < 0.005). Recombinant human growth hormone has more favorable therapeutic effects on the healing of ischemic colonic anastomoses than nandrolone phenylpropionate. Recombinant human growth hormone also improves healing of nonischemic colonic anastomosis.

Population-specific recombination sites within the human MHC region.

PubMed

Lam, T H; Shen, M; Chia, J-M; Chan, S H; Ren, E C

2013-08-01

Genetic rearrangement by recombination is one of the major driving forces for genome evolution, and recombination is known to occur in non-random, discreet recombination sites within the genome. Mapping of recombination sites has proved to be difficult, particularly, in the human MHC region that is complicated by both population variation and highly polymorphic HLA genes. To overcome these problems, HLA-typed individuals from three representative populations: Asian, European and African were used to generate phased HLA haplotypes. Extended haplotype homozygosity (EHH) plots constructed from the phased haplotype data revealed discreet EHH drops corresponding to recombination events and these signatures were observed to be different for each population. Surprisingly, the majority of recombination sites detected are unique to each population, rather than being common. Unique recombination sites account for 56.8% (21/37 of total sites) in the Asian cohort, 50.0% (15/30 sites) in Europeans and 63.2% (24/38 sites) in Africans. Validation carried out at a known sperm typing recombination site of 45 kb (HLA-F-telomeric) showed that EHH was an efficient method to narrow the recombination region to 826 bp, and this was further refined to 660 bp by resequencing. This approach significantly enhanced mapping of the genomic architecture within the human MHC, and will be useful in studies to identify disease risk genes.

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.

Kinetics of Inclusion Body Formation and Its Correlation with the Characteristics of Protein Aggregates in Escherichia coli

PubMed Central

Upadhyay, Arun K.; Murmu, Aruna; Singh, Anupam; Panda, Amulya K.

2012-01-01

The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH) and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm) and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2–3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low β-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies. PMID:22479486

A map of human PRDM9 binding provides evidence for novel behaviors of PRDM9 and other zinc-finger proteins in meiosis

PubMed Central

Noor, Nudrat; Bitoun, Emmanuelle; Tumian, Afidalina; Imbeault, Michael; Chapman, J Ross; Aricescu, A Radu

2017-01-01

PRDM9 binding localizes almost all meiotic recombination sites in humans and mice. However, most PRDM9-bound loci do not become recombination hotspots. To explore factors that affect binding and subsequent recombination outcomes, we mapped human PRDM9 binding sites in a transfected human cell line and measured PRDM9-induced histone modifications. These data reveal varied DNA-binding modalities of PRDM9. We also find that human PRDM9 frequently binds promoters, despite their low recombination rates, and it can activate expression of a small number of genes including CTCFL and VCX. Furthermore, we identify specific sequence motifs that predict consistent, localized meiotic recombination suppression around a subset of PRDM9 binding sites. These motifs strongly associate with KRAB-ZNF protein binding, TRIM28 recruitment, and specific histone modifications. Finally, we demonstrate that, in addition to binding DNA, PRDM9's zinc fingers also mediate its multimerization, and we show that a pair of highly diverged alleles preferentially form homo-multimers. PMID:29072575

The human interleukin-1 alpha gene is located on the long arm of chromosome 2 at band q13.

PubMed

Lafage, M; Maroc, N; Dubreuil, P; de Waal Malefijt, R; Pébusque, M J; Carcassonne, Y; Mannoni, P

1989-01-01

Interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) are two biochemically distinct, but distantly related, polypeptidic cytokines that play a key role in inflammation, immunologic reactions, and tissue repair. Recently, it has been shown that IL-1 alpha is identical to hematopoietin 1, which was described as a hematopoietic growth factor acting on early progenitor cells in synergy with other hematopoietic growth factors. In this report we discuss our use of in situ hybridization on human prometaphase cells with a human IL-1 alpha cDNA probe to localize the human IL-1 alpha gene on the proximal part of the long arm of chromosome 2 at band q13, in the same chromosomal region as the IL-1 beta gene.

The alpha-spectrin gene is on chromosome 1 in mouse and man.

PubMed Central

Huebner, K; Palumbo, A P; Isobe, M; Kozak, C A; Monaco, S; Rovera, G; Croce, C M; Curtis, P J

1985-01-01

By using alpha-spectrin cDNA clones of murine and human origin and somatic cell hybrids segregating either mouse or human chromosomes, the gene for alpha-spectrin has been mapped to chromosome 1 in both species. This assignment of the mouse alpha-spectrin gene to mouse chromosome 1 by DNA hybridization strengthens the previous identification of the alpha-spectrin locus in mouse with the sph locus, which previously was mapped by linkage analysis to mouse chromosome 1, distal to the Pep-3 locus. By in situ hybridization to human metaphase chromosomes, the human alpha-spectrin gene has been localized to 1q22-1q25; interestingly, the locus for a non-Rh-linked form of elliptocytosis has been provisionally mapped to band 1q2 by family linkage studies. Images PMID:2987946

A defective retroviral vector encoding human interferon-alpha2 can transduce human leukemic cell lines.

PubMed

Austruy, E; Bagnis, C; Carbuccia, N; Maroc, C; Birg, F; Dubreuil, P; Mannoni, P; Chabannon, C

1998-01-01

Using the LXSN backbone, a defective retroviral vector (LISN) was constructed that encodes the human interferon (IFN)-alpha2 (hIFN-alpha2) gene and the neomycin resistance gene; the hIFN-alpha2 gene was cloned from human placental genomic DNA. High titers of the LISN retrovirus were produced by the amphotropic packaging cell line GP+envAM12. LISN is able to infect three human hematopoietic and leukemic cell lines: K562, LAMA-84, and TF-1. G418-resistant cells were detected in a similar proportion after infection with either the LISN retroviral vector or the LnLSN retroviral vector (encoding the nlsLacZ gene instead of hIFN-alpha2), suggesting that hIFN-alpha2 does not inhibit (or only partially inhibits) the production of retroviral particles by the packaging cell line and the infection of human cells. LISN-infected cells express and secrete hIFN-alpha2 as demonstrated by Northern blot analysis of poly(A)+ RNA, detection of the intracellular protein by fluorescence-activated cell sorter analysis, and detection of secreted hIFN-alpha in cell supernatants using an enzyme-linked immunosorbent assay. Retrovirally produced hIFN-alpha2 is biologically active, as demonstrated by the partial inhibition of the growth of K562 and TF-1, the modulation of the expression of cell surface antigens, the induction of the (2'-5') oligoadenylate synthetase, and, for LAMA-84, the down-modulation of the BCR-ABL protein. We conclude that the infection of human leukemic cell lines with a retroviral vector encoding hIFN-alpha2 is feasible and induces the expected biological effects. This experimental model will be useful in investigating the possibility of transducing normal and leukemic cells and hematopoietic progenitors and in determining the consequences of the autocrine production of hIFN-alpha2 on the behavior of these cells.

Therapeutic compositions and uses of alpha1-antitrypsin: a patent review (2012 - 2015).

PubMed

Lior, Yotam; Geyra, Assaf; Lewis, Eli C

2016-05-01

Identified as a circulating serine-protease inhibitor, the genetic deficiency of which predisposes to the development of lung emphysema, alpha1-antitrypsin (AAT) has recently been found to possess various anti-inflammatory and immunomodulatory activities outside the biochemical inhibition of serine-proteases. AAT is presently extracted from human plasma to supply life-long infusions to patients with genetic AAT deficiency. However, its newly appreciated functions point to extended therapeutic uses; these, alongside modified production attempts, represent a novel and dynamic niche of drug repurposing, set apart from addressing lung emphysema in AAT-deficient individuals. The review provides a comprehensive summary of patent-protected inventions in the field of novel clinical indications for AAT and innovations in AAT production. A molecule no longer patentable per se, presents with novel clinical applications; its mechanism still unfolding. While modified protein sequences are patentable and potentially superior, they are burdened by regulatory setbacks. Thus, recent approaches in the context of AAT appear in patents that describe combinations with other drugs, redefined clinical subclasses, and unique recombinant entities, carefully skirting saturated areas of AAT patentology. It will be fascinating to follow technologies and creative patenting as AAT navigates the trying trades of pharmaceutical industry towards an increasing lineup of unmet clinical needs.

Non-specific gastrointestinal features: Could it be Fabry disease?

PubMed

Hilz, Max J; Arbustini, Eloisa; Dagna, Lorenzo; Gasbarrini, Antonio; Goizet, Cyril; Lacombe, Didier; Liguori, Rocco; Manna, Raffaele; Politei, Juan; Spada, Marco; Burlina, Alessandro

2018-05-01

Non-specific gastrointestinal symptoms, including pain, diarrhoea, nausea, and vomiting, can be the first symptoms of Fabry disease. They may suggest more common disorders, e.g. irritable bowel syndrome or inflammatory bowel disease. The confounding clinical presentation and rarity of Fabry disease often cause long diagnostic delays and multiple misdiagnoses. Therefore, specialists involved in the clinical evaluation of non-specific upper and lower gastrointestinal symptoms should recognize Fabry disease as a possible cause of the symptoms, and should consider Fabry disease as a possible differential diagnosis. When symptoms or family history suggest Fabry disease, in men, low alpha-galactosidase A enzyme levels, and in women, specific Fabry mutations confirm the diagnosis. In addition to symptomatic treatments, disease-specific enzyme replacement therapy with recombinant human alpha-galactosidase A enzyme or chaperone therapy (migalastat) in patients with amenable mutations can improve the disease, including gastrointestinal symptoms, and should be initiated as early as possible after Fabry disease has been confirmed; starting enzyme replacement therapy at as young an age as possible after diagnosis improves long-term clinical outcomes. Improved diagnostic tools, such as a modified gastrointestinal symptom rating scale, may facilitate diagnosing Fabry disease in patients with gastrointestinal symptoms of unknown cause and thus assure timely initiation of disease-specific treatment. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

International Validation of Two Human Recombinant Estrogen Receptor (ERa) Binding Assays

EPA Science Inventory

An international validation study has been successfully completed for 2 competitive binding assays using human recombinant ERa. Assays evaluated included the Freyberger-Wilson (FW) assay using a full length human ER, and the Chemical Evaluation and Research Institute (CERI) assay...

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

Lundasen, Thomas; Molecular Nutrition Unit, Department of Biosciences and Nutrition, NOVUM, Karolinska Institutet, Huddinge, SE-141 86 Stockholm; Hunt, Mary C.

The metabolic regulator fibroblast growth factor 21 (FGF21) has antidiabetic properties in animal models of diabetes and obesity. Using quantitative RT-PCR, we here show that the hepatic gene expression of FGF21 is regulated by the peroxisome proliferator-activated receptor alpha (PPAR{alpha}). Fasting or treatment of mice with the PPAR{alpha} agonist Wy-14,643 induced FGF21 mRNA by 10-fold and 8-fold, respectively. In contrast, FGF21 mRNA was low in PPAR{alpha} deficient mice, and fasting or treatment with Wy-14,643 did not induce FGF21. Obese ob/ob mice, known to have increased PPAR{alpha} levels, displayed 12-fold increased hepatic FGF21 mRNA levels. The potential importance of PPAR{alpha} formore » FGF21 expression also in human liver was shown by Wy-14,643 induction of FGF21 mRNA in human primary hepatocytes, and PPAR{alpha} response elements were identified in both the human and mouse FGF21 promoters. Further studies on the mechanisms of regulation of FGF21 by PPAR{alpha} in humans will be of great interest.« less

Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgenic cattle.

PubMed

Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning

2011-03-16

There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale.

Characterization of Bioactive Recombinant Human Lysozyme Expressed in Milk of Cloned Transgenic Cattle

PubMed Central

Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning

2011-01-01

Background There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. Methodology/Principal Findings We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Conclusions/Significance Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale. PMID:21436886

Macrocyclic peptide inhibitors for the protein-protein interaction of Zaire Ebola virus protein 24 and karyopherin alpha 5.

PubMed

Song, Xiao; Lu, Lu-Yi; Passioura, Toby; Suga, Hiroaki

2017-06-21

Ebola virus infection leads to severe hemorrhagic fever in human and non-human primates with an average case fatality rate of 50%. To date, numerous potential therapies are in development, but FDA-approved drugs or vaccines are yet unavailable. Ebola viral protein 24 (VP24) is a multifunctional protein that plays critical roles in the pathogenesis of Ebola virus infection, e.g. innate immune suppression by blocking the interaction between KPNA and PY-STAT1. Here we report macrocyclic peptide inhibitors of the VP24-KPNA5 protein-protein interaction (PPI) by means of the RaPID (Random non-standard Peptides Integrated Discovery) system. These macrocyclic peptides showed remarkably high affinity to recombinant Zaire Ebola virus VP24 (eVP24), with a dissociation constant in the single digit nanomolar range, and could also successfully disrupt the eVP24-KPNA interaction. This work provides for the first time a chemical probe capable of modulating this PPI interaction and is the starting point for the development of unique anti-viral drugs against the Ebola virus.

Nicotinic Receptor Alpha7 Expression during Tooth Morphogenesis Reveals Functional Pleiotropy

PubMed Central

Rogers, Scott W.; Gahring, Lorise C.

2012-01-01

The expression of nicotinic acetylcholine receptor (nAChR) subtype, alpha7, was investigated in the developing teeth of mice that were modified through homologous recombination to express a bi-cistronic IRES-driven tau-enhanced green fluorescent protein (GFP); alpha7GFP) or IRES-Cre (alpha7Cre). The expression of alpha7GFP was detected first in cells of the condensing mesenchyme at embryonic (E) day E13.5 where it intensifies through E14.5. This expression ends abruptly at E15.5, but was again observed in ameloblasts of incisors at E16.5 or molar ameloblasts by E17.5–E18.5. This expression remains detectable until molar enamel deposition is completed or throughout life as in the constantly erupting mouse incisors. The expression of alpha7GFP also identifies all stages of innervation of the tooth organ. Ablation of the alpha7-cell lineage using a conditional alpha7Cre×ROSA26-LoxP(diphtheria toxin A) strategy substantially reduced the mesenchyme and this corresponded with excessive epithelium overgrowth consistent with an instructive role by these cells during ectoderm patterning. However, alpha7knock-out (KO) mice exhibited normal tooth size and shape indicating that under normal conditions alpha7 expression is dispensable to this process. The function of ameloblasts in alpha7KO mice is altered relative to controls. High resolution micro-computed tomography analysis of adult mandibular incisors revealed enamel volume of the alpha7KO was significantly reduced and the organization of enamel rods was altered relative to controls. These results demonstrate distinct and varied spatiotemporal expression of alpha7 during tooth development, and they suggest that dysfunction of this receptor would have diverse impacts upon the adult organ. PMID:22666322

Chimpanzees Immunized with Recombinant Soluble CD4 Develop Anti-Self CD4 Antibody Responses with Anti-Human Immunodeficiency Virus Activity

NASA Astrophysics Data System (ADS)

Watanabe, Mamoru; Boyson, Jonathan E.; Lord, Carol I.; Letvin, Norman L.

1992-06-01

In view of the efficiency with which human immunodeficiency virus replication can be blocked in vitro with anti-CD4 antibodies, the elicitation of an anti-CD4 antibody response through active immunization might represent a useful therapeutic strategy for AIDS. Here we demonstrate that immunization of chimpanzees with recombinant soluble human CD4 elicited an anti-CD4 antibody response. The elicited antibody bound self CD4 on digitonin-treated but not freshly isolated lymphocytes. Nevertheless, this antibody blocked human immunodeficiency virus replication in chimpanzee and human lymphocytes. These observations suggest that immunization with recombinant soluble CD4 from human immunodeficiency virus-infected humans may be feasible and therapeutically beneficial.

Bacterial RecA Protein Promotes Adenoviral Recombination during In Vitro Infection

PubMed Central

Lee, Jeong Yoon; Lee, Ji Sun; Materne, Emma C.; Rajala, Rahul; Ismail, Ashrafali M.; Seto, Donald; Dyer, David W.

2018-01-01

ABSTRACT Adenovirus infections in humans are common and sometimes lethal. Adenovirus-derived vectors are also commonly chosen for gene therapy in human clinical trials. We have shown in previous work that homologous recombination between adenoviral genomes of human adenovirus species D (HAdV-D), the largest and fastest growing HAdV species, is responsible for the rapid evolution of this species. Because adenovirus infection initiates in mucosal epithelia, particularly at the gastrointestinal, respiratory, genitourinary, and ocular surfaces, we sought to determine a possible role for mucosal microbiota in adenovirus genome diversity. By analysis of known recombination hot spots across 38 human adenovirus genomes in species D (HAdV-D), we identified nucleotide sequence motifs similar to bacterial Chi sequences, which facilitate homologous recombination in the presence of bacterial Rec enzymes. These motifs, referred to here as ChiAD, were identified immediately 5′ to the sequence encoding penton base hypervariable loop 2, which expresses the arginine-glycine-aspartate moiety critical to adenoviral cellular entry. Coinfection with two HAdV-Ds in the presence of an Escherichia coli lysate increased recombination; this was blocked in a RecA mutant strain, E. coli DH5α, or upon RecA depletion. Recombination increased in the presence of E. coli lysate despite a general reduction in viral replication. RecA colocalized with viral DNA in HAdV-D-infected cell nuclei and was shown to bind specifically to ChiAD sequences. These results indicate that adenoviruses may repurpose bacterial recombination machinery, a sharing of evolutionary mechanisms across a diverse microbiota, and unique example of viral commensalism. IMPORTANCE Adenoviruses are common human mucosal pathogens of the gastrointestinal, respiratory, and genitourinary tracts and ocular surface. Here, we report finding Chi-like sequences in adenovirus recombination hot spots. Adenovirus coinfection in the presence of bacterial RecA protein facilitated homologous recombination between viruses. Genetic recombination led to evolution of an important external feature on the adenoviral capsid, namely, the penton base protein hypervariable loop 2, which contains the arginine-glycine-aspartic acid motif critical to viral internalization. We speculate that free Rec proteins present in gastrointestinal secretions upon bacterial cell death facilitate the evolution of human adenoviruses through homologous recombination, an example of viral commensalism and the complexity of virus-host interactions, including regional microbiota. PMID:29925671

Tryptophan W207 in transducin T alpha is the fluorescence sensor of the G protein activation switch and is involved in the effector binding.

PubMed Central

Faurobert, E; Otto-Bruc, A; Chardin, P; Chabre, M

1993-01-01

We have produced a recombinant transducin alpha subunit (rT alpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N-terminal increased the solubility and allowed the purification of rT alpha. When reconstituted with excess T beta gamma on retinal membrane, rT alpha displayed functional characteristics of wild-type T alpha vis à vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]T alpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor-catalysed GTP gamma S binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector-activating interaction we measured the affinity of [W207F]T alpha GDP-AlFx for PDE gamma, the effector subunit that binds most tightly to T alpha. [W207F]T alpha still bound in an activation-dependent way to PDE gamma, but with a 100-fold lower affinity than rT alpha. This suggests that W207 contributes to the G protein effector binding. Images PMID:8223434

Rhizomucor miehei triglyceride lipase is processed and secreted from transformed Aspergillus oryzae.

