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Sample records for beta 2-microglobulin gene

  1. Characterization of the beta2-microglobulin gene of the horse.

    PubMed

    Tallmadge, Rebecca L; Lear, Teri L; Johnson, Amanda K; Guérin, Gérard; Millon, Lee V; Carpenter, Susan L; Antczak, Douglas F

    2003-01-01

    A clone containing beta(2)-microglobulin (beta(2)-m), the light chain of the major histocompatibility complex class I cell surface molecule, was isolated from an equine bacterial artificial chromosome library. This clone was used as a template for polymerase chain reaction (PCR) and unidirectional sequencing to elucidate the genomic sequence and intron/exon boundaries. We obtained 7,000 bases of sequence, extending from 1,100 nucleotides (nt) upstream of the coding region start through 1,698 nt downstream of the stop codon. The sequence contained regulatory elements in the region upstream of the coding sequence similar to those of the beta(2)-m gene of other species. The beta(2)-m gene was localized to horse chromosome ECA1q23-q25 by fluorescent in situ hybridization. This was confirmed by synteny mapping on a (horse x mouse) somatic cell hybrid panel. The sequence and intron/exon boundaries determined were used to design PCR primers to amplify and sequence the coding region of the beta(2)-m gene in other equids, including five breeds of domestic horse, one Przewalski's horse, five domestic donkeys and five zebras. A high degree of conservation was found among equids, illustrated by >98% (349/354) identity at the nucleotide level and 95% (113/118) at the amino acid level, because of non-synonymous nucleotide substitutions. The promoter detected in the region upstream of the coding sequence was subcloned and used in chloramphenicol acetyl transferase (CAT) assays to demonstrate the presence of a functional promoter. This study provides tools for the analysis of regulation of not only the horse beta(2)-m gene, but also for any genes dependent upon beta(2)-m for expression.

  2. Beta-2 Microglobulin Kidney Disease Test

    MedlinePlus

    ... Serum or Urine Related tests: Albumin , Creatinine , BUN , Heavy Metals Were you looking for Beta-2 Microglobulin Tumor ... to high levels of cadmium and/or other heavy metals like mercury, such as may occur with occupational ...

  3. The chicken beta 2-microglobulin gene is located on a non-major histocompatibility complex microchromosome: a small, G+C-rich gene with X and Y boxes in the promoter.

    PubMed

    Riegert, P; Andersen, R; Bumstead, N; Döhring, C; Dominguez-Steglich, M; Engberg, J; Salomonsen, J; Schmid, M; Schwager, J; Skjødt, K; Kaufman, J

    1996-02-06

    beta 2-Microglobulin is an essential subunit of major histocompatibility complex (Mhc) class I molecules, which present antigenic peptides to T lymphocytes. We sequenced a number of cDNAs and two genomic clones corresponding to chicken beta 2-microglobulin. The chicken beta 2-microglobulin gene has a similar genomic organization but smaller introns and higher G+C content than mammalian beta 2-microglobulin genes. The promoter region is particularly G+C-rich and contains, in addition to interferon regulatory elements, potential S/W, X, and Y boxes that were originally described for mammalian class II but not class I alpha or beta 2-microglobulin genes. There is a single chicken beta 2-microglobulin gene that has little polymorphism in the coding region. Restriction fragment length polymorphisms from Mhc homozygous lines, Mhc congenic lines, and backcross families, as well as in situ hybridization, show that the beta 2-microglobulin gene is located on a microchromosome different from the one that contains the chicken Mhc. We propose that the structural similarities between the beta 2-microglobulin and Mhc genes in the chicken are due to their presence on microchromosomes and suggest that these features and the microchromosomes appeared by deletion of DNA in the lineage leading to the birds.

  4. The chicken beta 2-microglobulin gene is located on a non-major histocompatibility complex microchromosome: a small, G+C-rich gene with X and Y boxes in the promoter.

    PubMed Central

    Riegert, P; Andersen, R; Bumstead, N; Döhring, C; Dominguez-Steglich, M; Engberg, J; Salomonsen, J; Schmid, M; Schwager, J; Skjødt, K; Kaufman, J

    1996-01-01

    beta 2-Microglobulin is an essential subunit of major histocompatibility complex (Mhc) class I molecules, which present antigenic peptides to T lymphocytes. We sequenced a number of cDNAs and two genomic clones corresponding to chicken beta 2-microglobulin. The chicken beta 2-microglobulin gene has a similar genomic organization but smaller introns and higher G+C content than mammalian beta 2-microglobulin genes. The promoter region is particularly G+C-rich and contains, in addition to interferon regulatory elements, potential S/W, X, and Y boxes that were originally described for mammalian class II but not class I alpha or beta 2-microglobulin genes. There is a single chicken beta 2-microglobulin gene that has little polymorphism in the coding region. Restriction fragment length polymorphisms from Mhc homozygous lines, Mhc congenic lines, and backcross families, as well as in situ hybridization, show that the beta 2-microglobulin gene is located on a microchromosome different from the one that contains the chicken Mhc. We propose that the structural similarities between the beta 2-microglobulin and Mhc genes in the chicken are due to their presence on microchromosomes and suggest that these features and the microchromosomes appeared by deletion of DNA in the lineage leading to the birds. Images Fig. 4 Fig. 5 PMID:8577748

  5. [Plasma beta-2-microglobulin in rheumatoid arthritis].

    PubMed

    Fioravanti, A; Giordano, N; Loi, F; D'Amato, S; Castagna, M L; Frati, E; Marcolongo, R

    1984-09-30

    The beta 2 microglobulin (beta 2m) is a low molecular weight protein, recognized on the cellular membranes of numerous nucleated cells and strictly correlated to the antigens of Major Histocompatibility Complex. Many authors have demonstrated an increase of the plasmatic beta 2m in different inflammatory diseases and, particularly in rheumatic ones, as Rheumatoid Arthritis (RA), Reiter's syndrome, Ankylosing Spondylitis, Systemic lupus erythematosus. We have also investigated the behaviour of the plasmatic beta 2m in 52 RA patients and in 17 healthy subjects. The beta 2m was measured in serum, by radioimmunoassay. We have demonstrated that the plasmatic beta 2m has moderately increased in the serum of RA patients, even if there is not a significant difference when compared to the normal subjects.

  6. Amino acid sequences and structures of chicken and turkey beta 2-microglobulin.

    PubMed

    Welinder, K G; Jespersen, H M; Walther-Rasmussen, J; Skjødt, K

    1991-01-01

    The complete amino acid sequences of chicken and turkey beta 2-microglobulins have been determined by analyses of tryptic, V8-proteolytic and cyanogen bromide fragments, and by N-terminal sequencing. Mass spectrometric analysis of chicken beta 2-microglobulin supports the sequence-derived Mr of 11,048. The higher apparent Mr obtained for the avian beta 2-microglobulins as compared to human beta 2-microglobulin by SDS-PAGE is not understood. Chicken and turkey beta 2-microglobulin consist of 98 residues and deviate at seven positions: 60, 66, 74-76, 78 and 82. The chicken and turkey sequences are identical to human beta 2-microglobulin at 46 and 47 positions, respectively, and to bovine beta 2-microglobulin at 47 positions, i.e. there is about 47% identity between avian and mammalian beta 2-microglobulins. The known X-ray crystallographic structures of bovine beta 2-microglobulin and human HLA-A2 complex suggest that the seven chicken to turkey differences are exposed to solvent in the avian MHC class I complex. The key residues of beta 2-microglobulin involved in alpha chain contacts within the MHC class I molecule are highly conserved between chicken and man. This explains that heterologous human beta 2-microglobulin can substitute the chicken beta 2-microglobulin in exchange studies with B-F (chicken MHC class I molecule), and suggests that the MHC class I structure is conserved over long evolutionary distances.

  7. Beta-2-microglobulin gene expression is maintained in rainbow trout and Atlantic salmon kept at low temperatures.

    PubMed

    Kales, Stephen; Parks-Dely, Julie; Schulte, Patricia; Dixon, Brian

    2006-08-01

    Finfish in the wild are regularly subjected to low temperatures, which have been shown to cause a loss of Major Histocompatibility receptor expression in common carp kept at 6 degrees C. This is similar to what was seen in a mammalian cell line cultured at 26 degrees C. Loss of expression of this critical viral recognition protein may provide one mechanism for the increased frequency of fish diseases at low temperatures. This report demonstrates that unlike carp and mammals, beta(2)m transcript levels in both rainbow trout and Atlantic salmon do not decrease after 10 days at temperatures as low as 2 degrees C. Reverse transcriptase (RT)-PCR indicated that transcript steady-state levels of trout beta(2)m were maintained in both tissues and peripheral blood leucocytes, whether freshly isolated or in primary culture. Polyclonal antibodies raised against a recombinant form of trout beta(2)m, demonstrated cross-reactivity to both rainbow trout and Atlantic salmon protein lysates. Use of these antibodies in western blot analyses indicated that cellular protein levels are also maintained at low temperatures in both species while qualitative epifluorescence analysis of freshly isolated peripheral blood leucocytes indicated persistent cell surface expression of trout beta(2)m even after 10 days at 2 degrees C. Rainbow trout and Atlantic salmon may therefore utilise an alternative mode of immune gene regulation than the common carp and mammals allowing them to maintain viral recognition machinery at low temperatures, possibly due to selection for survival in cold climates.

  8. Beta-2 microglobulin is an amyloidogenic protein in man.

    PubMed Central

    Gorevic, P D; Casey, T T; Stone, W J; DiRaimondo, C R; Prelli, F C; Frangione, B

    1985-01-01

    Curvilinear fibrils with the tinctorial properties of amyloid were isolated from a patient with bone and joint involvement complicating chronic dialysis for renal disease. Subunit fractions of 24,000 and 12,000 mol wt were identified after gel filtration under dissociating conditions, the latter containing a significant amount of a dimer of the former. This was confirmed by Edman degradation of each fraction, which yielded the amino terminal sequence of normal human beta-2 microglobulin (B2M) to residues 20 and 30, respectively. The size of the subunit protein (12,000 mol wt) and the amino acid composition make it likely that intact B2M is a major constituent of the fibrils. B2M is thus another example of a low molecular weight serum protein, with a prominent beta-pleated sheet structure, that may adopt the fibrillar configuration of amyloid in certain pathologic states. Images PMID:3908488

  9. Increased production of beta2-microglobulin after heart transplantation.

    PubMed

    Erez, E; Aravot, D; Erman, A; Sharoni, E; van Oyk, D J; Raanani, E; Abramov, D; Sulkes, J; Vidne, B A

    1998-05-01

    Serum beta2-microglobulin (beta2m) levels were measured to evaluate the state of immunoactivation in stable heart transplant recipients. Serum beta2m and renal function of 29 heart transplant recipients were compared with 16 control subjects, who were age and sex matched, and 11 patients with chronic kidney failure. Serum creatinine and 24-hour urine collection for albuminuria were used as markers of renal impairment. Heart transplant recipients with normal renal function (n = 7) had significantly elevated beta2m levels compared with control subjects: 2.6 +/- 0.9 vs 1.66 +/- 0.32 microg/ml, p < or = 0.05. Heart transplant recipients with impaired renal function (n = 22) had significantly elevated beta2m compared with the chronic kidney failure group: 4.42 +/- 1.3 vs 3.49 +/- 0.66 microg/ml (p < or = 0.05); although there was no significant difference in serum creatinine levels. Albuminuria excretion was significantly elevated in the chronic kidney failure group compared with the heart transplant recipients with impaired renal function (p < or = 0.05). Elevated serum beta2m in heart transplant recipients suggests increased beta2m production, reflecting increased immunoactivation. This observation could be useful in monitoring long-term immunosuppressive therapy.

  10. Beta 2-microglobulin clearance in neonates: index of tubular maturation.

    PubMed

    Assadi, F K; John, E G; Justice, P; Fornell, L

    1985-08-01

    Serum and urinary beta 2-microglobulin (beta 2M) were studied by enzyme immunoassay in 28 normal neonates at day 1 and day 4 of life in relation to gestational age (GA) and postnatal age (PNA). The infants were grouped according to GA; 10 with GA ranging from 32 to 35 weeks (mean 33.5 weeks) and 18 with GA ranging from 36 to 41 weeks (mean 38.3 weeks). Serum beta 2M varied directly with both GA and PNA. When values for serum beta 2M were related to conceptional age (CA), a significant positive correlation was present for all the infants studied (r = 0.68, P less than 0.01). Fractional excretion of beta 2M (FE beta 2M) decreased as a function of both GA and PNA. When a comparison of FE beta 2M was made in infants of all CA, a significant inverse correlation was noted for infants with CA less than or equal to 35 weeks (r = -0.89, P less than 0.001). The fall in FE beta 2M reached a plateau by 36 weeks. The highest FE beta 2M (33%) was observed in infants of 32 weeks CA who had the lowest filtered beta 2M (F beta 2M). No statistically significant relationship between changes in FE beta 2M and fractional urine flow rate was observed within each of the CA categories (infants less than or equal to 35 weeks, r = 0.21, P = 0.28; infants greater than or equal to 36 weeks, r = 0.25, P = 0.18).(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Clearance and synthesis rates of beta 2-microglobulin in patients undergoing hemodialysis and in normal subjects

    SciTech Connect

    Floege, J.; Bartsch, A.; Schulze, M.; Shaldon, S.; Koch, K.M.; Smeby, L.C. )

    1991-08-01

    Retention of {beta} 2-microglobulin in patients undergoing hemodialysis is associated with a {beta} 2-microglobulin-derived amyloidosis. Removal of {beta} 2-microglobulin by renal replacement therapy has been proposed for the prevention of this amyloidosis. Currently, however, data on the {beta} 2-microglobulin synthesis rate in patients undergoing hemodialysis are scarce, and consequently it remains speculative how much removal would be necessary to counterbalance synthesis. The plasma kinetics of iodine 131-labeled {beta} 2-microglobulin were therefore examined in 11 patients with anuria who were undergoing long-term hemodialysis. Five healthy persons served as controls. Kinetic modeling of the plasma curves showed that the data fitted a two-pool model (r2 greater than 0.96) consisting of a rapid 2 to 4 hour distribution phase followed by a less steep curve, described by the plasma (metabolic) clearance (Clp). Synthetic rates were calculated from Clp and the {beta} 2-microglobulin steady state plasma concentration (plus {beta} 2-microglobulin removal during hemodialysis in the case of high flux hemodialysis). The results showed a significantly higher Clp in normal controls as compared with patients undergoing hemodialysis (65.5 {plus minus} 12.8 ml/min (mean {plus minus} SD) versus 3.4 {plus minus} 0.7 ml/min). In contrast, the {beta} 2-microglobulin synthesis rate in the patient group (3.10 {plus minus} 0.79 mg/kg/day) was not significantly different from that of normal controls (2.40 {plus minus} 0.67 mg/kg/day), which was due to markedly elevated {beta} 2-microglobulin plasma concentrations in the patients (37.6 {plus minus} 14.1 mg/L vs 1.92 {plus minus} 0.27 mg/L). These findings suggest that the presence of end-stage renal disease does not have a significant impact on the beta 2-microglobulin generation rate.

  12. MHC class I and class I-like gene product expression by malignant T cells: relationships between CD1a, HLA-ABC and beta 2-microglobulin.

    PubMed Central

    Jones, R A; Scott, C S; Katz, F E; Child, J A

    1988-01-01

    Beta 2-microglobulin (beta 2m) forms the invariant light chain of the MHC-encoded HLA-ABC and the non-MHC-encoded CD1 molecules. While HLA-ABC (MHC Class I) molecules are virtually ubiquitous in tissue distribution, CD1 determinants by contrast are more restricted. We have assessed, by indirect immunoenzymeassay, the relative membrane densities of these molecules on malignant thymic and post-thymic T cells. It was found that the T cells of mature post-thymic proliferations expressed significantly more beta 2m-associated protein, predominantly HLA-ABC in nature, than thymic-ALL blasts. This parallels the situation found in normal peripheral T cells and thymocytes. In contrast to post-thymic T cells, thymic-ALL blasts showed considerable case to case variation with respect to non-HLA-associated beta 2m and, of particular interest, not all of this excess beta 2m could be accounted for by CD1a. We therefore conclude that other beta 2m-containing molecules may be expressed on thymic-ALL blasts and possibly also on post-thymic leukaemic T cells. In addition, it was found that T cells from CD4+ cases of post-thymic proliferations expressed more beta 2m-associated determinants than other T cells, whether of either normal or malignant origin, and that certain post-thymic malignancies express significantly increased levels of beta 2m-associated protein relative to normal peripheral T-cells. This is in direct contrast to the situation seen in many solid malignancies. PMID:2466592

  13. Urinary beta 2-microglobulin concentration and mortality in a cadmium-polluted area

    SciTech Connect

    Nakagawa, H.; Nishijo, M.; Morikawa, Y.; Tabata, M.; Senma, M.; Kitagawa, Y.; Kawano, S.; Ishizaki, M.; Sugita, N.; Nishi, M. )

    1993-11-01

    A 9-y follow-up study of 3,178 persons who lived in a cadmium-polluted area was conducted to assess the influence of environmental cadmium exposure on long-term outcome. The standardized mortality ratios of the urinary beta 2-microglobulin-positive subjects (> 1,000 micrograms/g creatinine) of both sexes were higher than those of the general Japanese population, whereas the cumulative survival curves were lower than those of the urinary beta 2-microglobulin-negative group. A significant association was also found between urinary beta 2-microglobulin and mortality, using a Cox's proportional hazards model. Moreover, mortality rates increased in proportion to increases in the amount of urinary beta 2-microglobulin excreted. These results suggest that the prognosis for cadmium-exposed subjects with proximal tubular dysfunction is unfavorable. The mortality rate tended to become higher as the severity of renal dysfunction progressed.

  14. Beta2-microglobulin causes abnormal phosphatidylserine exposure in human red blood cells.

    PubMed

    Pavone, Barbara; Bucci, Sonia; Sirolli, Vittorio; Merlini, Giampaolo; Del Boccio, Piero; Di Rienzo, Marianna; Felaco, Paolo; Amoroso, Luigi; Sacchetta, Paolo; Di Ilio, Carmine; Federici, Giorgio; Urbani, Andrea; Bonomini, Mario

    2011-03-01

    The exposure of the aminophospholipid phosphatidylserine on the external leaflet of red blood cell plasma membrane can have several pathophysiological consequences with particular regard to the processes of cell phagocytosis, haemostasis and cell-cell interaction. A significant increase in phosphatidylserine-exposing erythrocytes has been reported in chronic haemodialysis patients and found to be strongly influenced by the uraemic milieu. To identify uraemic compound(s) enhancing phosphatidylserine externalization in erythrocytes, we fractionated by chromatographic methods the ultrafiltrate obtained during dialysis, and examined by flow cytometry the effect of the resulting fractions on phosphatidylserine exposure in human red cells. Chromatographic procedures disclosed a homogeneous fraction able to increase erythrocyte phosphatidylserine exposure. The inducer of such externalization was identified by monodimensional gel electrophoresis and mass spectrometry investigations as beta2-microglobulin. To confirm the beta2-microglobulin effect and to examine the influence of protein glycation (as it occurs in uraemia) on phosphatidylserine erythrocyte exposure, erythrocytes from normal subjects were incubated with recombinant beta2-microglobulin (showing no glycation sites at mass analysis), commercial beta2-microglobulin (8 glycation sites), or with in vitro glycated recombinant beta2-microglobulin (showing multiple glycation sites). Elevated concentrations of beta2-microglobulin (corresponding to plasma levels reached in dialysis patients) increased slightly but significantly the protein's ability to externalize phosphatidylserine on human erythrocytes. Such an effect was markedly enhanced by glycated forms of the protein. Beta2-microglobulin is recognized as a surrogate marker of middle-molecule uraemic toxins and represents a key component of dialysis-associated amyloidosis. Our study adds further evidence to the potential pathophysiologic consequences of beta2

  15. Molten globule precursor states are conformationally correlated to amyloid fibrils of human beta-2-microglobulin.

    PubMed

    Skora, Lukasz; Becker, Stefan; Zweckstetter, Markus

    2010-07-14

    Misfolding intermediates play a key role in defining aberrant protein aggregation and amyloid formation in more than 15 different human diseases. However, their experimental characterization is challenging due to the transient nature and conformational heterogeneity of the involved states. Here, we demonstrate that direct carbon-detected NMR experiments allow observation, assignment, and structural analysis of molten globule amyloid intermediates that are severely broadened by conformational exchange. The method is used to characterize the structure and dynamics of partially unfolded intermediates of the 99-residue protein beta-2-microglobulin, which is the major component of insoluble aggregates occurring in dialysis-related amyloidosis. Comparison of the conformational properties of the molten globule-like intermediates with levels of deuterium incorporation into amyloid fibrils of beta-2-microglobulin revealed a close relationship between the conformational properties of the metastable intermediates and the beta-sheet-rich insoluble aggregates of beta-2-microglobulin.

  16. Defective iron homeostasis in beta 2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man.

    PubMed

    Santos, M; Schilham, M W; Rademakers, L H; Marx, J J; de Sousa, M; Clevers, H

    1996-11-01

    Previously, hepatic iron overload resembling that in hereditary hemachromatosis (HH) has been found in beta 2-microglobulin knockout (beta 2m-/-) mice. We have now characterized iron metabolism in beta 2m-/- mice. The mutant mice fail to limit the transfer of iron from mucosal cells into the plasma. Transferrin saturation is abnormally high. Pathologic iron depositions occur predominantly in liver parenchymal cells. Reconstitution with normal hematopoietic cells redistributes the iron from parenchymal to Kupffer cells, but does not correct the mucosal defect. We conclude that (a) iron metabolism is defective in the gut mucosa as well as the liver of beta 2m-/- mice; and (b) a beta 2m-dependent gene product is involved in iron homeostasis. Recently, a novel gene of the major histocompatibility complex class I family, HLA-H, has been found to be mutated in a large proportion of HH patients. Our data provide functional support for the proposed causative role of HLA-H mutations in HH.

  17. Defective iron homeostasis in beta 2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man

    PubMed Central

    1996-01-01

    Previously, hepatic iron overload resembling that in hereditary hemachromatosis (HH) has been found in beta 2-microglobulin knockout (beta 2m-/-) mice. We have now characterized iron metabolism in beta 2m- /- mice. The mutant mice fail to limit the transfer of iron from mucosal cells into the plasma. Transferrin saturation is abnormally high. Pathologic iron depositions occur predominantly in liver parenchymal cells. Reconstitution with normal hematopoietic cells redistributes the iron from parenchymal to Kupffer cells, but does not correct the mucosal defect. We conclude that (a) iron metabolism is defective in the gut mucosa as well as the liver of beta 2m-/- mice; and (b) a beta 2m-dependent gene product is involved in iron homeostasis. Recently, a novel gene of the major histocompatibility complex class I family, HLA-H, has been found to be mutated in a large proportion of HH patients. Our data provide functional support for the proposed causative role of HLA-H mutations in HH. PMID:8920884

  18. 21 CFR 866.5630 - Beta-2-microglobulin immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Beta-2-microglobulin immunological test system. 866.5630 Section 866.5630 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  19. 21 CFR 866.5630 - Beta-2-microglobulin immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Beta-2-microglobulin immunological test system. 866.5630 Section 866.5630 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  20. 21 CFR 866.5630 - Beta-2-microglobulin immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Beta-2-microglobulin immunological test system. 866.5630 Section 866.5630 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  1. 21 CFR 866.5630 - Beta-2-microglobulin immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Beta-2-microglobulin immunological test system. 866.5630 Section 866.5630 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  2. Serum beta2-microglobulin in cadmium exposed workers.

    PubMed

    Piscator, M

    1978-09-01

    In cadmium exposed workers with renal tubular dysfunction the determination of beta2m in urine is an important diagnostic test. Cadmium exposure's influence on serum beta2m levels and its relationship to urinary excretion of beta2m were studied in 24 cadmium exposed workers with normal serum creatinine levels (less than 10 mg/l)) and no obvious tubular dysfunction. With increasing blood levels of cadmium beta2m was found to increase in serum. There was no concomitant increase in the urinary excretion of beta2m. Serum beta2m was not dependent on serum creatinine within the range studied. The results suggest that for evaluating renal glomerular function in cadmium exposed workers, it might be better to use the serum creatinine level, creatinine clearance or inulin clearance since beta2m might give some false positive results.

  3. Expression and temperature-dependent regulation of the beta2-microglobulin (Cyca-B2m) gene in a cold-blooded vertebrate, the common carp (Cyprinus carpio L.).

    PubMed

    Rodrigues, P N; Dixon, B; Roelofs, J; Rombout, J H; Egberts, E; Pohajdak, B; Stet, R J

    1998-01-01

    Expression of beta2-microglobulin (beta2m) in the common carp was studied using a polyclonal antibody raised against a recombinant protein obtained from eukaryotic expression of the Cyca-B2m gene. Beta2m is expressed on peripheral blood Ig+ and Ig lymphocytes, but not on erythrocytes and thrombocytes. In spleen and pronephros, dull- and bright-positive populations could be identified correlating with the presence of erythrocytes, thrombocytes, and mature leucocytes or immature and mature cells from the lympho-myeloid lineage, respectively. Thymocytes were shown to be comprised of a single bright-positive population. The Cyca-B2m polyclonal antiserum was used in conjunction with a similarly produced polyclonal antiserum to an MHC class I (Cyca-UA) alpha chain to investigate the expression of class I molecules on peripheral blood leucocytes (PBL) at different permissive temperatures. At 12 degrees C, a temporary downregulation of class I molecules was demonstrated, which recovered to normal levels within 3 days. However, at 6 degrees C, a lasting absence of class I cell-surface expression was observed, which could be restored slowly by transfer to 12 degrees C. The expression of immunoglobulin molecules on B cells was unaffected by temperature changes. The absence of the class I cell-surface expression was shown to be the result of a lack of sufficient Cyca-B2m gene transcription, although Cyca-UA mRNA was present at comparable levels at all temperatures. This suggests that class I expression is regulated by a temperature-sensitive transcription of the Cyca-B2m gene.

  4. Exothermic effects observed upon heating of beta2-microglobulin monomers in the presence of amyloid seeds.

    PubMed

    Sasahara, Kenji; Naiki, Hironobu; Goto, Yuji

    2006-07-25

    To understand the initial stages in the formation of amyloid fibrils of beta(2)-microglobulin, a protein responsible for dialysis-related amyloidosis, the effects of heat on the acid-unfolded monomer at pH 2.5 were studied. In the presence of a low concentration of seed fibrils, differential scanning calorimetric thermograms of acid-unfolded beta(2)-microglobulin monomers showed a large decrease in heat capacity with a sigmoidal temperature-dependence, which was subsequently released at higher temperature. Measurements of circular dichroism, atomic force microscopy, ultracentrifugation, and repeated differential scanning calorimetry indicated that the exothermic sigmoidal transition is accompanied by the conversion of about 12% of the monomeric beta(2)-microglobulin molecules into amyloid fibrils, which subsequently dissociate into monomers at high temperature. Interestingly, amyloid fibrils, formed partly after the sigmoidal transition, exhibited a heating rate-dependent, kinetically controlled thermal response, indicating that 12% of the total protein is enough to exhibit the unique thermal response. On the other hand, the salt-induced protofibrils did not show such a calorimetric response, indicating that the kinetic thermal response is unique to the particular structure of fibrils. Taken together, although the calorimetric behavior of amyloid fibrils remains elusive, it may be interpreted in terms of the effects of heat associated with the formation, the association, and the unfolding of fibrils, in which the interactions between specific beta-sheet structures and water molecules play a crucial role and are sensitively reflected in the heat capacity change in protein solution.