PubMed

Huge-Jensen, B; Andreasen, F; Christensen, T; Christensen, M; Thim, L; Boel, E

1989-09-01

The cDNA encoding the precursor of the Rhizomucor miehei triglyceride lipase was inserted in an Aspergillus oryzae expression vector. In this vector the expression of the lipase cDNA is under control of the Aspergillus oryzae alpha-amylase gene promoter and the Aspergillus niger glucoamylase gene terminator. The recombinant plasmid was introduced into Aspergillus oryzae, and transformed colonies were selected and screened for lipase expression. Lipase-positive transformants were grown in a small fermentor, and recombinant triglyceride lipase was purified from the culture broth. The purified enzymatically active recombinant lipase (rRML) secreted from A. oryzae was shown to have the same characteristics with respect to mobility on reducing SDS-gels and amino acid composition as the native enzyme. N-terminal amino acid sequencing indicated that approximately 70% of the secreted rRML had the same N-terminal sequence as the native Rhizomucor miehei enzyme, whereas 30% of the secreted rRML was one amino acid residue shorter in the N-terminal. The recombinant lipase precursor, which has a 70 amino acid propeptide, is thus processed in and secreted from Aspergillus oryzae. We have hereby demonstrated the utility of this organism as a host for the production of recombinant triglyceride lipases.

Fabrication, characterization and simulation of 4H-SiC Schottky diode alpha particle detectors for pyroprocessing actinide monitoring

NASA Astrophysics Data System (ADS)

Garcia, Timothy Richard

Pyroprocessing is a method of using high-temperature molten salts and electric fields to separate and collect fuel isotopes of used nuclear fuel. It has been has been tested in the U.S. at Idaho National Laboratory as a key step in closing the nuclear fuel cycle. One technical problem with the pyroprocessing method is a lack of knowledge regarding the actinide concentrations in the salt bath during operation, since on-line techniques for measuring these concentrations are not presently available. 4H-SiC Schottky diode detectors can potentially fulfill this need. Such detectors would operate in contact with the molten salt, and measure concentrations via alpha-particle spectroscopy. This work seeks to fabricate and characterize 4H-SiC Schottky diode detectors at high temperature, model the alpha particle spectrum expected in a molten salt, and model the operation of the detectors to confirm the physics of operation is as expected. In this work, 4H-SiC Schottky diode detectors were fabricated at OSU Nanotech West. After fabrication, these detectors were characterized using both I-V curves and Am-241 alpha-particle energy spectra. All measurements were made as a function of temperature, from room temperature up to 500°C. The average energy required to create an electron-hole pair was observed to decrease with an increase of temperature, due to a decrease of both the 4H-SiC bandgap and non-linear energy loss terms. Furthermore, the FWHM of the spectra was observed to be dependent on the leakage current at a certain temperature, and not dependent on the temperature itself. Secondly, the alpha particle energy spectrum in the pyroprocessing environment was modeled using SRIM. The molten salt was modeled in 3 different geometries, with or without a protective cover material on top of the detector. Due to the loss of alpha-particle energy in the molten salt itself, a high-energy alpha emitter may completely cover the spectrum from a lower-energy alpha emitter. Each of the geometries simulated showed a different sensitivity to the lower-energy alpha emitter. Regardless of which geometry was modeled, it was observed that it is possible to measure both the emission energy of the alpha particles, as well as the concentration of the alpha emitter in the liquid. Lastly, Sentaurus TCAD was used to simulate the detection of alpha-particle charge collection in situations that are relevant to the molten salt alpha particle energy spectra. The effect of electric field negation was investigated, as well as velocity saturation. Finally, the dependence of charge recombination on temperature, alpha particle energy, and angle of incidence was investigated. These simulations captured the measurements performed at room temperature. With changed angle of incidence, the change in the amount of charge collected was less than 1 percent, indicating a weak dependence. Also, the amount of charge lost to Auger recombination was seen to increase with temperature. This disagrees with observations from experiment, indicating that the temperature dependence of one or more parameters of the model may not be accurate.

Evidence for human meiotic recombination interference obtained through construction of a short tandem repeat-polymorphism linkage map of chromosome 19

PubMed Central

Weber, James L.; Wang, Zhenyuan; Hansen, Kevin; Stephenson, Matt; Kappel, Clarisse; Salzman, Sherry; Wilkie, Patricia J.; Keats, Bronya; Dracopoli, Nicholas C.; Brandriff, Brigitte F.; Olsen, Anne S.

1993-01-01

An improved linkage map for human chromosome 19 containing 35 short tandem repeat polymorphisms (STRPs) and one VNTR (D19S20) was constructed. The map included 12 new (GATA)n tetranucleotide STRPs. Although total lengths of the male (114 cM) and female (128 cM) maps were similar, at both ends of the chromosome male recombination exceeded female recombination, while in the interior portion of the map female recombination was in excess. Cosmid clones containing the STRP sequences were identified and were positioned along the chromosome by fluorescent in situ hybridization. Four rounds of careful checking and removal of genotyping errors allowed biologically relevant conclusions to be made concerning the numbers and distributions of recombination events on chromosome 19. The average numbers of recombinations per chromosome matched closely the lengths of the genetic maps computed by using the program CRIMAP. Significant numbers of chromosomes with zero, one, two, or three recombinations were detected as products of both female and male meioses. On the basis of the total number of observed pairs of recombination events in which only a single informative marker was situated between the two recombinations, a maximal estimate for the rate of meiotic STRP “gene” conversion without recombination was calculated as 3 × 10−4/meiosis. For distances up to 30 cM between recombinations, many fewer chromosomes which had undergone exactly two recombinations were observed than were expected on the basis of the assumption of independent recombination locations. This strong new evidence for human meiotic interference will help to improve the accuracy of interpretation of clinical DNA test results involving polymorphisms flanking a genetic abnormality. PMID:8213834

Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine

Cancer.gov

This page contains brief information about recombinant human papillomavirus (HPV) quadrivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine

Cancer.gov

This page contains brief information about recombinant human papillomavirus (HPV) nonavalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

Recombinant Human Papillomavirus (HPV) Bivalent Vaccine

Cancer.gov

This page contains brief information about recombinant human papillomavirus (HPV) bivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

Enzyme replacement therapy with agalsidase beta in kidney transplant patients with Fabry disease: a pilot study.

PubMed

Mignani, Renzo; Panichi, Vincenzo; Giudicissi, Antonio; Taccola, Daniele; Boscaro, Francesca; Feletti, Carlo; Moneti, Gloriano; Cagnoli, Leonardo

2004-04-01

We sought to assess the safety and efficacy of enzyme replacement therapy (ERT) with recombinant human-alpha-galactosidase A (rh-alpha-Gal A) in kidney transplant recipients with Fabry disease, a previously unstudied population. Three male kidney transplant recipients with biochemically, genetically, and histologically confirmed Fabry disease and documented Fabry myocardiopathy received the rh-alpha-Gal A, agalsidase beta, 1 mg/kg of body weight every 2 weeks by intravenous infusion and were monitored biochemically, clinically, and electrocardiographically and echocardiographically for 18 months. Patients showed biochemical, clinical/functional, and morphologic response to ERT. Plasma globotriaosylceramide decreased 23% to 50%. Extremity pain resolved within 2 months in the patient with this manifestation. On echocardiography, left ventricular mass, end diastolic diameter (EDD), and cardiac contractility, shown by ejection fraction (EF), improved in 2 of the 3 patients receiving essentially all planned infusions. EDD and EF remained basically stable, but cardiac morphologic abnormalities progressed in the other patient, who had a 5-month interruption in ERT after the initial month. Mild mitral insufficiency persisted in all patients, as did atrial fibrillation in the affected individual. After a combined total of 116 infusions, no treatment-related adverse event, intolerance, or seroconversion was seen. Renal function remained stable and the immunosuppression regimen unchanged in all patients. Our pilot study provides preliminary evidence that ERT with agalsidase beta, 1 mg/kg every 2 weeks, is safe and often effective against extra-renal manifestations in kidney transplant patients with Fabry disease. Studies with longer courses of this and higher doses of ERT are merited in this population.

Cytokine production by oral and peripheral blood neutrophils in adult periodontitis.

PubMed

Galbraith, G M; Hagan, C; Steed, R B; Sanders, J J; Javed, T

1997-09-01

Proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) also possess bone-resorptive properties, and are generally considered to play a role in the pathogenesis of periodontal disease. In the present study, TNF-alpha and IL-1 beta production by oral and peripheral blood polymorphonuclear leukocytes (PMN) was examined in 40 patients with adult periodontitis and 40 orally healthy matched controls. Oral PMN released considerable amounts of both cytokines in unstimulated culture, and there was no difference between patients and controls when the cytokine levels were corrected for cell number. However, when the effect of disease activity was examined, cytokine release by oral PMN was found to be greatest in patients with advanced periodontitis. Within the healthy control group, IL-1 beta production by oral PMN was significantly higher in males (Mann-Whitney test, P = 0.0008). Examination of IL-1 beta production by peripheral blood PMN exposed to recombinant human granulocyte-macrophage colony stimulating factor revealed no difference between the patient and control groups. In contrast, IL-1 beta production by peripheral blood PMN was significantly reduced in patients with advanced disease (Mann-Whitney test, P = 0.02), and peripheral PMN IL-1 beta synthesis was greater in female controls (Mann-Whitney test, P = 0.054). No effect of race on cytokine production could be discerned in patients or controls. These results indicate that several factors influence cytokine production in oral health and disease, and that a dichotomy in cytokine gene expression exists between oral and peripheral blood PMN in adult periodontitis.

A novel role for the integrin-binding III-10 module in fibronectin matrix assembly.

PubMed

Hocking, D C; Smith, R K; McKeown-Longo, P J

1996-04-01

Fibronectin matrix assembly is a cell-dependent process which is upregulated in tissues at various times during development and wound repair to support the functions of cell adhesion, migration, and differentiation. Previous studies have demonstrated that the alpha 5 beta 1 integrin and fibronectin's amino terminus and III-1 module are important in fibronectin polymerization. We have recently shown that fibronectin's III-1 module contains a conformationally sensitive binding site for fibronectin's amino terminus (Hocking, D.C., J. Sottile, and P.J. McKeown-Longo. 1994. J. Biol. Chem. 269: 19183-19191). The present study was undertaken to define the relationship between the alpha 5 beta 1 integrin and fibronectin polymerization. Solid phase binding assays using recombinant III-10 and III-1 modules of human plasma fibronectin indicated that the III-10 module contains a conformation-dependent binding site for the III-1 module of fibronectin. Unfolded III-10 could support the formation of a ternary complex containing both III-1 and the amino-terminal 70-kD fragment, suggesting that the III-1 module can support the simultaneous binding of III-10 and 70 kD. Both unfolded III-10 and unfolded III-1 could support fibronectin binding, but only III-10 could promote the formation of disulfide-bonded multimers of fibronectin in the absence of cells. III-10-dependent multimer formation was inhibited by both the anti-III-1 monoclonal antibody, 9D2, and amino-terminal fragments of fibronectin. A fragment of III-10, termed III-10/A, was able to block matrix assembly in fibroblast monolayers. Similar results were obtained using the III-10A/RGE fragment, in which the RGD site had been mutated to RGE, indicating that III-I0/A was blocking matrix assembly by a mechanism distinct from disruption of integrin binding. Texas red-conjugated recombinant III-1,2 localized to beta 1-containing sites of focal adhesions on cells plated on fibronectin or the III-9,10 modules of fibronectin. Monoclonal antibodies against the III-1 or the III-9,10 modules of fibronectin blocked binding of III-1,2 to cells without disrupting focal adhesions. These data suggest that a role of the alpha 5 beta 1 integrin in matrix assembly is to regulate a series of sequential self-interactions which result in the polymerization of fibronectin.

New measurements of W-values for protons and alpha particles.

PubMed

Giesen, U; Beck, J

2014-10-01

The increasing importance of ion beams in cancer therapy and the lack of experimental data for W-values for protons and heavy ions in air require new measurements. A new experimental set-up was developed at PTB and consistent measurements of W-values in argon, nitrogen and air for protons and alpha particles with energies from 0.7 to 3.5 MeV u(-1) at PTB, and for carbon ions between 3.6 and 7.0 MeV u(-1) at GSI were carried out. This publication concentrates on the measurements with protons and alpha particles at PTB. The experimental methods and the determination of corrections for recombination effects, beam-induced background radiation and additional effects are presented. © The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

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

McCormick, D.J.; Griesmann, G.E.; Huang, Z.

1986-03-05

A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 ..mu..g of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodiesmore » to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.« less

Tumor cell alpha-N-acetylgalactosaminidase activity and its involvement in GcMAF-related macrophage activation.

PubMed

Mohamad, Saharuddin B; Nagasawa, Hideko; Uto, Yoshihiro; Hori, Hitoshi

2002-05-01

Alpha-N-acetyl galactosaminidase (alpha-NaGalase) has been reported to accumulate in serum of cancer patients and be responsible for deglycosylation of Gc protein, which is a precursor of GcMAF-mediated macrophage activation cascade, finally leading to immunosuppression in advanced cancer patients. We studied the biochemical characterization of alpha-NaGalase from several human tumor cell lines. We also examined its effect on the potency of GcMAF to activate mouse peritoneal macrophage to produce superoxide in GcMAF-mediated macrophage activation cascade. The specific activity of alpha-NaGalases from human colon tumor cell line HCT116, human hepatoma cell line HepG2, and normal human liver cells (Chang liver cell line) were evaluated using two types of substrates; GalNAc-alpha-PNP (exo-type substrate) and Gal-beta-GalNAc-alpha-PNP (endo-type substrate). Tumor-derived alpha-NaGalase having higher activity than normal alpha-NaGalase, had higher substrate specificity to the exo-type substrate than to the endo-type substrate, and still maintained its activity at pH 7. GcMAF enhance superoxide production in mouse macrophage, and pre-treatment of GcMAF with tumor cell lysate reduce the activity. We conclude that tumor-derived alpha-NaGalase is different in biochemical characterization compared to normal alpha-NaGalase from normal Chang liver cells. In addition, tumor cell-derived alpha-NaGalase decreases the potency of GcMAF on macrophage activation.

A simplified bioprocess for human alpha-fetoprotein production from inclusion bodies.

PubMed

Leong, Susanna S J; Middelberg, Anton P J

2007-05-01

A simple and effective Escherichia coli (E. coli) bioprocess is demonstrated for the preparation of recombinant human alpha-fetoprotein (rhAFP), a pharmaceutically promising protein that has important immunomodulatory functions. The new rhAFP process employs only unit operations that are easy to scale and validate, and reduces the complexity embedded in existing inclusion body processing methods. A key requirement in the establishment of this process was the attainment of high purity rhAFP prior to protein refolding because (i) rhAFP binds easily to hydrophobic contaminants once refolded, and (ii) rhAFP aggregates during renaturation, in a contaminant- dependent way. In this work, direct protein extraction from cell suspension was coupled with a DNA precipitation-centrifugation step prior to purification using two simple chromatographic steps. Refolding was conducted using a single-step, redox-optimized dilution refolding protocol, with refolding success determined by reversed phase HPLC analysis, ELISA, and circular dichroism spectroscopy. Quantitation of DNA and protein contaminant loads after each unit operation showed that contaminant levels were reduced to levels comparable to traditional flowsheets. Protein microchemical modification due to carbamylation in this urea-based process was identified and minimized, yielding a final refolded and purified product that was significantly purified from carbamylated variants. Importantly, this work conclusively demonstrates, for the first time, that a chemical extraction process can substitute the more complex traditional inclusion body processing flowsheet, without compromising product purity and yield. This highly intensified and simplified process is expected to be of general utility for the preparation of other therapeutic candidates expressed as inclusion bodies. (c) 2006 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.

A naturally occurring Lgr4 splice variant encodes a soluble antagonist useful for demonstrating the gonadal roles of Lgr4 in mammals.

PubMed

Hsu, Pei-Jen; Wu, Fang-Ju; Kudo, Masataka; Hsiao, Chih-Lun; Hsueh, Aaron J W; Luo, Ching-Wei

2014-01-01

Leucine-rich repeat containing G protein-coupled receptor 4 (LGR4) promotes the Wnt signaling through interaction with R-spondins or norrin. Using PCR amplification from rat ovarian cDNAs, we identified a naturally occurring Lgr4 splice variant encoding only the ectodomain of Lgr4, which was named Lgr4-ED. Lgr4-ED can be detected as a secreted protein in the extracts from rodent and bovine postnatal gonads, suggesting conservation of Lgr4-ED in mammals. Recombinant Lgr4-ED purified from the conditioned media of transfected 293T cells was found to dose-dependently inhibit the LGR4-mediated Wnt signaling induced by RSPO2 or norrin, suggesting that it is capable of ligand absorption and could have a potential role as an antagonist. Intraperitoneal injection of purified recombinant Lgr4-ED into newborn mice was found to significantly decrease the testicular expression of estrogen receptor alpha and aquaporin 1, which is similar to the phenotype found in Lgr4-null mice. Administration of recombinant Lgr4-ED to superovulated female rats can also decrease the expression of estrogen receptor alpha, aquaporin 1, LH receptor and other key steroidogenic genes as well as bring about the suppression of progesterone production. Thus, these findings suggest that endogenously expressed Lgr4-ED may act as an antagonist molecule and help to fine-tune the R-spondin/norrin-mediated Lgr4-Wnt signaling during gonadal development.

cap alpha. /sub i/-3 cDNA encodes the. cap alpha. subunit of G/sub k/, the stimulatory G protein of receptor-regulated K/sup +/ channels

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

Codina, J.; Olate, J.; Abramowitz, J.

1988-05-15

cDNA cloning has identified the presence in the human genome of three genes encoding ..cap alpha.. subunits of pertussis toxin substrates, generically called G/sub i/. They are named ..cap alpha../sub i/-1, ..cap alpha../sub i/-2 and ..cap alpha../sub i/-3. However, none of these genes has been functionally identified with any of the ..cap alpha.. subunits of several possible G proteins, including pertussis toxin-sensitive G/sub p/'s, stimulatory to phospholipase C or A/sub 2/, G/sub i/, inhibitory to adenylyl cyclase, or G/sub k/, stimulatory to a type of K/sup +/ channels. The authors now report the nucleotide sequence and the complete predicted aminomore » acid sequence of human liver ..cap alpha../sub i/-3 and the partial amino acid sequence of proteolytic fragments of the ..cap alpha.. subunit of human erythrocyte G/sub k/. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of ..cap alpha../sub i/-3, thus identifying it as ..cap alpha../sub k/. The probable identity of ..cap alpha../sub i/-1 with ..cap alpha../sub p/ and possible roles for ..cap alpha../sub i/-2, as well as additional roles for ..cap alpha../sub i/-1 and ..cap alpha../sub i/-3 (..cap alpha../sub k/) are discussed.« less

Tumor necrosis factor-alpha inhibits differentiation of myogenic cells in human urethral rhabdosphincter.

PubMed

Shinohara, Mayuka; Sumino, Yasuhiro; Sato, Fuminori; Kiyono, Tohru; Hashimoto, Naohiro; Mimata, Hiromitsu

2017-06-01

To examine the inhibitory effects of tumor necrosis factor-α on myogenic differentiation of human urethral rhabdosphincter cells. A rhabdosphincter sample was obtained from a patient who underwent total cystectomy. To expand the lifespan of the primary cultured cells, rhabdosphincter myogenic cells were immortalized with mutated cyclin-dependent kinase 4, cyclin D1 and telomerase. The differential potential of the cells was investigated. The transfected human rhabdosphincter cells were induced for myogenic differentiation with recombinant human tumor necrosis factor-α and/or the tumor necrosis factor-α antagonist etanercept at different concentrations, and activation of signaling pathways was monitored. Human rhabdosphincter cells were selectively cultured for at least 40 passages. Molecular analysis confirmed the expression of myosin heavy chain, which is a specific marker of differentiated muscle cells, significantly increased after differentiation induction. Although tumor necrosis factor-α treatment reduced the myosin heavy chain expression in a concentration-dependent manner, etanercept inhibited this suppression. Tumor necrosis factor-α suppressed phosphorylation of protein kinase B and p38, whereas etanercept pretreatment promoted phosphorylation and myosin heavy chain expression in a concentration-dependent manner. Tumor necrosis factor-α inhibits differentiation of urethral rhabdosphincter cells in part through the p38 mitogen-activated protein kinase and phosphoinositide 3-kinase pathways. Inhibition of tumor necrosis factor-α might be a useful strategy to treat stress urinary incontinence. © 2017 The Japanese Urological Association.

Variability in response to nicotine in the LSxSS RI strains: potential role of polymorphisms in alpha4 and alpha6 nicotinic receptor genes.

PubMed

Tritto, Theresa; Stitzel, Jerry A; Marks, Michael J; Romm, Elena; Collins, Allan C

2002-04-01

Several studies have shown that genetic factors influence the effects of nicotine on respiration, acoustic startle, Y-maze crosses and rears, heart rate and body temperature in the mouse. Recently, we identified restriction fragment length polymorphisms (RFLPs) associated with the alpha4 (Chrna4) and alpha6 (Chrna6) nicotinic cholinergic receptor genes in the recombinant inbred (RI) strains derived from the Long-Sleep (LS) and Short-Sleep (SS) mouse lines. The alpha4 polymorphism has been identified as a point-mutation at position 529 (threonine to alanine) and the alpha6 polymorphism has not yet been identified. The studies described here evaluated the potential role of these polymorphisms in regulating sensitivity to nicotine by constructing dose-response curves for the effects of nicotine on six responses in the LSxSS RI strains. The results obtained suggest that both of the polymorphisms may play a role in regulating variability in sensitivity to nicotine. Those RI strains carrying the LS-like alpha4 RFLP were significantly more sensitive to the effects of nicotine on Y-maze crosses and rears, temperature and respiration and were less sensitive to the effects of nicotine on acoustic startle than those strains carrying the SS-like alpha4 RFLP. Those RI strains carrying the LS-like alpha6 RFLP were more sensitive to the effects of nicotine on respiration and acoustic startle, and less sensitive to the effects of nicotine on Y-maze crosses than those strains carrying the SS-like alpha6 RFLP. These results suggest that genetically determined differences in sensitivity to nicotine may be explained, in part, by variability associated with at least two of the neuronal nicotinic receptor genes, alpha4 and alpha6.

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

78 FR 78838 - Grant of Interim Extension of the Term of U.S. Patent No. 5,496,801; Recombinant Human...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-12-27

... Hormone AGENCY: United States Patent and Trademark Office, Commerce. ACTION: Notice of interim patent term... No. 5,496,801. The patent claims the human biological product recombinant human parathyroid hormone... human parathyroid hormone, was filed on October 24, 2013, and is currently undergoing regulatory review...

Beneficial effects of interleukin-6 in neonatal mouse models of group B streptococcal disease.

PubMed Central

Mancuso, G; Tomasello, F; Migliardo, M; Delfino, D; Cochran, J; Cook, J A; Teti, G

1994-01-01

Previous studies have shown that tumor necrosis factor alpha (TNF-alpha) plays a pathophysiologic role in sepsis induced in rat pups by group B streptococci (GBS). In this model, TNF-alpha is also partially responsible for the induction of interleukin-6 (IL-6). The present study was undertaken to investigate the role of IL-6 in neonatal BALB/c mice infected with type III GBS. The effect of anti-IL-6 monoclonal antibodies and recombinant IL-6 on lethality and TNF-alpha production was investigated. In mouse pups infected with GBS strain COH1, plasma IL-6 reached levels of 3,067 +/- 955 and 1,923 +/- 891 U/ml when measured at 22 and 48 h, respectively (P < 0.05 compared with uninfected controls). Pretreatment with 25 micrograms of anti-IL-6 antibodies totally prevented the increase in circulating IL-6 bioactivity at both 22 and 48 h after infection (P < 0.05). Treatment with anti-IL-6 also induced a moderate decrease in survival time of mice infected with lethal doses of strains COH1 and COH31, as evidenced by increased lethality (P < 0.05) at 24 to 48 h but not at 96 h. Mouse recombinant IL-6 (12,500 U) given 6 h before challenge with strains COH1 and COH31 consistently increased survival time, as evidenced by decreased (P < 0.05) lethality at 48 to 72 h but not at 96 h. The effects of IL-6 pretreatment were dose dependent, since no protection was observed with doses lower than 12,500 U. In addition, no effects on lethality were noted when IL-6 was given at the time of challenge or at later times. TNF-alpha elevations (P < 0.05 compared with uninfected controls) were measured at 12, 22, and 48 h after challenge with strain COH1 (68 +/- 28, 233 +/- 98, and 98 +/- 34 U, respectively). Pretreatment with IL-6 significantly (P < 0.05) decreased plasma TNF-alpha levels at 12 and 22 h, with 55 and 69% inhibitions, respectively. Anti-IL-6 had an opposite effect, as evidenced by a 145% increase (P < 0.05) in TNF-alpha levels at 48 h after challenge. Collectively, our data are compatible with the hypothesis that IL-6 is involved in negative feedback regulation of plasma TNF-alpha levels in experimental GBS sepsis. In this model, IL-6 pretreatment can increase survival time. Future studies will be needed to investigate the mechanisms underlying this effect. PMID:7927780

A reanalysis of the indirect evidence for recombination in human mitochondrial DNA.

PubMed

Piganeau, G; Eyre-Walker, A

2004-04-01

In an attempt to resolve the controversy about whether recombination occurs in human mtDNA, we have analysed three recently published data sets of complete mtDNA sequences along with 10 RFLP data sets. We have analysed the relationship between linkage disequilibrium (LD) and distance between sites under a variety of conditions using two measures of LD, r2 and /D'/. We find that there is a negative correlation between r2 and distance in the majority of data sets, but no overall trend for /D'/. Five out of six mtDNA sequence data sets show an excess of homoplasy, but this could be due to either recombination or hypervariable sites. Two additional recombination detection methods used, Geneconv and Maximum Chi-Square, showed nonsignificant results. The overall significance of these findings is hard to quantify because of nonindependence, but our results suggest a lack of evidence for recombination in human mtDNA.

Characterization of recombinant human HBP/CAP37/azurocidin, a pleiotropic mediator of inflammation-enhancing LPS-induced cytokine release from monocytes.

PubMed

Rasmussen, P B; Bjørn, S; Hastrup, S; Nielsen, P F; Norris, K; Thim, L; Wiberg, F C; Flodgaard, H

1996-07-15

Neutrophil-derived heparin-binding protein (HBP) is a strong chemoattractant for monocytes. We report here for the first time the expression of recombinant HBP. A baculovirus containing the human HBP cDNA mediated in insect cells the secretion of a 7-residue N-terminally extended HBP form (pro-HBP). Deletion of the pro-peptide-encoding cDNA sequence resulted in correctly processed HBP at the N-terminus. Electrospray mass spectrum analysis of recombinant HBP yielded a molecular weight of 27.237 +/- 3 amu. Consistent with this mass is a HBP form of 225 amino acids (mature part +3 amino acid C-terminal extension). The biological activity of recombinant HBP was confirmed by its chemotactic action towards monocytes. Furthermore, we have shown that recombinant HBP stimulates in a dose-dependent manner the lipopolysaccharide (LPS)-induced cytokine release from human monocytes.

Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells.

PubMed

Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani; Streicher, Werner; Wikström, Mats; Cazzamali, Giuseppe

2015-04-01

Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure and the final protein yields were between 1 and 12mg protein per liter culture media. The recombinant IGFBPs contained PTMs and exhibited high-affinity interactions with their natural ligands IGF-1 and IGF-2. Copyright © 2014 Elsevier Inc. All rights reserved.

Bimatoprost and prostaglandin F(2 alpha) selectively stimulate intracellular calcium signaling in different cat iris sphincter cells.

PubMed

Spada, Clayton S; Krauss, Achim H-P; Woodward, David F; Chen, June; Protzman, Charles E; Nieves, Amelia L; Wheeler, Larry A; Scott, David F; Sachs, George

2005-01-01

Bimatoprost is a synthetic analog of prostaglandin F(2 alpha) ethanolamide (prostamide F(2 alpha)), and shares a pharmacological profile consistent with that of the prostamides. Like prostaglandin F(2 alpha) carboxylic acid, bimatoprost potently lowers intraocular pressure in dogs, primates and humans. In order to distinguish its mechanism of action from prostaglandin F(2 alpha), fluorescence confocal microscopy was used to examine the effects of bimatoprost, prostaglandin F(2 alpha) and 17-phenyl prostaglandin F(2 alpha) on calcium signaling in resident cells of digested cat iris sphincter, a tissue which exhibits contractile responses to both agonists. Constant superfusion conditions obviated effective conversion of bimatoprost. Serial challenge with 100 nM bimatoprost and prostaglandin F(2 alpha) consistently evoked responses in different cells within the same tissue preparation, whereas prostaglandin F(2 alpha) and 17-phenyl prostaglandin F(2 alpha) elicited signaling responses in the same cells. Bimatoprost-sensitive cells were consistently re-stimulated with bimatoprost only, and prostaglandin F(2 alpha) sensitive cells could only be re-stimulated with prostaglandin F(2 alpha). The selective stimulation of different cells in the same cat iris sphincter preparation by bimatoprost and prostaglandin F(2 alpha), along with the complete absence of observed instances in which the same cells respond to both agonists, strongly suggests the involvement of distinct receptors for prostaglandin F(2 alpha) and bimatoprost. Further, prostaglandin F(2 alpha) but not bimatoprost potently stimulated calcium signaling in isolated human embryonic kidney cells stably transfected with the feline- and human-prostaglandin F(2 alpha) FP-receptor and in human dermal fibroblast cells, and only prostaglandin F(2 alpha) competed with radioligand binding in HEK-feFP cells. These studies provide further evidence for the existence of a bimatoprost-sensitive receptor that is distinct from any of the known prostaglandin receptor types.

Precise Correction of Disease Mutations in Induced Pluripotent Stem Cells Derived From Patients With Limb Girdle Muscular Dystrophy.

PubMed

Turan, Soeren; Farruggio, Alfonso P; Srifa, Waracharee; Day, John W; Calos, Michele P

2016-04-01

Limb girdle muscular dystrophies types 2B (LGMD2B) and 2D (LGMD2D) are degenerative muscle diseases caused by mutations in the dysferlin and alpha-sarcoglycan genes, respectively. Using patient-derived induced pluripotent stem cells (iPSC), we corrected the dysferlin nonsense mutation c.5713C>T; p.R1905X and the most common alpha-sarcoglycan mutation, missense c.229C>T; p.R77C, by single-stranded oligonucleotide-mediated gene editing, using the CRISPR/Cas9 gene-editing system to enhance the frequency of homology-directed repair. We demonstrated seamless, allele-specific correction at efficiencies of 0.7-1.5%. As an alternative, we also carried out precise gene addition strategies for correction of the LGMD2B iPSC by integration of wild-type dysferlin cDNA into the H11 safe harbor locus on chromosome 22, using dual integrase cassette exchange (DICE) or TALEN-assisted homologous recombination for insertion precise (THRIP). These methods employed TALENs and homologous recombination, and DICE also utilized site-specific recombinases. With DICE and THRIP, we obtained targeting efficiencies after selection of ~20%. We purified iPSC corrected by all methods and verified rescue of appropriate levels of dysferlin and alpha-sarcoglycan protein expression and correct localization, as shown by immunoblot and immunocytochemistry. In summary, we demonstrate for the first time precise correction of LGMD iPSC and validation of expression, opening the possibility of cell therapy utilizing these corrected iPSC.

Short-term effects of recombinant human growth hormone and feeding on gluconeogenesis in humans

USDA-ARS?s Scientific Manuscript database

After a short-term fast, lactating women have increased rates of glucose production but not gluconeogenesis (GNG) despite relative hypoinsulinemia. We explored the effects of non-insulin-dependent increase in glucose utilization and recombinant human growth hormone (rhGH) on glucose production, glyc...

Site-specific genetic recombination: hops, flips, and flops.

PubMed

Sadowski, P D

1993-06-01

Genetic recombination plays a key role in the life of organisms as diverse as bacteriophages and humans. Contrary to our idea that chromosomes are stable structures, studies of recombination over the past few decades have shown that in fact DNA replicons are remarkably plastic, undergoing frequent recombination-induced rearrangements. This review summarizes our recent knowledge of the biochemistry of the two major classes of site-specific recombination: 1) transpositional recombination, and 2) conservative site-specific recombination.

Negative feedback regulation of human platelets via autocrine activation of the platelet-derived growth factor alpha-receptor.

PubMed

Vassbotn, F S; Havnen, O K; Heldin, C H; Holmsen, H

1994-05-13

Human platelets contain platelet-derived growth factor (PDGF) in their alpha-granules which is released during platelet exocytosis. We show by immunoprecipitation and 125I-PDGF binding experiments that human platelets have functionally active PDGF alpha-receptors, but not beta-receptors. The PDGF alpha-receptor (PDGFR-alpha) was identified as a 170-kDa glycosylated protein-tyrosine kinase as found in other cell types. Stimulation of platelets with 0.1 unit/ml thrombin resulted in a significant increase (2-5-fold) of the tyrosine phosphorylation of the PDGFR-alpha, as determined by immunoprecipitation with phosphotyrosine antiserum as well as with PDGFR-alpha antiserum. The observed thrombin-induced autophosphorylation of the PDGFR-alpha was inhibited by the addition of a neutralizing monoclonal PDGF antibody. Thus, our results suggest that the platelet PDGFR-alpha is stimulated in an autocrine manner by PDGF secreted during platelet activation. Preincubation of platelets with PDGF inhibited thrombin-induced platelet aggregation and secretion of ATP + ADP and beta-hexosaminidase. Thrombin-induced platelet aggregation was also reversed when PDGF was added 30 s after thrombin stimulation. Inhibition of the autocrine PDGF pathway during platelet activation by the PDGF antibody led to a potentiation of thrombin-induced beta-hexosaminidase secretion. Thus, the PDGFR-alpha takes part in a negative feedback regulation during platelet activation. Our demonstration of PDGF alpha-receptors on human platelets and its inhibitory function during platelet activation identifies a new possible role of PDGF in the regulation of thrombosis.

The chorionic gonadotropin alpha-subunit gene is on human chromosome 18 in JEG cells.

PubMed Central

Hardin, J W; Riser, M E; Trent, J M; Kohler, P O

1983-01-01

The gene for the alpha subunit of human chorionic gonadotropin (hCG) has been tentatively assigned to human chromosome 18. This localization was accomplished through the use of Southern blot analysis. A full-length cDNA probe for the hCG alpha subunit and DNA isolated from a series of somatic hybrids between mouse and human cells were utilized to make this assignment. In addition, in situ hybridization with normal human peripheral blood lymphocytes as a source of human chromosomes and with the same cDNA probe confirmed this result. The presence of human chromosome 18 was required for the detection of DNA fragments characteristic of the alpha-hCG gene. These results are consistent with our previous observation that human chromosomes 10 and 18 are required for the production of hCG in cultured cells. Images PMID:6578509

Regulation of 1-alpha, 25-dihydroxyvitamin D3 on interleukin-6 and interleukin-8 induced by sulfur mustard (HD) on human skin cells.

PubMed

Arroyo, Carmen M; Kan, Robert K; Burman, Damon L; Kahler, David W; Nelson, Marian R; Corun, Charlene M; Guzman, Juanita J; Broomfield, Clarence A

2003-05-01

The regulatory effects of the active form of vitamin D, 1-alpha, 25-dihydroxyvitamin D3 (1-alpha, 25 (OH)2D3) were assessed on the cytokine and chemokine secretion induced by sulfur mustard on human skin fibroblasts and human epidermal keratinocytes. Stimulation of human skin fibroblasts with sulfur mustard (10(-4) M for 24 hr at 37 degrees ) resulted in approximately a 5 times increase in the secretion of interleukin-6 and over a 10 times increase for interleukin-8, which was inhibited by 1-alpha, 25 (OH)2D3, at

Finding trans-regulatory genes and protein complexes modulating meiotic recombination hotspots of human, mouse and yeast.

PubMed

Wu, Min; Kwoh, Chee-Keong; Li, Xiaoli; Zheng, Jie

2014-09-11

The regulatory mechanism of recombination is one of the most fundamental problems in genomics, with wide applications in genome wide association studies (GWAS), birth-defect diseases, molecular evolution, cancer research, etc. Recombination events cluster into short genomic regions called "recombination hotspots". Recently, a zinc finger protein PRDM9 was reported to regulate recombination hotspots in human and mouse genomes. In addition, a 13-mer motif contained in the binding sites of PRDM9 is found to be enriched in human hotspots. However, this 13-mer motif only covers a fraction of hotspots, indicating that PRDM9 is not the only regulator of recombination hotspots. Therefore, the challenge of discovering other regulators of recombination hotspots becomes significant. Furthermore, recombination is a complex process. Hence, multiple proteins acting as machinery, rather than individual proteins, are more likely to carry out this process in a precise and stable manner. Therefore, the extension of the prediction of individual trans-regulators to protein complexes is also highly desired. In this paper, we introduce a pipeline to identify genes and protein complexes associated with recombination hotspots. First, we prioritize proteins associated with hotspots based on their preference of binding to hotspots and coldspots. Second, using the above identified genes as seeds, we apply the Random Walk with Restart algorithm (RWR) to propagate their influences to other proteins in protein-protein interaction (PPI) networks. Hence, many proteins without DNA-binding information will also be assigned a score to implicate their roles in recombination hotspots. Third, we construct sub-PPI networks induced by top genes ranked by RWR for various species (e.g., yeast, human and mouse) and detect protein complexes in those sub-PPI networks. The GO term analysis show that our prioritizing methods and the RWR algorithm are capable of identifying novel genes associated with recombination hotspots. The trans-regulators predicted by our pipeline are enriched with epigenetic functions (e.g., histone modifications), demonstrating the epigenetic regulatory mechanisms of recombination hotspots. The identified protein complexes also provide us with candidates to further investigate the molecular machineries for recombination hotspots. Moreover, the experimental data and results are available on our web site http://www.ntu.edu.sg/home/zhengjie/data/RecombinationHotspot/NetPipe/.