  5. Serum beta-2 microglobulin levels for predicting acute kidney injury complicating aortic valve replacement.

    PubMed

    Zaleska-Kociecka, Marta; Skrobisz, Anna; Wojtkowska, Izabela; Grabowski, Maciej; Dabrowski, Maciej; Kusmierski, Krzysztof; Piotrowska, Katarzyna; Imiela, Jacek; Stepinska, Janina

    2017-10-01

    Acute kidney injury complicating both transcatheter and surgical aortic valve replacement is associated with high rates of morbidity and mortality. The aim of this study was to investigate the role of serum beta 2 (β2) microglobulin, cystatin C and neutrophil gelatinase-associated lipocalin levels in detecting periprocedural acute kidney injury. Eighty consecutive patients who were 70 years of age or older and who were having surgical (n = 40) or transcatheter (n = 40) aortic valve replacement were recruited in a prospective study. The biomarkers were tested before the procedure, 6 times afterwards, at discharge and at a 6-month follow-up visit. The baseline β2-microglobulin level was the strongest predictor of acute kidney injury as a complication of transcatheter aortic valve replacement [odds ratio (OR) 5.277, P = 0.009]. Its level 24 h after the procedure reached the largest area under the curve (AUC) of 0.880 (P < 0.001) for detecting acute kidney injury. In multivariate logistic regression analysis, the levels of β2-microglobulin and cystatin C 24 h after the procedure were significantly associated with acute kidney injury after transcatheter valve replacement (OR 38.15, P = 0.044; OR 1782, P = 0.019, respectively). In the surgical aortic valve replacement group, the highest AUCs belonged to β2-microglobulin and cystatin C at 24 h (AUC = 0.808, P = 0.003 and AUC = 0.854, P = 0.001, respectively). Their higher values were also associated with acute kidney injury (OR 17.2, P = 0.018; OR 965.6, P = 0.02, respectively). A persistent increase in the postoperative levels of β2-microglobulin following acute kidney injury was associated with the progression of chronic kidney disease for 6 months after both transcatheter (OR 6.56, P = 0.030) and surgical (OR 7.67, P = 0.03) aortic valve replacements. Serum β2-microglobulin had the potential to predict acute kidney injury complicating transcatheter

  6. beta2-microglobulin dependence of the lupus-like autoimmune syndrome of MRL-lpr mice.

    PubMed

    Christianson, G J; Blankenburg, R L; Duffy, T M; Panka, D; Roths, J B; Marshak-Rothstein, A; Roopenian, D C

    1996-06-15

    MRL-lpr/lpr mice develop a distinctive immunologic disease characterized by accumulation of unusually large numbers of T cells in the peripheral lymphoid organs. Most of the accumulating T cells express an alpha beta-TCR but are peculiar in that they express neither CD4 nor CD8 co-ligands. Concurrent with lymphoaccumulation of such double negative (DN) T cells, MRL-lpr/lpr mice develop a lethal systemic lupus erythematosus-like autoimmune syndrome. This study focuses on the role of MHC class I molecules in this latter pathologic process. Highly backcrossed class I molecule-deficient MRL and MRL-lpr mice carrying a functionally defective allele of the gene beta 2-microglobulin (B2m) were produced. Class I deficient MRL-lpr/lpr mice demonstrated a substantial reduction in DN T cells, confirming other reports indicating that most DN T cells arise from progenitors positively selected on MHC class I molecules. Significantly, class I-deficient MRL-lpr/lpr mice also demonstrated a diminution of every autoimmune disease indicator analyzed including hypergammaglobulinemia; autoantibodies including anti-DNA, anti-Smith antigen, and rheumatoid factor; and glomerulonephritis. The results indicate that class I-dependent T cells are crucial not only for the development of DN T cells, but for multiple features of the MRL-lpr/lpr systemic lupus erythematosus syndrome. Moreover, the pattern of hypergammaglobulinemia suggests that the requirement for MHC class I proteins is restricted temporally to later stages of the disease.

  7. [Prognostic value of beta-2-microglobulin in Hodgkin disease in young adults].

    PubMed

    Fleury, J; Tortochaux, J; Legros, M; Cure, H; Kwiatkowski, F; Ferrière, J P; Travade, P; Dionet, C; Gaillard, G; Chassagne, J

    1994-07-01

    From 1977 to 1989, we measured serum beta-2-microglobulin (beta 2-MG) levels from 64 unselected and untreated patients, between 18 to 50-year-old, affected by Hodgkin's disease. Serum beta 2-MG level was measured by radioimmunoassay (Phadebas beta 2 microtest). Then, all patients received a chemotherapy such as MOPP or alternating MOPP/ABVD followed or not by radiotherapy. Elevated serum beta 2-MG level (> 2.4 mg/l) is associated with advanced stage disease (stage III-IV), presence of systemic symptoms and bulky tumor. Nevertheless, a multivariate analysis shows that the serum beta 2-MG level is the most significant prognostic indicator for disease free survival. The prognostic value of serum beta 2-MG is demonstrated for myeloma and non Hodgkin's lymphoma. A few authors have evaluated the prognostic impact of serum beta 2-MG in Hodgkin's disease. This study requires confirmation by multicentric and prospective trial.

  8. Characterization of arrangement and expression of the beta-2 microglobulin locus in the sandbar and nurse shark.

    PubMed

    Chen, Hao; Kshirsagar, Sarika; Jensen, Ingvill; Lau, Kevin; Simonson, Caitlin; Schluter, Samuel F

    2010-02-01

    Beta 2 microglobulin (beta2m) is an essential subunit of major histocompatibility complex (MHC) type I molecules. In this report, beta2m cDNAs were identified and sequenced from sandbar shark spleen cDNA library. Sandbar shark beta2m gene encodes one amino acid less than most teleost beta2m genes, and 3 amino acids less than mammal beta2m genes. Although sandbar shark beta2m protein contains one beta sheet less than that of human in the predicted protein structure, the overall structure of beta2m proteins is conserved during evolution. Germline gene for the beta2m in sandbar and nurse shark is present as a single locus. It contains three exons and two introns. CpG sites are evenly distributed in the shark beta2m loci. Several DNA repeat elements were also identified in the shark beta2m loci. Sequence analysis suggests that the beta2m locus is not linked to the MHC I loci in the shark genome.

  9. Thermal response with exothermic effects of beta2-microglobulin amyloid fibrils and fibrillation.

    PubMed

    Sasahara, Kenji; Yagi, Hisashi; Naiki, Hironobu; Goto, Yuji

    2009-06-12

    Calorimetric measurements were carried out using a differential scanning calorimeter to characterize the thermal response of beta(2)-microglobulin amyloid fibrils, the deposition of which results in dialysis-related amyloidosis. The fibril solution showed a large decrease in heat capacity (exothermic effect) before the temperature-induced depolymerization of the fibrils, which was characterized by a definite dependence on heating rate. To understand the factors that determine the heating-rate-dependent thermal response, the concentration dependence of polyethylene glycol, which inhibits the association of amyloid fibrils with heating, on exothermic effect was examined in detail and showed a causal link between the exothermic effect and fibril association. The results suggest that the transient association driven by a spatial approach and the concomitant dehydration of hydrophobic areas of amyloid fibrils may be significant factors determining the thermal response with exothermic effect, which has not been observed in calorimetric studies of monomolecular globular proteins. The heating-rate-dependent thermal response with the exothermic effect was observed not only for other amyloid fibrils formed from amyloid beta-peptides but also during the processes of the temperature-induced conversion of beta(2)-microglobulin protofibrils and hen egg-white lysozyme into amyloid fibrils. These results highlight the physics related to the heating-rate-dependent behaviors of heat capacity in terms of interactions between the specific structures of amyloid fibrils and water molecules.

  10. [Renal handling of beta2 microglobulin. Its significance in carriers of adolescent nephronophthisis (NPH3)].

    PubMed

    Fernández, Carmen; Araque, Carolina; Méndez, Jorge; Angulo, Luisa; Fargier, Bernardo

    2007-06-01

    The adolescent nephronophthisis (NPH3) is a variant of the nephronophthisis. In Venezuela, one to three patients have been registered each year, all of them belonging to the same family tree. The objective of this study was to evaluate the function of the proximal convoluted tubule in NPHP3 carriers; using the beta2M as biological marker. Eight carriers, 7 heterozygotes and 1 homozygote, with normal renal function were compared with a 10 healthy subjects (control group). Serum beta2 microglobulin (beta2M), urinary beta2M, the quotient urinary beta2M/urinary creatinine and the beta2M fractional excretion were determinated. The filtered beta2M and the percentage of reabsortion were calculated. We observed an increase in the plasmatic concentration of beta2M but not related with a decrease of the glomerular filtration. The urinary beta2M, the beta2M/urinary creatinine relation and the fractional excretion of beta2M were normal. The filtered load of beta2M was elevated without increase in the excretion or percentage of reabsortion. We conclude that in our group of NPH3 carriers, functional changes in the proximal convoluted tubule, when measured by urinary excretion of beta2M, were absent. This finding suggests the existence of other mechanism of uptake or degradation of the substance in the proximal convoluted tubule, which have yet to be elucidated.

  11. A {beta}{sub 2}-microglobulin cleavage variant fibrillates at near-physiological pH

    SciTech Connect

    Corlin, Dorthe B. Johnsen, Christina K.; Nissen, Mogens H.; Heegaard, Niels H.H.

    2009-04-03

    {beta}{sub 2}-microglobulin ({beta}{sub 2}m) deposits as amyloid in dialysis-related amyloidosis (DRA), predominantly in joints. The molecular mechanisms underlying the amyloidogenicity of {beta}{sub 2}m are still largely unknown. In vitro, acidic conditions, pH < 4.5, induce amyloid fibrillation of native {beta}{sub 2}m within several days. Here, we show that amyloid fibrils are generated in less than an hour when a cleavage variant of {beta}{sub 2}m-found in the circulation of many dialysis patients-is exposed to pH levels (pH 6.6) occurring in joints during inflammation. Aggregation and fibrillation, including seeding effects with intact, native {beta}{sub 2}m were studied by Thioflavin T fluorescence spectroscopy, turbidimetry, capillary electrophoresis, and electron microscopy. We conclude that a biologically relevant variant of {beta}{sub 2}m is amyloidogenic at slightly acidic pH. Also, only a very small amount of preformed fibrils of this variant is required to induce fibrillation of native {beta}{sub 2}m. This may explain the apparent lack of detectable amounts of the variant {beta}{sub 2}m in extracts of amyloid from DRA patients.

  12. Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme.

    PubMed Central

    Larsson, S; Hotchkiss, G; Andäng, M; Nyholm, T; Inzunza, J; Jansson, I; Ahrlund-Richter, L

    1994-01-01

    We have generated three artificial hammerhead ribozymes, denoted 'Rz-b', 'Rz-c' and 'Rz-d', with different specificities for exon II of the mouse beta-2-microglobulin (beta 2M) mRNA. In this study we tested for ribozyme mediated reduction of beta 2M mRNA in a cell line and in transgenic mice. Transfections of either of the Rz-b, Rz-c or Rz-d plasmids into a mouse cell-line (NIH/3T3) revealed reductions of beta 2M mRNA substrate in each case. Ribozyme expression in individual transfected clones was accompanied with an up to 80% reduction of beta 2M mRNA levels. Rz-c was selected for a transgenic study. Seven Rz-c transgenic founder animals were identified from which three ribozyme expressing families were established and analysed. Expression of the ribozyme transgene was tested for and detected in lung, kidney and spleen. Expression was accompanied with reduction of the beta 2M mRNA levels of heterozygous (Rz+/-) animals compared to non-transgenic litter mates. The effect was most pronounced in lung with more than 90% beta 2M mRNA reduction in individual mice. In summary, expression of our ribozymes in a cell free system, in a cell-line and in transgenic mice were all accompanied with reductions of beta 2M mRNA levels. Images PMID:8036151

  13. Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme.

    PubMed

    Larsson, S; Hotchkiss, G; Andäng, M; Nyholm, T; Inzunza, J; Jansson, I; Ahrlund-Richter, L

    1994-06-25

    We have generated three artificial hammerhead ribozymes, denoted 'Rz-b', 'Rz-c' and 'Rz-d', with different specificities for exon II of the mouse beta-2-microglobulin (beta 2M) mRNA. In this study we tested for ribozyme mediated reduction of beta 2M mRNA in a cell line and in transgenic mice. Transfections of either of the Rz-b, Rz-c or Rz-d plasmids into a mouse cell-line (NIH/3T3) revealed reductions of beta 2M mRNA substrate in each case. Ribozyme expression in individual transfected clones was accompanied with an up to 80% reduction of beta 2M mRNA levels. Rz-c was selected for a transgenic study. Seven Rz-c transgenic founder animals were identified from which three ribozyme expressing families were established and analysed. Expression of the ribozyme transgene was tested for and detected in lung, kidney and spleen. Expression was accompanied with reduction of the beta 2M mRNA levels of heterozygous (Rz+/-) animals compared to non-transgenic litter mates. The effect was most pronounced in lung with more than 90% beta 2M mRNA reduction in individual mice. In summary, expression of our ribozymes in a cell free system, in a cell-line and in transgenic mice were all accompanied with reductions of beta 2M mRNA levels.

  14. Beta 2-microglobulin levels in serum and urine of cadmium exposed rabbits.

    PubMed

    Piscator, M; Björck, L; Nordberg, M

    1981-07-01

    Male rabbits were exposed to cadmium during 16 weeks by subcutaneous injections of either 0.25 mg or 0.5 md Cd as cadmium chloride per kg body weight 3 times per week. beta 2-microglobulin (beta 2-m) and creatinine in serum, cadmium in blood, as well as total protein, creatinine, beta 2-m and cadmium in urine were determined before exposure and after 3 and 7 weeks of exposure. Measurements were also made at 19 weeks, 3 weeks after the last exposure. During exposure, there was a slight increase in the serum beta 2-m/creatinine ratio among rabbits with the highest exposure, while no effect of the cadmium burden could be observed once exposure had ceased. Urinary excretion of beta 2-m was related to urinary pH, which appeared to be the case also for excretion of total protein. In the high exposure group, a significant increase in urinary beta 2-m excretion, indicative of renal tubular dysfunction was seen after 7 weeks of exposure. This was, however, not related to serum beta 2-m levels. It was concluded that before renal damage has occurred even heavy cadmium exposure has very little influence on serum beta 2-m levels.

  15. Prognostic significance of serum beta-2 microglobulin in patients with diffuse large B-cell lymphoma in the rituximab era

    PubMed Central

    Yoon, Shinkyo; Yoo, Changhoon; Park, Ji Hyun; Lee, Jung Bok; Park, Chan-sik; Huh, Jooryung; Lee, Yoonse; Kim, Kyung Won; Ryu, Jin-Sook; Kim, Seok Jin; Kim, Won Seog; Yoon, Dok Hyun; Suh, Cheolwon

    2016-01-01

    The prognostic value of serum beta-2 microglobulin for diffuse large B-cell lymphoma (DLBCL) is not well known in the rituximab era. A retrospective registry data analysis of 833 patients with de novo DLBCL treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) was conducted to establish the prognostic significance of serum beta-2 microglobulin at a ≥2.5 mg/L cutoff. Five-year progression-free survival (PFS, 76.1% vs. 41.0%; p < 0.001) and overall survival (OS, 83.8% vs. 49.2%; p < 0.001) were significantly worse in patients with elevated serum beta-2 microglobulin (n = 290, 34.8%). Furthermore, the five parameters of the International Prognostic Index, accompanying B symptoms, bone marrow involvement and impaired renal function were associated with worse PFS and OS. In multivariate analysis, elevated beta-2 microglobulin was a significant poor prognostic factor for PFS (hazard ratio [HR], 1.70; 95% confidence interval [CI], 1.29–2.24; p < 0.001) and OS (HR, 2.0; 95% CI, 1.47–2.75; p < 0.001). In an independent validation cohort of 258 R-CHOP treated patients with de novo DLBCL, elevated beta-2 microglobulin levels remained a significant poor prognostic factor for PFS (HR, 2.03; 95% CI, 1.23–3.32; p = 0.005) and exhibited a strong trend of association with worse OS (HR, 1.64; 95% CI, 0.98–2.75; p = 0.062). The significance of serum beta-2 microglobulin levels as an independent prognostic factor for patients with DLBCL receiving R-CHOP is confirmed. PMID:27764777

  16. [Interaction of the mechanisms of beta 2-microglobulin convection, diffusion, and adsorption in line hemodiafiltration].

    PubMed

    García, H; Hernández-Jaras, J; Maduell, F; Yago, M; Calvo, C; Ferrero, J A

    2000-01-01

    The in vivo contribution of diffusion, convection ad adsorption to beta 2-microglobulin (beta 2-m) elimination by hemodiafiltration (HDF) was investigated. 11 patients (8M/3W), with a mean age of 59 +/- 10 years and weighing 62.7 +/- 8.7 kg were studied. A 1.89 m2 polysulphone membrane was used in 180 min postdilution HDF. Samples at blood inlet (bi), blood autlet (bo), dialysate outlet (do) and ultrafiltrate (uf) were taken to determine beta 2-m concentrations at 30 and 150 min. Rates of flow (Q, ml(min) prescribed were: infusion, Qinf = 103.6 +/- 12.3, Quf = 14.6 +/- 4.0 y Qb = 465 +/- 5.0. Effective Qbi was automatically measured by the machine and Qdo = 800 + Quf. The removed beta 2-m mass (M, mg/min) was obtained by multiplying rates of flow (Q, L/min) by beta 2-m concentrations (mg/L) at each sampling point. From mass balance, we calculated the mass of beta 2-m removed (mg/min) by adsorption 0.23 +/- 0.2, by convection 0.7 +/- 0.3 and by diffusion 1.0 +/- 0.4, at 30 min. At 150 min, the beta 2-m mass removed was -0.06 +/- 0.1 by adsorption 0.4 +/- 0.1 by convection and 0.3 +/- 0.1 by diffusion. In HDF, these beta 2-m eliminating mechanisms play a variable role throughout the session. The more significant conclusion is that diffusion of beta 2-m with a synthetic "open" membrane is an important method of removing beta 2-m, comparable to convection over the whole procedure. That result explain the relative efficacy of beta 2-m clearance by HDF convection, and also explain why isolated diffusion is an efficient mechanism for beta 2-m removal by high-flux hemodialysis.

  17. [beta subsccript 2]-microglobulin forms three-dimensional domain-swapped amyloid fibrils with disulfide linkages

    SciTech Connect

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

    2011-08-09

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

  18. Removal of the N-terminal hexapeptide from human beta2-microglobulin facilitates protein aggregation and fibril formation.

    PubMed Central

    Esposito, G.; Michelutti, R.; Verdone, G.; Viglino, P.; Hernández, H.; Robinson, C. V.; Amoresano, A.; Dal Piaz, F.; Monti, M.; Pucci, P.; Mangione, P.; Stoppini, M.; Merlini, G.; Ferri, G.; Bellotti, V.

    2000-01-01

    The solution structure and stability of N-terminally truncated beta2-microglobulin (deltaN6beta2-m), the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that deltaN6beta2-m has a free energy of stabilization that is reduced by 2.5 kcal/mol compared to the intact protein. Hydrogen exchange of a mixture of the truncated and full-length proteins at microM concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that deltaN6beta2-m is significantly less protected than its wild-type counterpart. Analysis of deltaN6beta2-m by NMR shows that this loss of protection occurs in beta strands I, III, and part of II. At mM concentration gel filtration analysis shows that deltaN6beta2-m forms a series of oligomers, including trimers and tetramers, and NMR analysis indicates that strand V is involved in intermolecular interactions that stabilize this association. The truncated species of beta2-microglobulin was found to have a higher tendency to self-associate than the intact molecule, and unlike wild-type protein, is able to form amyloid fibrils at physiological pH. Limited proteolysis experiments and analysis by mass spectrometry support the conformational modifications identified by NMR and suggest that deltaN6beta2-m could be a key intermediate of a proteolytic pathway of beta2-microglobulin. Overall, the data suggest that removal of the six residues from the N-terminus of beta2-microglobulin has a major effect on the stability of the overall fold. Part of the tertiary structure is preserved substantially by the disulfide bridge between Cys25 and Cys80, but the pairing between beta-strands far removed from this constrain is greatly perturbed. PMID:10850793

  19. Cartilage biomarkers in hemodialysis patients and the effect of beta2-microglobulin on articular chondrocytes.

    PubMed

    Sunk, I-G; Demetriou, D; Szendroedi, J; Amoyo, L; Raffetseder, A; Hörl, W H; Sunder-Plassmann, G; Smolen, J S; Bobacz, K

    2008-11-01

    Dialysis-related amyloidosis (DRA) is a severe complication of maintenance hemodialysis (HD). Given the predominant deposition of beta(2)-microglobulin (beta2m) fibrils on articular cartilage in early DRA, we investigated the significance of beta2m and its relationship to distinct cartilage biomarkers in early DRA diagnosis in HD patients. Furthermore, we assessed the effects of beta2m on articular chondrocytes in vitro. Serum samples from 133 patients were collected before and after HD. Type II collagen cleavage product (C2C), procollagen II c-propeptide (CPII), aggrecan chondroitin sulfate 846 epitope (CS-486) and cartilage oligomeric matrix protein (COMP) levels were determined by enzyme-linked immunosorbent assay. Primary bovine articular chondrocytes were cultured as monolayers and incubated with beta2m at 1.5mg/l and 20mg/l. Cartilage glucosaminoglycan synthesis was measured by [(35)S]sulfate incorporation. mRNA expression of interleukin (IL)-1beta, matrix metalloproteinases (MMPs)-3 and -9 was measured by reverse-transcriptase polymerase chain reaction (RT-PCR). Incubation with beta2m at 20mg/l significantly decreased matrix biosynthesis. PCR analysis revealed an increase of IL-1beta, as well as MMPs-3 and -9 on the mRNA level. C2C/CPII, CS-486 and COMP levels were increased only in a subset of patients without a significant correlation with beta2m concentrations. A subgroup analysis elucidated an increase in type II collagen degradation during the first years of HD, as shown by the elevation of C2C/CPII ratio. beta2m exerted anti-anabolic effects on articular chondrocytes in vitro and might be involved in cartilage degradation in HD patients. beta2m serum levels, however, did not reflect cartilage degradation in DRA. The assessment of C2C/CPII, CS-486 or COMP concentrations apparently has minor relevance in DRA diagnosis in HD patients. However, the increased type II collagen breakdown within 5 years after HD onset possibly mirrors the early stages of DRA

  20. Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells

    PubMed Central

    Quan, Yuan; Yan, Qing; Morales, John E.

    2015-01-01

    Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2-microglobulin on both gene alleles in hESCs. Because β2-microglobulin serves as the HLA-I light chain, disruption of the β2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with β2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, β2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. Significance This study reports the generation of a novel β2-microglobulin (B2M)−/− human embryonic stem cell (hESC) line. Differentiated mature cells

  1. Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells.

    PubMed

    Wang, Dachun; Quan, Yuan; Yan, Qing; Morales, John E; Wetsel, Rick A

    2015-10-01

    Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2-microglobulin on both gene alleles in hESCs. Because β2-microglobulin serves as the HLA-I light chain, disruption of the β2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with β2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, β2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. This study reports the generation of a novel β2-microglobulin (B2M)-/- human embryonic stem cell (hESC) line. Differentiated mature cells from this line

  2. Relationship between serum beta 2-microglobulin, bone histology, and dialysis membranes in uraemic patients.

    PubMed

    Ferreira, A; Ureña, P; Ang, K S; Simon, P; Morieux, C; Souberbielle, J C; de Vernejoul, M C; Drüeke, T B

    1995-01-01

    beta 2-Microglobulin (beta 2M) is the main constituent of osteoarticular amyloid deposits in haemodialysis patients. When dialysed with cellulosic (C) membrane such patients present a higher incidence of beta 2M-related amyloid arthropathy than with synthetic high-flux (SHF) membrane, and they have higher serum levels of beta 2M. This could favour beta 2M deposition as amyloid fibrils and/or modify bone and cartilage metabolism. We examined 56 uraemic patients dialysed in the same centre for 7.5 +/- 4.8 years (mean +/- SD). Based on bone histomorphometry criteria they were classified into either high-turnover bone disease (HTBD, 45 patients) or normal/low-turnover bone disease (N/LTBD, 11 patients). A subgroup of 30 patients had been dialysed with the same dialysis membrane for at least 18 months prior to study, 8 on C and 22 on SHF membrane. Serum intact parathyroid hormone levels were not different between the two patient subgroups. In contrast, serum beta 2M levels were higher in patients on C than on SHF membrane: 59.8 +/- 14.1 versus 32.8 +/- 8.7 mg/l, and so were serum total alkaline phosphatase and osteocalcin levels: 323 +/- 167 versus 173 +/- 50 IU/l, and 656 +/- 395 versus 288 +/- 263 ng/ml respectively. The increase of these serum markers of bone formation was associated with a higher bone cell number: osteoblast surface, 21.7 +/- 5.1 versus 9.8 +/- 11%; osteoclast surface, 4.27 +/- 1.86 versus 1.96 +/- 1.34%; and osteoclast number/mm2, 2.85 +/- 1.26 versus 1.27 +/- 0.88 respectively. Serum beta 2M was positively correlated with serum osteocalcin (r = 0.58, P < 0.001), bone-specific alkaline phosphatase (bAP) (r = 0.46, P < 0.008), and free pyridinoline (PYD) (r = 0.62, P < 0.002), and negatively correlated, only for HTBD, with osteoid volume: r = -0.40, P < 0.006. Serum beta 2M was higher in patients with HTBD than N/LTBD: The bone metabolism of chronic haemodialysis patients may be influenced by dialysis membrane biocompatibility. Moreover, the

  3. Association of Beta-2 Microglobulin with Inflammation and Dislipidemia in High-Flux Membrane Hemodialysis Patients.

    PubMed

    Topçiu-Shufta, Valdete; Miftari, Ramë; Haxhibeqiri, Valdete; Haxhibeqiri, Shpend

    2016-10-01

    Higher than expected cardiovascular mortality in hemodialysis patients, has been attributed to dyslipidemia as well as inflammation. Beta2-Microglobulin (β2M) is an independent predictor of outcome for hemodialysis patients and a representative substance of middle molecules. In 40 patients in high-flux membrane hemodialysis, we found negative correlation of β2M with high density lipoprotein (r=-0.73, p<0.001) and albumin (r= -0.53, p<0.001) and positive correlation with triglycerides (r=0.69, p<0.001), parathyroid hormone (r=0.58, p < 0.05) and phosphorus (r= 0.53, p<0.001). There was no correlation of β2M with C- reactive protein (CRP) and interleukin-6 (IL-6). During the follow-up period of three years, 6 out of 40 patients have died from cardiovascular events. In high-flux membrane hemodialysis patients, we observed a significant relationship of β2M with dyslipidemia and mineral bone disorders, but there was no correlation with inflammation.