Localization of alpha 1-adrenoceptors in rat and human hearts by immunocytochemistry.

PubMed

Schulze, W; Fu, M L

1996-01-01

The localization of the alpha 1 adrenoceptors (alpha 1-AR) in the heart tissues from rat and human and in the cultured heart cells from neonatal rats was studied by indirect immunofluorescence and postembedding electronmicroscopical immuno-gold technique. With antipeptide antibodies directed against the second extracellular loop of the human alpha 1-AR (AS sequence 192-218), this receptor was found to be localized along the sarcolemma in both human and rat hearts. Similar localization sites were detected in cultivated rat neonatal cardiomyocytes. Beside the localization in cardiomyocytes, alpha 1-AR were identified in endothelial cells of capillaries and smooth muscle cells of coronary vessels, in neuronal endings, in mast cells of cultivated heart cells but not, or in less amount in fibroblasts. Interestingly, in the right atrium of rat heart the localization of alpha 1-AR was found to be near or on atrial natriuretic factor (ANF) granules, providing the basis for the alpha-adrenergic influence on ANF release. The immunocytochemical studies further confirm and complete the findings known by using autoradiographic binding studies with specific ligands.

Enhancement of Recombinant Protein Production in Transgenic Nicotiana benthamiana Plant Cell Suspension Cultures with Co-Cultivation of Agrobacterium Containing Silencing Suppressors.

PubMed

Huang, Ting-Kuo; Falk, Bryce W; Dandekar, Abhaya M; McDonald, Karen A

2018-05-24

We have previously demonstrated that the inducible plant viral vector (CMViva) in transgenic plant cell cultures can significantly improve the productivity of extracellular functional recombinant human alpha-1-antiryspin (rAAT) compared with either a common plant constitutive promoter ( Cauliflower mosaic virus (CaMV) 35S) or a chemically inducible promoter (estrogen receptor-based XVE) system. For a transgenic plant host system, however, viral or transgene-induced post-transcriptional gene silencing (PTGS) has been identified as a host response mechanism that may dramatically reduce the expression of a foreign gene. Previous studies have suggested that viral gene silencing suppressors encoded by a virus can block or interfere with the pathways of transgene-induced PTGS in plant cells. In this study, the capability of nine different viral gene silencing suppressors were evaluated for improving the production of rAAT protein in transgenic plant cell cultures (CMViva, XVE or 35S system) using an Agrobacterium -mediated transient expression co-cultivation process in which transgenic plant cells and recombinant Agrobacterium carrying the viral gene silencing suppressor were grown together in suspension cultures. Through the co-cultivation process, the impacts of gene silencing suppressors on the rAAT production were elucidated, and promising gene silencing suppressors were identified. Furthermore, the combinations of gene silencing suppressors were optimized using design of experiments methodology. The results have shown that in transgenic CMViva cell cultures, the functional rAAT as a percentage of total soluble protein is increased 5.7 fold with the expression of P19, and 17.2 fold with the co-expression of CP, P19 and P24.

Alpha-crystallin-mediated protection of lens cells against heat and oxidative stress-induced cell death.

PubMed

Christopher, Karen L; Pedler, Michelle G; Shieh, Biehuoy; Ammar, David A; Petrash, J Mark; Mueller, Niklaus H

2014-02-01

In addition to their key role as structural lens proteins, α-crystallins also appear to confer protection against many eye diseases, including cataract, retinitis pigmentosa, and macular degeneration. Exogenous recombinant α-crystallin proteins were examined for their ability to prevent cell death induced by heat or oxidative stress in a human lens epithelial cell line (HLE-B3). Wild type αA- or αB-crystallin (WT-αA and WT-αB) and αA- or αB-crystallins, modified by the addition of a cell penetration peptide (CPP) designed to enhance the uptake of proteins into cells (gC-αB, TAT-αB, gC-αA), were produced by recombinant methods. In vitro chaperone-like assays were used to assay the ability of α-crystallins to protect client proteins from chemical or heat induced aggregation. In vivo viability assays were performed in HLE-B3 to determine whether pre-treatment with α-crystallins reduced death after exposure to oxidative or heat stress. Most of the five recombinant α-crystallin proteins tested conferred some in vitro protection from protein aggregation, with the greatest effect seen with WT-αB and gC-αB. All α-crystallins displayed significant protection to oxidative stress induced cell death, while only the αB-crystallins reduced cell death induced by thermal stress. Our findings indicate that the addition of the gC tag enhanced the protective effect of αB-crystallin against oxidative but not thermally-induced cell death. In conclusion, modifications that increase the uptake of α-crystallin proteins into cells, without destroying their chaperone-like activity and anti-apoptotic functions, create the potential to use these proteins therapeutically. Copyright © 2013 Elsevier B.V. All rights reserved.

Biochemical properties of the matrix metalloproteinase NtMMP1 from Nicotiana tabacum cv. BY-2 suspension cells.

PubMed

Mandal, Manoj K; Fischer, Rainer; Schillberg, Stefan; Schiermeyer, Andreas

2010-09-01

A zinc-dependent matrix metalloproteinase (NtMMP1) found in the plasma membrane of Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells is thought to be responsible for the degradation of recombinant proteins secreted into the culture supernatant. We have characterized the proteolytic activity of NtMMP1 by expressing a recombinant derivative lacking the C-terminal transmembrane domain in yeast. After purifying the protein by affinity chromatography, its autocatalytic activity was analyzed using monoclonal antibodies raised against its N-terminal and C-terminal portions. Both the unprocessed and processed forms of NtMMP1 displayed caseinolytic activity and N-terminal sequencing identified an autocatalytic cleavage site within the sequence motif HFSFFP, which is similar to the corresponding sequences of the human matrix metalloproteinases stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10). Unlike all other matrix metalloproteinases investigated so far, NtMMP1 contains a disulfide bond within its propeptide thus rendering the proenzyme catalytically active. Kinetic analysis of NtMMP1 with a synthetic substrate revealed a K(m) of 10.55 +/- 0.9 microM, a k(cat) of 0.6 +/- 0.01 s(-1) and maximum activity at pH 7.5. We found that NtMMP1 degrades Desmodus rotundus salivary plasminogen activator alpha 1 (DSPAalpha1), a biopharmaceutical protein, that has proven difficult to produce in tobacco BY-2 cells. This provides a likely explanation for the frequent instability of secreted recombinant biopharmaceuticals produced in plant suspension cell cultures. Our data suggest new avenues that can be explored to improve the production of pharmaceutical proteins in plants and plant cells.

Distinct symmetry and limited peptide refolding activity of the thermosomes from the acidothermophilic archaea Acidianus tengchongensis S5{sup T}

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

Wang, Li; Hu, Zhong-jun; Luo, Yuan-ming

Recombinant thermosomes from the Acidianus tengchongensis strain S5{sup T} were purified to homogeneity and assembled in vitro into homo-oligomers (rATcpn{alpha} or rATcpn{beta}) and hetero-oligomers (rATcpn{alpha}{beta}). The symmetries of these complexes were determined by electron microscopy and image analysis. The rATcpn{alpha} homo-oligomer was shown to possess 8-fold symmetry while both rATcpn{beta} and rATcpn{alpha}{beta} oligomers adopted 9-fold symmetry. rATcpn{alpha}{beta} oligomers were shown to contain the {alpha} and {beta} subunits in a 1:2 ratio. All of the complexes prevented the irreversible inactivation of yeast alcohol dehydrogenase at 55 {sup o}C and completely prevented the formation of aggregates during thermal inactivation of citrate synthasemore » at 45 {sup o}C. All rATcpn complexes showed trace ATP hydrolysis activity. Furthermore, rATcpn{beta} sequestered fully chemically denatured substrates (GFP and thermophilic malic dehydrogenase) in vitro without refolding them in an ATP-dependent manner. This property is similar to previously reported properties of chaperonins from Sulfolobus tokodaii and Sulfolobus acidocaldarius. These features are consistent with the slow growth rates of these species of archaea in their native environment.« less

Combined effect of tumor necrosis factor (TNF)-alpha and heat shock protein (HSP)-70 in reducing apoptotic injury in hypoxia: a cell culture study.

PubMed

Goel, Gunjan; Guo, Miao; Ding, Jamie; Dornbos, David; Ali, Ahmer; Shenaq, Mohammed; Guthikonda, Murali; Ding, Yuchuan

2010-10-15

Studies have demonstrated neuroprotective effects of either TNF-alpha or HSP-70 in ischemia/reperfusion injury following exercise. However, the protective mechanisms involving combined effect of the two proteins, particularly in neuronal apoptosis, remain unclear. This study aims to elucidate the beneficial role of TNF-alpha and HSP-70 in the regulation of apoptotic proteins and ERK signaling in hypoxic injury. Cortical neurons from 20 Sprague-Dawley rat embryos were isolated and cultured in five groups with or without pretreatment with recombinant TNF-alpha, HSP-70 protein or both prior to hypoxic conditions: (1) control; (2) control/hypoxia; (3) TNF-alpha/hypoxia; (4) HSP-70/hypoxia and (5) TNF-alpha/HSP-70/hypoxia. Western blotting was used to detect pro- and anti-apoptotic proteins, including Bax, AIF, Bcl-xL, Bcl-2, and pERK1/2 protein. TNF-alpha and HSP-70 significantly (p<0.05) reduced the levels of pro-apoptotic proteins, Bax and AIF. Also, pretreatment of hypoxic brain tissue with TNF-alpha and HSP-70 significantly (p<0.05) enhanced the levels of anti-apoptotic protein, Bcl-xL. TNF-alpha and HSP-70 together increased Bcl-2 levels by 70%. Hypoxia caused a significant (p<0.05) increase in ERK1/2 phosphorylation levels by 224%. The most effective inhibition of ERK levels was obtained by the combined administration of TNF-alpha and HSP-70. This study suggested that TNF-alpha and HSP-70 together enhance the decrease in pro-apoptotic protein levels and the increase in anti-apoptotic protein levels in the event of neuronal hypoxia through ERK1/2 signal transduction. 2010. Published by Elsevier Ireland Ltd.

Integrin alpha 3 beta 1 participates in the phagocytosis of extracellular matrix molecules by human breast cancer cells.

PubMed

Coopman, P J; Thomas, D M; Gehlsen, K R; Mueller, S C

1996-11-01

The mechanisms and receptors involved in phagocytosis by nonhematopoietic cells are not well understood. The involvement of the alpha 3 beta 1 integrin in phagocytosis of the extracellular matrix by human breast cancer cells was studied. The possible role of this integrin was suggested since alpha 3 and beta 1 but not alpha 2 subunits are concentrated at membrane sites where local degradation of fluorescently labeled gelatin occurs. Strikingly, anti-alpha 3 integrin monoclonal antibodies (mAbs) stimulate the phagocytosis of fluorescently labeled gelatin films, gelatin beads, and Matrigel films in a quantitative phagocytosis assay. Stimulation of the gelatin uptake by the anti-alpha 3 mAb is dose responsive, saturable, and time dependent. Antibodies against other integrin subunits have a lower stimulatory effect (anti-beta 1) or no significant effect (anti-alpha 2, -alpha 5, -alpha 6, and -alpha v) on gelatin phagocytosis. The synthetic HGD-6 human laminin peptide that binds specifically the alpha 3 beta 1 integrin, but not the scrambled HSGD-6 control peptide, also markedly stimulates gelatin uptake in a dose-responsive way. Furthermore, the stimulatory effects of the HGD-6 peptide and the anti-alpha 3 mAb are additive, suggesting that they might promote phagocytosis in different ways. Other laminin (YIGSR, IKVAV) and fibronectin (GRGDS) peptides have no effect on gelatin phagocytosis. Immunofluorescence shows that the alpha 3 and the beta 1, but not the alpha 2 integrin subunit, concentrate into patches on the cell surface after treatment with their respective mAbs. And, both gelatin and the alpha 3 beta 1 but not the alpha 2 beta 1 integrin are cointernalized and routed to acidic vesicles such as lysosomes. In conclusion, we demonstrate that human breast cancer cells locally degrade and phagocytose the extracellular matrix and show for the first time that the alpha 3 beta 1 integrin participates in this phagocytosis. We hypothesize that the anti-alpha 3 antibodies and the laminin peptide HGD-6 activate the alpha 3 beta 1 integrin, which results in a downstream signaling cascade stimulating phagocytosis.

Immunologic Intervention in HIV Infection: Anti-Polymerase Responses and Hormonal Regulation

DTIC Science & Technology

1993-09-01

chronic human immunodeficiency virus infection is blocked in vitro by a methylphosphonate oligodeoxynucleoside targeted to a U3/enhancer element. J...Grimison B, Gonenne A. 1992. Effect of recombinant human growth hormone on acute and chronic human immunodeficiency virus infection in vitro. Blood 79...Kong X-B, Chou T-C. Interactions of recombinant human growth hormone with dideoxynucleoside inhibitors of human immunodeficiency virus. Blood, in

Identification of two novel mammalian genes establishes a subfamily of KH-domain RNA-binding proteins.

PubMed

Makeyev, A V; Liebhaber, S A

2000-08-01

We have identified two novel human genes encoding proteins with a high level of sequence identity to two previously characterized RNA-binding proteins, alphaCP-1 and alphaCP-2. Both of these novel genes, alphaCP-3 and alphaCP-4, are predicted to encode proteins with triplicated KH domains. The number and organization of the KH domains, their sequences, and the sequences of the contiguous regions are conserved among all four alphaCP proteins. The common evolutionary origin of these proteins is substantiated by conservation of exon-intron organization in the corresponding genes. The map positions of alphaCP-1 and alphaCP-2 (previously reported) and those of alphaCP-3 and alphaCP-4 (present report) reveal that the four alphaCP loci are dispersed in the human genome; alphaCP-3 and alphaCP-4 mapped to 21q22.3 and 3p21, and the respective mouse orthologues mapped to syntenic regions of the mouse genome, 10B5 and 9F1-F2, respectively. Two additional loci in the human genome were identified as alphaCP-2 processed pseudogenes (PCBP2P1, 21q22.3, and PCBP2P2, 8q21-q22). Although the overall levels of alphaCP-3 and alphaCP-4 mRNAs are substantially lower than those of alphaCP-1 and alphaCP-2, transcripts of alphaCP-3 and alphaCP-4 were found in all mouse tissues tested. These data establish a new subfamily of genes predicted to encode closely related KH-containing RNA-binding proteins with potential functions in posttranscriptional controls. Copyright 2000 Academic Press.

The contribution of alu elements to mutagenic DNA double-strand break repair.

PubMed

Morales, Maria E; White, Travis B; Streva, Vincent A; DeFreece, Cecily B; Hedges, Dale J; Deininger, Prescott L

2015-03-01

Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both the rate and nature of DNA repair events.

The excitation of the O(1S) state by the dissociative recombination of O2(+) ions - Electron temperature dependence

NASA Technical Reports Server (NTRS)

Zipf, Edward C.

1988-01-01

The rate coefficient for the excitation of the O(1S) state due to the dissociative recombination of O2(+) (v of not greater than 3) ions has been determined as a function of the electron temperature from 300-3500 K. In agreement with the work of Guberman (1987), the results suggest that the absolute magnitude of alpha(1S) is nearly the same for a wide variety of O2(+) vibrational distributions over the electron temperature range normally encountered in the nocturnal F-region. It is noted that previous studies which modeled 5577-A airglow data using a fixed value for f(1S) may be misleading.

[Culture conditions of engineered strain of L-asparaginase and the recombinant plasmid stability].

PubMed

Wang, Y; Qian, S; Ye, J; Meng, G; Zhang, S

1999-12-01

The growth curves of engineered strain JM105(pASN) were different in LB and M-3 media. The expression level and activity of L-asparaginase were affected apparently by both biomass and induction time. Glucose repression of production of L-asparaginase was found. The stability of the recombinant plasmid pASN in different host strains and in LB and M-3 media was determined. After cultivation inLB broth and M-3 media at 30 degrees C for more than 50 generations without antibiotic selection, then induced at 42 degrees C for up to 5 h, the engineered strains were proved to be stable, except for DHA alpha (pASN).

Imbalance of tumor necrosis factor receptors during progression in bovine leukemia virus infection

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

Konnai, Satoru; Usui, Tatsufumi; Ikeda, Manabu

2005-09-01

Previously, we found an up-regulation of tumor necrosis factor alpha (TNF)-{alpha} and an imbalance of TNF receptors in sheep experimentally infected with bovine leukemia virus (BLV). In order to investigate the different TNF-{alpha}-induced responses, in this study we examined the TNF-{alpha}-induced proliferative response and the expression levels of two distinct TNF receptors on peripheral blood mononuclear cells (PBMC) derived from BLV-uninfected cattle and BLV-infected cattle that were aleukemic (AL) or had persistent lymphocytosis (PL). The proliferative response of PBMC isolated from those cattle with PL in the presence of recombinant bovine TNF-{alpha} (rTNF-{alpha}) was significantly higher than those from ALmore » cattle and uninfected cattle and the cells from PL cattle expressed significantly higher mRNA levels of TNF receptor type II (TNF-RII) than those from AL and BLV-uninfected cattle. No difference was found in TNF-RI mRNA levels. Most cells expressing TNF-RII in PL cattle were CD5{sup +} or sIgM{sup +} cells and these cells showed resistance to TNF-{alpha}-induced apoptosis. Additionally, there were significant positive correlations between the changes in provirus load and TNF-RII mRNA levels, and TNF-{alpha}-induced proliferation and TNF-RII mRNA levels. These data suggest that imbalance in the expression of TNF receptors could at least in part contribute to the progression of lymphocytosis in BLV infection.« less

Induction of human airway hyperresponsiveness by tumour necrosis factor-alpha.

PubMed

Anticevich, S Z; Hughes, J M; Black, J L; Armour, C L

1995-09-15

Tumour necrosis factor-alpha (TNF alpha) is implicated in the pathogenesis of asthma; however, little is known of its direct effect on smooth muscle reactivity. We investigated the effect of TNF alpha on the responsiveness of human bronchial tissue to electrical field stimulation in vitro. Incubation of non-sensitized tissue with 1 nM, 3 nM and 10 nM TNF alpha significantly increased responsiveness to electrical field stimulation (113 +/- 8, 110 +/- 4 and 112 +/- 2% respectively) compared to control (99 +/- 2%) (P < 0.05, n = 6). Responses were not increased in sensitized tissue (101 +/- 3% versus 105 +/- 5%, n = 3, P > 0.05) nor were responses to exogenous acetylcholine (93 +/- 4% versus 73 +/- 7%, n = 3, P = 0.38). These results show that TNF alpha causes an increase in responsiveness of human bronchial tissue and that this occurs prejunctionally on the parasympathetic nerve pathway. This is the first report of a cytokine increasing human airway tissue responsiveness.