  4. Association of Beta-2 Microglobulin with Inflammation and Dislipidemia in High-Flux Membrane Hemodialysis Patients

    PubMed Central

    Topçiu–Shufta, Valdete; Miftari, Ramë; Haxhibeqiri, Valdete; Haxhibeqiri, Shpend

    2016-01-01

    Background: Higher than expected cardiovascular mortality in hemodialysis patients, has been attributed to dyslipidemia as well as inflammation. Beta2-Microglobulin (β2M) is an independent predictor of outcome for hemodialysis patients and a representative substance of middle molecules. Results: In 40 patients in high-flux membrane hemodialysis, we found negative correlation of β2M with high density lipoprotein (r=-0.73, p<0.001) and albumin (r= -0.53, p<0.001) and positive correlation with triglycerides (r=0.69, p<0.001), parathyroid hormone (r=0.58, p < 0.05) and phosphorus (r= 0.53, p<0.001). There was no correlation of β2M with C- reactive protein (CRP) and interleukin-6 (IL-6). During the follow-up period of three years, 6 out of 40 patients have died from cardiovascular events. Conclusion: In high-flux membrane hemodialysis patients, we observed a significant relationship of β2M with dyslipidemia and mineral bone disorders, but there was no correlation with inflammation. PMID:27994294

  5. [Analytical performances of SPAPLUS® turbidimeter for the dosage of immunoglobulins and beta-2 microglobulin in serum].

    PubMed

    Thevenet, Isabelle; Benat, Clarisse; Chauzeix, Jasmine; Blancher, Antoine; Puissant-Lubrano, Bénédicte

    2014-01-01

    We evaluated the analytical performances of the SPAPLUS(®) immunoturbidimeter assays manufactured by The Binding Site(®) for the quantification of thirteen immunological parameters in serum: IgG, IgA, IgM and IgD immunoglobulins, IgG subclasses (1 to 4), IgA subclasses (1 and 2), beta-2 microglobulin, free light chains kappa and lambda. The within-day precision (repeatability) and the between-day precision (reproducibility) were obtained for two or three concentration levels depending of the parameter and were below the recommendations of the manufacturer, except for the repeatability of IgG1 (at a level of concentration of 6.7 g/L). An agreement above 90% (with Bland and Altman analysis) was observed between the results obtained with SPAPLUS(®) and those obtained with the nephelometer IMMAGE(®) 800 or radial immunodiffusion. The evaluation confirmed the linearity of the assays and the absence of contamination for all the parameters tested. We also assessed the practicability of the SPAPLUS(®) in terms of maintenance, frequency of calibration and cadence tests. The SPAPLUS(®) immunoturbidimeter displays good analytical performances for the immunological parameters evaluated in the present work.

  6. [The dynamics of the humoral immunity indices and of the beta 2-microglobulin level in children with chronic hepatitis].

    PubMed

    Volosianko, A B

    2000-03-01

    Studied in children with chronic hepatitis B (n = 38) were indices for humoral immunity, such as blood serum content of immunoglobulins A, G, M, and beta 2-microglobulin. The revealed increase in the level of the indices under study suggest to us an apparent tension of the humoral link of immunity. The polyenzymic drug preparation wobenzym in the complex of therapeutic measures instituted in the above children was found out to have a positive effect on humoral homeostasis.

  7. Imaging of dialysis-related amyloid (AB-amyloid) deposits with sup 131 I-beta 2-microglobulin

    SciTech Connect

    Floege, J.; Burchert, W.; Brandis, A.; Gielow, P.; Nonnast-Daniel, B.; Spindler, E.; Hundeshagen, H.; Shaldon, S.; Koch, K.M. )

    1990-12-01

    The diagnosis of dialysis-related amyloid (AB-amyloid) has been based usually on clinical and radiological criteria. Following the discovery that beta 2-microglobulin was the major protein of this amyloid, we isolated and radiolabelled uremic plasma beta 2-microglobulin. After intravenous injection, gamma-camera images of selected joint areas were obtained from 42 patients who were on regular hemodialysis therapy. Positive scans involving the shoulder, hip, knee and carpal regions were found in 13 of 14 patients treated for more than 10 years and 10 of 16 patients treated for 5 to 10 years. Patients treated for less time had negative scans. Specificity was indicated by negative scans in non-amyloid inflammatory lesions in control hemodialysis patients. Up to 48-fold tracer enrichment was detected in excised AB-amyloid containing tissue as compared to amyloid-free tissue. These findings suggest that circulating radiolabelled beta 2-microglobulin is taken up by the amyloid deposits. This method may non-invasively detect tissue infiltrates of amyloid. It may also permit prospective evaluation of the efficacy of prophylactic dialysis strategies which are designed to prevent or delay the onset of this complication of long-term dialysis.

  8. Serum Beta 2-Microglobulin/Cystatin C Index: A Useful Biomarker in Lupus Nephritis?

    PubMed Central

    Madureira Silva, Marcus Vinicius; Moscoso-Solorzano, Grace T.; Nishida, Sonia K.; Mastroianni-Kirsztajn, Gianna

    2012-01-01

    Background Systemic lupus erythematosus (SLE) is a chronic multisystem autoimmune disease with frequent flares. Our aim was to evaluate the beta 2-microglobulin/cystatin C (β2M/CysC) index versus other markers as a predictor factor for assessment of SLE reactivation. Methods We prospectively analyzed 42 patients with lupus nephritis. Disease activity was classified using SLEDAI-2K and BILAG. Routine renal function and laboratory markers of SLE activity were performed, as well as serum β2M (Sβ2M)/serum CysC (SCysC) and Sβ2M/serum creatinine (SCreat) indexes determinations. Results The 42 enrolled patients had a mean age of 37.7 ± 13.1 years, 88% were female and 67% Caucasians; mean estimated glomerular filtration rate was 61.9 ± 20.0 ml/min/1.73 m2. There was a strong correlation between SCreat versus SCysC (r = 0.887), SCreat versus Sβ2M (r = 0.865), and SCysC versus Sβ2M (r = 0.880). Multivariate analysis showed that the Sβ2M/SCreat index is a prognostic factor predicting active lupus nephritis. Conclusion As SCysC is a good marker of renal function, it would be expected that the Sβ2M/SCysC index could be a better indicator of renal activity than Sβ2M/SCreat, but in the present study it did not add relevant clinical information in the assessment of renal activity in SLE. PMID:22811690

  9. Rediscovering Beta-2 Microglobulin As a Biomarker across the Spectrum of Kidney Diseases

    PubMed Central

    Argyropoulos, Christos P.; Chen, Shan Shan; Ng, Yue-Harn; Roumelioti, Maria-Eleni; Shaffi, Kamran; Singh, Pooja P.; Tzamaloukas, Antonios H.

    2017-01-01

    There is currently an unmet need for better biomarkers across the spectrum of renal diseases. In this paper, we revisit the role of beta-2 microglobulin (β2M) as a biomarker in patients with chronic kidney disease and end-stage renal disease. Prior to reviewing the numerous clinical studies in the area, we describe the basic biology of β2M, focusing in particular on its role in maintaining the serum albumin levels and reclaiming the albumin in tubular fluid through the actions of the neonatal Fc receptor. Disorders of abnormal β2M function arise as a result of altered binding of β2M to its protein cofactors and the clinical manifestations are exemplified by rare human genetic conditions and mice knockouts. We highlight the utility of β2M as a predictor of renal function and clinical outcomes in recent large database studies against predictions made by recently developed whole body population kinetic models. Furthermore, we discuss recent animal data suggesting that contrary to textbook dogma urinary β2M may be a marker for glomerular rather than tubular pathology. We review the existing literature about β2M as a biomarker in patients receiving renal replacement therapy, with particular emphasis on large outcome trials. We note emerging proteomic data suggesting that β2M is a promising marker of chronic allograft nephropathy. Finally, we present data about the role of β2M as a biomarker in a number of non-renal diseases. The goal of this comprehensive review is to direct attention to the multifaceted role of β2M as a biomarker, and its exciting biology in order to propose the next steps required to bring this recently rediscovered biomarker into the twenty-first century. PMID:28664159

  10. Serum beta-2 microglobulin as a prognostic biomarker in patients with mantle cell lymphoma.

    PubMed

    Yoo, Changhoon; Yoon, Dok Hyun; Kim, Shin; Huh, Jooryung; Park, Chan-Sik; Park, Chan-Jeong; Lee, Sang-Wook; Suh, Cheolwon

    2016-03-01

    Although serum beta-2 microglobulin (B2M) has been suggested as a prognostic factor for mantle cell lymphoma (MCL), additional data are necessary to confirm its role. Between November 2005 and July 2014, a total of 52 patients with MCL were identified from the database of Asan Medical Center, Seoul, Korea. Pretreatment serum B2M information was available in 50 patients (96%). Overall survival (OS) was compared according to the serum B2M level with a cut-off value of 2.5 mg/L. The median MCL international prognostic index (MIPI) score was 5.84 (range 4.72-7.80), and the median biologic MIPI (MIPI-b) score was 6.27 (4.93-8.47). Pretreatment serum B2M was elevated in 30 patients (60%) and was significantly related to advanced stage (p = 0.02) and high MIPI (p = 0.03) and MIPI-b (p = 0.03) scores. With median follow-up duration of 29.8 months (range 0.8-87.0 months), the median OS was 56.2 months [95% confidence interval (CI) 36.6-75.9 months] in all patients, and serum B2M was significantly associated with OS (p = 0.001). In multivariate analyses adjusted for MIPI or MIPI-b scores and rituximab, elevated serum B2M was significantly associated with poor OS (when adjusting MIPI, hazard ratio = 26.4, 95% CI 2.9-241.3, p = 0.004; when adjusting MIPI-b, hazard ratio = 20.1, 95% CI 2.4-170.1, p = 0.006). Thus, pretreatment serum B2M may be an independent and significant prognostic factor in patients with MCL.

  11. Organic solvent mediated self-association of an amyloid forming peptide from beta2-microglobulin: an atomic force microscopy study.

    PubMed

    Chaudhary, Nitin; Singh, Shashi; Nagaraj, Ramakrishnan

    2008-01-01

    Human beta(2)-microglobulin (beta(2)m) forms amyloid fibrils in hemodialysis related amyloidosis. Peptides spanning the beta strands of beta(2)m have been shown to form amyloid fibrils in isolation. We have studied the self-association of a 13-residue peptide Ac-DWSFYLLYYTEFT-am (Pbeta(2)m) spanning one of the beta-strands of human beta(2)-microglobulin when dissolved in various organic solvents such as methanol (MeOH), trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), and dimethylsulfoxide. We have observed that Pbeta(2)m forms amyloid fibrils when diluted from organic solvents into aqueous buffer at pH 7.0 as judged by increase in thioflavin T fluorescence. Fibril formation was observed to depend on the solvents in which peptide stock solutions were prepared. Circular dichroism spectra indicated propensity for helical conformation in MeOH, TFE, and HFIP. In buffer, beta-structure was observed irrespective of the solvent in which the peptide stock solutions were prepared. Atomic force microscopy images obtained by drying the peptide on mica from organic solvents indicated the ability of Pbeta(2)m to self-associate to form nonfibrillar structures. Morphology of the structures was dependent on the solvent in which the peptide was dissolved. Peptides that have the ability to self-associate such as amyloid-forming peptides would be attractive candidates for the generation of self-assembled structures with varying morphologies by appropriate choice of surfaces and solvents for dissolution. Copyright 2008 Wiley Periodicals, Inc.

  12. Factors Other than the Glomerular Filtration Rate That Determine the Serum Beta-2-Microglobulin Level

    PubMed Central

    Medina-Escobar, Pedro; Nydegger, Urs E.; Risch, Martin; Risch, Lorenz

    2013-01-01

    Background β2-microglobulin has been increasingly investigated as a diagnostic marker of kidney function and a prognostic marker of adverse outcomes. To date, non-renal determinants of β2-microglobulin levels have not been well described. Non-renal determinants are important for the interpretation and appraisal of the diagnostic and prognostic value of any endogenous kidney function marker. Methods This cross-sectional analysis was performed within the framework of the www.seniorlabor.ch study, which includes subjectively healthy individuals aged ≥60 years. Factors known or suspected to have a non-renal association with kidney function markers were investigated for a non-renal association with serum β2-microglobulin. As a marker of kidney function, the Berlin Initiative Study equation 2 for the estimation of the estimated glomerular filtration rate (eGFRBIS2) in the elderly was employed. Results A total of 1302 participants (714 females and 588 males) were enrolled in the study. The use of a multivariate regression model adjusting for age, gender and kidney function (eGFRBIS2) revealed age, male gender, and C-reactive protein level to be positively associated with β2-microglobulin levels. In addition, there was an inverse non-renal relationship between systolic blood pressure, total cholesterol and current smoking status. No association with markers of diabetes mellitus, body stature, nutritional risk, thyroid function or calcium and phosphate levels was observed. Conclusions Serum β2-microglobulin levels in elderly subjects are related to several non-renal factors. These non-renal factors are not congruent to those known from other markers (i.e. cystatin C and creatinine) and remind of classical cardiovascular risk factors. PMID:23991042

  13. Pseudotumoral amyloidosis of beta 2-microglobulin origin in the buttock of a patient receiving long term haemodialysis.

    PubMed Central

    Fernández-Alonso, J; Rios-Camacho, C; Valenzuela-Castaño, A; Rocha-Castilla, J L

    1993-01-01

    A 52 year old man who had been receiving haemodialysis for 13 years, with a history of renal tuberculosis, right ischial tuberculous osteomyelitis, and dialysis arthropathy, developed a soft tissue tumour in his left buttock. Histological analysis, immunohistological staining, and electron microscopic examination of the surgically removed tumour showed massive deposits of beta 2-microglobulin (beta 2-M) amyloid. This case shows the expanding clinical spectrum of this type of amyloidosis, and it is suggested that amyloid infiltration should be considered in the differential diagnosis of gluteal tumours in these patients. Images PMID:8408708

  14. The Relationship between Salivary Beta-2 Microglobulin and Uremia Intensity in Men with Chronic Renal Failure

    PubMed Central

    Vahedi, Mohammad; Malekzadeh, Hossein; Haybar, Habib; Soltanian, Ali Reza; Abdollahzadeh, Shermin; Yoosefi, Hojjat; Seyedian, Masoud; Yazdanpanah, Leila; Saeid, Abrotan; Shabanpour Fooladi, Maryam; Ghasemi, Marziyeh

    2013-01-01

    Objective: This study defines the relationship between salivary beta-2 microglobulin (β2-M) and intensity of uremia in male patients diagnosed with chronic renal failure (CRF). Materials and Methods: In total of 42 males were enrolled in a case-control study. There were 21 cases of CRF and 21 control cases. We collected 10cc of saliva plus 5 cc of blood from all patients to determine β2-M, blood urea nitrogen (BUN) and creatinine (Cr) levels. Results: There was a correlation between the level of serum BUN and salivary urea in controls and patients, which was statistically significant for controls (p=0.028).The correlation between serum and salivary Cr was 0.195 in controls (p=0.398) and 0.598 in patients (p=0.006), which was statistically significant in patients. The correlation between serum and saliva was 0.133 (p=0.566) in controls and 0.078 (p=0.737) in patients, which was not statistically significant. The correlation between serum BUN and β2-M was 0.168 (p=0.469) in the control group and 0.629 (p=0.002) in patients, which was statistically significant in patients. The correlation between serum Cr and β2-M was 0.110 (p=0.635) in the control group and 0.678 (p=0.001) in patients, which was statistically significant in patients. The correlation between serum BUN and salivary β2-M was 0.093 (p=0.0690) in controls and 0.152 (p=0.152) in patients, which was not statistically significant. The correlation between serum Cr and salivary β2-M was 0.072 (p=0.070) in the control group and 0.286 (p=0.209) in patients, which was not statistically significant in either group. Conclusion: The results of the study indicated that salivary β2-M cannot be used as a noninvasive indicator to detect the severity of renal failure. PMID:23577307

  15. Characterization of beta(2)-microglobulin coding sequence from three non-placental mammals: the duckbill platypus, the short-beaked echidna, and the grey short-tailed opossum.

    PubMed

    Miska, Katarzyna B; Hellman, Lars; Miller, Robert D

    2003-03-01

    To further characterize genes of immunological importance from non-placental mammals, cDNAs encoding beta(2)-microglobulin (beta(2)m) were isolated from two prototherians, the platypus and an echidna, and one metatherian, a grey short-tailed opossum. In addition, a second allele of beta(2)m was identified in another metatherian species, the brushtail possum. Analysis of the deduced translations revealed conservation of key residues in these molecules over a long evolutionary history. The types of nucleotide substitutions present among the various taxa are also consistent with purifying selection at this conserved locus. An evolutionary tree of beta(2)m was constructed that supports the classic view of evolution with prototherians as the basal mammalian group.

  16. [Determination of beta 2-microglobulin in the serum and cerebrospinal fluid using radial immunodiffusion (comparison with the Elisa Pharmacia method)].

    PubMed

    Králová, E; Novotná, H; Adam, Z

    1993-12-20

    The authors elaborated a method of radial immunodiffusion for assessment of beta 2-microglobulin serum and cerebrospinal fluid levels in patients. The method is based on a modified procedure of staining and decolouration. The antibody produced by USOL Co. which was used for the purpose was not modified. Comparison of 90 sera and 27 cerebrospinal fluid samples where also the reference method ELISA Pharmacia was used revealed almost absolute agreement, the correlation coefficient was 0.98. The method is used for clinical monitoring in patients with myelomas and renal insufficiencies.

  17. Translocation of peptides through microsomal membranes is a rapid process and promotes assembly of HLA-B27 heavy chain and beta 2- microglobulin translated in vitro

    PubMed Central

    1991-01-01

    We have translated major histocompatibility complex (MHC) class I heavy chains and human beta 2-microglobulin in vitro in the presence of microsomal membranes and a peptide from the nucleoprotein of influenza A. This peptide stimulates assembly of HLA-B27 heavy chain and beta 2- microglobulin about fivefold. By modifying this peptide to contain biotin at its amino terminus, we could precipitate HLA-B27 heavy chains with immobilized streptavidin, thereby directly demonstrating class I heavy chain-peptide association under close to physiological conditions. The biotin-modified peptide stimulates assembly to the same extent as the unmodified peptide. Both peptides bind to the same site on the HLA-B27 molecule. Immediately after synthesis of the HLA-B27 heavy chain has been completed, it assembles with beta 2-microglobulin and peptide. These interactions occur in the lumen of the microsomes (endoplasmic reticulum), demonstrating that the peptide must cross the microsomal membrane in order to promote assembly. The transfer of peptide across the microsomal membrane is a rapid process, as peptide binding to heavy chain-beta 2-microglobulin complexes is observed in less than 1 min after addition of peptide. By using microsomes deficient of beta 2-microglobulin (from Daudi cells), we find a strict requirement of beta 2-microglobulin for detection of peptide interaction with the MHC class I heavy chain. Furthermore, we show that heavy chain interaction with beta 2-microglobulin is likely to precede peptide binding. Biotin-modified peptides are likely to become a valuable tool in studying MHC antigen interaction and assembly. PMID:1955465

  18. Neopterin and beta-2 microglobulin relations to immunity and inflammatory status in nonischemic dilated cardiomyopathy patients.

    PubMed

    Wojciechowska, Celina; Wodniecki, Jan; Wojnicz, Romuald; Romuk, Ewa; Jacheć, Wojciech; Tomasik, Andrzej; Skrzep-Poloczek, Bronisława; Spinczyk, Beata; Nowalany-Kozielska, Ewa

    2014-01-01

    The aim of the study was to assess the relationships among serum neopterin (NPT), β2-microglobulin (β2-M) levels, clinical status, and endomyocardial biopsy results of dilated cardiomyopathy patients (DCM). Serum NPT and β-2 M were determined in 172 nonischaemic DCM patients who underwent right ventricular endomyocardial biopsy and 30 healthy subjects (ELISA test). The cryostat biopsy specimens were assessed using histology, immunohistology, and immunochemistry methods (HLA ABC, HLA DR expression, CD3 + lymphocytes, and macrophages counts). The strong increase of HLA ABC or HLA DR expression was detected in 27.2% patients-group A-being low in 72.8% patients-group B. Neopterin level was increased in patients in group A compared to healthy controls 8.11 (4.50-12.57) versus 4.99 (2.66-8.28) nmol/L (P < 0.05). β-2 microglobulin level was higher in DCM groups A (2.60 (1.71-3.58)) and B (2.52 (1.51-3.72)) than in the control group 1.75 (1.28-1.96) mg/L, P < 0.001. Neopterin correlated positively with the number of macrophages in biopsy specimens (P < 0.05) acute phase proteins: C-reactive proteins (P < 0.05); fibrinogen (P < 0.01); and NYHA functional class (P < 0.05) and negatively with left ventricular ejection fraction (P < 0.05). Neopterin but not β-2 microglobulin concentration reflected immune response in biopsy specimens. Neopterin correlated with acute phase proteins and stage of heart failure and may indicate a general immune and inflammatory activation in heart failure.

  19. Lacking prognostic significance of beta 2-microglobulin, MHC class I and class II antigen expression in breast carcinomas.

    PubMed Central

    Wintzer, H. O.; Benzing, M.; von Kleist, S.

    1990-01-01

    To evaluate the impact of MHC antigen expression on the survival of patients with cancer, 77 human breast carcinomas were investigated for the expression of beta 2-microglobulin (beta 2m), HLA-A,B,C and HLA-DR. Thirty-one benign breast tumours were stained for comparison. The results for the carcinomas were related to the survival data of the cancer patients. The expression of beta 2m, HLA-A,B,C and HLA-DR was significantly lower in malignant tumours compared to the benign lesions. Whereas all benign tumours were positive for beta 2m and HLA-A,B,C and 28/31 positive for HLA-DR the following positivity rates were found in carcinomas: 74/77 for beta 2m, 57/77 for HLA-A,B,C and 10/77 for HLA-DR. The follow-up (median 45 months) of 66 cancer patients for overall survival and of 65 patients for disease-free survival revealed no influence of beta 2m, HLA-A,B,C or HLA-DR expression on the prognosis of this cancer. In conclusion, experimental data indicating the importance of MHC antigens in anti-tumour responses are not confirmed by the analysis of cancer patient survival data. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2201398

  20. Reassociation with beta 2-microglobulin is necessary for Db class I major histocompatibility complex binding of an exogenous influenza peptide.

    PubMed Central

    Rock, K L; Gamble, S; Rothstein, L; Benacerraf, B

    1991-01-01

    A synthetic peptide corresponding to residues 365-380 of the influenza nucleoprotein (NP365-380) has been previously shown to associate with class I major histocompatibility complex-encoded molecules and to stimulate cytotoxic T lymphocytes [Townsend, A. R. M., Rothbard, J., Gotch, F. M., Bahadur, G., Wraith, D. & McMichael, A. J. (1986) Cell 44, 959-968]. We find that intact Db class I heterodimers on the cell surface are unreceptive to binding this antigen. However, NP365-380 readily associates with Db molecules on the plasma membrane in the presence of exogenous beta 2-microglobulin. In addition, there is a second pathway through which this peptide associates with class I molecules that requires energy and de novo protein synthesis. These findings have implications for maintaining the immunological identity of cells and for the use of peptides as vaccines for priming cytolytic T-cell immunity. Images PMID:1986378

  1. Interaction of human TNF and beta2-microglobulin with Tanapox virus-encoded TNF inhibitor, TPV-2L.

    PubMed

    Rahman, Masmudur M; Jeng, David; Singh, Rajkumari; Coughlin, Jake; Essani, Karim; McFadden, Grant

    2009-04-10

    Tanapox virus (TPV) encodes and expresses a secreted TNF-binding protein, TPV-2L or gp38, that displays inhibitory properties against TNF from diverse mammalian species, including human, monkey, canine and rabbit. TPV-2L also has sequence similarity with the MHC-class I heavy chain and interacts differently with human TNF as compared to the known cellular TNF receptors or any of the known virus-encoded TNF receptor homologs derived from many poxviruses. In order to determine the TNF binding region in TPV-2L, various TPV-2L C-terminal truncations and internal deletions were created and the muteins were expressed using recombinant baculovirus vectors. C-terminal deletions from TPV-2L resulted in reduced binding affinity for human TNF and specific mutants of TNF that discriminate between TNF-R1 and TNF-R2. However, deletion of C-terminal 42 amino acid residues totally abolished the binding of human TNF and its mutants. Removal of any of the predicted internal domains resulted in a mutant TPV-2L protein incapable of binding to human TNF. Deletion of C-terminal residues also affected the ability of TPV-2L to block TNF-induced cellular cytotoxicity. In addition to TNF, TPV-2L can also form complexes with human beta2-microglobulin to form a novel macromolecular complex. In summary, the TPV-2L protein is a bona fide MHC-1 heavy chain family member that binds and inhibits human TNF in a fashion very distinct from other known poxvirus-encoded TNF inhibitors, and also can form a novel complex with the human MHC-1 light chain, beta2-microglobulin.

  2. Circulating Beta-2 Microglobulin and Risk of Cancer: The Atherosclerosis Risk in Communities Study (ARIC).

    PubMed

    Prizment, Anna E; Linabery, Amy M; Lutsey, Pamela L; Selvin, Elizabeth; Nelson, Heather H; Folsom, Aaron R; Church, Timothy R; Drake, Charles G; Platz, Elizabeth A; Joshu, Corinne

    2016-04-01

    Serum β-2 microglobulin (B2M), a major histocompatibility complex class I molecule that is a biomarker of kidney filtration and increased cell turnover, is elevated at the time of diagnosis in hematological and some solid cancers. However, serum B2M was not examined prospectively as a marker for cancer risk. We hypothesized that in a population without a prior cancer diagnosis, serum B2M is associated with risk of cancer (n = 2,436), including colorectal (n = 255), lung (n = 298), breast (n = 424), and prostate (n = 524) cancers, and hematological (n = 176) malignancies. The analytical cohort (n = 12,300) was followed for incident cancers from 1990 through 2006. B2M (range, 0.9-57.8 mg/L) was measured in stored serum collected in 1990-1992. Cox proportional hazards models were used to estimate hazard ratios (HR) and 95% confidence intervals for cancer incidence and mortality in relation to quartiles of B2M. Adjusting for age, sex, race, center, education, body mass index, smoking, aspirin, and hormone therapy (in women) and comparing highest to lowest B2M quartiles, HRs were 1.25 (1.06-1.47; Ptrend = 0.002) for total cancer risk and 2.21 (1.32-3.70; Ptrend=0.001) for colorectal cancer risk, with similar HRs for colon and rectal cancers. These associations remained after adjustment for an inflammatory biomarker, C-reactive protein, and after excluding the first three years of follow-up. Significant associations were also observed for mortality from total, lung, and hematological cancers. These findings provide the first evidence that higher serum B2M is associated with increased colorectal cancer risk. This study supports B2M as a potential biomarker for colorectal cancer risk. Cancer Epidemiol Biomarkers Prev; 25(4); 657-64. ©2016 AACR. ©2016 American Association for Cancer Research.

  3. B cell antigens of the HLA system: a simple serotyping technique based on non-cytotoxic anti-beta2-microglobulin reagents.

    PubMed

    Bernoco, D; Bernoco, M; Ceppellini, R; Poulik, M D; Leeuwen, A; Rood, J J

    1976-10-01

    A modification of the NIH cytotoxicity test for recognizing B cell (D-associated) antigens and antibodies, when sera also contain anti-HLA(ABC) activity is described. The method is based on the observation that anti-beta2-microglobulin reagents are able to block lympholysis only when due to HLA(ABC) antigens.

  4. Unusual association of beta 2-microglobulin with certain class I heavy chains of the murine major histocompatibility complex.

    PubMed Central

    Bushkin, Y; Tung, J S; Pinter, A; Michaelson, J; Boyse, E A

    1986-01-01

    Class I products of the major histocompatibility complex (MHC) comprise a heavy chain of about 45 kDa noncovalently linked to a 12-kDa beta 2-microglobulin (beta 2m) light chain encoded on a different chromosome. We find that class I products of some mouse strains include an additional 62-kDa molecule which on the following evidence consists of a heavy chain linked covalently with beta 2m. Production of the 62-kDa protein invariably accorded with the occurrence of cysteine at position 121 of the heavy chain (Kb,Kbm1,Kbm3,Dd, and Ld). Substitution of arginine at position 121 invariably accorded with absence of the 62-kDa protein (Kbm6,Kbm7,Kbm9,Kd, and Db). On the basis of observed production versus nonproduction of the 62-kDa molecule, predictions are made regarding residue 121 in class I products for which this is not yet known; namely, Kk, Ks, and Dk, which produce the 62-kDa molecule, as compared with Kj, Qa-2, and TL, which do not. Reported differences in immunologic reactivity between Kb mutant strains with Arg-121 in place of Cys-121 imply that the occurrence of 62-kDa class I products in mice of Cys-121 genotype has functional consequences. Images PMID:3510435

  5. Advanced glycation end products of beta2-microglobulin in uremic patients as determined by high resolution mass spectrometry.

    PubMed

    Bertoletti, Laura; Regazzoni, Luca; Altomare, Alessandra; Colombo, Raffaella; Colzani, Mara; Vistoli, Giulio; Marchese, Loredana; Carini, Marina; De Lorenzi, Ersilia; Aldini, Giancarlo

    2014-03-01

    By using a high resolution top-down and bottom-up approach we identified and characterized the AGEs of beta2-microglobulin (β2-m) formed by incubating the protein in the presence of glucose and of the main reactive carbonyl species. Glucose induced glycation on the N-terminal residue, while glyoxal (GO) and methylglyoxal (MGO) covalently reacted with Arg3. Carboxymethyl (CM-R) and imidazolinone (R-GO) derivatives were identified in the case of GO and carboxyethyl arginine (CE-R) and methyl-imidazolinone (R-MGO) for MGO. Interestingly, α,β-unsaturated aldehydes [4-hydroxy-2-nonenal (HNE); 4-oxo-2-nonenal (ONE); acrolein (ACR)] did not induce any covalent modifications up to 100μM. The different reactivity of β2-m towards the different RCS was then rationalized by molecular modeling studies. The MS method was then applied to fully characterize the AGEs of β2-m isolated from the urine of uremic subjects. CM-R, CE-R and R-MGO were easily identified on Arg3 and their relative abundance in respect to the native protein determined by a semi-quantitative approach. Overall, the AGEs content of urinary β2-m ranged from 0.2 to 1% in uremic subjects. The results here reported offer novel insights and technical achievements for a potential biological role of AGEs-β2-m in pathological conditions.

  6. Expression of beta2-microglobulin and c-fos mRNA: is there an influence of high- or low-flux dialyzer membranes?

    PubMed

    Haufe, C C; Eismann, U; Deppisch, R M; Stein, G

    2001-02-01

    Dialysis-related amyloidosis is an important complication of long-term hemodialysis (HD) therapy with several pathogenetic factors. One of them is the influence of the dialyzer membrane type on the synthesis of beta2-microglobulin (beta2m). In vitro results are controversial. Thus, the hypothesis of whether in vivo beta2m generation is induced by the HD procedure and whether this induction depends on the type of the used dialyzer membrane should be tested. The aim of the present study was to investigate the influence of "biocompatible" high-flux versus "bioincompatible" low-flux HD on in vivo beta2m generation as well as the induction of the early activation gene c-fos in peripheral blood cells. Six nondiabetic HD patients [mean age 46 (21 to 69) years; Kt/V> 1.2] were included in a randomized crossover study using either a low-flux (cellulosic/cuprophan) or a high-flux (polyamide) dialyzer membrane. At the end of a four-week run-in period for each membrane, whole blood samples were taken before, immediately at, and four hours after the end of the dialysis session. MRNA was extracted, and after transcription to cDNA, quantitative polymerase chain reaction was performed for the beta2m gene, the early response gene c-fos, and the GAP-DH housekeeping gene. Based on the applied method for detection of specific mRNA, the results were given as ratio of beta2m or c-fos cDNA per GAP-DH cDNA. General cell activation during HD was indicated by increasing mRNA expression of c-fos related to the time course of the dialysis session, whereas beta2m did not change significantly. However, no difference was found when comparing the low-flux and the high-flux dialyzer membranes. Despite the evidence for activation of peripheral blood cells, as indicated by increasing c-fos message, no sign of beta2m mRNA induction during HD procedure with different dialyzer membranes was seen. Our results suggest that there is post-transcriptional regulation of beta2m generation and/or release as

  7. Molecular basis for the Cu2+ binding-induced destabilization of beta2-microglobulin revealed by molecular dynamics simulation.

    PubMed

    Deng, Nan-Jie; Yan, Lisa; Singh, Deepak; Cieplak, Piotr

    2006-06-01

    According to experimental data, binding of the Cu(2+) ions destabilizes the native state of beta2-microglobulin (beta2m). The partial unfolding of the protein was generally considered an early step toward fibril formation in dialysis-related amyloidosis. Recent NMR studies have suggested that the destabilization of the protein might be achieved through increased flexibility upon Cu(2+) binding. However, the molecular mechanism of destabilization due to Cu(2+), its role in amyloid formation, and the relative contributions of different potential copper-binding sites remain unclear. To elucidate the effect of ion ligation at atomic detail, a series of molecular dynamics simulations were carried out on apo- and Cu(2+)-beta2m systems in explicit aqueous solutions, with varying numbers of bound ions. Simulations at elevated temperatures (360 K) provide detailed pictures for the process of Cu(2+)-binding-induced destabilization of the native structure at the nanosecond timescale, which are in agreement with experiments. Conformational transitions toward partially unfolded states were observed in protein solutions containing bound copper ions at His-31 and His-51, which is marked by an increase in the protein vibrational entropy, with TDeltaS(vibr) ranging from 30 to 69 kcal/mol. The binding of Cu(2+) perturbs the secondary structure and the hydrogen bonding pattern disrupts the native hydrophobic contacts in the neighboring segments, which include the beta-strand D2 and part of the beta-strand E, B, and C and results in greater exposure of the D-E loop and the B-C loop to the water environment. Analysis of the MD trajectories suggests that the changes in the hydrophobic environment near the copper-binding sites lower the barrier of conformational transition and stabilize the more disordered conformation. The results also indicate that the binding of Cu(2+) at His-13 has little effect on the conformational stability, whereas the copper-binding site His-31, and to a lesser

  8. Enzyme immunoassay of serum beta-2-microglobulin levels in various histological forms of leprosy with special reference to its elevation in type I and type II lepra reactions.

    PubMed

    Saha, K; Bhatnagar, A; Sharma, V K; Chakrabarty, A K

    1985-04-01

    The mean beta-2-microglobulin level in serum (3,362 +/- 2,494 micrograms/liter) for 76 leprosy patients, including 9 borderline-tuberculoid, 8 borderline-borderline, 9 borderline-lepromatous, and 16 lepromatous-lepromatous patients and 34 patients with type I or type II lepra reactions, was significantly higher (P less than 0.001) than that (2,122 +/- 1,844 micrograms/liter) for 35 normal subjects. It decreased significantly (P less than 0.001) as the disease glided down from borderline tuberculoid (3,173 +/- 899 micrograms/liter) to the lepromatous end (1,813 +/- 1,391 micrograms/liter). At the onset of type I or type II reaction, the mean beta-2-microglobulin level in serum increased (4,447 +/- 2,863 micrograms/liter), and it remained unchanged (4,433 +/- 2,623 micrograms/liter) after clinical remission. The beta-2-microglobulin level in serum decreased in 55.5% of the patients tested after subsidence of reaction. The level was significantly higher in patients with type II reactions (5,433 +/- 3,299 micrograms/liter) than in patients with type I reactions (3,558 +/- 2,171 micrograms/liter).

  9. Self-assembly of the beta2-microglobulin NHVTLSQ peptide using a coarse-grained protein model reveals a beta-barrel species.

    PubMed

    Song, Wei; Wei, Guanghong; Mousseau, Normand; Derreumaux, Philippe

    2008-04-10

    Although a wide variety of proteins can assemble into amyloid fibrils, the structure of the early oligomeric species on the aggregation pathways remains unknown at an atomic level of detail. In this paper we report, using molecular dynamics simulations with the OPEP coarse-grained force field, the free energy landscape of a tetramer and a heptamer of the beta2-microglobulin NHVTLSQ peptide. On the basis of a total of more than 17 ns trajectories started from various states, we find that both species are in equilibrium between amorphous and well-ordered aggregates with cross-beta-structure, a perpendicular bilayer beta-sheet, and, for the heptamer, six- or seven-stranded closed and open beta-barrels. Moreover, analysis of the heptamer trajectories shows that the perpendicular bilayer beta-sheet is one possible precursor of the beta-barrel, but that this barrel can also be formed from a twisted monolayer beta-sheet with successive addition of chains. Comparison with previous aggregation simulations and the fact that nature constructs transmembrane beta-sheet proteins with pores open the possibility that beta-barrels with small inner diameters may represent a common intermediate during the early steps of aggregation.

  10. [Clinical usefulness of Beta2microglobulin in patients with Primary Sjögren Syndrome].

    PubMed

    Rozzatti, M S; Fontaneto, E; Pedano, V; Racca, A; Pelosso, M; Gobbi, C; Alba, P; Demarchi, M

    2015-01-01

    Las manifestaciones extranglandulares y desórdenes linfoproliferativos son complicaciones que pueden comprometer el curso benigno del Síndrome de Sjögren Primario (SSp). Existen escasos marcadores serológicos con comprobada utilidad para predecirlas y/o diagnosticarlas. Objetivos: Evaluar la utilidad de Beta2microglobulina (β2m) en pacientes con SSp y correlacionarlos con parámetros séricos predictivos de manifestaciones extraglandulares y enfermedades linfoproliferativas (Factor Reumatoideo (FR), Inmunoglobulinas séricas (Igs), C3 y C4). Materiales y métodos: Se realizó una revisión retrospectiva de historias clínicas de pacientes que consultaron en la Unidad de Reumatología del Hospital Córdoba desde enero de 2010 a octubre 2013 y que fueron derivados a la Sección de Inmunología del Servicio de Bioquímica para la determinación de pruebas de laboratorio . Los pacientes fueron clasificados de acuerdo a los Criterios Diagnósticos de patologías autoinmunes en pacientes con diagnóstico de SSp según el Grupo Consenso Americano-Europeo, otras enfermedades autoinmunes y los controles sanos. Se estudiaron las IgG, IgA e IgM, factores del complemento C3, C4 séricos y FR por inmunoturbidimetría y β2m por ELISA. Resultados: 19 pacientes con SSp (Grupo SSp), 28 pacientes con patologías autoinmunes distintas a SSp (Grupo PAD), y 24 controles sanos (Grupo C) fueron incluidos en este estudio. Se evidenció un aumento estadísticamente significativo de β2m en el Grupo SSp respecto al Grupo C (6.19mg/dl vs 2.53mg/dl p<0.001) y respecto al Grupo PAD (6.19 vs 4.38mg/dl p<0.01). En el grupo SSp se observó aumento estadísticamente significativo de IgA (p<0.05) y G (p<0.001) y disminución de C4 (p<0.05) respecto al Grupo C. No se observó correlación entre β2m y el resto de parámetros séricos determinados. Conclusión: β2m permitió discriminar pacientes con SSp de aquellos con otras patologías autoinmunes y sujetos sanos. El aumento de β2m en

  11. Alpha 1-microglobulin, beta 2-microglobulin and retinol binding protein in childhood febrile illness and renal disease.

    PubMed

    Donaldson, M D; Chambers, R E; Woolridge, M W; Whicher, J T

    1990-07-01

    Serum and urinary levels of alpha-1-microglobulin (A1M), beta-2-microglobulin (B2M) and retinol binding protein (RBP) were measured using a Mancini radial immunodiffusion technique in 52 children with renal disease, 36 with non-renal febrile illness and 29 controls. In controls the mean serum level for A1M was 25 +/- 4.6 (SD) mg/l for B2M 1.7 +/- 0.5 mg/l and for RBP 31 +/- 8 mg/l. A1M levels were not significantly altered by febrile illness while B2M was elevated and RBP markedly depressed. Serum A1M and B2M were elevated in the nephrotic syndrome, while serum B2M was also raised during infancy. Coefficients of log-transformed data with creatinine-derived glomerular filtration rate (GFR) were -0.87 for B2M, -0.71 for RBP, and -0.62 for A1M. In the urine A1M was always measurable in controls while B2M and RBP were undetectable in all but a small number. The urine levels of all three proteins increased in response to non-renal febrile illness, and rose invariably when GFR fell to below 40-50 ml/min per 1.73 m2. Of the three proteins A1M was most frequently elevated in the urine with febrile and renal illness. RBP was rarely detectable when the other two proteins were not. Urinary A1M was consistently elevated in the nephrotic syndrome in contrast to B2M, possibly as a reflection of the increased glomerular permeability. We conclude that serum B2M is superior to A1M and RBP as an index of glomerular filtration, although its levels should be interpreted with caution in febrile disease.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. The significance of urinary beta-2 microglobulin level for differential diagnosis of familial Mediterranean fever and acute appendicitis.

    PubMed

    Ugan, Yunus; Korkmaz, Hakan; Dogru, Atalay; Koca, Yavuz Savas; Balkarlı, Ayse; Aylak, Firdevs; Tunc, Sevket Ercan

    2016-07-01

    The clinical and laboratory parameters widely used are not specific to discriminate the abdominal pain due to FMF attack from that of acute appendicitis. The present study aims to investigate the urinary beta-2 microglobulin (U-β2M) level as a potential parameter to identify these two diseases mimicking each other. A total of 51 patients with established FMF diagnosis due to Tel Hashomer criteria on colchicine treatment (1-1.5 mg/day), 15 patients with acute appendicitis who had appropriate clinical picture and were also supported pathologically after the surgery, and 20 healthy controls were enrolled in the study. Of the 51 patients with FMF, 25 were at an attack period, while remaining 26 were not. For the diagnosis of acute attack, as well as physical examination, laboratory tests including white blood cell count, C-reactive protein, and erythrocyte sedimentation rate were performed. From urine specimens U-β2M, microalbumin, and N-acetyl glucosaminidase (U-NAG) were measured. U-β2M levels were significantly higher in acute appendicitis group compared to FMF attack, FMF non-attack, and control groups (p < 0.001, p < 0.001, and p < 0.001, respectively). U-NAG and microalbuminuria were significantly higher in acute appendicitis, FMF attack, and FMF non-attack groups compared to controls (U-NAG p < 0.001, p = 0.016, p = 0.004, microalbuminuria p < 0.001, p < 0.001, p < 0.001, respectively). Microalbuminuria was significantly higher in acute appendicitis group compared to the FMF attack group (p = 0.004). Determination of U-β2M levels may be helpful for differential diagnosis of peritonitis attacks of FMF patients on colchicine treatment and acute appendicitis. However, this finding should be substantiated with other studies.

  13. Beta 2-microglobulin-, CD8+ T-cell-deficient mice survive inoculation with high doses of vaccinia virus and exhibit altered IgG responses.

    PubMed Central

    Spriggs, M K; Koller, B H; Sato, T; Morrissey, P J; Fanslow, W C; Smithies, O; Voice, R F; Widmer, M B; Maliszewski, C R

    1992-01-01

    Transgenic mice lacking an intact beta 2-microglobulin (beta 2m) gene fail to express major histocompatibility complex (MHC) class I proteins on the cell surface and, as a result, are virtually devoid of CD4- CD8+ lymphocytes. These animals provide a unique model system for directly assessing the role of CD8+ lymphocytes in the modulation of viral infection in vivo. beta 2m- CD8- mice and their normal littermates were inoculated at the base of the tail with the WR strain of vaccinia virus and monitored for serum antibody and lesion formation. Both groups developed similar lesions in response to a broad virus dose range, and all animals had completely recovered by day 28 after inoculation. Isotype-specific immunoglobulin levels were determined for each animal on day 7 and day 14 after primary inoculation, and again 7 days after a virus challenge. The virus-specific IgG1, IgG2a, and IgG2b levels were significantly different in the beta 2m-/- group (20-, 9-, and 30-fold lower, respectively, on day 7 after challenge) compared with the beta 2m+/- group. Virus-specific serum IgM levels for both groups remained similar throughout the experiment. In a separate experiment, beta 2m-/- mice were immunized with a nonviral antigen, 2,4,6-trinitrophenyl-conjugated keyhole limpet hemocyanin, and both total and antigen-specific isotype-specific immunoglobulin titers were determined. Total IgG1, IgG2a, IgG2b, and IgG3 tended to be lower overall in the beta 2m-/- mice compared with beta 2m+/- littermates. In contrast, total and antigen-specific IgE titers were similar in the two groups. These data indicate that CD8+ lymphocytes are not required to clear high doses of vaccinia virus, and they suggest that beta 2m-/- mice are less efficient at antigen-specific IgG production than their beta 2m+/- littermates. PMID:1631092

  14. The beta2-microglobulin mRNA in human Daudi cells has a mutated initiation codon but is still inducible by interferon.

    PubMed Central

    Rosa, F; Berissi, H; Weissenbach, J; Maroteaux, L; Fellous, M; Revel, M

    1983-01-01

    The human Burkitt lymphoma cell line Daudi does not synthesize beta2-microglobulin (beta2m) and lacks the cell surface histocompatibility antigens. The cells, however, contain RNA hybridizing to a cloned human beta2m cDNA probe. cDNA from this Daudi beta2m RNA, was cloned and sequenced. By comparison with cDNA prepared from Ramos cells, which synthesized microglobulin, we determined the sequence of the 20 amino acid long leader peptide of pre-beta2m and show that in Daudi cells the initiator ATG has been mutated to ATC. Although Daudi beta2m RNA cannot be translated, interferon induces the beta2m RNA in Daudi cells as well as in normal human cells. Images Fig. 1. PMID:11894933

  15. C-reactive protein, Neopterin and Beta2 microglobulin levels pre and post TB treatment in The Gambia.

    PubMed

    Mendy, Joseph; Togun, Toyin; Owolabi, Olumuyiwa; Donkor, Simon; Ota, Martin O C; Sutherland, Jayne S

    2016-03-08

    Tuberculosis is one of the leading causes of morbidity and mortality in developing countries. Analysis of the host immune response may help with generating point-of-care tests for personalised monitoring. Thus, the aim of this study was to assess the relationship between immune activation markers: C-reactive protein (CRP), Beta2 microglobulin (B2M) and Neopterin, disease severity prior to treatment and response to therapy in adult pulmonary TB patients. HIV negative adult pulmonary TB index cases (n = 91) were recruited from the TB clinic at MRC, The Gambia. Plasma samples were collected at enrolment and at 2 and 6 months following TB treatment initiation. An enzyme linked immunosorbent assay (ELISA) was performed for evaluation of CRP, B2M and Neopterin levels and correlated with clinical and microbiological parameters including strain of infection. Disease severity was determined using Chest X-ray (CXR), Body Mass Index (BMI) and sputum smear grade. Plasma levels of all three markers were highly elevated in patients at recruitment and declined significantly during TB therapy. No correlation with disease severity was seen at recruitment. CRP showed the most significant decrease by 2 months of treatment (p < 0.0001) whereas levels of B2M and Neopterin showed little change by 2 months but a significant decrease by 6 months of treatment (p = 0.0002 and p < 0.0001 respectively). At recruitment, B2M levels were significantly higher in subjects infected with Mycobacterium africanum (Maf) compared with those infected with Mycobacterium tuberculosis sensu stricto (Mtb) (p = 0.0075). In addition, while CRP and Neopterin showed a highly significant decline post-treatment regardless of strain (p < 0.0001 for all), B2M showed differential decline depending on strain (p = 0.0153 for Mtb and p = 0.0048 for Maf) and levels were still significantly higher at 6 months in Maf compared to Mtb infected subjects (p = 0.0051). Our findings suggest that activation markers, particularly

  16. Evaluation of salivary beta-2 microglobulin as HBV proliferation marker in HBS Ag+, HBV DNA PCR+ and HBV DNA PCR− subjects

    PubMed Central

    Abdolsamadi, Hamidreza; Eini, Peiman; Kaboli, Seyed Alireza; Hajilooei, Mehrdad; MoghimBeigi, Abbas; Davoudi, Poorandokht; AhmadiMotemayel, Fatemeh; Shalmani, Hamid Mohaghegh

    2013-01-01

    Aim The aim of this study was to determine the concentration of salivary B2M as a marker of viral proliferation in HBS Ag+, HBV DNA PCR+ and Hbs Ag+ and HBV DNA PCR− subjects. Background Beta-2 microglobulin (B2M) is responsible for transmission of viral antigens such as Hepatitis B (HBV) on the surface of liver cells as part of an HLA complex. Patients and methods In this case control study, 25 PCR+ and 2 PCR− patients were included. 5 mL of the saliva sample was obtained from all patients and salivary B2M level was measured using nephelometer. The data was evaluated by the descriptive, chi square and t tests. Results 72% of the PCR+ patients received medications and in contrast, 85.7% of the patients with PCR− did not take any medication (P < 0.001). The average salivary concentration ofBeta-2 microglobulin in the PCR+ group (5.28 ± 5.45 mg/deciliter) was more than PCR− group (1.51±0.77) and this difference was statistically significant (P = 0.003). Conclusion The salivary B2Mlevel can be used as a marker of viral proliferation in patients with hepatitis B. PMID:24834278

  17. The adjusted International Prognostic Index and beta-2-microglobulin predict the outcome after autologous stem cell transplantation in relapsing/refractory peripheral T-cell lymphoma.

    PubMed

    Rodríguez, José; Conde, Eulogio; Gutiérrez, Antonio; Lahuerta, Juan José; Arranz, Reyes; Sureda, Anna; Zuazu, Javier; Fernández de Sevilla, Alberto; Bendandi, Maurizio; Solano, Carlos; León, Angel; Varela, María Rosario; Caballero, María Dolores

    2007-08-01

    Preliminary data on the use of autologous stem cell transplantation (ASCT) as a salvage therapy for peripheral T-cell lymphoma (PTCL) indicate that the results are similar to those obtained in aggressive B-cell lymphomas. The aim of our study was to analyze outcomes of a large series of patients with PTCL with a prolonged follow-up who received ASCT as salvage therapy. Between 1990 and 2004, 123 patients in this situation were registered in the GELTAMO database. The median age at transplantation was 43.5 years; in 91% of patients the disease was chemosensitive. Seventy-three percent of the patients achieved complete remission, 11% partial remission and the procedure failed in 16%. At a median follow-up of 61 months, the 5-year overall and progression-free survival rates were 45% and 34%, respectively. The presence of more than one factor of the adjusted International Prognostic Index (a-IPI) and a high beta2-microglobulin at transplantation were identified as adverse prognostic factors for both overall and progression-free survival and allowed the population to be stratified into three distinct risk groups. Our data show that approximately one third of patients with PTCL in the salvage setting may enjoy prolonged survival following ASCT, provided they are transplanted in a chemosensitive disease state. The a-IPI and beta2-microglobulin level predict the outcome after ASCT in relapsing/refractory PTCL.

  18. Separation and characterisation of beta2-microglobulin folding conformers by ion-exchange liquid chromatography and ion-exchange liquid chromatography-mass spectrometry.

    PubMed

    Bertoletti, Laura; Regazzoni, Luca; Aldini, Giancarlo; Colombo, Raffaella; Abballe, Franco; Caccialanza, Gabriele; De Lorenzi, Ersilia

    2013-04-10

    In this work we present for the first time the use of ion-exchange liquid chromatography to separate the native form and a partially structured intermediate of the folding of the amyloidogenic protein beta2-microglobulin. Using a strong anion-exchange column that accounts for the differences in charge exposure of the two conformers, a LC-UV method is initially optimised in terms of mobile phase pH, composition and temperature. The preferred mobile phase conditions that afford useful information were found to be 35 mM ammonium formate, pH 7.4 at 25°C. The dynamic equilibrium of the two species is demonstrated upon increasing the concentration of acetonitrile in the protein sample. Then, the chromatographic method is transferred to MS detection and the respective charge state distributions of the separated conformers are identified. The LC-MS results demonstrate that one of the conformers is partially unfolded, compared with the native and more compact species. The correspondence with previous results obtained in free solution by capillary electrophoresis suggest that strong ion exchange LC-MS does not alter beta2-microglobulin conformation and maintains the dynamic equilibrium already observed between the native protein and its folding intermediate.

  19. Genomic characterization and sequence diversity of the β2-microglobulin gene in the miiuy croaker, Miichthys miiuy.

    PubMed

    Sun, Y N; Su, X R; Xu, T J; Li, T W

    2015-08-28

    β2-Microglobulin (β2m) is related to major histocompatibility complex class I alpha chains, and forms cell-surface glycoproteins that mediate a variety of functions in immune defense. In general, β2m has no isoforms and is not polymorphic in higher vertebrates, but polymorphisms between different alleles have been found in some fish species. In this study, full-length β2m cDNA and genomic sequences were cloned from the miiuy croaker (Miichthys miiuy). The miiuy croaker β2m gene shares many of the same characteristics as other fish species. Three exons and two introns were identified in the miiuy croaker β2m gene; these genomic structural features are similar to those present in other fish. The deduced β2m amino acid sequence exhibited 34.7-90.1% identity with mammal and teleost β2m amino acid sequences. Sequence polymorphism analysis in six individuals identified three alleles that encoded two proteins, confirming that β2m polymorphisms exist in this species. Phylogenetic analysis elucidated the evolutionary history of the β2m protein among warm-blooded vertebrates and bony fish.