Cloning and sequence analysis of a cDNA encoding the alpha-subunit of mouse beta-N-acetylhexosaminidase and comparison with the human enzyme.

PubMed Central

Beccari, T; Hoade, J; Orlacchio, A; Stirling, J L

1992-01-01

cDNAs encoding the mouse beta-N-acetylhexosaminidase alpha-subunit were isolated from a mouse testis library. The longest of these (1.7 kb) was sequenced and showed 83% similarity with the human alpha-subunit cDNA sequence. The 5' end of the coding sequence was obtained from a genomic DNA clone. Alignment of the human and mouse sequences showed that all three putative N-glycosylation sites are conserved, but that the mouse alpha-subunit has an additional site towards the C-terminus. All eight cysteines in the human sequence are conserved in the mouse. There are an additional two cysteines in the mouse alpha-subunit signal peptide. All amino acids affected in Tay-Sachs-disease mutations are conserved in the mouse. Images Fig. 1. PMID:1379046

Stimulation of the follicular bulge LGR5+ and LGR6+ stem cells with the gut-derived human alpha defensin 5 results in decreased bacterial presence, enhanced wound healing, and hair growth from tissues devoid of adnexal structures.

PubMed

Lough, Denver; Dai, Hui; Yang, Mei; Reichensperger, Joel; Cox, Lisa; Harrison, Carrie; Neumeister, Michael W

2013-11-01

Discovery of leucine-rich repeat-containing G-protein-coupled receptors 5 and 6 (LGR5 and LGR6) as markers of adult epithelial stem cells of the skin and intestine permits researchers to draw on the intrinsic cellular fundamentals of wound healing and proliferation dynamics of epithelial surfaces. In this study, the authors use the intestine-derived human alpha defensin 5 to stimulate epithelial proliferation, bacterial reduction, and hair production in burn wound beds to provide the field with initial insight on augmenting wound healing in tissues devoid of adnexal stem cells. Murine third-degree burn wound beds were treated with (1) intestine-derived human alpha defensin 5, (2) skin-derived human beta defensin 1, and (3) sulfadiazine to determine their roles in wound healing, bacterial reduction, and hair growth. The human alpha defensin 5 peptide significantly enhanced wound healing and reduced basal bacterial load compared with human beta defensin 1 and sulfadiazine. Human alpha defensin 5 was the only therapy to induce LGR stem cell migration into the wound bed. In addition, gene heat mapping showed significant mRNA up-regulation of key wound healing and Wnt pathway transcripts such as Wnt1 and Wisp1. Ex vivo studies showed enhanced cell migration in human alpha defensin 5-treated wounds compared with controls. Application of human alpha defensin 5 increases LGR stem cell migration into wound beds, leading to enhanced healing, bacterial reduction, and hair production through the augmentation of key Wnt and wound healing transcripts. These findings can be used to derive gut protein-based therapeutics in wound healing.

Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome

PubMed Central

Erliandri, Indri; Fu, Haiqing; Nakano, Megumi; Kim, Jung-Hyun; Miga, Karen H.; Liskovykh, Mikhail; Earnshaw, William C.; Masumoto, Hiroshi; Kouprina, Natalay; Aladjem, Mirit I.; Larionov, Vladimir

2014-01-01

In human chromosomes, centromeric regions comprise megabase-size arrays of 171 bp alpha-satellite DNA monomers. The large distances spanned by these arrays preclude their replication from external sites and imply that the repetitive monomers contain replication origins. However, replication within these arrays has not previously been profiled and the role of alpha-satellite DNA in initiation of DNA replication has not yet been demonstrated. Here, replication of alpha-satellite DNA in endogenous human centromeric regions and in de novo formed Human Artificial Chromosome (HAC) was analyzed. We showed that alpha-satellite monomers could function as origins of DNA replication and that replication of alphoid arrays organized into centrochromatin occurred earlier than those organized into heterochromatin. The distribution of inter-origin distances within centromeric alphoid arrays was comparable to the distribution of inter-origin distances on randomly selected non-centromeric chromosomal regions. Depletion of CENP-B, a kinetochore protein that binds directly to a 17 bp CENP-B box motif common to alpha-satellite DNA, resulted in enrichment of alpha-satellite sequences for proteins of the ORC complex, suggesting that CENP-B may have a role in regulating the replication of centromeric regions. Mapping of replication initiation sites in the HAC revealed that replication preferentially initiated in transcriptionally active regions. PMID:25228468

HTLV-I Tax protein binds to MEKK1 to stimulate IkappaB kinase activity and NF-kappaB activation.

PubMed

Yin, M J; Christerson, L B; Yamamoto, Y; Kwak, Y T; Xu, S; Mercurio, F; Barbosa, M; Cobb, M H; Gaynor, R B

1998-05-29

NF-kappaB, a key regulator of the cellular inflammatory and immune response, is activated by the HTLV-I transforming and transactivating protein Tax. We show that Tax binds to the amino terminus of the protein kinase MEKK1, a component of an IkappaB kinase complex, and stimulates MEKK1 kinase activity. Tax expression increases the activity of IkappaB kinase beta (IKKbeta) to enhance phosphorylation of serine residues in IkappaB alpha that lead to its degradation. Dominant negative mutants of both IKKbeta and MEKK1 prevent Tax activation of the NF-kappaB pathway. Furthermore, recombinant MEKK1 stimulates IKKbeta phosphorylation of IkappaB alpha. Thus, Tax-mediated increases in NF-kappaB nuclear translocation result from direct interactions of Tax and MEKK1 leading to enhanced IKKbeta phosphorylation of IkappaB alpha.

A case of reversible dilated cardiomyopathy after alpha-interferon therapy in a patient with renal cell carcinoma.

PubMed

Kuwata, Akiko; Ohashi, Masuo; Sugiyama, Masaya; Ueda, Ryuzo; Dohi, Yasuaki

2002-12-01

A 47-year-old man with renal cell carcinoma underwent nephrectomy, and postoperative chemotherapy was performed with recombinant alpha-interferon. Five years later, he experienced dyspnea during physical exertion. An echocardiogram revealed dilatation and systolic dysfunction of the left ventricle, and thallium-201 myocardial scintigraphy showed diffuse heterogeneous perfusion. We diagnosed congestive heart failure because of cardiomyopathy induced by alpha-interferon therapy. Withdrawal of interferon therapy and the combination of an angiotensin-converting enzyme inhibitor, diuretics, and digitalis improved left ventricular systolic function. Furthermore, myocardial scintigraphy using [123I] beta-methyl-p-iodophenylpentadecanoic acid (123I-BMIPP) or [123 I]metaiodobenzylguanidine (123I-MIBG) revealed normal perfusion after the improvement of congestive heart failure. This is a rare case of interferon-induced cardiomyopathy that resulted in normal myocardial images in 123I-BMIPP and 123I-MIBG scintigrams after withdrawal of interferon therapy.

Identification of a major continuous epitope of human alpha crystallin

NASA Technical Reports Server (NTRS)

Takemoto, L.; Emmons, T.; Spooner, B. S. (Principal Investigator)

1992-01-01

Human lens proteins were digested with trypsin or V8 protease, and the resulting peptides resolved on a C18 reverse phase column. Fractions from this column were probed with polyclonal antiserum made against the whole alpha crystallin molecule. Peptides in the seropositive fraction were purified to homogeneity, then characterized by mass spectral analysis and partial Edman degradation. The tryptic and V8 digests contained only one seropositive peptide that was derived from the C-terminal region of the alpha-A molecule. To determine the exact boundaries of the epitope, various size analogues of this region were synthesized and probed with anti-alpha serum. Together, these studies demonstrate that the major continuous epitope of the alpha-A chain includes the sequence KPTSAPS, corresponding to residues 166-172 of the human alpha-A crystallin chain.

Saw palmetto extracts potently and noncompetitively inhibit human alpha1-adrenoceptors in vitro.

PubMed

Goepel, M; Hecker, U; Krege, S; Rübben, H; Michel, M C

1999-02-15

We wanted to test whether phytotherapeutic agents used in the treatment of lower urinary tract symptoms have alpha1-adrenoceptor antagonistic properties in vitro. Preparations of beta-sitosterol and extracts of stinging nettle, medicinal pumpkin, and saw palmetto were obtained from several pharmaceutical companies. They were tested for their ability to inhibit [3H]tamsulosin binding to human prostatic alpha1-adrenoceptors and [3H]prazosin binding to cloned human alpha1A- and alpha1B-adrenoceptors. Inhibition of phenylephrine-stimulated [3H]inositol phosphate formation by cloned receptors was also investigated. Up to the highest concentration which could be tested, preparations of beta-sitosterol, stinging nettle, and medicinal pumpkin were without consistent inhibitory effect in all assays. In contrast, all tested saw palmetto extracts inhibited radioligand binding to human alpha1-adrenoceptors and agonist-induced [3H]inositol phosphate formation. Saturation binding experiments in the presence of a single saw palmetto extract concentration indicated a noncompetitive antagonism. The relationship between active concentrations in vitro and recommended therapeutic doses for the saw palmetto extracts was slightly lower than that for several chemically defined alpha1-adrenoceptor antagonists. Saw palmetto extracts have alpha1-adrenoceptor-inhibitory properties. If bioavailability and other pharmacokinetic properties of these ingredients are similar to those of the chemically defined alpha1-adrenoceptor antagonists, alpha1-adrenoceptor antagonism might be involved in the therapeutic effects of these extracts in patients with lower urinary tract symptoms suggestive of benign prostatic obstruction.

Absence of integrin alpha 7 causes a novel form of muscular dystrophy.

PubMed

Mayer, U; Saher, G; Fässler, R; Bornemann, A; Echtermeyer, F; von der Mark, H; Miosge, N; Pöschl, E; von der Mark, K

1997-11-01

Integrin alpha 7 beta 1 is a specific cellular receptor for the basement membrane protein laminin-1 (refs 1,2), as well as for the laminin isoforms -2 and -4 (ref. 3). The alpha 7 subunit is expressed mainly in skeletal and cardiac muscle and has been suggested to be involved in differentiation and migration processes during myogenesis. Three cytoplasmic and two extracellular splice variants that have been described are developmentally regulated and expressed in different sites in the muscle. In adult muscle, the alpha 7A and alpha 7B subunits are concentrated in myotendinous junctions but can also be detected in neuromuscular junctions and along the sarcolemmal membrane. To study the potential involvement of alpha 7 integrin, during myogenesis and its role in muscle integrity and function, we generated a null allele of the alpha 7 gene (Itga7) in the germline of mice by homologous recombination in embryonic stem (ES) cells. Surprisingly, mice homozygous for the mutation are viable and fertile, indicating that the alpha 7 beta 1 integrin is not essential for myogenesis. However, histological analysis of skeletal muscle revealed typical symptoms of a progressive muscular dystrophy starting soon after birth, but with a distinct variability in different muscle types. The observed histopathological changes strongly indicate an impairment of function of the myotendinous junctions. These findings demonstrate that alpha 7 beta 1 integrin represents an indispensable linkage between the muscle fibre and the extracellular matrix that is independent of the dystrophin-dystroglycan complex-mediated interaction of the cytoskeleton with the muscle basement membrane.

Cloning and Characterization of an alpha-amylase Gene from the Hyperthermophilic Archaeon Thermococcus Thioreducens

NASA Technical Reports Server (NTRS)

Bernhardsdotter, Eva C. M. J.; Pusey, Mark L.; Ng, Joseph D.; Garriott, Owen K.

2004-01-01

The gene encoding an extracellular alpha-amylase, TTA, from the hyperthermophilic archaeon Thermococcus thioreducens was cloned and expressed in Escherichia coli. Primary structural analysis revealed high similarity with other a-amylases from the Thermococcus and Pyrococcus genera, as well as the four highly conserved regions typical for a-amylases. The 1374 bp gene encodes a protein of 457 amino acids, of which 435 constitute the mature protein preceded by a 22 amino acid signal peptide. The molecular weight of the purified recombinant enzyme was estimated to be 43 kDa by denaturing gel electrophoresis. Maximal enzymatic activity of recombinant TTA was observed at 90 C and pH 5.5 in the absence of exogenous Ca(2+), and the enzyme was considerably stable even after incubation at 90 C for 2 hours. The thermostability at 90 and 102 C was enhanced in the presence of 5 mM Ca(2+). The extraordinarily high specific activity (about 7.4 x 10(exp 3) U/mg protein at 90 C, pH 5.5 with soluble starch as substrate) together with its low pH optimum makes this enzyme an interesting candidate for starch processing applications.

Intraperitoneal implant of recombinant encapsulated cells overexpressing alpha-L-iduronidase partially corrects visceral pathology in mucopolysaccharidosis type I mice.

PubMed

Baldo, Guilherme; Mayer, Fabiana Quoos; Martinelli, Barbara; Meyer, Fabiola Schons; Burin, Maira; Meurer, Luise; Tavares, Angela Maria Vicente; Giugliani, Roberto; Matte, Ursula

2012-08-01

Mucopolysaccharidosis type I (MPS I) is characterized by deficiency of the enzyme alpha-L-iduronidase (IDUA) and storage of glycosaminoglycans (GAG) in several tissues. Current available treatments present limitations, thus the search for new therapies. Encapsulation of recombinant cells within polymeric structures combines gene and cell therapy and is a promising approach for treating MPS I. We produced alginate microcapsules containing baby hamster kidney (BHK) cells overexpressing IDUA and implanted these capsules in the peritoneum of MPS I mice. An increase in serum and tissue IDUA activity was observed at early time-points, as well as a reduction in GAG storage; however, correction in the long term was only partially achieved, with a drop in the IDUA activity being observed a few weeks after the implant. Analysis of the capsules obtained from the peritoneum revealed inflammation and a pericapsular fibrotic process, which could be responsible for the reduction in IDUA levels observed in the long term. In addition, treated mice developed antibodies against the enzyme. The results suggest that the encapsulation process is effective in the short term but improvements must be achieved in order to reduce the immune response and reach a stable correction.

[Development of an orphan drug to treat a genetic disease: the paradigm of agalsidase beta].

PubMed

Germain, Dominique P; Benistan, Karelle

2007-03-01

Preclinical and phase I/II studies gave the proof of principle of enzyme replacement therapy (ERT) with recombinant alpha-galactosidase A through the demonstration of the clearance of the accumulated subtrate from plasma and tissues. In a multicenter, randomized, placebo-controlled, double-blind phase Ill study, the biological efficacy of recombinant alpha-galactosidose A (agalsidase beta 1 mg/kg/714 days) was demonstrated on the basis of complete clearance of accumulated globotriaosylceramide from the endothelia of the kidney, heart and skin. The phase III extension study data gives additional results: kidney function appears to be stabilized after 54 to 60 months of treatment with agolsidase beta in most patients. Intent-to-treat analysis of a double-blind, randomized, placebo-controlled, phase IV study, showed that, adjusted for on imbalance in baseline proteinuria, agalsidase beta significantly reduces by 53% the risk of a first clinical event (renal, cardiac and cerebrovascular), compared with placebo. Clinical benefits of ERT depend on patients' clinical status at baseline, therefore prompting for onset of ERT before irreversible damage occur and underlying the need to stratify patients' populations to better understand the outcome of ERT.

Correction of the enzymatic and functional deficits in a model of Pompe disease using adeno-associated virus vectors.

PubMed

Fraites, Thomas J; Schleissing, Mary R; Shanely, R Andrew; Walter, Glenn A; Cloutier, Denise A; Zolotukhin, Irene; Pauly, Daniel F; Raben, Nina; Plotz, Paul H; Powers, Scott K; Kessler, Paul D; Byrne, Barry J

2002-05-01

Pompe disease is a lysosomal storage disease caused by the absence of acid alpha-1,4 glucosidase (GAA). The pathophysiology of Pompe disease includes generalized myopathy of both cardiac and skeletal muscle. We sought to use recombinant adeno-associated virus (rAAV) vectors to deliver functional GAA genes in vitro and in vivo. Myotubes and fibroblasts from Pompe patients were transduced in vitro with rAAV2-GAA. At 14 days postinfection, GAA activities were at least fourfold higher than in their respective untransduced controls, with a 10-fold increase observed in GAA-deficient myotubes. BALB/c and Gaa(-/-) mice were also treated with rAAV vectors. Persistent expression of vector-derived human GAA was observed in BALB/c mice up to 6 months after treatment. In Gaa(-/-) mice, intramuscular and intramyocardial delivery of rAAV2-Gaa (carrying the mouse Gaa cDNA) resulted in near-normal enzyme activities. Skeletal muscle contractility was partially restored in the soleus muscles of treated Gaa(-/-) mice, indicating the potential for vector-mediated restoration of both enzymatic activity and muscle function. Furthermore, intramuscular treatment with a recombinant AAV serotype 1 vector (rAAV1-Gaa) led to nearly eight times normal enzymatic activity in Gaa(-/-) mice, with concomitant glycogen clearance as assessed in vitro and by proton magnetic resonance spectroscopy.

Exclusion of candidate genes from the chromosome 1q juvenile glaucoma region and mapping of the peripheral cannabis receptor gene (CNR2) to chromosome 1

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

Sunden, S.L.F.; Nichols, B.E.; Alward, W.L.M.

Juvenile onset primary open angle glaucoma has been mapped by linkage to 1q21-q31. Several candidate genes were evaluated in the same family used to identify the primary linkage. Atrionatriuretic peptide receptor A (NPR1) and laminin C1 (LAMC1) have been previously mapped to this region and could putatively play a role in the pathogenesis of glaucoma. A third gene, the peripheral cannabis receptor (CNR2) was not initially mapped in humans but was a candidate because of the relief that cannabis affords some patients with primary open angle glaucoma. Microsatellites associated with NPR1 and LAMC1 revealed multiple recombinations in affected members ofmore » this pedigree. CNR2 was shown to be on chromosome 1 by PCR amplification of a 150 bp fragment of the 3{prime} untranslated region in monochromosomal somatic cell hybrids (NIGMS panel No. 2). These primers also revealed a two allele single strand conformation polymorphism which showed multiple recombinants with juvenile onset primary open angle glaucoma in large pedigrees, segregating this disorder. The marker was then mapped to 1p34-p36 by linkage, with the most likely location between liver alkaline phosphatase (ALPL) and alpha-L-1 fucosidase (FUCA1).« less

Expression of biologically active recombinant porcine interleukin-12 from Escherichia coli.