  20. Discovery and Verification of Osteopontin and Beta-2-microglobulin as Promising Markers for Staging Human African Trypanosomiasis*

    PubMed Central

    Tiberti, Natalia; Hainard, Alexandre; Lejon, Veerle; Robin, Xavier; Ngoyi, Dieudonné Mumba; Turck, Natacha; Matovu, Enock; Enyaru, John; Ndung'u, Joseph Mathu; Scherl, Alexander; Dayon, Loïc; Sanchez, Jean-Charles

    2010-01-01

    Human African trypanosomiasis, or sleeping sickness, is a parasitic disease endemic in sub-Saharan Africa, transmitted to humans through the bite of a tsetse fly. The first or hemolymphatic stage of the disease is associated with presence of parasites in the bloodstream, lymphatic system, and body tissues. If patients are left untreated, parasites cross the blood-brain barrier and invade the cerebrospinal fluid and the brain parenchyma, giving rise to the second or meningoencephalitic stage. Stage determination is a crucial step in guiding the choice of treatment, as drugs used for S2 are potentially dangerous. Current staging methods, based on counting white blood cells and demonstrating trypanosomes in cerebrospinal fluid, lack specificity and/or sensitivity. In the present study, we used several proteomic strategies to discover new markers with potential for staging human African trypanosomiasis. Cerebrospinal fluid (CSF) samples were collected from patients infected with Trypanosoma brucei gambiense in the Democratic Republic of Congo. The stage was determined following the guidelines of the national control program. The proteome of the samples was analyzed by two-dimensional gel electrophoresis (n = 9), and by sixplex tandem mass tag (TMT) isobaric labeling (n = 6) quantitative mass spectrometry. Overall, 73 proteins were overexpressed in patients presenting the second stage of the disease. Two of these, osteopontin and β-2-microglobulin, were confirmed to be potential markers for staging human African trypanosomiasis (HAT) by Western blot and ELISA. The two proteins significantly discriminated between S1 and S2 patients with high sensitivity (68% and 78%, respectively) for 100% specificity, and a combination of both improved the sensitivity to 91%. The levels of osteopontin and β-2-microglobulin in CSF of S2 patients (μg/ml range), as well as the fold increased concentration in S2 compared with S1 (3.8 and 5.5 respectively) make the two markers good

  1. [Amyloidosis associated with chronic hemodialysis. The prognostic significance of beta 2-microglobulin changes in patients with terminal chronic kidney failure treated by repeated hemodialysis].

    PubMed

    Manasia, M; Meseşan, M; Gherman-Căprioară, M; Malide, D; Paţiu, I M; Pârvu, L; Vlăduţiu, D; Spânu, C S

    1991-01-01

    In patients with chronic renal insufficiency (CRI) treated by programmed haemodialysis (HD) were detected, during the last years, amyloid stores at the level of carpal tunnel, of some joints, bones etc., finding which permitted to describe a new type of amyloid, the so-called "dialysis associated amyloid". The immunochemical structure of this amyloid is similar to that of the beta-2-microglobulin (beta-2m). Patients display various clinical manifestations. The variations of serum and urinary beta-2m were studied in 51 uraemic patients chronically dialyzed by means of dialyzers with cuprophan membrane, the average duration of the HD treatment being of 51.5 months. The pre- and postdialysis values of the beta-2-m were determined by Mancini radial immunodiffusion. A considerable increase--about 25 times--of serum beta-2-m was observed, which was more marked in anuric patients and those with a duration of more than 5 years of HD treatment. Among these, 15.7% show various articular manifestations (detected clinically and radiologically): a carpal tunnel syndrome (one patient required surgery) and arthropathies with various sites (scapulohumeral, knee). During a HD sitting with cuprophan membrane dialyzers, an increase of beta-Z-m was recorded, but it was statistically non-significant.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Urinary excretion of albumin and beta-2-microglobulin in hypertensive and normotensive renal transplant recipients during urinary diluting and concentrating tests.

    PubMed

    Jespersen, B; Pedersen, E B; Danielsen, H; Kornerup, H J; Knudsen, F; Mogensen, C E; Nielsen, A H

    1986-11-01

    Urinary excretion of albumin and beta-2-microglobulin was measured in nine hypertensive and nine normotensive renal transplant recipients and 10 healthy control subjects before and after an oral water load of 20 ml (kg body weight)-1 (study 1) and in eight hypertensive and 11 normotensive renal transplant recipients and 11 healthy control subjects during 24-h water deprivation (study 2). In both studies 1 and 2 urinary albumin excretion was significantly higher (p less than 0.01) in the hypertensive renal transplant recipients that in the normotensive patients and the control subjects (levels before loading; hypertensives: 23.9 micrograms/min (median), range 7.5-58.7; normotensives: 3.4 micrograms/min, range 1.0-49.3; controls: 2.9 micrograms/min, range 1.3-10.3). Urinary albumin excretion was significantly positive correlated to both systolic, diastolic and mean blood pressure (for mean blood pressure: rho = 0.625, n = 18, p less than 0.01) in transplanted patients. Albumin excretion tended to increase after water loading and to decrease during water deprivation in all groups. Beta-2-microglobulin excretion was approximately the same in all groups in both studies 1 and 2 and was not correlated to blood pressure. During a follow-up period of at least 18 months, none of the renal transplant recipients developed signs of chronic graft failure. Increased urinary albumin excretion in hypertensive renal transplant recipients thus appears to be caused by increased glomerular permeability that may be due to glomerular damage induced by arterial hypertension corresponding to the findings in essential hypertension.

  3. Beta 2-microglobulin-deficient mice are protected from hypergammaglobulinemia and have defective antibody responses because of increased IgG catabolism.

    PubMed

    Christianson, G J; Brooks, W; Vekasi, S; Manolfi, E A; Niles, J; Roopenian, S L; Roths, J B; Rothlein, R; Roopenian, D C

    1997-11-15

    The goal of this study was to determine whether class I proteins play an important role in the regulation of Ig and to elucidate the mechanism(s) involved. We analyzed the phenotype imposed by a null allele of beta 2-microglobulin (beta 2m). Serum Ig levels of several mouse strains showed a beta 2m dependence that was most evident in mice genetically predisposed to develop chronic systemic lupus erythematosus, was preferential to IgG isotypes, and was greatly exaggerated in aging mice that normally develop hypergammaglobulinemia. Beta 2m-deficient mice, regardless of genetic background, also displayed a substantial reduction of specific Ab in response to a prototypic T cell-dependent Ag and a prototypic T cell-independent 2 Ag. This reduction could be accounted for by a selective diminution of Abs of the IgG class. Therefore, class I proteins play a considerable role in the regulation of Ig. The beta 2m dependence could not be explained by class I-dependent immunoregulatory cells (CD8+ cells, NK1.1+ T cells, or conventional NK+ cells) or by the transfer of maternal IgG into the prenatal/neonatal mouse made possible by the beta 2m-dependent Fc receptor (FcRn). However, a beta 2m-dependent increase in the half-lives of IgG, presumably conferred by lifelong FcRn expression, was observed in all mice regardless of genetic background and age. We conclude that FcRn-mediated protection of IgG from catabolism is a generic mechanism that best explains the lifelong beta 2m dependence of Ig in both normal and pathologic situations.

  4. The heavy chain of neonatal Fc receptor for IgG is sequestered in endoplasmic reticulum by forming oligomers in the absence of beta2-microglobulin association.

    PubMed Central

    Zhu, Xiaoping; Peng, Junmin; Raychowdhury, Raktima; Nakajima, Atsushi; Lencer, Wayne I; Blumberg, Richard S

    2002-01-01

    The heavy chain (HC) of the neonatal Fc receptor (FcRn) for IgG is non-convalently associated with beta(2)-microglobulin (beta(2)m). In beta(2)m(-/-) mice, FcRn functions are greatly impaired. We sought to determine how FcRn HC, particularly its structure and biogenesis, is affected by the absence of beta(2)m. Human FcRn HC, expressed from the beta(2)m-null cell line FO-1(FcRn), was present as a monomeric 45-kDa protein under reducing conditions but primarily as a 92-kDa oligomeric protein under non-reducing conditions. Two-dimensional electrophoresis and MS analysis showed that the 92-kDa protein was a dimer of the 45-kDa HC. Immunostaining showed that FcRn HC in FO-1(FcRn) was co-localized with the endoplasmic reticulum (ER) protein Bip/GRP78 but not with an endosome protein, EEA1. In contrast, FcRn HC in FO-1(FcRn+beta2m) was detected in both the ER and endosome. The dimeric HC in FcRn oligomers was free of beta(2)m association in FO-1(FcRn+beta2m). Mutation of non-paired cysteine residues at positions 48 and 251 within the human FcRn cDNA failed to eliminate the oligomers. The FcRn HC oligomers could be reduced by reconstitution of FO-1(FcRn) with beta(2)m or by balanced expression of FcRn HC with beta(2)m, or beta(2)m fused with a KDEL retention sequence. Similarly, the majority of FcRn HC isolated from neonatal beta(2)m(-/-) mice was in a dimeric form under non-reducing conditions. The amount of FcRn HC was significantly decreased in beta(2)m(-/-) mice and FO-1(FcRn). Furthermore, beta(2)m-free FcRn HC was sensitive to endoglycosidase digestion. These results indicate that FcRn HC alone can form disulphide-bonded oligomers in the ER, which may represent a misfolded protein. The beta(2)m association with FcRn HC is critical for correct folding of FcRn and exiting the ER for routing to endosomes and the cell surface. PMID:12162790

  5. Relationship between effluent levels of beta(2)-microglobulin and peritoneal injury markers in 7.5% icodextrin-based peritoneal dialysis solution.

    PubMed

    Minami, Satoshi; Hora, Kazuhiko; Kamijo, Yuji; Higuchi, Makoto

    2007-08-01

    The removal of low molecular weight proteins such as beta(2)-microglobulin (beta(2)MG) is accelerated by using a 7.5% icodextrin-based peritoneal dialysis solution (ICO) dwell. To examine the possibility of peritoneal injury in ICO, we investigated the relationship between beta(2)MG and the injury markers in effluent. Sixteen ICO-treated patients (11 male and five female, mean age 50.1 +/- 10.9 years) with continuous ambulatory peritoneal dialysis (CAPD; mean duration 54.6 +/- 30.8 months) were studied. The patients were treated with ICO 2 L and 2.27% glucose-based solution 2 L for an 8-h dwell and the effluent was collected. We investigated the correlations between beta(2)MG and the injury markers (e.g. hyaluronic acid [HA], interleukin-6 [IL-6], matrix metalloproteinase-2 [MMP-2]) in each effluent sample. The beta(2)MG level in the ICO effluent was 8978 +/- 2431 microg/L, significantly higher than in the 2.27% glucose-based solution effluent (6454 +/- 2956 microg/L; P = 0.0032). The levels of HA and MMP-2 in ICO effluent were significantly higher than those in the 2.27% glucose-based solution effluent (P = 0.00214, P = 0.0113, respectively). There was a trend toward higher IL-6-values in ICO effluent, although no significant differences were seen. There were positive correlations between levels of various injury markers and beta(2)MG. We propose that the subclinical injury of the peritoneum by ICO treatment may accelerate peritoneal permeability to increase beta(2)MG in effluent. ICO's biocompatibility might not be superior to that of glucose-based solution.

  6. Beta 2-microglobulin-dependent NK1.1+ T cells are not essential for T helper cell 2 immune responses

    PubMed Central

    1996-01-01

    A number of investigations have established the critical role of interleukin 4 (IL-4) in mediating the development of T helper (Th)2 effector cells in vitro and in vivo. Despite intensive study, the origin of the IL-4 required for Th2 priming and differentiation remains unclear. Natural killer (NK)1.1+ alpha/beta T cell receptor+ T(NT) cells, a unique lineage of cells capable of producing large amounts of IL-4 after activation in vivo, are important candidates for directing Th2 priming. These cells are selected by the nonpolymorphic major histocompatibility complex (MHC) class I molecule, CD1, and are deficient in beta 2-microglobulin (beta 2m)-null mice. We used beta 2m- deficient mice on both BALB/c and C57BL/6 backgrounds to examine their capacity to mount Th2 immune responses after challenge with a number of well-characterized antigens administered by a variety of routes. As assessed by immunization with protein antigen, infection with Leishmania major, embolization with eggs of Schistosoma mansoni, intestinal infection with Nippostrongylus brasiliensis, or induction of airway hyperreactivity to aerosolized antigen, beta 2m-deficient mice developed functional type 2 immune responses that were not substantially different than those in wild-type mice. Production of IL- 4 and the generation of immunoglobulin E (IgE) and eosinophil responses were preserved as assessed by a variety of assays. Collectively, these results present a comprehensive analysis of type 2 immune responses in beta 2m-deficient mice, and indicate that beta 2m-dependent NT cells are not required for Th2 development in vivo. PMID:8879201

  7. Beta2-microglobulin is a better predictor of treatment-free survival in patients with chronic lymphocytic leukaemia if adjusted according to glomerular filtration rate.

    PubMed

    Delgado, Julio; Pratt, Guy; Phillips, Neil; Briones, Javier; Fegan, Chris; Nomdedeu, Josep; Pepper, Chris; Aventin, Anna; Ayats, Ramon; Brunet, Salut; Martino, Rodrigo; Valcarcel, David; Milligan, Donald; Sierra, Jorge

    2009-06-01

    Even in the era of newer and sophisticated prognostic markers, beta(2)-microglobulin (B2M) remains a simple but very powerful predictor of treatment-free survival (TFS) and overall survival (OS) in patients with chronic lymphocytic leukaemia (CLL). However, B2M levels are heavily influenced by the patient's glomerular filtration rate (GFR) and this study aimed to evaluate whether GFR-adjusted B2M (GFR-B2M) had improved prognostic value compared to unadjusted B2M in a cohort of over 450 consecutive CLL patients from two separate institutions. Multivariate analysis identified a significantly shorter TFS in patients who were ZAP-70 + (P < 0.001), with increased GFR-B2M (P < 0.001), and del(11q) or del(17p) as detected by fluorescence in situ hybridization (FISH; P < 0.001). When OS was evaluated by multivariate analysis, age 65 years or older (P < 0.001) and poor risk FISH abnormalities (P < 0.001) had a confirmed adverse prognostic impact, but the predictive value of GFR-B2M was lost in the validation analysis. In all survival models, B2M did not attain independent significance unless GFR-B2M was eliminated from the analysis. In conclusion, GFR-B2M is a better predictor of TFS than unadjusted B2M in CLL patients.

  8. Determination of beta-2 microglobulin levels in plasma using a high-throughput mass spectrometric immunoassay system.

    PubMed

    Niederkofler, E E; Tubbs, K A; Gruber, K; Nedelkov, D; Kiernan, U A; Williams, P; Nelson, R W

    2001-07-15

    A high-throughput mass spectrometric immunoassay (MSIA) system for the analysis of proteins directly from biological fluids is reported. A 96-well-format robotic workstation equipped with antibody-derivatized affinity pipet tips was used for the parallel extraction of specific proteins from samples and subsequent deposition onto 96-well arrayed matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) targets. Interferences from nonspecifically bound proteins were minimized through choice of appropriate affinity pipet tip derivatization chemistries. Sample preparation for MALDI-TOFMS was enhanced through the use of hydrophobic/hydrophilic contrasting targets, which also presented functionalities found to promote matrix/analyte crystal growth. Automated mass spectrometry was used in the unattended acquisition of data, resulting in an analysis rate of approximately 100 samples/h (biological fluid-->data). The quantitative MSIA of beta2m levels present in human plasma samples is given as illustration.

  9. Total loss of MHC class I in colorectal tumors can be explained by two molecular pathways: beta2-microglobulin inactivation in MSI-positive tumors and LMP7/TAP2 downregulation in MSI-negative tumors.

    PubMed

    Cabrera, C M; Jiménez, P; Cabrera, T; Esparza, C; Ruiz-Cabello, F; Garrido, F

    2003-03-01

    The mechanisms that lead to loss of MHC class I expression in different types of tumors are not yet fully known. Accordingly, we studied colorectal carcinomas to elucidate the specific mechanisms of evasion of the T-cell immune response. We selected tumors with total loss of MHC class I expression and studied 124 colorectal carcinomas with immunohistochemical staining and anti-HLA monoclonal antibodies (mAb). Fourteen of 124 (11%) tumors exhibited a phenotype with HLA class I total loss. Microsatellite instability (MSI) analysis was also carried out in the same tumor samples. The expression of beta2-microglobulin (beta2m), HLA-A, B, and C antigens, transporter associated with antigen processing 1 (TAP1), TAP2, low-molecular-weight protein 2 (LMP2), and LMP7 were analyzed using reverse-transcription polymerase chain reaction (RT-PCR) in microdissected tumor samples. Four of 14 microsatellite instability-positive (MSI+) and W6/32 mAb-negative tumors showed biallelic inactivation of beta2m and accumulation of HLA class I heavy chain in the cytoplasm. MSI-negative (MSI-)/W6/32 mAb-negative tumors presented alterations in the expression of components of the antigen processing machinery (APM). Nine of 10 tumor samples showed LMP7 gene downregulation, and four of 10 presented TAP2 dysregulation. This group apparently expressed normal levels of heavy chain and beta2m mRNA. Two major mechanisms in colorectal cancer appear to be responsible for the total loss of MHC surface expression (beta2m mutations and LMP7/TAP2 downregulation) that may contribute to the failure of T lymphocyte recognition during an immune response. The precise identification of the molecular defects that underlie HLA class I abnormalities will have important implications for patients receiving T-cell-based specific immunotherapy.

  10. The ESAT-6 protein of Mycobacterium tuberculosis interacts with beta-2-microglobulin (β2M) affecting antigen presentation function of macrophage.

    PubMed

    Sreejit, Gopalkrishna; Ahmed, Asma; Parveen, Nazia; Jha, Vishwanath; Valluri, Vijaya Lakshmi; Ghosh, Sudip; Mukhopadhyay, Sangita

    2014-10-01

    ESAT-6, an abundantly secreted protein of Mycobacterium tuberculosis (M. tuberculosis) is an important virulence factor, inactivation of which leads to reduced virulence of M. tuberculosis. ESAT-6 alone, or in complex with its chaperone CFP-10 (ESAT-6:CFP-10), is known to modulate host immune responses; however, the detailed mechanisms are not well understood. The structure of ESAT-6 or ESAT-6:CFP-10 complex does not suggest presence of enzymatic or DNA-binding activities. Therefore, we hypothesized that the crucial role played by ESAT-6 in the virulence of mycobacteria could be due to its interaction with some host cellular factors. Using a yeast two-hybrid screening, we identified that ESAT-6 interacts with the host protein beta-2-microglobulin (β2M), which was further confirmed by other assays, like GST pull down, co-immunoprecipitation and surface plasmon resonance. The C-terminal six amino acid residues (90-95) of ESAT-6 were found to be essential for this interaction. ESAT-6, in complex with CFP-10, also interacts with β2M. We found that ESAT-6/ESAT-6:CFP-10 can enter into the endoplasmic reticulum where it sequesters β2M to inhibit cell surface expression of MHC-I-β2M complexes, resulting in downregulation of class I-mediated antigen presentation. Interestingly, the ESAT-6:β2M complex could be detected in pleural biopsies of individuals suffering from pleural tuberculosis. Our data highlight a novel mechanism by which M. tuberculosis may undermine the host adaptive immune responses to establish a successful infection. Identification of such novel interactions may help us in designing small molecule inhibitors as well as effective vaccine design against tuberculosis.

  11. The ESAT-6 Protein of Mycobacterium tuberculosis Interacts with Beta-2-Microglobulin (β2M) Affecting Antigen Presentation Function of Macrophage

    PubMed Central

    Parveen, Nazia; Jha, Vishwanath; Valluri, Vijaya Lakshmi; Ghosh, Sudip; Mukhopadhyay, Sangita

    2014-01-01

    ESAT-6, an abundantly secreted protein of Mycobacterium tuberculosis (M. tuberculosis) is an important virulence factor, inactivation of which leads to reduced virulence of M. tuberculosis. ESAT-6 alone, or in complex with its chaperone CFP-10 (ESAT-6:CFP-10), is known to modulate host immune responses; however, the detailed mechanisms are not well understood. The structure of ESAT-6 or ESAT-6:CFP-10 complex does not suggest presence of enzymatic or DNA-binding activities. Therefore, we hypothesized that the crucial role played by ESAT-6 in the virulence of mycobacteria could be due to its interaction with some host cellular factors. Using a yeast two-hybrid screening, we identified that ESAT-6 interacts with the host protein beta-2-microglobulin (β2M), which was further confirmed by other assays, like GST pull down, co-immunoprecipitation and surface plasmon resonance. The C-terminal six amino acid residues (90–95) of ESAT-6 were found to be essential for this interaction. ESAT-6, in complex with CFP-10, also interacts with β2M. We found that ESAT-6/ESAT-6:CFP-10 can enter into the endoplasmic reticulum where it sequesters β2M to inhibit cell surface expression of MHC-I-β2M complexes, resulting in downregulation of class I-mediated antigen presentation. Interestingly, the ESAT-6:β2M complex could be detected in pleural biopsies of individuals suffering from pleural tuberculosis. Our data highlight a novel mechanism by which M. tuberculosis may undermine the host adaptive immune responses to establish a successful infection. Identification of such novel interactions may help us in designing small molecule inhibitors as well as effective vaccine design against tuberculosis. PMID:25356553

  12. Beta-2 Microglobulin Tumor Marker

    MedlinePlus

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  13. Revisiting the Middle Molecule Hypothesis of Uremic Toxicity: A Systematic Review of Beta 2 Microglobulin Population Kinetics and Large Scale Modeling of Hemodialysis Trials In Silico

    PubMed Central

    Roumelioti, Maria Eleni; Nolin, Thomas; Unruh, Mark L.; Argyropoulos, Christos

    2016-01-01

    Background Beta-2 Microglobulin (β2M) is a prototypical “middle molecule” uremic toxin that has been associated with a higher risk of death in hemodialysis patients. A quantitative description of the relative importance of factors determining β2M concentrations among patients with impaired kidney function is currently lacking. Methods Herein we undertook a systematic review of existing studies reporting patient level data concerning generation, elimination and distribution of β2M in order to develop a population model of β2M kinetics. We used this model and previously determined relationships between predialysis β2M concentration and survival, to simulate the population distribution of predialysis β2M and the associated relative risk (RR) of death in patients receiving conventional thrice-weekly hemodialysis with low flux (LF) and high flux (HF) dialyzers, short (SD) and long daily (LD) HF hemodialysis sessions and on-line hemodiafiltration at different levels of residual renal function (RRF). Results We identified 9 studies of 106 individuals and 156 evaluations of or more compartmental kinetic parameters of β2M. These studies used a variety of experimental methods to determine β2M kinetics ranging from isotopic dilution to profiling of intra/inter dialytic concentration changes. Most of the patients (74/106) were on dialysis with minimal RRF, thus facilitating the estimation of non-renal elimination kinetics of β2M. In large scale (N = 10000) simulations of individuals drawn from the population of β2M kinetic parameters, we found that, higher dialytic removal materially affects β2M exposures only when RRF (renal clearance of β2M) was below 2 ml/min. In patients initiating conventional HF hemodialysis, total loss of RRF was predicted to be associated with a RR of death of more than 20%. Hemodiafiltration and daily dialysis may decrease the high risk of death of anuric patients by 10% relative to conventional, thrice weekly HF dialysis. Only daily

  14. Revisiting the Middle Molecule Hypothesis of Uremic Toxicity: A Systematic Review of Beta 2 Microglobulin Population Kinetics and Large Scale Modeling of Hemodialysis Trials In Silico.

    PubMed

    Roumelioti, Maria Eleni; Nolin, Thomas; Unruh, Mark L; Argyropoulos, Christos

    2016-01-01

    Beta-2 Microglobulin (β2M) is a prototypical "middle molecule" uremic toxin that has been associated with a higher risk of death in hemodialysis patients. A quantitative description of the relative importance of factors determining β2M concentrations among patients with impaired kidney function is currently lacking. Herein we undertook a systematic review of existing studies reporting patient level data concerning generation, elimination and distribution of β2M in order to develop a population model of β2M kinetics. We used this model and previously determined relationships between predialysis β2M concentration and survival, to simulate the population distribution of predialysis β2M and the associated relative risk (RR) of death in patients receiving conventional thrice-weekly hemodialysis with low flux (LF) and high flux (HF) dialyzers, short (SD) and long daily (LD) HF hemodialysis sessions and on-line hemodiafiltration at different levels of residual renal function (RRF). We identified 9 studies of 106 individuals and 156 evaluations of or more compartmental kinetic parameters of β2M. These studies used a variety of experimental methods to determine β2M kinetics ranging from isotopic dilution to profiling of intra/inter dialytic concentration changes. Most of the patients (74/106) were on dialysis with minimal RRF, thus facilitating the estimation of non-renal elimination kinetics of β2M. In large scale (N = 10,000) simulations of individuals drawn from the population of β2M kinetic parameters, we found that, higher dialytic removal materially affects β2M exposures only when RRF (renal clearance of β2M) was below 2 ml/min. In patients initiating conventional HF hemodialysis, total loss of RRF was predicted to be associated with a RR of death of more than 20%. Hemodiafiltration and daily dialysis may decrease the high risk of death of anuric patients by 10% relative to conventional, thrice weekly HF dialysis. Only daily long sessions of hemodialysis

  15. Estimation of benchmark dose as the threshold levels of urinary cadmium, based on excretion of total protein, {beta} {sub 2}-microglobulin, and N-acetyl-{beta}-D-glucosaminidase in cadmium nonpolluted regions in Japan

    SciTech Connect

    Kobayashi, Etsuko . E-mail: ekoba@faculty.chiba-u.jp; Suwazono, Yasushi; Uetani, Mirei; Inaba, Takeya; Oishi, Mitsuhiro; Kido, Teruhiko; Nishijo, Muneko; Nakagawa, Hideaki; Nogawa, Koji

    2006-07-15

    Previously, we investigated the association between urinary cadmium (Cd) concentration and indicators of renal dysfunction, including total protein, {beta} {sub 2}-microglobulin ({beta} {sub 2}-MG), and N-acetyl-{beta}-D-glucosaminidase (NAG). In 2778 inhabitants {>=}50 years of age (1114 men, 1664 women) in three different Cd nonpolluted areas in Japan, we showed that a dose-response relationship existed between renal effects and Cd exposure in the general environment without any known Cd pollution. However, we could not estimate the threshold levels of urinary Cd at that time. In the present study, we estimated the threshold levels of urinary Cd as the benchmark dose low (BMDL) using the benchmark dose (BMD) approach. Urinary Cd excretion was divided into 10 categories, and an abnormality rate was calculated for each. Cut-off values for urinary substances were defined as corresponding to the 84% and 95% upper limit values of the target population who have not smoked. Then we calculated the BMD and BMDL using a log-logistic model. The values of BMD and BMDL for all urinary substances could be calculated. The BMDL for the 84% cut-off value of {beta} {sub 2}-MG, setting an abnormal value at 5%, was 2.4 {mu}g/g creatinine (cr) in men and 3.3 {mu}g/g cr in women. In conclusion, the present study demonstrated that the threshold level of urinary Cd could be estimated in people living in the general environment without any known Cd-pollution in Japan, and the value was inferred to be almost the same as that in Belgium, Sweden, and China.

  16. Induction of cross clade reactive specific antibodies in mice by conjugates of HGP-30 (peptide analog of HIV-1(SF2) p17) and peptide segments of human beta-2-microglobulin or MHC II beta chain.