PubMed

Morris, K R; Bruce, M P; Janardhana, V; Thomas, J D; Bean, A G D; Strom, D G

2008-12-15

The control of viral infections is of critical importance to livestock industries worldwide and is highlighted by costly infection outbreaks, such as that seen with foot and mouth disease virus. To ameliorate the impact of increasing problems with viral infections, new vaccine and anti-viral strategies are required and a greater understanding of the anti-viral response is essential. Furthermore, in pigs, evidence is still being gathered on the components of a defined anti-viral immune response. However, this has been greatly improved by the recent cloning and expression of critical cytokines involved in the anti-viral response. To assess the use of recombinant porcine interleukin-12 (rPoIL-12) as an immunotherapeutic and immunomodulator of swine, we have cloned and expressed rPoIL-12 as a single-chain fusion protein from Esherichia coli (E. coli). The fusion encodes the p40 and p35 subunits, linked by a glycine-serine linker and expressed as a C-terminal 6xHis tagged protein. rPoIL-12 stimulated the proliferation of human lymphoblasts and its activity on porcine cells was demonstrated by the ability of rPoIL-12 to increase the mRNA expression of porcine interleukin-18 receptor-alpha (poIL-18Ralpha) from porcine peripheral blood mononuclear cells (PoPMBCs). This data supports the inclusion of E. coli produced rPoIL-12 in immunomodulation strategies in the pig.

Inhibition of recombinant Ca(v)3.1 (alpha(1G)) T-type calcium channels by the antipsychotic drug clozapine.

PubMed

Choi, Kee-Hyun; Rhim, Hyewhon

2010-01-25

Low voltage-activated T-type calcium channels are involved in the regulation of the neuronal excitability, and could be subject to many antipsychotic drugs. The effects of clozapine, an atypical antipsychotic drug, on recombinant Ca(v)3.1 T-type calcium channels heterologously expressed in human embryonic kidney 293 cells were examined using whole-cell patch-clamp recordings. At a standard holding potential of -100 mV, clozapine inhibited Ca(v)3.1 currents with an IC(50) value of 23.7+/-1.3 microM in a use-dependent manner. However, 10 microM clozapine inhibited more than 50% of the Ca(v)3.1 currents in recordings at a more physiologically relevant holding potential of -75 mV. Clozapine caused a significant hyperpolarizing shift in the steady-state inactivation curve of the Ca(v)3.1 channels, which is presumably the main mechanism accounting for the inhibition of the Ca(v)3.1 currents. In addition, clozapine slowed Ca(v)3.1 deactivation and inactivation kinetics but not activation kinetics. Clozapine-induced changes in deactivation and inactivation rates of the Ca(v)3.1 channel gating would likely facilitate calcium influx via Ca(v)3.1 T-type calcium channels. Thus, clozapine may exert its therapeutic and/or side effects by altering cell's excitability and firing properties through actions on T-type calcium channels.

D-ribulose-5-phosphate 3-epimerase: Cloning and heterologous expression of the spinach gene, and purification and characterization of the recombinant enzyme

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

Chen, Y.R.; Hartman, F.C.; Lu, T.Y.S.

The authors have achieved, to their knowledge, the first high-level heterologous expression of the gene encoding D-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by DL-{alpha}-glycerophosphate or ethanol and destabilized by D-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deducedmore » from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.« less

Current Ebola vaccines

PubMed Central

Hoenen, Thomas; Groseth, Allison; Feldmann, Heinz

2012-01-01

Introduction Ebolaviruses cause severe viral hemorrhagic fever in humans and non-human primates, with case fatality rates of up to 90%. Currently, neither a specific treatment nor a vaccine licensed for use in humans is available. However, a number of vaccine candidates have been developed in the last decade that are highly protective in non-human primates, the gold standard animal model for Ebola hemorrhagic fever. Areas covered This review analyzes a number of scenarios for the use of ebolavirus vaccines, discusses the requirements for ebolavirus vaccines in these scenarios, and describes current ebolavirus vaccines. Among these vaccines are recombinant Adenoviruses, recombinant Vesicular Stomatitis viruses, recombinant Human Parainfluenza viruses and virus-like particles. Interestingly, one of these vaccine platforms, based on recombinant Vesicular Stomatitis viruses, has also demonstrated post-exposure protection in non-human primates. Expert opinion The most pressing remaining challenge is now to move these vaccine candidates forward into human trials and towards licensure. In order to achieve this, it will be necessary to establish the mechanisms and correlates of protection for these vaccines, and to continue to demonstrate their safety, particularly in potentially immunocompromised populations. However, already now there is sufficient evidence that, from a scientific perspective, a vaccine protective against ebolaviruses is possible. PMID:22559078

Interconversion of the Specificities of Human Lysosomal Enzymes Associated with Fabry and Schindler Diseases

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

Tomasic, Ivan B.; Metcalf, Matthew C.; Guce, Abigail I.

2010-09-03

The human lysosomal enzymes {alpha}-galactosidase ({alpha}-GAL, EC 3.2.1.22) and {alpha}-N-acetylgalactosaminidase ({alpha}-NAGAL, EC 3.2.1.49) share 46% amino acid sequence identity and have similar folds. The active sites of the two enzymes share 11 of 13 amino acids, differing only where they interact with the 2-position of the substrates. Using a rational protein engineering approach, we interconverted the enzymatic specificity of {alpha}-GAL and {alpha}-NAGAL. The engineered {alpha}-GAL (which we call {alpha}-GALSA) retains the antigenicity of {alpha}-GAL but has acquired the enzymatic specificity of {alpha}-NAGAL. Conversely, the engineered {alpha}-NAGAL (which we call {alpha}-NAGAL{sup EL}) retains the antigenicity of {alpha}-NAGAL but has acquired themore » enzymatic specificity of the {alpha}-GAL enzyme. Comparison of the crystal structures of the designed enzyme {alpha}-GAL{sup SA} to the wild-type enzymes shows that active sites of {alpha}-GAL{sup SA} and {alpha}-NAGAL superimpose well, indicating success of the rational design. The designed enzymes might be useful as non-immunogenic alternatives in enzyme replacement therapy for treatment of lysosomal storage disorders such as Fabry disease.« less

The Past, Present, and Future of Human Centromere Genomics

PubMed Central

Aldrup-MacDonald, Megan E.; Sullivan, Beth A.

2014-01-01

The centromere is the chromosomal locus essential for chromosome inheritance and genome stability. Human centromeres are located at repetitive alpha satellite DNA arrays that compose approximately 5% of the genome. Contiguous alpha satellite DNA sequence is absent from the assembled reference genome, limiting current understanding of centromere organization and function. Here, we review the progress in centromere genomics spanning the discovery of the sequence to its molecular characterization and the work done during the Human Genome Project era to elucidate alpha satellite structure and sequence variation. We discuss exciting recent advances in alpha satellite sequence assembly that have provided important insight into the abundance and complex organization of this sequence on human chromosomes. In light of these new findings, we offer perspectives for future studies of human centromere assembly and function. PMID:24683489

The hematopoietic effect of Epotin (recombinant human erythropoietin-alpha) on maintenance hemodialysis end-stage kidney disease patients.

PubMed

Al-Shohaib, S; Shaker, D S; Ghaedi, B B; Alyarim, M; Emara, S; Behairy, M

2010-04-01

Recombinant human erythropoietin (rHuEpo) has revolutionized the management of renal anemia, significantly improving patient quality of life. Great attention has been paid lately on how to optimally use this potent anti-anemic agent. Aiming to overview anemic patient management with Epotin (Julphar's rHuEpo) according to the new guidelines, we included in the study anemic (hemoglobin [Hb]or=18 years who were of iron replete (transeferene saturation (TSAT)>or=20% and serum ferritin>or=100 microg/L) with no evidence of serious inflammation (c-reactive protein (CRP)<30 mg/L) on thrice-weekly hemodialysis. The mean age and dialysis duration of 50.8+/-17 and 3.8+/-2.8 years, respectively, included 88.6% (n=31) de novo patients in the corrective phase with no previous exposure to erythropoietin. Safety-efficacy parameters showed insignificant changes throughout the 4-month study period, including iron profile that was maintained according to Kidney Disease Outcome Quality Initiative guidelines. Efficacy parameters revealed a significant increase (P<.0001) of Hb levels from a baseline of 8.5+/-1.0 to 11.1+/-1.1. Targeting an absolute increase of 2.5 g/dL in Hb throughout 3 months of the study period resulted in a 90.3% success rate. There were no dropouts due to intolerance, whereas all the recorded adverse events were classified as unrelated to the test product. In conclusion, Epotin was clinically effective to correct and maintain Hb levels in ESKD anemic patients on maintenance hemodialysis within the current recommended range and with a satisfactory safety profile consistent with previous international reports. Copyright (c) 2010 Elsevier Inc. All rights reserved.

CCR5 signal transduction in macrophages by human immunodeficiency virus and simian immunodeficiency virus envelopes.

PubMed

Arthos, J; Rubbert, A; Rabin, R L; Cicala, C; Machado, E; Wildt, K; Hanbach, M; Steenbeke, T D; Swofford, R; Farber, J M; Fauci, A S

2000-07-01

The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1beta. Distinct differences in the capacity of envelopes to mediate calcium mobilization were observed. Envelopes derived from viruses capable of replicating in macrophages mobilized relatively high levels of calcium, while envelopes derived from viruses incapable of replicating in macrophages mobilized relatively low levels of calcium. The failure to efficiently mobilize calcium was not restricted to envelopes derived from CXCR4-utilizing isolates but also included envelopes derived from CCR5-utilizing isolates that fail to replicate in macrophages. We characterized one CCR5-utilizing isolate, 92MW959, which entered macrophages but failed to replicate. A recombinant envelope derived from this virus mobilized low levels of calcium. When macrophages were inoculated with 92MW959 in the presence of MIP-1alpha, viral replication was observed, indicating that a CC chemokine-mediated signal provided the necessary stimulus to allow the virus to complete its replication cycle. Although the role that envelope-CCR5 signal transduction plays in viral replication is not yet understood, it has been suggested that envelope-mediated signals facilitate early postfusion events in viral replication. The data presented here are consistent with this hypothesis and suggest that the differential capacity of viral envelopes to signal through CCR5 may influence their ability to replicate in macrophages.

Impact of cysteine variants on the structure, activity, and stability of recombinant human α-galactosidase A

PubMed Central

Qiu, Huawei; Honey, Denise M; Kingsbury, Jonathan S; Park, Anna; Boudanova, Ekaterina; Wei, Ronnie R; Pan, Clark Q; Edmunds, Tim

2015-01-01

Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity. PMID:26044846

Petaloid recombinant peptide enhances in vitro cartilage formation by synovial mesenchymal stem cells.

PubMed

Naritomi, Mana; Mizuno, Mitsuru; Katano, Hisako; Ozeki, Nobutake; Otabe, Koji; Komori, Keiichiro; Fujii, Shizuka; Ichinose, Shizuko; Tsuji, Kunikazu; Koga, Hideyuki; Muneta, Takeshi; Sekiya, Ichiro

2018-05-07

In vitro chondrogenesis of mesenchymal stem cells (MSCs) mimics in vivo chondrogenesis of MSCs. However, the size of the cartilage pellets that can be attained in vitro is limited by current methods; therefore, some modifications are required to obtain larger pellets. Petaloid pieces of recombinant peptide (petaloid RCP) have the advantage of creating spaces between cells in culture. The RCP used here is based on the alpha-1 sequence of human collagen type I and contains 12 Arg-Gly-Asp motifs. We examined the effect and mechanisms of adding petaloid RCP on the in vitro chondrogenesis of human synovial MSCs by culturing 125k cells with or without 0.125 mg petaloid RCP in chondrogenic medium for 21 days. The cartilage pellets were sequentially analyzed by weight, sulfated glycosaminoglycan content, DNA retention, and histology. Petaloid RCP significantly increased the weight of the cartilage pellets: the petaloid RCP group weighed 7.7 ± 1.2 mg (n = 108), whereas the control group weighed 5.3 ± 1.6 mg. Sulfated glycosaminoglycan and DNA contents were significantly higher in the petaloid RCP group than in the control group. Light and transmission electron microscopy images showed that the petaloid RCP formed the framework of the pellet at day 1, the framework was broken by production of cartilage matrix by the synovial MSCs at day 7, and the cartilage pellet grew larger, with diffuse petaloid RCP remaining, at day 21. Therefore, petaloid RCP formed a framework for the pellet, maintained a higher cell number, and promoted in vitro cartilage formation of synovial MSCs. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Intrathecal enzyme replacement therapy reduces lysosomal storage in the brain and meninges of the canine model of MPS I.

PubMed

Kakkis, E; McEntee, M; Vogler, C; Le, S; Levy, B; Belichenko, P; Mobley, W; Dickson, P; Hanson, S; Passage, M

2004-01-01

Enzyme replacement therapy (ERT) has been developed for several lysosomal storage disorders, including mucopolysaccharidosis I (MPS I), and is effective at reducing lysosomal storage in many tissues and in ameliorating clinical disease. However, intravenous ERT does not adequately treat storage disease in the central nervous system (CNS), presumably due to effects of the blood-brain barrier on enzyme distribution. To circumvent this barrier, we studied whether intrathecal (IT) recombinant human alpha-L-iduronidase (rhIDU) could penetrate and treat the brain and meninges. An initial dose-response study showed that doses of 0.46-4.14 mg of IT rhIDU successfully penetrated the brain of normal dogs and reached tissue levels 5.6 to 18.9-fold normal overall and 2.7 to 5.9-fold normal in deep brain sections lacking CSF contact. To assess the efficacy and safety in treating lysosomal storage disease, four weekly doses of approximately 1 mg of IT rhIDU were administered to MPS I-affected dogs resulting in a mean 23- and 300-fold normal levels of iduronidase in total brain and meninges, respectively. Quantitative glycosaminoglycan (GAG) analysis showed that the IT treatment reduced mean total brain GAG to normal levels and achieved a 57% reduction in meningeal GAG levels accompanied by histologic improvement in lysosomal storage in all cell types. The dogs did develop a dose-dependent immune response against the recombinant human protein and a meningeal lymphocytic/plasmacytic infiltrate. The IT route of ERT administration may be an effective way to treat the CNS disease in MPS I and could be applicable to other lysosomal storage disorders.

Distribution of recombination hotspots in the human genome--a comparison of computer simulations with real data.

PubMed

Mackiewicz, Dorota; de Oliveira, Paulo Murilo Castro; Moss de Oliveira, Suzana; Cebrat, Stanisław

2013-01-01

Recombination is the main cause of genetic diversity. Thus, errors in this process can lead to chromosomal abnormalities. Recombination events are confined to narrow chromosome regions called hotspots in which characteristic DNA motifs are found. Genomic analyses have shown that both recombination hotspots and DNA motifs are distributed unevenly along human chromosomes and are much more frequent in the subtelomeric regions of chromosomes than in their central parts. Clusters of motifs roughly follow the distribution of recombination hotspots whereas single motifs show a negative correlation with the hotspot distribution. To model the phenomena related to recombination, we carried out computer Monte Carlo simulations of genome evolution. Computer simulations generated uneven distribution of hotspots with their domination in the subtelomeric regions of chromosomes. They also revealed that purifying selection eliminating defective alleles is strong enough to cause such hotspot distribution. After sufficiently long time of simulations, the structure of chromosomes reached a dynamic equilibrium, in which number and global distribution of both hotspots and defective alleles remained statistically unchanged, while their precise positions were shifted. This resembles the dynamic structure of human and chimpanzee genomes, where hotspots change their exact locations but the global distributions of recombination events are very similar.

Distribution of Recombination Hotspots in the Human Genome – A Comparison of Computer Simulations with Real Data

PubMed Central

Mackiewicz, Dorota; de Oliveira, Paulo Murilo Castro; Moss de Oliveira, Suzana; Cebrat, Stanisław

2013-01-01

Recombination is the main cause of genetic diversity. Thus, errors in this process can lead to chromosomal abnormalities. Recombination events are confined to narrow chromosome regions called hotspots in which characteristic DNA motifs are found. Genomic analyses have shown that both recombination hotspots and DNA motifs are distributed unevenly along human chromosomes and are much more frequent in the subtelomeric regions of chromosomes than in their central parts. Clusters of motifs roughly follow the distribution of recombination hotspots whereas single motifs show a negative correlation with the hotspot distribution. To model the phenomena related to recombination, we carried out computer Monte Carlo simulations of genome evolution. Computer simulations generated uneven distribution of hotspots with their domination in the subtelomeric regions of chromosomes. They also revealed that purifying selection eliminating defective alleles is strong enough to cause such hotspot distribution. After sufficiently long time of simulations, the structure of chromosomes reached a dynamic equilibrium, in which number and global distribution of both hotspots and defective alleles remained statistically unchanged, while their precise positions were shifted. This resembles the dynamic structure of human and chimpanzee genomes, where hotspots change their exact locations but the global distributions of recombination events are very similar. PMID:23776462

Human Prostate Side Population Cells Demonstrate Stem Cell Properties in Recombination with Urogenital Sinus Mesenchyme

PubMed Central

Foster, Barbara A.; Gangavarapu, Kalyan J.; Mathew, Grinu; Azabdaftari, Gissou; Morrison, Carl D.; Miller, Austin; Huss, Wendy J.

2013-01-01

Stem cell enrichment provides a tool to examine prostate stem cells obtained from benign and malignant tissue. Functional assays can enrich stem cells based on common stem cell phenotypes, such as high ATP binding cassette (ABC) transporter mediated efflux of Hoechst substrates (side population assay). This functional assay is based upon mechanisms that protect cells from environmental insult thus contributing to the survival and protection of the stem cell population. We have isolated and analyzed cells digested from twelve clinical prostate specimens based on the side population assay. Prostate stem cell properties of the isolated cells were tested by serial recombination with rat urogenital mesenchyme. Recombinants with side population cells demonstrate an increase in the frequency of human ductal growth and the number of glands per recombinant when compared to recombinants with non-side population cells. Isolated cells were capable of prostatic growth for up to three generations in the recombination assay with as little as 125 sorted prostate cells. The ability to reproducibly use cells isolated by fluorescence activated cell sorting from human prostate tissue is an essential step to a better understanding of human prostate stem cell biology. ABC transporter G2 (ABCG2) was expressed in recombinants from side population cells indicating the side population cells have self-renewal properties. Epithelial cell differentiation of recombinants was determined by immunohistochemical analysis for expression of the basal, luminal, and neuroendocrine markers, p63, androgen receptor, prostate specific antigen, and chromogranin A, respectively. Thus, the ABCG2 expressing side population demonstrates multipotency and self-renewal properties indicating stem cells are within this population. PMID:23383057

Production of transgenic cattle highly expressing human serum albumin in milk by phiC31 integrase-mediated gene delivery.