    PubMed

    Zimmerman, D H; Lloyd, J P; Heisey, D; Winship, M D; Siwek, M; Talor, E; Sarin, P S

    2001-09-14

    HGP-30, a 30 amino acid synthetic peptide homologous to a conserved region of HIV-1(SF2) p17 (aa86-115), has previously been shown to elicit both cellular and humoral immune responses when conjugated to KLH and adsorbed to alum. However, the free HGP-30 peptide is not immunogenic in animals. In order to improve the immunogenicity of HGP-30, peptide conjugates consisting of a modified HGP-30 sequence (m-HGP-30/aa82-111) and a peptide segment, residues 38-50, of the MHC I accessory molecule, human beta-2-microglobulin (beta-2-M), referred to as Peptide J, or a peptide from the MHC II beta chain (peptide G) were evaluated in mice. The effects of carriers and adjuvants on serum antibody titers, specificities to various HIV-1 clade peptides similar to HGP-30 and isotype patterns were examined. Peptides J or especially G conjugated to modified-HGP-30 (LEAPS 102 and LEAPS 101, respectively) generated comparable or better immune responses to modified HGP-30 than KLH conjugates as judged by the induction of: (1) similar antibody titers; (2) broader HIV clade antigen binding; and (3) antibody isotype response patterns indicative of a TH1 pathway (i.e. increased amounts of IgG2a and IgG2b antibodies). The ISA 51 and MPL(R)-SE adjuvants induced higher antibody responses than alum, with the ISA 51 being more potent. Immune responses to LEAPS 102, as compared to LEAPS 101, were weaker and slower to develop as determined by antibody titers and cross clade reactivity of the antibodies induced. Compared to KLH conjugates which induced significant anti-KLH antibody titers, minimal antibody responses were observed to peptide G, the more immunogenic conjugate, and peptide J. These results suggest that modified HGP-30 L.E.A.P.S. constructs may be useful as HIV vaccine candidates for preferential induction of TH1 directed cell mediated immune responses.

  17. Structural Analysis of H2-Db Class I Molecules Containing Two Different Allelic Forms of the type 1 Diabetes Susceptibility Factor beta-2 Microglobulin: Implications for the Mechanism Underlying Viriations in Antigen Presentation

    SciTech Connect

    Roden,M.; Brims, D.; Fedorov, A.; DiLorenzo, T.; Almo, S.; Nathenson, s.; Anovitz, L.; Wesolowski, D.

    2006-01-01

    Beta-2 microglobulin ({beta}2m) is a member of the immunoglobulin-like domain superfamily that is an essential structural subunit of the MHC class I (MHC-I) molecule. {beta}2m was previously identified as a susceptibility factor for the development of type 1 diabetes (T1D) in NOD mice, whereby transgenic expression of the {beta}2m{sup a} variant, but not the {beta}2mb variant, restored diabetes susceptibility to normally resistant NOD.{beta}2m{sup null} mice. Here we report the crystal structures and thermodynamic stabilities of the NOD MHC-I molecule H2-D{sup b} containing these two variants. Our results reveal subtle differences in the structures of the {beta}2m variants, namely in minor loop shifts and in variations in the hydrogen bonding networks at the interfaces between the components of the ternary complex. We also demonstrate that the thermodynamic stabilities of the {beta}2m variants in isolation differ. However, the conformation of the peptide in the MHC cleft is unchanged in {beta}2m allelic Db complexes, as are the TCR recognition surfaces. Thus, despite modest structural differences between allelic complexes, the evidence indicates that D{sup b} peptide presentation of the representative peptide is unchanged in the context of either {beta}2m allelic variant. These data suggest that other mechanisms, such as differential association of MHC-I in multiprotein complexes, are likely responsible for the effect of {beta}2m on T1D development.

  18. The relationship between concentrations of magnesium and oxidized low-density lipoprotein and Beta2-microglobulin in the serum of patients on the end-stage of renal disease.

    PubMed

    Raikou, Vaia D; Kyriaki, Despina

    2016-05-01

    The end-stage of renal disease is associated with increased oxidative stress and oxidative modification of low-density lipoproteins (LDLs). Beta2 microglobulin (beta2M) is accumulated in the serum of dialysis patients. Magnesium (Mg) plays a protective role in the development of oxidative stress in healthy subjects. We studied the relationship between concentrations of magnesium and oxidized LDL (ox-LDL) and beta2M in the serum of patients on the end stage of renal disease. In 96 patients on on-line- predilution hemodiafiltration, beta2M and intact parathormone were measured by radioimmunoassays. High-sensitivity C-reactive protein (hsCRP) and ox-LDL were measured using ΕLISA. Serum bicarbonate levels were measured in the blood gas analyser gas machine. We performed logistic regression analysis models to investigate Mg as an important independent predictor of elevated ox-LDL and high beta2M serum concentrations, after adjustment to traditional and specific for dialysis patients' factors. We observed a positive correlation of Mg with ox-LDL (r = 0.383, P = 0.001), but the association of Mg with beta2M, hsCRP, and serum bicarbonate levels was significantly inverse (r = -0.252, P = 0.01, r = -0.292, P = 0.004, and r = -0.282, P = 0.04 respectively). The built logistic-regression analysis showed that Mg act as a significant independent factor for the elevated ox-LDL and beta2M serum concentrations adjusting to traditional and specific factors for these patients. We observed a positive relationship between magnesium and acidosis status- related ox-LDL concentrations, but the inverse association between magnesium and beta2M serum concentrations in hemodialysis patients.

  19. Calcium Binding to Beta-2-Microglobulin at Physiological Ph Drives the Occurrence of Conformational Changes Which Cause the Protein to Precipitate into Amorphous Forms That Subsequently Transform into Amyloid Aggregates

    PubMed Central

    Kumar, Sukhdeep; Sharma, Prerna; Arora, Kanika; Raje, Manoj; Guptasarma, Purnananda

    2014-01-01

    Using spectroscopic, calorimetric and microscopic methods, we demonstrate that calcium binds to beta-2-microglobulin (β2m) under physiological conditions of pH and ionic strength, in biological buffers, causing a conformational change associated with the binding of up to four calcium atoms per β2m molecule, with a marked transformation of some random coil structure into beta sheet structure, and culminating in the aggregation of the protein at physiological (serum) concentrations of calcium and β2m. We draw attention to the fact that the sequence of β2m contains several potential calcium-binding motifs of the DXD and DXDXD (or DXEXD) varieties. We establish (a) that the microscopic aggregation seen at physiological concentrations of β2m and calcium turns into actual turbidity and visible precipitation at higher concentrations of protein and β2m, (b) that this initial aggregation/precipitation leads to the formation of amorphous aggregates, (c) that the formation of the amorphous aggregates can be partially reversed through the addition of the divalent ion chelating agent, EDTA, and (d) that upon incubation for a few weeks, the amorphous aggregates appear to support the formation of amyloid aggregates that bind to the dye, thioflavin T (ThT), resulting in increase in the dye's fluorescence. We speculate that β2m exists in the form of microscopic aggregates in vivo and that these don't progress to form larger amyloid aggregates because protein concentrations remain low under normal conditions of kidney function and β2m degradation. However, when kidney function is compromised and especially when dialysis is performed, β2m concentrations probably transiently rise to yield large aggregates that deposit in bone joints and transform into amyloids during dialysis related amyloidosis. PMID:24755626

  20. Calcium binding to beta-2-microglobulin at physiological pH drives the occurrence of conformational changes which cause the protein to precipitate into amorphous forms that subsequently transform into amyloid aggregates.

    PubMed

    Kumar, Sukhdeep; Sharma, Prerna; Arora, Kanika; Raje, Manoj; Guptasarma, Purnananda

    2014-01-01

    Using spectroscopic, calorimetric and microscopic methods, we demonstrate that calcium binds to beta-2-microglobulin (β2m) under physiological conditions of pH and ionic strength, in biological buffers, causing a conformational change associated with the binding of up to four calcium atoms per β2m molecule, with a marked transformation of some random coil structure into beta sheet structure, and culminating in the aggregation of the protein at physiological (serum) concentrations of calcium and β2m. We draw attention to the fact that the sequence of β2m contains several potential calcium-binding motifs of the DXD and DXDXD (or DXEXD) varieties. We establish (a) that the microscopic aggregation seen at physiological concentrations of β2m and calcium turns into actual turbidity and visible precipitation at higher concentrations of protein and β2m, (b) that this initial aggregation/precipitation leads to the formation of amorphous aggregates, (c) that the formation of the amorphous aggregates can be partially reversed through the addition of the divalent ion chelating agent, EDTA, and (d) that upon incubation for a few weeks, the amorphous aggregates appear to support the formation of amyloid aggregates that bind to the dye, thioflavin T (ThT), resulting in increase in the dye's fluorescence. We speculate that β2m exists in the form of microscopic aggregates in vivo and that these don't progress to form larger amyloid aggregates because protein concentrations remain low under normal conditions of kidney function and β2m degradation. However, when kidney function is compromised and especially when dialysis is performed, β2m concentrations probably transiently rise to yield large aggregates that deposit in bone joints and transform into amyloids during dialysis related amyloidosis.

  1. Urinary {alpha}{sub 1}-microglobulin, {beta}{sub 2}-microglobulin, and retinol-binding protein levels in general populations in Japan with references to cadmium in urine, blood, and 24-hour food duplicates

    SciTech Connect

    Ikeda, Masayuki; Moon, Chan-Seok; Zhang, Zuo-Wen

    1995-07-01

    Possible cadmium (Cd) exposure-associated changes in urinary levels of low-molecular-weight proteins were studied in nonsmoking and nondrinking female members of the general Japanese population (378 subjects with no known occupational heavy metal exposure) who lived at 19 study sites (all without any known environmental heavy metal pollution) in 13 prefectures throughout Japan. The external Cd dose was evaluated in terms of daily Cd intake via food (Cd-F), whereas Cd levels in blood (Cd-B) and urine (Cd-U) were taken as internal dose indicators. When the subjects were classified according to Cd-F into three groups with {open_quotes}low{close_quotes} (20.4 {mu}g/day as a geometric mean of 97 women), {open_quotes}middle{close_quotes} (35.0 {mu}g/day, 120 women) and {open_quotes}high{close_quotes} (67.0 {mu}g/day, 66 women) exposure, both Cd-B and Cd-U increased in parallel with the changes in Cd-F. However, there were no dose-dependent changes in {beta}{sub 2}-microglobulin or retinol-binding protein levels in urine. {alpha}{sub 1}-Microglobulin levels appeared to increase, but the distribution of the cases above the two cutoff levels of 9.6 and 15.8 {mu}g/mg creatinine among the three Cd-F groups did not show any bias. Overall, it was concluded that there was no apparent Cd exposure-associated elevation in urinary low-molecular-weight protein levels in the study population. 41 refs., 2 figs., 7 tabs.

  2. Preparation of an epitope-imprinted polymer with antibody-like selectivity for beta2-microglobulin and application in serum sample analysis with a facile method of on-line solid-phase extraction coupling with high performance liquid chromatography.

    PubMed

    Yang, Fangfang; Deng, Dandan; Dong, Xiangchao; Lin, Shen

    2017-03-09

    Molecularly imprinted polymers (MIPs) for protein recognition have great application potential in the biological analysis. However, preparation of protein imprinted polymer is still facing challenge. Beta2-microglobulin (β2m) is a protein biomarker that can be used in diagnosis of different diseases. In this research, a novel MIP with ability of β2m recognition has been developed by epitope and surface-confined imprinting approaches. A peptide with sequence of MIQRTPKIQ was selected as template. A strategy of combination of hierarchical imprinting and template immobilization was employed in the β2m-MIP synthesis. Imprinted binding sites with open-entrance have been created that have good accessibility for β2m and facilitated fast reversible binding kinetics. The experimental results demonstrated that the MIP has good selectivity. It can differentiate the template from peptide with different sequence and distinguish the β2m from other proteins with similar size and pI values. After binding property study of the β2m-MIP, a method of β2m determination in serum was established in which β2m was on-line extracted by MIP and analyzed by HPLC process. The recoveries for spiked serum was ≥83% with RSD <1.1%, indicating that the method has good accuracy and precisions. The LOD and LOQ were 0.058 and 0.195mgL(-1) respectively, which meet the requirements of the β2m analysis. The successful application of the β2m-MIP demonstrated that β2m has reversible binding on the MIP with a kinetics that can meet the requirements of the HPLC analysis. It also indicated that the β2m-MIP has good mechanical strength and reusability that can be applied reliably in the practical analysis. As a synthetic antibody, β2m-MIP is advantageous compared to the biological molecules.

  3. Selective development of T helper (Th)2 cells induced by continuous administration of low dose soluble proteins to normal and beta(2)- microglobulin-deficient BALB/c mice

    PubMed Central

    1996-01-01

    Continuous administration of soluble proteins, delivered over a 10-d period by a mini-osmotic pump implanted subcutaneously, induces a long- lasting inhibition of antigen-specific T cell proliferation in lymph node cells from BALB/c mice subsequently primed with antigen in adjuvant. The decreased T cell proliferative response is associated with a down-regulation of the T helper cell (Th)1 cytokines interleukin (IL)-2 and interferon (IFN)-gamma and with a strong increase in the secretion of the Th2 cytokines IL-4 and IL-5 by antigen specific CD4+ T cells. This is accompanied by predominant inhibition of antigen- specific antibody production of IgG2a and IgG2b, rather than IgG1 isotype. Interestingly, inhibition of Th1 and priming of Th2 cells is also induced in beta(2) microglobulin-deficient BALB/c mice, indicating that neither CD8+ nor CD4+ NK1.1+ T cells, respectively, are required. The polarization in Th2 cells is stably maintained by T cell lines, all composed of CD4+/CD8- cells expressing T cell receptor for antigen (TCR) alpha/beta chains, derived from BALB/c mice treated with continuous antigen administration, indicating that they originate from Th2 cells fully differentiated in vivo. This polarization is induced in BALB/c mice by continuous administration of any protein antigen tested, including soluble extracts from pathogenic microorganisms. Priming of Th2 cells is dose dependent and it is optimal for low rather than high doses of protein. Blocking endogenous IL-4 in vivo inhibits expansion of antigen-specific Th2 cells, but does not restore IFN-gamma production by T cells from mice treated with soluble antigen-specific Th2 cells, but does not restore IFN-gamma production by T cells from mice treated with soluble antigen, indicating the involvement of two independent mechanisms. Consistent with this, Th2 cell development, but not inhibition of Th1 cells, depends on non-major histocompatibility complex genetic predisposition, since the Th2 response is

  4. THE RELATIONSHIP BETWEEN β2-MICROGLOBULIN AND IMMUNOGLOBULIN IN CULTURED HUMAN LYMPHOID CELL LINES

    PubMed Central

    Hütteroth, T. H.; Cleve, H.; Litwin, S. D.; Poulik, M. D.

    1973-01-01

    β2-microglobulin was detected on the cell surface and in the medium of human lymphoid cells established in long-term culture. The secretion of β2-microglobulin was relatively uniform when different cell lines were compared, whereas IgG production varied widely. κ- and µ-membrane antigens were modulated by specific antibody; β2-microglobulin was not modulated. Anti-κ and anti-µ antisera had no effect on the expression of membrane β2-microglobulin, nor had anti-β2-microglobulin antiserum any effect on the expression of κ- and µ-membrane antigens. PMID:4120289

  5. Can small hydrophobic gold nanoparticles inhibit β2-microglobulin fibrillation?

    NASA Astrophysics Data System (ADS)

    Brancolini, Giorgia; Toroz, Dimitrios; Corni, Stefano

    2014-06-01

    Inorganic nanoparticles stabilized by a shell of organic ligands can enhance or suppress the natural propensity of proteins to form fibrils. Functionalization facilitates targeted delivery of the nanoparticles to various cell types, bioimaging, drug delivery and other therapeutic and diagnostic applications. In this study, we provide a computational model of the effect of a prototypical thiol-protected gold nanoparticle, Au25L18- (L = S(CH2)2Ph) on the β2-microglobulin natural fibrillation propensity. To reveal the molecular basis of the protein-nanoparticle association process, we performed various simulations at multiple levels (Classical Molecular Dynamics and Brownian Dynamics) that cover multiple length- and timescales. The results provide a model of the ensemble of structures constituting the protein-gold nanoparticle complexes, and insights into the driving forces for the binding of β2-microglobulin to hydrophobic small size gold nanoparticles. We have found that the small nanoparticles can bind the protein to form persistent complexes. This binding of nanoparticles is able to block the active sites of domains from binding to another protein, thus leading to potential inhibition of the fibrillation activity. A comparison with the binding patches identified for the interaction of the protein with a known inhibitor of fibrillation, supports our conclusion.Inorganic nanoparticles stabilized by a shell of organic ligands can enhance or suppress the natural propensity of proteins to form fibrils. Functionalization facilitates targeted delivery of the nanoparticles to various cell types, bioimaging, drug delivery and other therapeutic and diagnostic applications. In this study, we provide a computational model of the effect of a prototypical thiol-protected gold nanoparticle, Au25L18- (L = S(CH2)2Ph) on the β2-microglobulin natural fibrillation propensity. To reveal the molecular basis of the protein-nanoparticle association process, we performed various

  6. Foetal serum but not urinary β2-microglobulin correlates with histological injury to the kidney.

    PubMed

    Luton, D; Delezoide, A L; Leguy, M C; Gobeaux, C; Vuillard, E; Grangé, G; Guibourdenche, J

    2013-10-01

    In a context of foetal obstructive uropathies, biochemical markers can be helpful to assess the renal function, but most studies to date have focused on their correlation with ultrasound findings and neonatal outcome. Our aim was to evaluate foetal β2-microglobulin as an index of histological injury to the kidney. β2-microglobulin was measured in serum and/or urine from 27 foetuses with bilateral obstructive uropathy, and compared to the findings of kidney examination following the termination of pregnancy. In serum, increased β2-microglobulin levels correlated to a decreased number of glomeruli, a reduction in the blastema and the presence of primitive ducts reflecting renal hypoplasia and dysplasia. However, elevated β2-microglobulin levels in the urine correlated only to a decreased number of glomeruli.

  7. Clinical Utility of Urinary β2-Microglobulin in Detection of Early Nephropathy in African Diabetes Mellitus Patients

    PubMed Central

    Effa, E. E.; Akpan, E. E.; Obot, A. S.; Kadiri, S.

    2017-01-01

    Background. Studies have indicated that diabetic tubulopathy may occur earlier than glomerulopathy, therefore providing a potential avenue for earlier diagnosis of diabetic nephropathy. Urinary beta-2-microglobulin (β2m) was investigated in this study as a potential biomarker in the detection of early nephropathy in type 2 diabetics. Methods. One hundred and two diabetic subjects and 103 controls that met the inclusion criteria had data (sociodemographic, medical history, physical examination, and laboratory) collected. Urinary β2m levels and urinary albumin concentration (UAC) were determined. Results. Elevated urinary β2m was more frequent among the diabetics (52%, 95% CI: 42.1–61.8%) than among the controls (32%, 95% CI: 22.9–41.2%). The frequency of microalbuminuria was higher in the diabetics (35.3%, 95% CI: 25.9–44.7%) than in the controls (15.5%, 95% CI: 8.4–22.6%). There was a positive correlation between urinary β2m and UAC (rho = 0.38, p < 0.001). Multivariate analysis showed BMI (OR: 1.23, 95% CI: 1.05–1.45), eGFR (OR: 0.97, 95% CI: 0.94–0.99), and presence of microalbuminuria (OR: 3.94, 95% CI: 1.32–11.77) as independent predictors of elevated urinary beta-2-microglobulin among the diabetics. Conclusion. Urinary β2m may be useful, either as a single test or as a component of a panel of tests, in the early detection of diabetic nephropathy. PMID:28250988

  8. Ochratoxin A and β2-microglobulin in BEN patients and controls.

    PubMed

    Yordanova, Pavlina; Wilfried, Karmaus; Tsolova, Svetla; Dimitrov, Plamen

    2010-04-01

    Ochratoxin A (OTA) is a mycotoxin naturally occurring in different foods. OTA is arguably a risk factor for Balkan endemic nephropathy (BEN). The aims of this study are to (1) test the OTA-BEN association in BEN-groups and controls and (2) determine whether urine β2-microglobulin, a marker of impaired ability of the kidneys to re-absorb, is related to OTA. BEN patients had significantly higher OTA serum levels. Within the offspring, OTA was significantly related to higher β2-microglobulin excretion. OTA (2005/2006) was related to a higher incidence of BEN after 2008, providing further evidence that OTA is a risk factor for BEN.

  9. Multiple Common Properties of Human β2-Microglobulin and the Common Portion Fragment Derived from HL-A Antigen Molecules

    PubMed Central

    Nakamuro, K.; Tanigaki, N.; Pressman, D.

    1973-01-01

    11,000-Dalton common portion fragments derived from HL-A antigen molecules were isolated and found to have a significantly high homology to β2-microglobulin in amino-acid composition. Common portion fragments are also very similar to β2-microglobulin with respect to molecular size, charge, and distribution in tissues. Moreover, both are found in the spent culture media of human cell lines and in human plasma and urine. Thus it appears that β2-microglobulin may well be the same substance as the common portion fragment of HL-A antigen molecules. PMID:4517941

  10. Predictive accuracy of urinary β2-microglobulin for kidney injury in children with acute pyelonephritis.

    PubMed

    Kangari, Gholamreza; Esteghamati, Maryam; Ghasemi, Kambiz; Mahboobi, Hamidreza

    2015-01-01

    Leukocyte count, erythrocyte sediment rate and C-reactive protein are available laboratory markers which may be helpful in prediction of technetium Tc 99m dimercaptosuccinic acid (DMSA) renal scintigraphy results. None of these, however, have enough accuracy for prediction of renal injury and scar. This study was aimed to evaluate the diagnostic accuracy of urinary β2-microglobulin in detection of renal injury in children with acute pyelonephritis. Eighty-nine children between 2 months and 14 years old with the diagnosis of acute pyelonephritis that had no past history of infection in the urinary tract system were enrolled in the study. A standard urine sample according to patients' age was obtained for urine culture, urinalysis, and urinary β2-microglobulin tests. Blood sample was obtained for leukocyte count, creatinine, blood urea nitrogen, C-reactive protein, erythrocyte sediment rate, and electrolytes tests. All patients underwent DMSA scan. The cutoff point for urinary β2-microglubulin for prediction of positive DMSA scan was 0.8 mg with a sensitivity of 40.9% (95% CI, 26.3% to 56.8%) and a specificity of 84.1% (95% CI, 69.9% to 93.4%), a positive predictive value of 72.0% (95% CI, 50.6% to 87.9%) and an negative predictive value of 58.7% (95% CI, 45.6% to 71.0%). Urinary β2-microglobulin is not enough sensitive and specific to be used as a diagnostic marker for prediction of renal injury. Other common markers such as erythrocyte sediment rate, leukocyte count, and C-reactive protein can be used in combination to predict kidney injury in children with acute pyelonephritis.

  11. Upregulation of β2-microglobulin expression in progressive human oral squamous cell carcinoma.

    PubMed

    Jiang, Qian; Patima, Sdek; Ye, Dong-Xia; Pan, Hong-Ya; Zhang, Pin; Zhang, Zhi-Yuan

    2012-04-01

    The aim of the present study was to investigate β2-microglobulin (β2-M) expression in normal oral mucosa and progressive oral squamous cell carcinoma (OSCC) and to assess the clinical significance of β2-microglobulin expression. The study included 10 cases of normal oral mucosa epithelium specimens, 55 cases of primary OSCC specimens, and 25 cases of OSCC metastasis specimens. Immunohistochemistry was used to determine β2-M expression, and its correlation with clinicopathological factors in progressive OSCC was evaluated. Immunohistochemistry showed that strong β2-M expression was significantly asscociated with tumor size (T3, T4 vs. T1, T2; P=0.001), positive node status (N positive vs. N negative; P=0.000) and advanced clinical stage (Ⅲ, Ⅳ vs. Ⅰ, Ⅱ, P=0.000) in primary OSCC lesions. Compared to primary OSCC lesions, the frequency of β2-M expression was significantly increased in metastatic OSCC lesions (P=0.02). In addition, in vitro results from Western blotting showed increased β2-M expression in the two OSCC lines studied. Therefore, we speculate that the up-regulation of β2-M expression may contribute to the oncogenesis of human oral mucosa, tumor invasion and metastasis.

  12. Molecular cloning and characterization of sea bass (Dicentrarchus labrax, L.) MHC class I heavy chain and β2-microglobulin.

    PubMed

    Pinto, Rute D; Randelli, Elisa; Buonocore, Francesco; Pereira, Pedro J B; dos Santos, Nuno M S

    2013-03-01

    In this work, the gene and cDNA of sea bass (Dicentrarchus labrax) β2-microglobulin (Dila-β2m) and several cDNAs of MHC class I heavy chain (Dila-UA) were characterized. While Dila-β2m is single-copy, numerous Dila-UA transcripts were identified per individual with variability at the peptide-binding domain (PBD), but also with unexpected diversity from the connective peptide (CP) through the 3' untranslated region (UTR). Phylogenetic analysis segregates Dila-β2m and Dila-UA into each subfamily cluster, placing them in the fish class and branching Dila-MHC-I with lineage U. The α1 domains resemble those of the recently proposed L1 trans-species lineage. Although no Dila-specific α1, α2 or α3 sub-lineages could be observed, two highly distinct sub-lineages were identified at the CP/TM/CYT regions. The three-dimensional homology model of sea bass MHC-I complex is consistent with other characterized vertebrate structures. Furthermore, basal tissue-specific expression profiles were determined for both molecules, and expression of β2m was evaluated after poly I:C stimulus. Results suggest these molecules are orthologues of other β2m and teleost classical MHC-I and their basic structure is evolutionarily conserved, providing relevant information for further studies on antigen presentation in this fish species.