PubMed

Luo, Yan; Wang, Yongsheng; Liu, Jun; Lan, Hui; Shao, Minghao; Yu, Yuan; Quan, Fusheng; Zhang, Yong

2015-10-01

Transgenic cattle expressing high levels of recombinant human serum albumin (HSA) in their milk may as an alternative source for commercial production. Our objective was to produce transgenic cattle highly expressing HSA in milk by using phiC31 integrase system and somatic cell nuclear transfer (SCNT). The mammary-specific expression plasmid pIACH(-), containing the attB recognition site for phiC31 integrase, were co-transfected with integrase expression plasmid pCMVInt into bovine fetal fibroblast cells (BFFs). PhiC31 integrase-mediated integrations in genome of BFFs were screened by nested inverse PCR. After analysis of sequence of the PCR products, 46.0% (23/50) of the both attB-genome junction sites (attL and attR) were confirmed, and four pseudo attP sites were identified. The integration rates in BF3, BF11, BF19 and BF4 sites were 4.0% (2/50), 6.0% (3/50), 16.0% (8/50) and 20.0% (10/50), respectively. BF3 is located in the bovine chromosome 3 collagen alpha-3 (VI) chain isomer 2 gene, while the other three sites are located in the non-coding region. The transgenic cell lines from BF11, BF19 and BF4 sites were used as donors for SCNT. Two calves from transgenic cells BF19 were born, one died within a few hours after birth, and another calf survived healthy. PCR and Southern blot analysis revealed integration of the transgene in the genome of cloned calves. The nested reverse PCR confirmed that the integration site in cloned calves was identical to the donor cells. The western blotting assessment indicated that recombinant HSA was expressed in the milk of transgenic cattle and the expression level was about 4-8 mg/mL. The present study demonstrated that phiC31 integrase system was an efficient and safety gene delivery tool for producing HSA transgenic cattle. The production of recombinant HSA in the milk of cattle may provide a large-scale and cost-effective resource.

Deletion of alpha-synuclein decreases impulsivity in mice.

PubMed

Peña-Oliver, Y; Buchman, V L; Dalley, J W; Robbins, T W; Schumann, G; Ripley, T L; King, S L; Stephens, D N

2012-03-01

The presynaptic protein alpha-synuclein, associated with Parkinson's Disease (PD), plays a role in dopaminergic neurotransmission and is implicated in impulse control disorders (ICDs) such as drug addiction. In this study we investigated a potential causal relationship between alpha-synuclein and impulsivity, by evaluating differences in motor impulsivity in the 5-choice serial reaction time task (5-CSRTT) in strains of mice that differ in the expression of the alpha-synuclein gene. C57BL/6JOlaHsd mice differ from their C57BL/6J ancestors in possessing a chromosomal deletion resulting in the loss of two genes, snca, encoding alpha-synuclein, and mmrn1, encoding multimerin-1. C57BL/6J mice displayed higher impulsivity (more premature responding) than C57BL/6JOlaHsd mice when the pre-stimulus waiting interval was increased in the 5-CSRTT. In order to ensure that the reduced impulsivity was indeed related to snca, and not adjacent gene deletion, wild type (WT) and mice with targeted deletion of alpha-synuclein (KO) were tested in the 5-CSRTT. Similarly, WT mice were more impulsive than mice with targeted deletion of alpha-synuclein. Interrogation of our ongoing analysis of impulsivity in BXD recombinant inbred mouse lines revealed an association of impulsive responding with levels of alpha-synuclein expression in hippocampus. Expression of beta- and gamma-synuclein, members of the synuclein family that may substitute for alpha-synuclein following its deletion, revealed no differential compensations among the mouse strains. These findings suggest that alpha-synuclein may contribute to impulsivity and potentially, to ICDs which arise in some PD patients treated with dopaminergic medication. © 2011 The Authors. Genes, Brain and Behavior © 2011 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.

Expression of recombinant human flavin monooxygenase and moclobemide-N-oxide synthesis on multi-mg scale.

PubMed

Hanlon, Steven P; Camattari, Andrea; Abad, Sandra; Glieder, Anton; Kittelmann, Matthias; Lütz, Stephan; Wirz, Beat; Winkler, Margit

2012-06-18

A panel of human flavin monooxygenases were heterologously expressed in E. coli to obtain ready-to-use biocatalysts for the in vitro preparation of human drug metabolites. Moclobemide-N-oxide (65 mg) was the first high-priced metabolite prepared with recombinant hFMO3 on the multi-milligram scale.

Nonhomologous Recombination between Defective Poliovirus and Coxsackievirus Genomes Suggests a New Model of Genetic Plasticity for Picornaviruses

PubMed Central

Holmblat, Barbara; Jégouic, Sophie; Muslin, Claire; Blondel, Bruno; Joffret, Marie-Line

2014-01-01

ABSTRACT Most of the circulating vaccine-derived polioviruses (cVDPVs) implicated in poliomyelitis outbreaks in Madagascar have been shown to be recombinants between the type 2 poliovirus (PV) strain of the oral polio vaccine (Sabin 2) and another species C human enterovirus (HEV-C), such as type 17 coxsackie A virus (CA17) in particular. We studied intertypic genetic exchanges between PV and non-PV HEV-C by developing a recombination model, making it possible to rescue defective type 2 PV RNA genomes with a short deletion at the 3′ end by the cotransfection of cells with defective or infectious CA17 RNAs. We isolated over 200 different PV/CA17 recombinants, using murine cells expressing the human PV receptor (PVR) and selecting viruses with PV capsids. We found some homologous (H) recombinants and, mostly, nonhomologous (NH) recombinants presenting duplications of parental sequences preferentially located in the regions encoding proteins 2A, 2B, and 3A. Short duplications appeared to be stable, whereas longer duplications were excised during passaging in cultured cells or after multiplication in PVR-transgenic mice, generating H recombinants with diverse sites of recombination. This suggests that NH recombination events may be a transient, intermediate step in the generation and selection of the fittest H recombinants. In addition to the classical copy-choice mechanism of recombination thought to generate mostly H recombinants, there may also be a modular mechanism of recombination, involving NH recombinant precursors, shaping the genomes of recombinant enteroviruses and other picornaviruses. PMID:25096874

Recombinant low-seroprevalent adenoviral vectors Ad26 and Ad35 expressing the respiratory syncytial virus (RSV) fusion protein induce protective immunity against RSV infection in cotton rats.

PubMed

Widjojoatmodjo, Myra N; Bogaert, Lies; Meek, Bob; Zahn, Roland; Vellinga, Jort; Custers, Jerome; Serroyen, Jan; Radošević, Katarina; Schuitemaker, Hanneke

2015-10-05

RSV is an important cause of lower respiratory tract infections in children, the elderly and in those with underlying medical conditions. Although the high disease burden indicates an urgent need for a vaccine against RSV, no licensed RSV vaccine is currently available. We developed an RSV vaccine candidate based on the low-seroprevalent human adenovirus serotypes 26 and 35 (Ad26 and Ad35) encoding the RSV fusion (F) gene. Single immunization of mice with either one of these vectors induced high titers of RSV neutralizing antibodies and high levels of F specific interferon-gamma-producing T cells. A Th1-type immune response was indicated by a high IgG2a/IgG1 ratio of RSV-specific antibodies, strong induction of RSV-specific interferon-gamma and tumor necrosis factor-alpha cytokine producing CD8 Tcells, and low RSV-specific CD4 T-cell induction. Both humoral and cellular responses were increased upon a boost with RSV-F expressing heterologous adenovirus vector (Ad35 boost after Ad26 prime or vice versa). Both single immunization and prime-boost immunization of cotton rats induced high and long-lasting RSV neutralizing antibody titers and protective immunity against lung and nasal RSV A2 virus load up to at least 30 weeks after immunization. Cotton rats were also completely protected against challenge with a RSV B strain (B15/97) after heterologous prime-boost immunization. Lungs from vaccinated animals showed minimal damage or inflammatory infiltrates post-challenge, in contrast to animals vaccinated with formalin-inactivated virus. Our results suggest that recombinant human adenoviral Ad26 and Ad35 vectors encoding the RSV F gene have the potential to provide broad and durable protection against RSV in humans, and appear safe to be investigated in infants. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

15 beta-hydroxysteroids (Part IV). Steroids of the human perinatal period: the synthesis of 3 alpha,15 beta,17 alpha-trihydroxy-5 alpha-pregnan-20-one and its A/B-ring configurational isomers.

PubMed

Reeder, A Y; Joannou, G E

1995-12-01

In recent years several 15 beta-hydroxysteroids have emerged pathognomonic of adrenal disorders in human neonates of which 3 alpha,15 beta,17 alpha-trihydroxy-5 beta-pregnan-20-one (2) was the first to be identified in the urine of newborn infants affected with congenital adrenal hyperplasia. In this investigation we report the synthesis of the three remaining 3 xi,5 xi-isomers, namely 3 alpha,15 beta,17 alpha-trihydroxy-5 alpha-pregnan-20-one (3), 3 beta,15 beta,17 alpha-trihydroxy-5 alpha-pregnan-20-one (7) and 3 beta,15 beta,17 alpha-trihydroxy-5 beta-pregnan-20-one (8) for their definitive identification in pathological conditions in human neonates. 3 beta,15 beta-Diacetoxy-17 alpha-hydroxy-5-pregnen-20-one (11), a product of chemical synthesis was converted to the isomeric 3 and 7, while conversion of 15 beta,17 alpha-dihydroxy-4-pregnen-3,20-dione (4), a product of microbiological transformation, resulted in the preparation of 8. In brief, selective acetate hydrolysis of 11 gave 15 beta-acetoxy-3 beta,17 alpha-dihydroxy-5-pregnen-20-one (12) which on catalytic hydrogenation gave 15 beta-acetoxy-3 beta,17 alpha-dihydroxy-5 alpha-pregnan-20-one (13) a common intermediate for the synthesis of the 3 beta(and alpha),5 alpha-isomers. Hydrolysis of the 15 beta-acetate gave 7, whereas oxidation with pyridinium chlorochromate gave 15 beta-acetoxy-17 alpha-hydroxy-5 alpha-pregnan-3,20-dione (14) which on reduction with L-Selectride and hydrolysis of the 15 beta-acetate gave 3. Finally, hydrogenation of 4 gave 15 beta, 17 alpha-dihydroxy-5 beta-pregnan-3,20-dione (10) which on reduction with L-Selectride gave 8.

A critical developmental role for tgfbr2 in myogenic cell lineages is revealed in mice expressing SM22-Cre, not SMMHC-Cre.

PubMed

Frutkin, Andrew D; Shi, Haikun; Otsuka, Goro; Levéen, Per; Karlsson, Stefan; Dichek, David A

2006-10-01

Smooth muscle cell (SMC)-specific deletion of transforming growth factor beta (TGF-beta) signaling would help elucidate the mechanisms through which TGF-beta signaling contributes to vascular development and disease. We attempted to generate mice with SMC-specific deletion of TGF-beta signaling by mating mice with a conditional ("floxed") allele for the type II TGF-beta receptor (tgfbr2flox) to mice with SMC-targeted expression of Cre recombinase. We bred male mice transgenic for smooth muscle myosin heavy chain (SMMHC)-Cre with females carrying tgfbr2flox. Surprisingly, SMMHC-Cre mice recombined tgfbr2flox at low levels in SMC and at high levels in the testis. Recombination of tgfbr2flox in testis correlated with high-level expression of SMMHC-Cre in testis and germline transmission of tgfbr2null. In contrast, mice expressing Cre from a SM22alpha promoter (SM22-Cre) efficiently recombined tgfbr2flox in vascular and visceral SMC and the heart, but not in testis. Use of the R26R reporter allele confirmed that Cre-mediated recombination in vascular SMC was inefficient for SMMHC-Cre mice and highly efficient for SM22-Cre mice. Breedings that introduced the SM22-Cre allele into tgfbr2flox/flox zygotes in order to generate adult mice that are hemizygous for SM22-Cre and homozygous for tgfbr2flox- and would have conversion of tgfbr2flox/flox to tgfbr2null/null in SMC-produced no live SM22-Cre : tgfbr2flox/flox pups (P<0.001). We conclude: (1) "SMC-targeted" Cre lines vary significantly in specificity and efficiency of Cre expression; (2) TGF-beta signaling in the subset of cells that express SM22alpha is required for normal development; (3) generation of adult mice with absent TGF-beta signaling in SMC remains a challenge.

Soluble antigens from group B streptococci induce cytokine production in human blood cultures.

PubMed Central

von Hunolstein, C; Totolian, A; Alfarone, G; Mancuso, G; Cusumano, V; Teti, G; Orefici, G

1997-01-01

Group B streptococcal antigens stimulated tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6 production in human blood cultures in a concentration- and time-dependent fashion. The minimal concentrations of type-specific polysaccharides, lipoteichoic acid, and group-specific polysaccharide required to produce these effects were, respectively, 0.01, 1, and 10 microg/ml. Cell separation experiments indicated that monocytes were the cell type mainly responsible for cytokine production. Time course studies indicated that TNF-alpha was released before the other cytokines. TNF-alpha, however, did not appear to directly induce IL-1beta, as shown by blockade experiments with anti-TNF-alpha antibodies. IL-6 levels were moderately but significantly decreased by anti-TNF-alpha. These data indicate that several products from group B streptococci are able to directly stimulate human monocytes to release TNF-alpha, IL-1beta, and IL-6. These findings may be clinically relevant, since proinflammatory cytokines can mediate pathophysiologic changes during sepsis. PMID:9317001

Alpha-dispersion in human tissue

NASA Astrophysics Data System (ADS)

Grimnes, Sverre; Martinsen, Ørjan G.

2010-04-01

Beta dispersion is found in living tissue in the kilohertz - megahertz range and is caused by the cellular structure of biological materials with low frequency properties caused by cell membranes. Alpha dispersion is found in the hertz range and the causes are not so well known. Alpha dispersions are the first to disappear when tissue dies. Tissue data have often been based upon excised specimen from animals and are therefore not necessarily representative for human tissue alpha dispersions. Here we present data obtained with non-invasive skin surface electrodes for different segments of the living human body. We found alpha dispersions in all cases; the ankle-wrist results had the smallest. Large alpha dispersions were found where the distance between the electrodes and muscle masses was small, e.g. on the calf. Further studies on electrode technique and reciprocity, electrode positioning, statistical variations, gender, age and bodily constitutions are necessary in order to reveal more about the alpha dispersion, its appearance and disappearance.

Mastering the game of Go without human knowledge.

PubMed

Silver, David; Schrittwieser, Julian; Simonyan, Karen; Antonoglou, Ioannis; Huang, Aja; Guez, Arthur; Hubert, Thomas; Baker, Lucas; Lai, Matthew; Bolton, Adrian; Chen, Yutian; Lillicrap, Timothy; Hui, Fan; Sifre, Laurent; van den Driessche, George; Graepel, Thore; Hassabis, Demis

2017-10-18

A long-standing goal of artificial intelligence is an algorithm that learns, tabula rasa, superhuman proficiency in challenging domains. Recently, AlphaGo became the first program to defeat a world champion in the game of Go. The tree search in AlphaGo evaluated positions and selected moves using deep neural networks. These neural networks were trained by supervised learning from human expert moves, and by reinforcement learning from self-play. Here we introduce an algorithm based solely on reinforcement learning, without human data, guidance or domain knowledge beyond game rules. AlphaGo becomes its own teacher: a neural network is trained to predict AlphaGo's own move selections and also the winner of AlphaGo's games. This neural network improves the strength of the tree search, resulting in higher quality move selection and stronger self-play in the next iteration. Starting tabula rasa, our new program AlphaGo Zero achieved superhuman performance, winning 100-0 against the previously published, champion-defeating AlphaGo.

Mastering the game of Go without human knowledge

NASA Astrophysics Data System (ADS)

Silver, David; Schrittwieser, Julian; Simonyan, Karen; Antonoglou, Ioannis; Huang, Aja; Guez, Arthur; Hubert, Thomas; Baker, Lucas; Lai, Matthew; Bolton, Adrian; Chen, Yutian; Lillicrap, Timothy; Hui, Fan; Sifre, Laurent; van den Driessche, George; Graepel, Thore; Hassabis, Demis

2017-10-01

A long-standing goal of artificial intelligence is an algorithm that learns, tabula rasa, superhuman proficiency in challenging domains. Recently, AlphaGo became the first program to defeat a world champion in the game of Go. The tree search in AlphaGo evaluated positions and selected moves using deep neural networks. These neural networks were trained by supervised learning from human expert moves, and by reinforcement learning from self-play. Here we introduce an algorithm based solely on reinforcement learning, without human data, guidance or domain knowledge beyond game rules. AlphaGo becomes its own teacher: a neural network is trained to predict AlphaGo’s own move selections and also the winner of AlphaGo’s games. This neural network improves the strength of the tree search, resulting in higher quality move selection and stronger self-play in the next iteration. Starting tabula rasa, our new program AlphaGo Zero achieved superhuman performance, winning 100-0 against the previously published, champion-defeating AlphaGo.

A synthetic peptide derived from A1 module in CRD4 of human TNF receptor-1 inhibits binding and proinflammatory effect of human TNF-alpha.

PubMed

Cao, Yingnan; Wang, Zhaohe; Bu, Xianzhang; Tang, Shu; Mei, Zhengrong; Liu, Peiqing

2009-06-01

Tumour necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine, which has been shown to be a causative factor in rheumatoid arthritis, inflammatory bowel disease and septic shock. Proinflammatory effect of TNF-alpha is activated mainly through human TNF receptor-1 (TNF-R1). However, the role of the fourth cystein-rich domain (CRD4) of TNF-R1 extracellular portion in the interaction of TNF-alpha with TNF-R1 is still unclear. In the present study, binding activity of TNF-alpha to TNF-R1 and protein levels of IkappaB-alpha and nuclear transcription factor kappa B (NF-kappaB) p65 subunit in HeLa cells were measured using enzyme-linked immunosorbent assay (ELISA) and western-blot analysis. Pep 3 (LRENECVS) which was derived from the hydrophilic region of A1 module in CRD4 remarkably inhibited the binding of TNF-alpha to TNF-R1, and also reversed TNF-alpha-induced degradation of IkappaB-alpha and nuclear translocation of NF-kappaB p65 subunit in HeLa cells. Our results confirmed that the hydrophilic region of A1 module in CRD4 participated in the interaction of TNF-alpha with TNF-R1, and demonstrated the potential of small-molecule TNF-alpha extracellular inhibitors targeting at A1 module in CRD4 of TNF-R1 in suppressing proinflammatory effect of TNF-alpha.

Recombinant Allergens in Structural Biology, Diagnosis, and Immunotherapy

PubMed Central

Tscheppe, Angelika; Breiteneder, Heimo

2017-01-01

The years 1988–1995 witnessed the beginning of allergen cloning and the generation of recombinant allergens, which opened up new avenues for the diagnosis and research of human allergic diseases. Most crystal and solution structures of allergens have been obtained using recombinant allergens. Structural information on allergens allows insights into their evolutionary biology, illustrates clinically observed cross-reactivities, and makes the design of hypoallergenic derivatives for allergy vaccines possible. Recombinant allergens are widely used in molecule-based allergy diagnosis such as protein microarrays or suspension arrays. Recombinant technologies have been used to produce well-characterized, noncontaminated vaccine components with known biological activities including a variety of allergen derivatives with reduced IgE reactivity. Such recombinant hypoallergens as well as wild-type recombinant allergens have been used successfully in several immunotherapy trials for more than a decade to treat birch and grass pollen allergy. As a more recent application, the development of antibody repertoires directed against conformational epitopes during immunotherapy has been monitored by recombinant allergen chimeras. Although much progress has been made, the number and quality of recombinant allergens will undoubtedly increase and keep improving our knowledge in basic scientific investigations, diagnosis, and therapy of human allergic diseases. PMID:28467993

Epigenetic functions enriched in transcription factors binding to mouse recombination hotspots.

PubMed

Wu, Min; Kwoh, Chee-Keong; Przytycka, Teresa M; Li, Jing; Zheng, Jie

2012-06-21

The regulatory mechanism of recombination is a fundamental problem in genomics, with wide applications in genome-wide association studies, birth-defect diseases, molecular evolution, cancer research, etc. In mammalian genomes, recombination events cluster into short genomic regions called "recombination hotspots". Recently, a 13-mer motif enriched in hotspots is identified as a candidate cis-regulatory element of human recombination hotspots; moreover, a zinc finger protein, PRDM9, binds to this motif and is associated with variation of recombination phenotype in human and mouse genomes, thus is a trans-acting regulator of recombination hotspots. However, this pair of cis and trans-regulators covers only a fraction of hotspots, thus other regulators of recombination hotspots remain to be discovered. In this paper, we propose an approach to predicting additional trans-regulators from DNA-binding proteins by comparing their enrichment of binding sites in hotspots. Applying this approach on newly mapped mouse hotspots genome-wide, we confirmed that PRDM9 is a major trans-regulator of hotspots. In addition, a list of top candidate trans-regulators of mouse hotspots is reported. Using GO analysis we observed that the top genes are enriched with function of histone modification, highlighting the epigenetic regulatory mechanisms of recombination hotspots.

Epigenetic functions enriched in transcription factors binding to mouse recombination hotspots

PubMed Central

2012-01-01

The regulatory mechanism of recombination is a fundamental problem in genomics, with wide applications in genome-wide association studies, birth-defect diseases, molecular evolution, cancer research, etc. In mammalian genomes, recombination events cluster into short genomic regions called "recombination hotspots". Recently, a 13-mer motif enriched in hotspots is identified as a candidate cis-regulatory element of human recombination hotspots; moreover, a zinc finger protein, PRDM9, binds to this motif and is associated with variation of recombination phenotype in human and mouse genomes, thus is a trans-acting regulator of recombination hotspots. However, this pair of cis and trans-regulators covers only a fraction of hotspots, thus other regulators of recombination hotspots remain to be discovered. In this paper, we propose an approach to predicting additional trans-regulators from DNA-binding proteins by comparing their enrichment of binding sites in hotspots. Applying this approach on newly mapped mouse hotspots genome-wide, we confirmed that PRDM9 is a major trans-regulator of hotspots. In addition, a list of top candidate trans-regulators of mouse hotspots is reported. Using GO analysis we observed that the top genes are enriched with function of histone modification, highlighting the epigenetic regulatory mechanisms of recombination hotspots. PMID:22759569

Integrin expression by human osteoblasts cultured on degradable polymeric materials applicable for tissue engineered bone.

PubMed

El-Amin, Saadiq F; Attawia, Mohamed; Lu, Helen H; Shah, Asist K; Chang, Richard; Hickok, Noreen J; Tuan, Rocky S; Laurencin, Cato T

2002-01-01

The use of biodegradable polymers in the field of orthopaedic surgery has gained increased popularity, as surgical pins and screws, and as potential biological scaffolds for repairing cartilage and bone defects. One such group of polymers that has gained considerable attention are the polyesters, poly(lactide-co-glycolide) (PLAGA) and polylactic acid (PLA), because of their minimal tissue inflammatory response, favorable biocompatibility and degradation characteristics. The objective of this study was to evaluate human osteoblastic cell adherence and growth on PLAGA and PLA scaffolds by examining integrin receptor (alpha2, alpha3, alpha4, alpha5, alpha6 and beta1) expression. Primary human osteoblastic cells isolated from trabecular bone adhered efficiently to both PLAGA and PLA, with the rate of adherence on PLAGA comparable to that of control tissue culture polystyrene (TCPS), and significantly higher than on PLA polymers at 3, 6 and 12 h. Human osteoblastic phenotypic expression, alkaline phosphatase (ALP) activity was positive on both degradable matrices, whereas osteocalcin levels were significantly higher on cells grown on PLAGA than on PLA composites. Interestingly, the integrin subunits, alpha2, alpha3, alpha4, alpha5, alpha6 and beta1 were all expressed at higher levels by osteoblasts cultured on PLAGA than those on PLA as analyzed by westerns blots and by flow cytometry. Among the integrins, alpha2, beta5 and beta1 showed the greatest difference in levels between the two surfaces. Thus, both PLA and PLAGA support osteoblastic adhesion and its accompanying engagement of integrin receptor and expression of osteocalcin and ALP. However PLAGA consistently appeared to be a better substrate for osteoblastic cells based on these parameters. This study is one of the first to investigate the ability of primary human osteoblastic cells isolated from trabecular bone to adhere to the biodegradable polymers PLAGA and PLA, and to examine the expression of their key adhesion receptors (integrins) on these substrates.

alpha-Adrenergic-mediated activation of human reconstituted fibrinogen receptor (integrin alphaIIbbeta3) in Chinese hamster ovary cells.

PubMed

Butta, Nora; Larrucea, Susana; Gonzalez-Manchon, Consuelo; Alonso, Sonia; Parrilla, Roberto

2004-12-01

This work reports the functional studies of CHO cells coexpressing alpha-adrenergic (alphaAR) and human fibrinogen (Fg) receptors (integrin alphaIIbbeta3). Stimulation of these cells with alpha-agonists produced a transient rise in the free cytosolic calcium (Ca(++)) accompanied by enhanced binding to soluble Fg, and these effects were prevented by specific alphaAR antagonists. The alpha-adrenergic-induced activation of alphaIIbbeta3 in CHO-alphaIIbbeta3-alphaAR increased the rate of adhesion and extension of cells onto Fg coated plates, and also induced a soluble Fg- and alphaIIbbeta3-dependent formation of cell aggregates, whereas no effects were observed by the stimulation of CHO-alphaIIbbeta3 cells. alpha-Adrenergic antagonists, the ligand mimetic peptide RGDS, pertussis toxin (PTX), or EDTA, they all prevented the alpha-adrenergic stimulation of adhesion and aggregation. However, inhibition of PKC prevented the alpha-adrenergic stimulation of cell adherence, whereas blocking the intracellular Ca(++) mobilization impeded the stimulation of cell aggregation. The alpha-adrenergic activation was associated with phosphorylation of a protein of approximately 100 kDa and proteins of the MAPK family. The former was selectively phosphorylated by alpha-adrenergic stimulation whereas the latter were phosphorylated by the binding of cells to Fg and markedly intensified by alpha-adrenergic stimulation.

Detection of 12 micron Mg I and OH lines in stellar spectra

NASA Technical Reports Server (NTRS)

Jennings, D. E.; Deming, D.; Wiedemann, G. R.; Keady, J. J.

1986-01-01

Infrared lines of Mg I and OH have been detected in stellar spectra near 12.3 microns. The Mg I 7i-6h transition was seen in Alpha Ori and Alpha Tau, and the R2e(23.5) and R1f(24.5) transitions of OH were seen in Alpha Ori. All lines appear in absorption, in contrast to the solar spectrum where the Mg I line shows a prominent emission core. The lack of emission in these low surface gravity stars is due to a greatly reduced volume recombination rate for the high-n states of Mg I, which is not fully compensated by the increased chromospheric scale height. The OH equivalent widths are sensitive to the temperature structure of the upper photosphere of Alpha Ori, and they indicate that the photosphere near tau 5000 of about 10 to the -5th is approximately 100 K hotter than is given by flux constant models. The OH measurements agree more closely with the 1981 semiemprical model of Basri, Linsky, and Eriksson (1981), which is based on Ca II and Mg II ultraviolet features.

Low latitude middle atmosphere ionization studies

NASA Technical Reports Server (NTRS)

Bassi, J. P.

1976-01-01

Low latitude middle atmosphere ionization was studied with data obtained from three blunt conductivity probes and one Gerdien condenser. An investigation was conducted into the effects of various ionization sources in the 40 to 65 Km altitude range. An observed enhancement of positive ion conductivity taking place during the night can be explained by an atmsopheric effect, with cosmic rays being the only source of ionization only if the ion-ion recombination coefficient (alpha sub i) is small(10 to the -7 power cu cm/s) and varies greatly with altitude. More generally accepted values of alpha sub i ( approximately equal to 3x10 to the -7 power cu cm/s) require an additional source of ionization peaking at about 65 Km, and corresponding approximately to the integrated effect of an X-ray flux measured on a rocket flown in conjunction with the ionization measurements. The reasonable assumption of an alpha sub i which does not vary with altitude in the 50-70 Km range implies an even greater value alpha sub i and a more intense and harder X-ray spectrum.

Effects of Inteferons on Human B-cell Differentiation in vitro

PubMed Central

Kim, Samyong; Stoetter, Hans; Heimpel, Herrman

1987-01-01

The effects of interferons (IFN) on in vitro differentiation of B-lymphocytes were studied. Peripheral lymphocytes from normal subjects were cultivated under polyclonal activator pokeweed mitogen (PWN) or Epstein-Barr virus (EBV) stimulation. The secreted Ig in the culture supernatants were measured for IgM by ELISA method. To determine the cellular level of IFN action T-cell enriched fraction (Te) or B-cell enriched fraction (Be) were preincubated with IFN prior to recombination culture. IFN had modulatory activities on Ig production; at low to moderately high doses (10–1000 U/ml of IFN-alpha or 12–120 U/ml of IFN-gamma) stimulating when IFN was added until 48 hr after the start of the culture, while after 72 hr from culture start IFN suppressed Ig production. Preincubation of Be-cells with moderately high doses of IFN (120 U/ml of IFN-gamma or 1000 U/ml of IFN-alpha) prior to PWM-stimulation suppressed Ig production. Likewise, in EBV-stimulated culture, high dose IFN suppressed Ig production. But low dose of IFN enhanced ig production in EBV-stimulated culture. Preincubation of Te-cells with IFN prior to PWM-stimulation with Be-cells enhanced the Ig production. The T-cell subset analysis at the end of these culture showed enhanced ratio of T-helper cell relative to T-suppressor cells, suggesting increased T-helper cell proliferation after incubation with IFN. Thus, it is concluded that IFNs have modulatory activities on B-cell differentiation. The mechanism seems to be direct effects on B-cells (in PWM and EBV system) as well as through T-helper cell mediation (PWM system). The IFN-gamma showed more potent (2-to 6-fold) stimulatory activities than IFN-alpha. PMID:2484953

Recombinant human-activated protein C inhibits cardiomyocyte apoptosis in a rat model of myocardial ischemia-reperfusion.

PubMed

Pirat, Bahar; Muderrisoglu, Haldun; Unal, Muge Tecder; Ozdemir, Handan; Yildirir, Aylin; Yucel, Muammer; Turkoglu, Suna

2007-02-01

Myocardial apoptosis is recognized as a major mechanism of cell death during ischemia-reperfusion. In this study, we assessed the hypothesis that activated protein C may have a cardioprotective effect via preventing apoptosis in a rat model of myocardial ischemia-reperfusion. Thirty male Sprague-Dawley rats were anesthetized, instrumented for hemodynamic measurements and ventilated mechanically. Twenty rats were subjected to 20 min of left anterior descending coronary artery occlusion and 2 h of reperfusion. They were randomly assigned to receive intravenous Ringer lactate (vehicle) or activated protein C (2 mg/kg/h) 10 min after occlusion and during reperfusion. The other 10 rats were sham-operated. At the end of the reperfusion period, serum samples were obtained for evaluation of creatine kinase, C-reactive protein and tumor necrosis factor-alpha. Apoptosis was measured quantitatively by the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling method. Serum creatine kinase, C-reactive protein and tumor necrosis factor-alpha values and percentage of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling- positive myocyte nuclei demonstrated negligible myocardial injury in sham-operated controls. During reperfusion, mean arterial pressures were significantly higher in activated protein C-treated rats than in the control group (68.2+/-10.3 vs. 55.4+/-11.6 mmHg, P=0.01). Number of apoptotic cells was significantly reduced from 47.7 to 24.8% with activated protein C administration (P=0.008). No difference was seen between activated protein C-treated and untreated animals with respect to creatine kinase, C-reactive protein and tumor necrosis factor-alpha levels. Treatment with activated protein C significantly improved hemodynamics after ischemia-reperfusion and reduced ischemia-reperfusion-induced myocardial apoptosis in rats.

Comprehensive long-term efficacy and safety of recombinant human alpha-mannosidase (velmanase alfa) treatment in patients with alpha-mannosidosis.

PubMed

Lund, Allan M; Borgwardt, Line; Cattaneo, Federica; Ardigò, Diego; Geraci, Silvia; Gil-Campos, Mercedes; De Meirleir, Linda; Laroche, Cécile; Dolhem, Philippe; Cole, Duncan; Tylki-Szymanska, Anna; Lopez-Rodriguez, Monica; Guillén-Navarro, Encarna; Dali, Christine I; Héron, Bénédicte; Fogh, Jens; Muschol, Nicole; Phillips, Dawn; Van den Hout, J M Hannerieke; Jones, Simon A; Amraoui, Yasmina; Harmatz, Paul; Guffon, Nathalie

2018-05-03

Long-term outcome data provide important insights into the clinical utility of enzyme replacement therapies. Such data are presented for velmanase alfa in the treatment of alpha-mannosidosis (AM). Patient data (n = 33; 14 adults, 19 paediatric) from the clinical development programme for velmanase alfa were integrated in this prospectively-designed analysis of long-term efficacy and safety. Patients who participated in the phase I/II or phase III trials and were continuing to receive treatment after completion of the trials were invited to participate in a comprehensive evaluation visit to assess long-term outcomes. Primary endpoints were changes in serum oligosaccharide and the 3-minute stair climb test (3MSCT). Mean (SD) treatment exposure was 29.3 (15.2) months. Serum oligosaccharide levels were significantly reduced in the overall population at 12 months (mean change: -72.7%, P < 0.001) and remained statistically significant at last observation (-62.8%, P < 0.001). A mean improvement of +9.3% in 3MSCT was observed at 12 months (P = 0.013), which also remained statistically significant at last observation (+13.8%, P = 0.004), with a more pronounced improvement detected in the paediatric subgroup. No treatment-emergent adverse events were reported leading to permanent treatment discontinuation. Patients treated with velmanase alfa experienced improvements in biochemical and functional measures that were maintained for up to 4 years. Long term follow-up is important and further supports the use of velmanase alfa as an effective and well-tolerated treatment for AM. Based on the currently available data set, no baseline characteristic can be predictive of treatment outcome. Early treatment during paediatric age showed better outcome in functional endpoints.

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

Function of Several Critical Amino Acids in Human Pyruvate Dehydrogenase Revealed by Its Structure

NASA Technical Reports Server (NTRS)

Korotchkina, Lioubov G.; Ciszak, E.; Patel, M.

2004-01-01

Pyruvate dehydrogenase (E1), an alpha 2 beta 2 tetramer, catalyzes the oxidative decarboxylation of pyruvate and reductive acetylation of lipoyl moieties of the dihydrolipoamide acetyltransferase. The roles of beta W135, alpha P188, alpha M181, alpha H15 and alpha R349 of E1 determined by kinetic analysis were reassessed by analyzing the three-dimensional structure of human E1. The residues identified above are found to play a structural role rather than being directly involved in catalysis: beta W135 is the center residue in the hydrophobic interaction between beta and beta' subunits; alpha P188 and alpha M181 are critical for the conformation of the TPP-binding motif and interaction between alpha and beta subunits; alpha H15, is necessary for the organization of the N-terminus of alpha and alpha'; subunits and alpha R349 supports the interaction of the C-terminus of the alpha subunits with the beta subunits. Analysis of several critical E1 residues confirms the importance of residues distant from the active site for subunit interactions and enzyme function.

Major psychological factors affecting acceptance of gene-recombination technology.

PubMed

Tanaka, Yutaka

2004-12-01

The purpose of this study was to verify the validity of a causal model that was made to predict the acceptance of gene-recombination technology. A structural equation model was used as a causal model. First of all, based on preceding studies, the factors of perceived risk, perceived benefit, and trust were set up as important psychological factors determining acceptance of gene-recombination technology in the structural equation model. An additional factor, "sense of bioethics," which I consider to be important for acceptance of biotechnology, was added to the model. Based on previous studies, trust was set up to have an indirect influence on the acceptance of gene-recombination technology through perceived risk and perceived benefit in the model. Participants were 231 undergraduate students in Japan who answered a questionnaire with a 5-point bipolar scale. The results indicated that the proposed model fits the data well, and showed that acceptance of gene-recombination technology is explained largely by four factors, that is, perceived risk, perceived benefit, trust, and sense of bioethics, whether the technology is applied to plants, animals, or human beings. However, the relative importance of the four factors was found to vary depending on whether the gene-recombination technology was applied to plants, animals, or human beings. Specifically, the factor of sense of bioethics is the most important factor in acceptance of plant gene-recombination technology and animal gene-recombination technology, and the factors of trust and perceived risk are the most important factors in acceptance of human being gene-recombination technology.

Interferon-alpha and interferon-gamma modulate Fas-mediated apoptosis in mitomycin-C-resistant human Tenon's fibroblasts.

PubMed

Wang, Xiao Yang; Crowston, Jonathan G; White, Andrew J R; Zoellner, Hans; Healey, Paul R

2014-08-01

The aim of the study was to investigate, using a native mitomycin-C-resistant human Tenon's fibroblast cell line, the possibility that interferon-alpha and gamma could be used with Fas agonists as an alternative anti-fibrotic strategy to mitomycin-C in trabeculectomy. A clinically resistant and in vitro verified mitomycin-C-resistant human Tenon's fibroblast cell line was pretreated with interferon-alpha and interferon-gamma for 48 h before stimulation with an agonistic Fas antibody (CH11) for 2 days to induce cell death. Cell death assays were undertaken. Changes in apoptosis-related proteins were determined by flow cytometry and Western blot. Pretreatment with interferon-alpha or interferon-gamma for 48 h increased Fas, Fas-associated protein with death domain and caspase-8 expression. Protein expression was further increased by combined exposure to interferon-alpha and gamma. Pretreatment with cytokines had no effect on Fas-L and Bcl-2. Interferon-alpha alone did not change the rate of induced cell death. A combination of interferon-alpha and gamma synergistically increased the sensitivity of mitomycin-C-resistant human Tenon's fibroblast cell line to induced cell death. An antagonistic anti-Fas antibody (ZB4) completely blocked induced cell death. Broad caspase inhibitors specific for caspases-8 and -3 reduced induced deaths in interferon pretreated mitomycin-C-resistant human Tenon's fibroblast cell line in a dose-dependent manner. Interferon-alpha and interferon-gamma render mitomycin-C-resistant human Tenon's fibroblast cell line sensitive to Fas-mediated apoptosis. The mechanism involves increased death-inducing signalling complex formation by upregulation of Fas, Fas-associated protein with death domain and caspase-8 expression. © 2013 Royal Australian and New Zealand College of Ophthalmologists.