  13. Beta 2-Microglobulin: A Novel Therapeutic Target for the Treatment of Human Prostate Cancer Bone Metastasis

    DTIC Science & Technology

    2009-03-14

    Marshall FF, Zhau HE, and Chung LWK. (2008). Receptor activator of NFkB ligand (RANKL) expression is associated with epithelial to mesenchymal...Chu G, Xu J, Shi C, Marshall FF, Zhau HE, and Chung LWK. (2008). Receptor activator of NFkB ligand (RANKL) expression is associated with epithelial...M, Eger A et al (2004) Molecular aspects of epithelial cell plasticity: implications for local tumor invasion and metastasis. Mutat Res 566(1):9–20

  14. Molecular Dynamics Simulation Suggests Possible Interaction Patterns at Early Steps of β2-Microglobulin Aggregation

    PubMed Central

    Fogolari, Federico; Corazza, Alessandra; Viglino, Paolo; Zuccato, Pierfrancesco; Pieri, Lidia; Faccioli, Pietro; Bellotti, Vittorio; Esposito, Gennaro

    2007-01-01

    Early events in aggregation of proteins are not easily accessible by experiments. In this work, we perform a 5-ns molecular dynamics simulation of an ensemble of 27 copies of β2-microglobulin in explicit solvent. During the simulation, the formation of intermolecular contacts is observed. The simulation highlights the importance of apical residues and, in particular, of those at the N-terminus end of the molecule. The most frequently found pattern of interaction involves a head-to-head contact arrangement of molecules. Hydrophobic contacts appear to be important for the establishment of long-lived (on the simulation timescale) contacts. Although early events on the pathway to aggregation and fibril formation are not directly related to the end-state of the process, which is reached on a much longer timescale, simulation results are consistent with experimental data and in general with a parallel arrangement of intermolecular β-strand pairs. PMID:17158575

  15. Equilibrium Unfolding Thermodynamics of β2-Microglobulin Analyzed through Native-State H/D Exchange

    PubMed Central

    Rennella, Enrico; Corazza, Alessandra; Fogolari, Federico; Viglino, Paolo; Giorgetti, Sofia; Stoppini, Monica; Bellotti, Vittorio; Esposito, Gennaro

    2009-01-01

    Abstract The exchange rates for the amide hydrogens of β2-microglobulin, the protein responsible for dialysis-related amyloidosis, were measured under native conditions at different temperatures ranging from 301 to 315 K. The pattern of protection factors within different regions of the protein correlates well with the hydrogen-bonding pattern of the deposited structures. Analysis of the exchange rates indicates the presence of mixed EX1- and EX2-limit mechanisms. The measured parameters are consistent with a two-process model in which two competing pathways, i.e., global unfolding in the core region and partial openings of the native state, determine the observed exchange rates. These findings are analyzed with respect to the amyloidogenic properties of the protein. PMID:18835891

  16. Effective RNAi-mediated β2-microglobulin loss of function by transgenesis in Xenopus laevis

    PubMed Central

    Nedelkovska, Hristina; Edholm, Eva-Stina; Haynes, Nikesha; Robert, Jacques

    2013-01-01

    Summary To impair MHC class I (class I) function in vivo in the amphibian Xenopus, we developed an effective reverse genetic loss of function approach by combining I-SceI meganuclease-mediated transgenesis with RNAi technology. We generated transgenic outbred X. laevis and isogenetic laevis/gilli cloned lines with stably silenced expression of β2-microglobulin (b2m) critical for class I function. Transgenic F1 frogs exhibited decreased surface class I expression on erythrocytes and lymphocytes, decreased frequency of peripheral CD8 T cells and impaired CD8 T cell-mediated skin allograft rejection. Additionally, b2m knockdown increased susceptibility to viral infection of F0 transgenic larvae. This loss of function strategy offers new avenues for studying ontogeny of immunity and other developmental processes in Xenopus. PMID:23519478

  17. Retinoic acid induction of major histocompatibility complex class I genes in NTera-2 embryonal carcinoma cells involves induction of NF-kappa B (p50-p65) and retinoic acid receptor beta-retinoid X receptor beta heterodimers.

    PubMed Central

    Segars, J H; Nagata, T; Bours, V; Medin, J A; Franzoso, G; Blanco, J C; Drew, P D; Becker, K G; An, J; Tang, T

    1993-01-01

    Retinoic acid (RA) treatment of human embryonal carcinoma (EC) NTera-2 (NT2) cells induces expression of major histocompatibility complex (MHC) class I and beta-2 microglobulin surface molecules. We found that this induction was accompanied by increased levels of MHC class I mRNA, which was attributable to the activation of the two conserved upstream enhancers, region I (NF-kappa B like) and region II. This activation coincided with the induction of nuclear factor binding activities specific for the two enhancers. Region I binding activity was not present in undifferentiated NT2 cells, but binding of an NF-kappa B heterodimer, p50-p65, was induced following RA treatment. The p50-p65 heterodimer was produced as a result of de novo induction of p50 and p65 mRNAs. Region II binding activity was present in undifferentiated cells at low levels but was greatly augmented by RA treatment because of activation of a nuclear hormone receptor heterodimer composed of the retinoid X receptor (RXR beta) and the RA receptor (RAR beta). The RXR beta-RAR beta heterodimer also bound RA responsive elements present in other genes which are likely to be involved in RA triggering of EC cell differentiation. Furthermore, transfection of p50 and p65 into undifferentiated NT2 cells synergistically activated region I-dependent MHC class I reporter activity. A similar increase in MHC class I reporter activity was demonstrated by cotransfection of RXR beta and RAR beta. These data show that following RA treatment, heterodimers of two transcription factor families are induced to bind to the MHC enhancers, which at least partly accounts for RA induction of MHC class I expression in NT2 EC cells. Images PMID:8413217

  18. The Extracellular Chaperone Haptoglobin Prevents Serum Fatty Acid-promoted Amyloid Fibril Formation of β2-Microglobulin, Resistance to Lysosomal Degradation, and Cytotoxicity

    PubMed Central

    Sultan, Abdullah; Raman, Bakthisaran; Rao, Ch. Mohan; Tangirala, Ramakrishna

    2013-01-01

    Fibril formation of β2-microglobulin and associated inflammation occur in patients on long term dialysis. We show that the plasma protein haptoglobin prevents the fatty acid-promoted de novo fibril formation of β2-microglobulin even at substoichiometric concentration. The fibrils are cytotoxic, and haptoglobin abolishes the cytotoxicity by preventing fibril formation. Haptoglobin does not alleviate the cytotoxicity of preformed fibrils. Fibrillar β2-microglobulin is resistant to lysosomal degradation. However, the species of β2-microglobulin populated in the presence of haptoglobin is susceptible to degradation. We observed that haptoglobin interacts with oligomeric prefibrillar species of β2-microglobulin but not with monomeric or fibrillar β2-microglobulin that may underlie the molecular mechanism. 1,1′-Bis(4-anilino)naphthalene-5,5′-disulfonic acid cross-linking to haptoglobin significantly compromises its chaperone activity, suggesting the involvement of hydrophobic surfaces. Haptoglobin is an acute phase protein whose level increases severalfold during inflammation, where local acidosis can occur. Our data show that haptoglobin prevents fibril formation of β2-microglobulin under conditions of physiological acidosis (between pH 5.5 and 6.5) but with relatively decreased efficiency. However, compromise in its chaperone activity under these conditions is more than compensated by its increased level of expression under inflammation. Erythrolysis is known to release hemoglobin into the plasma. Haptoglobin forms a 1:1 (mol/mol) complex with hemoglobin. This complex, like haptoglobin, interacts with the prefibrillar species of β2-microglobulin, preventing its fibril formation and the associated cytotoxicity and resistance to intracellular degradation. Thus, our study demonstrates that haptoglobin is a potential extracellular chaperone for β2-microglobulin even in moderately acidic conditions relevant during inflammation, with promising therapeutic

  19. Structure, Folding Dynamics, and Amyloidogenesis of D76N β2-Microglobulin

    PubMed Central

    Mangione, P. Patrizia; Esposito, Gennaro; Relini, Annalisa; Raimondi, Sara; Porcari, Riccardo; Giorgetti, Sofia; Corazza, Alessandra; Fogolari, Federico; Penco, Amanda; Goto, Yuji; Lee, Young-Ho; Yagi, Hisashi; Cecconi, Ciro; Naqvi, Mohsin M.; Gillmore, Julian D.; Hawkins, Philip N.; Chiti, Fabrizio; Rolandi, Ranieri; Taylor, Graham W.; Pepys, Mark B.; Stoppini, Monica; Bellotti, Vittorio

    2013-01-01

    Systemic amyloidosis is a fatal disease caused by misfolding of native globular proteins, which then aggregate extracellularly as insoluble fibrils, damaging the structure and function of affected organs. The formation of amyloid fibrils in vivo is poorly understood. We recently identified the first naturally occurring structural variant, D76N, of human β2-microglobulin (β2m), the ubiquitous light chain of class I major histocompatibility antigens, as the amyloid fibril protein in a family with a new phenotype of late onset fatal hereditary systemic amyloidosis. Here we show that, uniquely, D76N β2m readily forms amyloid fibrils in vitro under physiological extracellular conditions. The globular native fold transition to the fibrillar state is primed by exposure to a hydrophobic-hydrophilic interface under physiological intensity shear flow. Wild type β2m is recruited by the variant into amyloid fibrils in vitro but is absent from amyloid deposited in vivo. This may be because, as we show here, such recruitment is inhibited by chaperone activity. Our results suggest general mechanistic principles of in vivo amyloid fibrillogenesis by globular proteins, a previously obscure process. Elucidation of this crucial causative event in clinical amyloidosis should also help to explain the hitherto mysterious timing and location of amyloid deposition. PMID:24014031

  20. The asymmetry defect of hippocampal circuitry impairs working memory in β2-microglobulin deficient mice.

    PubMed

    Goto, Kazuhiro; Ito, Isao

    2017-03-01

    Left-right (L-R) asymmetry is a fundamental feature of brain function, but the mechanisms underlying functional asymmetry remain largely unknown. We previously identified structural and functional asymmetries in the circuitry of the mouse hippocampus that result from the asymmetrical distribution of NMDA receptor GluR ε2 (NR2B) subunits. By examining the synaptic distribution of ε2 subunits, we found that β2-microglobulin (β2m)-deficient mice that are defective in the stable cell surface expression of major histocompatibility complex class I (MHCI) lack this circuit asymmetry. To investigate the effect of hippocampal asymmetry defect on brain function, we examined working memory of β2m-deficient mice in a delayed nonmatching-to-position (DNMTP) task. Mice were trained to nosepoke either a left or right key of a sample, to retain the position of the key during a delay interval, and then to choose the key opposite from the sample. During training sessions in which no programmed delay interval was imposed, the β2m-deficient mice acquired the task as fast as control mice, suggesting that the discrimination of left and right positions is not impaired by the total loss of hippocampal asymmetry. In contrast, the β2m-deficient mice made fewer correct responses than control mice when variable delay was imposed, suggesting that the asymmetry of hippocampal circuitry plays an important role in working memory.

  1. Predictive value of plasma β2-microglobulin on human body function and senescence.

    PubMed

    Dong, X-M; Cai, R; Yang, F; Zhang, Y-Y; Wang, X-G; Fu, S-L; Zhang, J-R

    2016-06-01

    To explore the correlation between plasma β2-microglobulin (β2-MG) as senescence factor with age, heart, liver and kidney function as well as the predictive value of β2-MG in human metabolism function and senescence. 387 cases of healthy people of different ages were selected and the automatic biochemical analyzer was used to test β2-MG in plasma based on immunoturbidimetry and also all biochemical indexes. The correlation between β2-MG and age, gender and all biochemical indexes was analyzed. β2-MG was positively correlated to age, r = 0.373; and the difference was of statistical significance (p < 0.010). It was significantly negative correlated to HDL-C but positively correlated to LP (a), BUN, CREA, UA, CYS-C, LDH, CK-MB, HBDH, AST, GLB and HCY. β2-MG was closely correlated to age, heart, kidney and liver biochemical indexes, which can be taken as an important biomarker for human body function and anti-senescence and have significant basic research and clinical guidance values.

  2. Structural and Thermodynamic Characteristics of Amyloidogenic Intermediates of β-2-Microglobulin.

    PubMed

    Chong, Song-Ho; Hong, Jooyeon; Lim, Sulgi; Cho, Sunhee; Lee, Jinkeong; Ham, Sihyun

    2015-09-08

    β-2-microglobulin (β2m) self-aggregates to form amyloid fibril in renal patients taking long-term dialysis treatment. Despite the extensive structural and mutation studies carried out so far, the molecular details on the factors that dictate amyloidogenic potential of β2m remain elusive. Here we report molecular dynamics simulations followed by the solvation thermodynamic analyses on the wild-type β2m and D76N, D59P, and W60C mutants at the native (N) and so-called aggregation-prone intermediate (IT) states, which are distinguished by the native cis- and non-native trans-Pro32 backbone conformations. Three major structural and thermodynamic characteristics of the IT-state relative to the N-state in β2m protein are detected that contribute to the increased amyloidogenic potential: (i) the disruption of the edge D-strand, (ii) the increased solvent-exposed hydrophobic interface, and (iii) the increased solvation free energy (less affinity toward solvent water). Mutation effects on these three factors are shown to exhibit a good correlation with the experimentally observed distinct amyloidogenic propensity of the D76N (+), D59P (+), and W60C (-) mutants (+/- for enhanced/decreased). Our analyses thus identify the structural and thermodynamic characteristics of the amyloidogenic intermediates, which will serve to uncover molecular mechanisms and driving forces in β2m amyloid fibril formation.

  3. Aggregation Modulators Interfere with Membrane Interactions of β2-Microglobulin Fibrils

    PubMed Central

    Sheynis, Tania; Friediger, Anat; Xue, Wei-Feng; Hellewell, Andrew L.; Tipping, Kevin W.; Hewitt, Eric W.; Radford, Sheena E.; Jelinek, Raz

    2013-01-01

    Amyloid fibril accumulation is a pathological hallmark of several devastating disorders, including Alzheimer’s disease, prion diseases, type II diabetes, and others. Although the molecular factors responsible for amyloid pathologies have not been deciphered, interactions of misfolded proteins with cell membranes appear to play important roles in these disorders. Despite increasing evidence for the involvement of membranes in amyloid-mediated cytotoxicity, the pursuit for therapeutic strategies has focused on preventing self-assembly of the proteins comprising the amyloid plaques. Here we present an investigation of the impact of fibrillation modulators upon membrane interactions of β2-microglobulin (β2m) fibrils. The experiments reveal that polyphenols (epigallocatechin gallate, bromophenol blue, and resveratrol) and glycosaminoglycans (heparin and heparin disaccharide) differentially affect membrane interactions of β2m fibrils measured by dye-release experiments, fluorescence anisotropy of labeled lipid, and confocal and cryo-electron microscopies. Interestingly, whereas epigallocatechin gallate and heparin prevent membrane damage as judged by these assays, the other compounds tested had little, or no, effect. The results suggest a new dimension to the biological impact of fibrillation modulators that involves interference with membrane interactions of amyloid species, adding to contemporary strategies for combating amyloid diseases that focus on disruption or remodeling of amyloid aggregates. PMID:23931322

  4. Analysis of betaS and betaA genes in a Mexican population with African roots.

    PubMed

    Magaña, María Teresa; Ongay, Zoyla; Tagle, Juan; Bentura, Gilberto; Cobián, José G; Perea, F Javier; Casas-Castañeda, Maricela; Sánchez-López, Yoaly J; Ibarra, Bertha

    2002-01-01

    To investigate the origin of the beta(A) and beta(S) genes in a Mexican population with African roots and a high frequency of hemoglobin S, we analyzed 467 individuals (288 unrelated) from different towns in the states of Guerrero and Oaxaca in the Costa Chica region. The frequency of the sickle-cell trait was 12.8%, which may represent a public health problem. The frequencies of the beta-haplotypes were determined from 350 nonrelated chromosomes (313 beta(A) and 37 beta(S)). We observed 15 different beta(A) haplotypes, the most common of which were haplotypes 1 (48.9%), 2 (13.4%), and 3 (13.4%). The calculation of pairwise distributions and Nei's genetic distance analysis using 32 worldwide populations showed that the beta(A) genes are more closely related to those of Mexican Mestizos and North Africans. Bantu and Benin haplotypes and haplotype 9 were related to the beta(S) genes, with frequencies of 78.8, 18.2, and 3.0%, respectively. Comparison of these haplotypes with 17 other populations revealed a high similitude with the population of the Central African Republic. These data suggest distinct origins for the beta(A) and beta(S) genes in Mexican individuals from the Costa Chica region.

  5. Human globin gene analysis for a patient with beta-o/delta beta-thalassemia.

    PubMed Central

    Ottolenghi, S; Lanyon, W G; Williamson, R; Weatherall, D J; Clegg, J B; Pitcher, C S

    1975-01-01

    Complementary DNA (cDNA) was prepared with RNA-dependent DNA polymerase from human globin messenger RNA (mRNA). Annealing and translation experimenta with total mRNA from circulating cells from a patient with heterozygous beta/heterozygous beta-delta-o thalassemia (beta-o/delta beta-o-thalassemia) demonstrated no detectable mRNA for beta-globin. cDNA enriched in sequences homologous to beta-globin mRNA was prepared by hydroxylapatite fractionation of hybrids formed between beta-o/delta beta-o-thalassemic mRNA and cDNA made from mRNA from a patient with alpha-thalassemia (hemoglobin H disease). The rate of annealing of this beta-enriched cDNA to normal human nuclear DNA was that of a sequence present as only a single copy per haploid genome. The beta-enriched cDNA annealed to the beta-o-delta beta-o-thalassemia total DNA with approximately the same kinetics as to normal DNA, indicating that no total gene deletion of beta-globin genes from the diploid genome has occurred, although the accuracy of the technique could not exclude with certainty a partial deletion or a deletion of a beta-globin gene from only one of the haploid genomes. This demonstrates that at least one of the beta-o- or the delta beta-o-thalassemia haploid genomes in this case contains a substantially intact beta-globin gene. PMID:49057

  6. The nucleotide sequence of the human beta-globin gene.

    PubMed

    Lawn, R M; Efstratiadis, A; O'Connell, C; Maniatis, T

    1980-10-01

    We report the complete nucleotide sequence of the human beta-globin gene. The purpose of this study is to obtain information necessary to study the evolutionary relationships between members of the human beta-like globin gene family and to provide the basis for comparing normal beta-globin genes with those obtained from the DNA of individuals with genetic defects in hemoglobin expression.

  7. HLA-DR and ß2 microglobulin expression in medullary and atypical medullary carcinoma of the breast: histopathologically similar but biologically distinct entities

    PubMed Central

    Feinmesser, M.; Sulkes, A.; Morgenstern, S.; Sulkes, J.; Stern, S.; Okon, E.

    2000-01-01

    Aims—To examine the expression of HLA-DR and ß2 microglobulin in medullary carcinoma and atypical medullary carcinoma of the breast to determine if the effective presentation of tumour antigens to the immune system can differentiate between these two histopathologically similar entities. Methods—Expression of HLA-DR and ß2 microglobulin was examined by immunohistochemical methods in five samples of medullary carcinoma of the breast, which has a relatively favourable prognosis, six samples of atypical medullary carcinoma of the breast, which has a prognosis closer to that of regular invasive duct carcinoma, and 20 samples of invasive duct carcinomas, 10 with an accompanying lymphocytic infiltrate. Results—A positive and significant correlation was found between tumour type and both HLA-DR and ß2 microglobulin expression. Expression was most prominent in medullary carcinoma, followed by atypical medullary carcinoma and invasive duct carcinoma with and without lymphocytic infiltrates. The mean intensity and percentage of HLA-DR tumour immunostaining were significantly higher in medullary carcinoma than in the other three tumour groups, as was the mean intensity of ß2 microglobulin immunostaining. Mean percentage of ß2 microglobulin immunostaining was significantly higher in medullary carcinoma than in invasive duct carcinoma without lymphocytic infiltrates, and showed a trend to increase from invasive duct carcinoma with lymphocytic infiltrates to atypical medullary carcinoma and medullary carcinoma. Conclusions—Medullary carcinoma and atypical medullary carcinoma of the breast differ in their expression of HLA-DR and ß2 microglobulin. The relatively favourable prognosis of medullary carcinoma of the breast may be related to effective tumour antigen presentation to the immune system through MHC-I and MHC-II expression. Immunotherapy aimed at MHC-I and MHC-II induction might have a beneficial effect in breast cancer. Key Words: medullary carcinoma of the

  8. Chronic Lymphocytic Inflammation with Pontine Perivascular Enhancement Responsive to Steroids with a Significant Elevation of β-2 Microglobulin Levels

    PubMed Central

    Fujisawa, Naoaki; Mori, Harushi; Matsui, Toru

    2015-01-01

    Chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS) is a relapsing-remitting disorder for which steroid administration is a key to control the progression. CLIPPERS can exhibit radiological features similar to malignant lymphoma, whose diagnosis is confounded by prior steroid administration. We report a case of CLIPPERS accompanied by abnormal elevation of β-2 microglobulin in the cerebrospinal fluid (CSF). A 62-year-old man started to experience numbness in all fingers of his left hand one year ago, which gradually extended to his body trunk and legs on both sides. Magnetic resonance imaging demonstrated numerous small enhancing spots scattered in his brain and spinal cord. CSF levels of β-2 microglobulin were elevated; although this often indicates central nervous system involvement in leukemia and lymphoma, the lesions were diagnosed as CLIPPERS based on the pathological findings from a biopsy specimen. We emphasize the importance of biopsy to differentiate between CLIPPERS and malignant lymphoma because the temporary radiological response to steroid might be the same in both diseases but the treatment strategies regarding the use of steroid are quite different. PMID:26713153

  9. Chronic Lymphocytic Inflammation with Pontine Perivascular Enhancement Responsive to Steroids with a Significant Elevation of β-2 Microglobulin Levels.

    PubMed

    Fujisawa, Naoaki; Oya, Soichi; Mori, Harushi; Matsui, Toru

    2015-11-01

    Chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS) is a relapsing-remitting disorder for which steroid administration is a key to control the progression. CLIPPERS can exhibit radiological features similar to malignant lymphoma, whose diagnosis is confounded by prior steroid administration. We report a case of CLIPPERS accompanied by abnormal elevation of β-2 microglobulin in the cerebrospinal fluid (CSF). A 62-year-old man started to experience numbness in all fingers of his left hand one year ago, which gradually extended to his body trunk and legs on both sides. Magnetic resonance imaging demonstrated numerous small enhancing spots scattered in his brain and spinal cord. CSF levels of β-2 microglobulin were elevated; although this often indicates central nervous system involvement in leukemia and lymphoma, the lesions were diagnosed as CLIPPERS based on the pathological findings from a biopsy specimen. We emphasize the importance of biopsy to differentiate between CLIPPERS and malignant lymphoma because the temporary radiological response to steroid might be the same in both diseases but the treatment strategies regarding the use of steroid are quite different.

  10. The human thyrotropin beta-subunit gene differs in 5' structure from murine TSH-beta genes.

    PubMed

    Guidon, P T; Whitfield, G K; Porti, D; Kourides, I A

    1988-12-01

    The gene encoding the beta-subunit of human thyrotropin (hTSH-beta) was isolated, and its nucleotide sequence was determined. The gene is 4.3 kb in length, consists of three exons and two introns, and is present as a single copy as determined by Southern blot analysis of total genomic DNA. The protein coding portion of the gene, which includes exons 2 and 3, was isolated from a human genomic phage library, while exon 1, which encodes only 5' untranslated mRNA sequence, was isolated from a plasmid library of size-selected genomic DNA fragments. Here we describe the isolation of the 5' untranslated exon of the hTSH-beta subunit and 5'-flanking region. The structure of the hTSH-beta gene is very similar to the previously characterized TSH-beta genes from mouse and rat. The genes from all three species have two distinct promoter regions, but while both promoters are utilized by the murine TSH-beta genes, the human TSH-beta gene apparently utilizes only the proximal promoter for transcription initiation. A striking difference in hTSH-beta gene structure compared to the murine genes is that exon 1 of the human gene is 36 nucleotides. An analysis of the mouse, rat, and human exon 1 and 5'-flanking region shows a high percentage of sequence homology, with the exception of a 9-nucleotide insertion 13 bases 3' from the proximal TATA box found in the human gene but not found in the other two species. We propose that this insertion results in the additional length of human exon 1 compared to the mouse and rat genes. By isolating the promoter region of the hTSH-beta gene, we can begin to identify specific sequences involved in the regulation of hTSH gene expression.

  11. Gene encoding the human beta-hexosaminidase beta chain: extensive homology of intron placement in the alpha- and beta-chain genes.

    PubMed Central

    Proia, R L

    1988-01-01

    Lysosomal beta-hexosaminidase (EC 3.2.1.52) is composed of two structurally similar chains, alpha and beta, that are the products of different genes. Mutations in either gene causing beta-hexosaminidase deficiency result in the lysosomal storage disease GM2-gangliosidosis. To enable the investigation of the molecular lesions in this disorder and to study the evolutionary relationship between the alpha and beta chains, the beta-chain gene was isolated, and its organization was characterized. The beta-chain coding region is divided into 14 exons distributed over approximately 40 kilobases of DNA. Comparison with the alpha-chain gene revealed that 12 of the 13 introns interrupt the coding regions at homologous positions. This extensive sharing of intron placement demonstrates that the alpha and beta chains evolved by way of the duplication of a common ancestor. PMID:2964638

  12. Cloning and expression analysis of the murine lymphotoxin beta gene.

    PubMed Central

    Pokholok, D K; Maroulakou, I G; Kuprash, D V; Alimzhanov, M B; Kozlov, S V; Novobrantseva, T I; Turetskaya, R L; Green, J E; Nedospasov, S A

    1995-01-01

    Tumor necrosis factor alpha (TNF-alpha) and soluble lymphotoxin (LT) (also called LT-alpha or TNF-beta) are cytokines with similar biological activities that are encoded by related and closely linked genes. TNF-alpha, a mediator of the inflammatory response, exists in soluble and transmembrane forms. LT-alpha can be secreted or retained at the cell surface by binding to a 33-kDa transmembrane subunit, LT-beta. The recently cloned human LT-beta gene encodes another TNF family member and is linked to the TNF/LT locus within the major histocompatibility complex locus. The cell surface LT is a heterotrimer consisting of LT-alpha and LT-beta, whose physiological function is not yet clearly defined. We now report the sequence analysis of the genomic region and cDNA of murine LT-beta gene, which is closely associated with the TNF-alpha and LT-alpha genes within the murine major histocompatibility complex locus. Unlike the TNF-alpha, LT-alpha, and human LT-beta genes, which contain four exons, the murine LT-beta contains three exons and encodes a 244-amino acid polypeptide with a 66-amino acid insert that is absent from the human homologue. In situ hybridization demonstrates constitutive expression of LT-beta in lymphoid and hematopoietic tissues. LT-beta transcription is maximal in the thymic medulla and in splenic white pulp. LT-beta mRNA is also detected in the skin and in specific regions of the brain. The LT-beta promoter region contains putative Ets-binding sites, suggesting that the expression of LT-beta may be regulated in part by Ets transcription factors whose pattern of lymphoid expression overlaps that of LT-beta. Images Fig. 3 Fig. 4 PMID:7846035

  13. Characterization of a beta-tubulin gene and a beta-tubulin gene products of Brugia pahangi.

    PubMed

    Guénette, S; Prichard, R K; Klein, R D; Matlashewski, G

    1991-02-01

    A genomic clone containing a beta-tubulin gene from the parasitic nematode Brugia pahangi was isolated. This gene was sequenced to determine its size, structural organization, and corresponding primary amino acid sequence. The coding sequence of the beta-tubulin gene spans 3.8 kb, is organized into 9 exons and expresses an mNRA of 1.8 kb which codes for a protein of 448 amino acids. The predicted beta-tubulin amino acid sequence is 89%, 94%, 90% and 88% identical to the chicken beta 2, and the Caenorhabditis elegans ben-1, tub-1 and mec-7 gene products, respectively. Southern hybridization analyses demonstrated that there is only one copy of this gene isotype but that other distinct beta-tubulin genes may exist in the Brugia pahangi genome. A nematode specific antipeptide rabbit antiserum raised against the predicted amino acid sequence of the extreme carboxy-terminal region of the B. pahangi beta-tubulin was used to identify beta-tubulin isoforms in adult nematodes and microfilariae. Isoforms detected by this nematode-specific antipeptide antiserum were identical in both adult worms and microfilariae and did not differ from the isoform patterns detected by a monoclonal antibody recognizing a conserved beta-tubulin epitope. This suggests that this carboxy-terminal peptide is highly represented in the beta-tubulin isoforms of B. pahangi.

  14. Evolutionary ecology of beta-lactam gene clusters in animals.

    PubMed

    Suring, Wouter; Meusemann, Karen; Blanke, Alexander; Mariën, Janine; Schol, Tim; Agamennone, Valeria; Faddeeva-Vakhrusheva, Anna; Berg, Matty P; Brouwer, Abraham; van Straalen, Nico M; Roelofs, Dick

    2017-06-01

    Beta-lactam biosynthesis was thought to occur only in fungi and bacteria, but we recently reported the presence of isopenicillin N synthase in a soil-dwelling animal, Folsomia candida. However, it has remained unclear whether this gene is part of a larger beta-lactam biosynthesis pathway and how widespread the occurrence of penicillin biosynthesis is among animals. Here, we analysed the distribution of beta-lactam biosynthesis genes throughout the animal kingdom and identified a beta-lactam gene cluster in the genome of F. candida (Collembola), consisting of isopenicillin N synthase (IPNS), δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine synthetase (ACVS), and two cephamycin C genes (cmcI and cmcJ) on a genomic scaffold of 0.76 Mb. All genes are transcriptionally active and are inducible by stress (heat shock). A beta-lactam compound was detected in vivo using an ELISA beta-lactam assay. The gene cluster also contains an ABC transporter which is coregulated with IPNS and ACVS after heat shock. Furthermore, we show that different combinations of beta-lactam biosynthesis genes are present in over 60% of springtail families, but they are absent from genome- and transcript libraries of other animals including close relatives of springtails (Protura, Diplura and insects). The presence of beta-lactam genes is strongly correlated with an euedaphic (soil-living) lifestyle. Beta-lactam genes IPNS and ACVS each form a phylogenetic clade in between bacteria and fungi, while cmcI and cmcJ genes cluster within bacteria. This suggests a single horizontal gene transfer event most probably from a bacterial host, followed by differential loss in more recently evolving species. © 2017 John Wiley & Sons Ltd.

  15. Validation of the NCCN-IPI for diffuse large B-cell lymphoma (DLBCL): the addition of β2 -microglobulin yields a more accurate GELTAMO-IPI.

    PubMed

    Montalbán, Carlos; Díaz-López, Antonio; Dlouhy, Ivan; Rovira, Jordina; Lopez-Guillermo, Armando; Alonso, Sara; Martín, Alejandro; Sancho, Juan M; García, Olga; Sánchez, Jose M; Rodríguez, Mario; Novelli, Silvana; Salar, Antonio; Gutiérrez, Antonio; Rodríguez-Salazar, Maria J; Bastos, Mariana; Domínguez, Juan F; Fernández, Rubén; Gonzalez de Villambrosia, Sonia; Queizan, José A; Córdoba, Raul; de Oña, Raquel; López-Hernandez, Andrés; Freue, Julian M; Garrote, Heidys; López, Lourdes; Martin-Moreno, Ana M; Rodriguez, Jose; Abraira, Víctor; García, Juan F

    2017-03-01

    The study included 1848 diffuse large B-cell lymphoma (DLBCL)patients treated with chemotherapy/rituximab. The aims were to validate the National Comprehensive Cancer Network International Prognostic Index (NCCN-IPI) and explore the effect of adding high Beta-2 microglobulin (β2M), primary extranodal presentation and intense treatment to the NCCN-IPI variables in order to develop an improved index. Comparing survival curves, NCCN-IPI discriminated better than IPI, separating four risk groups with 5-year overall survival rates of 93%, 83%, 67% and 49%, but failing to identify a true high-risk population. For the second aim the series was split into training and validation cohorts: in the former the multivariate model identified age, lactate dehydrogenase, Eastern Cooperative Oncology Group performance status, Stage III-IV, and β2M as independently significant, whereas the NCCN-IPI-selected extranodal sites, primary extranodal presentation and intense treatments were not. These results were confirmed in the validation cohort. The Grupo Español de Linfomas/Trasplante de Médula ósea (GELTAMO)-IPI developed here, with 7 points, significantly separated four risk groups (0, 1-3, 4 or ≥5 points) with 11%, 58%, 17% and 14% of patients, and 5-year overall survival rates of 93%, 79%, 66% and 39%, respectively. In the comparison GELTAMO IPI discriminated better than the NCCN-IPI. In conclusion, GELTAMO-IPI is more accurate than the NCCN-IPI and has statistical and practical advantages in that the better discrimination identifies an authentic high-risk group and is not influenced by primary extranodal presentation or treatments of different intensity.

  16. Endogenous beta-cell CART regulates insulin secretion and transcription of beta-cell genes.

    PubMed

    Shcherbina, L; Edlund, A; Esguerra, J L S; Abels, M; Zhou, Y; Ottosson-Laakso, E; Wollheim, C B; Hansson, O; Eliasson, L; Wierup, N

    2017-05-15

    Impaired beta-cell function is key to the development of type 2 diabetes. Cocaine- and amphetamine-regulated transcript (CART) is an islet peptide with insulinotropic and glucagonostatic properties. Here we studied the role of endogenous CART in beta-cell function. CART silencing in INS-1 (832/13) beta-cells reduced insulin secretion and production, ATP levels and beta-cell exocytosis. This was substantiated by reduced expression of several exocytosis genes, as well as reduced expression of genes important for insulin secretion and processing. In addition, CART silencing reduced the expression of a network of transcription factors essential for beta-cell function. Moreover, in RNAseq data from human islet donors, CARTPT expression levels correlated with insulin, exocytosis genes and key beta-cell transcription factors. Thus, endogenous beta-cell CART regulates insulin expression and secretion in INS-1 (832/13) cells, via actions on the exocytotic machinery and a network of beta-cell transcription factors. We conclude that CART is important for maintaining the beta-cell phenotype. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. β2-microglobulin induces epithelial to mesenchymal transition and confers cancer lethality and bone metastasis in human cancer cells.

    PubMed

    Josson, Sajni; Nomura, Takeo; Lin, Jen-Tai; Huang, Wen-Chin; Wu, Daqing; Zhau, Haiyen E; Zayzafoon, Majd; Weizmann, M Neale; Gururajan, Murali; Chung, Leland W K

    2011-04-01

    Bone metastasis is one of the predominant causes of cancer lethality. This study demonstrates for the first time how β2-microglobulin (β2-M) supports lethal metastasis in vivo in human prostate, breast, lung, and renal cancer cells. β2-M mediates this process by activating epithelial to mesenchymal transition (EMT) to promote lethal bone and soft tissue metastases in host mice. β2-M interacts with its receptor, hemochromatosis (HFE) protein, to modulate iron responsive pathways in cancer cells. Inhibition of either β2-M or HFE results in reversion of EMT. These results demonstrate the role of β2-M in cancer metastasis and lethality. Thus, β2-M and its downstream signaling pathways are promising prognostic markers of cancer metastases and novel therapeutic targets for cancer therapy.

  18. A Novel Anti-Beta2-Microglobulin Antibody Inhibition of Androgen Receptor Expression, Survival, and Progression in Prostate Cancer Cells

    DTIC Science & Technology

    2009-05-31

    receptor signaling in prostate cancer. Expert Rev Anticancer Ther 2005;5(1):63-74. 2. Heinlein CA, Chang C. Androgen receptor in prostate cancer. Endocr...prostate cancer. Expert Rev Anticancer Ther 2005;5:63^74. 22. Heinlein CA, Chang C. Androgen receptor in pros- tate cancer. Endocr Rev 2004;25:276^308

  19. A Novel Anti-Beta2-Microglobulin Antibody Inhibition of Androgen Receptor Expression, Survival, and Progression in Prostate Cancer Cells

    DTIC Science & Technology

    2011-05-01

    lethal bone and soft tissue metastases in host mice. b2-M interacts with its receptor, hemochromatosis (HFE) protein, to modulate iron responsive...develop symptoms of hemochromatosis involving iron overload and its associated diseases (19, 20). Several studies demonstrate the interaction between...b2-M/HFE and its physical interaction with transferrin receptor, the primary mechanism for iron uptake in mammalian cells (21). In the present study

  20. Reassociation with beta 2-microglobulin is necessary for Kb class I major histocompatibility complex binding of exogenous peptides.

    PubMed Central

    Rock, K L; Rothstein, L E; Gamble, S R; Benacerraf, B

    1990-01-01

    T lymphocytes recognize endogenously produced antigenic peptides in association with major histocompatibility complex (MHC)-encoded molecules. Peptides from the extracellular fluid can be displayed in association with class I and class II MHC molecules. Here we report that mature Kb class I MHC molecules bind peptides upon dissociation and reassociation of their light chain. Intact Kb heterodimers, unlike class II MHC molecules, are relatively unreceptive to binding peptides. This property may maintain segregation of class I and class II MHC-restricted peptides and has implications for the use of peptides as vaccines. Images PMID:2217182

  1. A Novel Anti-Beta2-Microglobulin Antibody Inhibition of Androgen Receptor Expression, Survival, and Progression in Prostate Cancer Cells

    DTIC Science & Technology

    2012-01-01

    5B, the left panel). Additionally, expression of catalase , a key enzyme of hydrogen peroxide degradation, decreased in SREBP-1 overexpresssing H1...from -5400 to +580) in length, we further used a restriction enzyme , SacI, to generate a shorter promoter luciferase construct, the hAR/SacI vector (2...well as AR protein expression, but not SREBP-2 in LNCaP and C4-2B cells (Fig. 4C). 5) SREBP-1 induces oxidative stress, Nox5 and catalase

  2. A Novel Anti-Beta2-Microglobulin Antibody Inhibition of Androgen Receptor Expression, Survival, and Progression in Prostate Cancer Cells

    DTIC Science & Technology

    2010-05-01

    the levels of superoxide were not significantly changed by SREBP-1 (Fig. 4B, the left panel). Additionally, expression of catalase , a key enzyme of...hAR promoter reporter vector contains approximate 6 kb (from -5400 to +580) in length, we further used a restriction enzyme , SacI, to generate a...Fig. 3C). 4) SREBP-1 induces oxidative stress, Nox5 and catalase expression in prostate cancer cells. ROS and Nox (a ROS generator), have been

  3. The T-Cell Receptor Can Bind to the Peptide-Bound Major Histocompatibility Complex and Uncomplexed β2-Microglobulin through Distinct Binding Sites.

    PubMed

    Merkle, Patrick S; Irving, Melita; Hongjian, Song; Ferber, Mathias; Jørgensen, Thomas J D; Scholten, Kirsten; Luescher, Immanuel; Coukos, George; Zoete, Vincent; Cuendet, Michel A; Michielin, Olivier; Rand, Kasper D

    2017-08-01

    T-Cell receptor (TCR)-mediated recognition of the peptide-bound major histocompatibility complex (pMHC) initiates an adaptive immune response against antigen-presenting target cells. The recognition events take place at the TCR-pMHC interface, and their effects on TCR conformation and dynamics are controversial. Here, we have measured the time-resolved hydrogen/deuterium exchange (HDX) of a soluble TCR in the presence and absence of its cognate pMHC by mass spectrometry to delineate the impact of pMHC binding on solution-phase structural dynamics in the TCR. Our results demonstrate that while TCR-pMHC complex formation significantly stabilizes distinct CDR loops of the TCR, it does not trigger structural changes in receptor segments remote from the binding interface. Intriguingly, our HDX measurements reveal that the TCR α-constant domain (C- and F-strand) directly interacts with the unbound MHC light chain, β2-microglobulin (β2m). Surface plasmon resonance measurements corroborated a binding event between TCR and β2m with a dissociation constant of 167 ± 20 μM. We propose a model structure for the TCR-β2m complex based on a refined protein-protein docking approach driven by HDX data and information from molecular dynamics simulations. Using a biological assay based on TCR gene-engineered primary human T cells, we did not observe a significant effect of β2m on T-cell cytotoxicity, suggesting an alternate role for β2m binding. Overall, we show that binding of β2m to the TCR occurs in vitro and, as such, not only should be considered in structure-function studies of the TCR-pMHC complex but also could play a hitherto unidentified role in T-cell function in vivo.

  4. Nonstructural Protein 4 of Porcine Reproductive and Respiratory Syndrome Virus Modulates Cell Surface Swine Leukocyte Antigen Class I Expression by Downregulating β2-Microglobulin Transcription

    PubMed Central

    Qi, Pengfei; Liu, Ke; Wei, Jianchao; Li, Yuming; Li, Beibei; Shao, Donghua; Wu, Zhuanchang; Shi, Yuanyuan; Tong, Guangzhi

    2016-01-01

    ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which has important impacts on the pig industry. PRRSV infection results in disruption of the swine leukocyte antigen class I (SLA-I) antigen presentation pathway. In this study, highly pathogenic PRRSV (HP-PRRSV) infection inhibited transcription of the β2-microglobulin (β2M) gene (B2M) and reduced cellular levels of β2M, which forms a heterotrimeric complex with the SLA-I heavy chain and a variable peptide and plays a critical role in SLA-I antigen presentation. HP-PRRSV nonstructural protein 4 (Nsp4) was involved in the downregulation of β2M expression. Exogenous expression of Nsp4 downregulated β2M expression at both the mRNA and the protein level and reduced SLA-I expression on the cell surface. Nsp4 bound to the porcine B2M promoter and inhibited its transcriptional activity. Domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter were identified as essential for the interaction between Nsp4 and B2M. These findings demonstrate a novel mechanism whereby HP-PRRSV may modulate the SLA-I antigen presentation pathway and provide new insights into the functions of HP-PRRSV Nsp4. IMPORTANCE PRRSV modulates the host response by disrupting the SLA-I antigen presentation pathway. We show that HP-PRRSV downregulates SLA-I expression on the cell surface via transcriptional inhibition of B2M expression by viral Nsp4. The interaction between domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter is essential for inhibiting B2M transcription. These observations reveal a novel mechanism whereby HP-PRRSV may modulate SLA-I antigen presentation and provide new insights into the functions of viral Nsp4. PMID:28003480

  5. Gene expression in scrapie. Cloning of a new scrapie-responsive gene and the identification of increased levels of seven other mRNA transcripts.

    PubMed

    Dandoy-Dron, F; Guillo, F; Benboudjema, L; Deslys, J P; Lasmézas, C; Dormont, D; Tovey, M G; Dron, M

    1998-03-27

    To define genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies, we analyzed gene expression in scrapie-infected mouse brain using "mRNA differential display." The RNA transcripts of eight genes were increased 3-8-fold in the brains of scrapie-infected animals. Five of these genes have not previously been reported to exhibit increased expression in this disease: cathepsin S, the C1q B-chain of complement, apolipoprotein D, and two previously unidentified genes denominated scrapie-responsive gene (ScRG)-1 and ScRG-2, which are preferentially expressed in brain tissue. Increased expression of the three remaining genes, beta2 microglobulin, F4/80, and metallothionein II, has previously been reported to occur in experimental scrapie. Kinetic analysis revealed a concomitant increase in the levels of ScRG-1, cathepsin S, the C1q B-chain of complement, and beta2 microglobulin mRNA as well as glial fibrillary acidic protein and F4/80 transcripts, markers of astrocytosis and microglial activation, respectively. In contrast, the level of ScRG-2, apolipoprotein D, and metallothionein II mRNA was only increased at the terminal stage of the disease. ScRG-1 mRNA was found to be preferentially expressed in glial cells and to code for a short protein of 47 amino acids with a strong hydrophobic N-terminal region.

  6. Beta-amylase gene variability in introgressive wheat lines.

    PubMed

    Antonyuk, Maksym; Navalikhina, Anastasiia; Ternovska, Tamara

    2017-05-01

    Variability of the beta-amylase gene in bread wheat, artificial amphidiploids, and derived introgression wheat lines was analyzed. Variation in homeologous beta-amylase sequences caused by the presence of MITE (Miniature Inverted-Repeat Transposable Element) and its footprint has been identified in bread wheat. The previously unknown location of MITE in Triticum urartu and T. aestivum L. beta-amylase gene has been found. These species have a MITE sequence in the third intron of beta-amylase, as opposed to Aegilops comosa and a number of other Triticeae species, which have it in the fourth intron. These two MITEs from Ae. comosa and T. aestivum were shown to have low identity scores. Miosa, an artificial amphidiploid, which has the M genome from Ae. comosa was shown to lose the MITE sequences. This loss might be caused by genomic shock due to allopolyploidization.

  7. Nucleotide sequence of SHV-2 beta-lactamase gene

    SciTech Connect

    Garbarg-Chenon, A.; Godard, V.; Labia, R.; Nicolas, J.C. )

    1990-07-01

    The nucleotide sequence of plasmid-mediated beta-lactamase SHV-2 from Salmonella typhimurium (SHV-2pHT1) was determined. The gene was very similar to chromosomally encoded beta-lactamase LEN-1 of Klebsiella pneumoniae. Compared with the sequence of the Escherichia coli SHV-2 enzyme (SHV-2E.coli) obtained by protein sequencing, the deduced amino acid sequence of SHV-2pHT1 differed by three amino acid substitutions.

  8. β2-Microglobulin Amyloid Fibril-Induced Membrane Disruption Is Enhanced by Endosomal Lipids and Acidic pH

    PubMed Central

    Goodchild, Sophia C.; Sheynis, Tania; Thompson, Rebecca; Tipping, Kevin W.; Xue, Wei-Feng; Ranson, Neil A.; Beales, Paul A.; Hewitt, Eric W.; Radford, Sheena E.

    2014-01-01

    Although the molecular mechanisms underlying the pathology of amyloidoses are not well understood, the interaction between amyloid proteins and cell membranes is thought to play a role in several amyloid diseases. Amyloid fibrils of β2-microglobulin (β2m), associated with dialysis-related amyloidosis (DRA), have been shown to cause disruption of anionic lipid bilayers in vitro. However, the effect of lipid composition and the chemical environment in which β2m-lipid interactions occur have not been investigated previously. Here we examine membrane damage resulting from the interaction of β2m monomers and fibrils with lipid bilayers. Using dye release, tryptophan fluorescence quenching and fluorescence confocal microscopy assays we investigate the effect of anionic lipid composition and pH on the susceptibility of liposomes to fibril-induced membrane damage. We show that β2m fibril-induced membrane disruption is modulated by anionic lipid composition and is enhanced by acidic pH. Most strikingly, the greatest degree of membrane disruption is observed for liposomes containing bis(monoacylglycero)phosphate (BMP) at acidic pH, conditions likely to reflect those encountered in the endocytic pathway. The results suggest that the interaction between β2m fibrils and membranes of endosomal origin may play a role in the molecular mechanism of β2m amyloid-associated osteoarticular tissue destruction in DRA. PMID:25100247

  9. Uncovering the Early Assembly Mechanism for Amyloidogenic β2-Microglobulin Using Cross-linking and Native Mass Spectrometry*

    PubMed Central

    Hall, Zoe; Schmidt, Carla; Politis, Argyris

    2016-01-01

    β2-Microglobulin (β2m), a key component of the major histocompatibility class I complex, can aggregate into fibrils with severe clinical consequences. As such, investigating the structural aspects of the formation of oligomeric intermediates of β2m and their subsequent progression toward fibrillar aggregates is of great importance. However, β2m aggregates are challenging targets in structural biology, primarily due to their inherent transient and heterogeneous nature. Here we study the oligomeric distributions and structures of the early intermediates of amyloidogenic β2m and its truncated variant ΔN6-β2m. We established compact oligomers for both variants by integrating advanced mass spectrometric techniques with available electron microscopy maps and atomic level structures from NMR spectroscopy and x-ray crystallography. Our results revealed a stepwise assembly mechanism by monomer addition and domain swapping for the oligomeric species of ΔN6-β2m. The observed structural similarity and common oligomerization pathway between the two variants is likely to enable ΔN6-β2m to cross-seed β2m fibrillation and allow the formation of mixed fibrils. We further determined the key subunit interactions in ΔN6-β2m tetramer, revealing the importance of a domain-swapped hinge region for formation of higher order oligomers. Overall, we deliver new mechanistic insights into β2m aggregation, paving the way for future studies on the mechanisms and cause of amyloid fibrillation. PMID:26655720

  10. β2-microglobulin amyloid fibrils are nanoparticles that disrupt lysosomal membrane protein trafficking and inhibit protein degradation by lysosomes.

    PubMed

    Jakhria, Toral; Hellewell, Andrew L; Porter, Morwenna Y; Jackson, Matthew P; Tipping, Kevin W; Xue, Wei-Feng; Radford, Sheena E; Hewitt, Eric W

    2014-12-26

    Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of β2-microglobulin (β2m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented β2m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented β2m fibrils did not, however, cause cell death. Instead, fragmented β2m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Structural insights into the pre-amyloid tetramer of β-2-microglobulin from covalent labeling and mass spectrometry.

    PubMed

    Mendoza, Vanessa Leah; Barón-Rodríguez, Mario A; Blanco, Cristian; Vachet, Richard W

    2011-08-09

    The main pathogenic process underlying dialysis-related amyloidosis is the accumulation of β-2-microglobulin (β2m) as amyloid fibrils in the musculoskeletal system, and some evidence suggests that Cu(II) may play a role in β2m amyloid formation. Cu(II)-induced β2m fibril formation is preceded by the formation of discrete, oligomeric intermediates, including dimers, tetramers, and hexamers. In this work, we use selective covalent labeling reactions combined with mass spectrometry to investigate the amino acids responsible for mediating tetramer formation in wild-type β2m. By comparing the labeling patterns of the monomer, dimer, and tetramer, we find evidence that the tetramer interface is formed by the interaction of D strands from one dimer unit and G strands from another dimer unit. These covalent labeling data along with molecular dynamics calculations allow the construction of a tetramer model that indicates how the protein might proceed to form even higher-order oligomers.

  12. Heparin-induced amyloid fibrillation of β2 -microglobulin explained by solubility and a supersaturation-dependent conformational phase diagram.

    PubMed

    So, Masatomo; Hata, Yasuko; Naiki, Hironobu; Goto, Yuji

    2017-05-01

    Amyloid fibrils are fibrillar deposits of denatured proteins associated with amyloidosis and are formed by a nucleation and growth mechanism. We revisited an alternative and classical view of amyloid fibrillation: amyloid fibrils are crystal-like precipitates of denatured proteins formed above solubility upon breaking supersaturation. Various additives accelerate and then inhibit amyloid fibrillation in a concentration-dependent manner, suggesting that the combined effects of stabilizing and destabilizing forces affect fibrillation. Heparin, a glycosaminoglycan and anticoagulant, is an accelerator of fibrillation for various amyloidogenic proteins. By using β2 -microglobulin, a protein responsible for dialysis-related amyloidosis, we herein examined the effects of various concentrations of heparin on fibrillation at pH 2. In contrast to previous studies that focused on accelerating effects, higher concentrations of heparin inhibited fibrillation, and this was accompanied by amorphous aggregation. The two-step effects of acceleration and inhibition were similar to those observed for various salts. The results indicate that the anion effects caused by sulfate groups are one of the dominant factors influencing heparin-dependent fibrillation, although the exact structures of fibrils and amorphous aggregates might differ between those formed by simple salts and matrix-forming heparin. We propose that a conformational phase diagram, accommodating crystal-like amyloid fibrils and glass-like amorphous aggregates, is important for understanding the effects of various additives. © 2017 The Protein Society.

  13. Comparison of the aggregation of homologous β2-microglobulin variants reveals protein solubility as a key determinant of amyloid formation

    PubMed Central

    Pashley, Clare L.; Hewitt, Eric W.; Radford, Sheena E.

    2016-01-01

    The mouse and human β2-microglobulin protein orthologs are 70 % identical in sequence and share 88 % sequence similarity. These proteins are predicted by various algorithms to have similar aggregation and amyloid propensities. However, whilst human β2m (hβ2m) forms amyloid-like fibrils in denaturing conditions (e.g. pH 2.5) in the absence of NaCl, mouse β2m (mβ2m) requires the addition of 0.3 M NaCl to cause fibrillation. Here, the factors which give rise to this difference in amyloid propensity are investigated. We utilise structural and mutational analyses, fibril growth kinetics and solubility measurements under a range of pH and salt conditions, to determine why these two proteins have different amyloid propensities. The results show that, although other factors influence the fibril growth kinetics, a striking difference in the solubility of the proteins is a key determinant of the different amyloidogenicity of hβ2m and mβ2m. The relationship between protein solubility and lag time of amyloid formation is not captured by current aggregation or amyloid prediction algorithms, indicating a need to better understand the role of solubility on the lag time of amyloid formation. The results demonstrate the key contribution of protein solubility in determining amyloid propensity and lag time of amyloid formation, highlighting how small differences in protein sequence can have dramatic effects on amyloid formation. PMID:26780548

  14. Immunomodulation by mucosal gene transfer using TGF-beta DNA.

    PubMed Central

    Kuklin, N A; Daheshia, M; Chun, S; Rouse, B T

    1998-01-01

    This report evaluates the efficacy of DNA encoding TGF-beta administered mucosally to suppress immunity and modulate the immunoinflammatory response to herpes simplex virus (HSV) infection. A single intranasal administration of an eukaryotic expression vector encoding TGF-beta1 led to expression in the lung and lymphoid tissue. T cell-mediated immune responses to HSV infection were suppressed with this effect persisting as measured by the delayed-type hypersensitivity reaction for at least 7 wk. Treated animals were more susceptible to systemic infection with HSV. Multiple prophylactic mucosal administrations of TGF-beta DNA also suppressed the severity of ocular lesions caused by HSV infection, although no effects on this immunoinflammatory response were evident after therapeutic treatment with TGF-beta DNA. Our results demonstrate that the direct mucosal gene transfer of immunomodulatory cytokines provides a convenient means of modulating immunity and influencing the expression of inflammatory disorders. PMID:9664086

  15. Role of Residual Kidney Function and Convective Volume on Change in β2-Microglobulin Levels in Hemodiafiltration Patients

    PubMed Central

    Penne, E. Lars; van der Weerd, Neelke C.; Blankestijn, Peter J.; van den Dorpel, Marinus A.; Grooteman, Muriel P.C.; Nubé, Menso J.; Lévesque, Renée; Bots, Michiel L.

    2010-01-01

    Background and objectives: Removal of β2-microglobulin (β2M) can be increased by adding convective transport to hemodialysis (HD). The aim of this study was to investigate the change in β2M levels after 6-mo treatment with hemodiafiltration (HDF) and to evaluate the role of residual kidney function (RKF) and the amount of convective volume with this change. Design, setting, participants, & measurements: Predialysis serum β2M levels were evaluated in 230 patients with and 176 patients without RKF from the CONvective TRAnsport STudy (CONTRAST) at baseline and 6 mo after randomization for online HDF or low-flux HD. In HDF patients, potential determinants of change in β2M were analyzed using multivariable linear regression models. Results: Mean serum β2M levels decreased from 29.5 ± 0.8 (±SEM) at baseline to 24.3 ± 0.6 mg/L after 6 mo in HDF patients and increased from 31.9 ± 0.9 to 34.4 ± 1.0 mg/L in HD patients, with the difference of change between treatment groups being statistically significant (regression coefficient −7.7 mg/L, 95% confidence interval −9.5 to −5.6, P < 0.001). This difference was more pronounced in patients without RKF as compared with patients with RKF. In HDF patients, β2M levels remained unchanged in patients with GFR >4.2 ml/min/1.73 m2. The β2M decrease was not related to convective volume. Conclusions: This study demonstrated effective lowering of β2M levels by HDF, especially in patients without RKF. The role of the amount of convective volume on β2M decrease appears limited, possibly because of resistance to β2M transfer between body compartments. PMID:19965537

  16. β2-microglobulin Normalization Within 6 months of Ibrutinib-based Treatment is Associated with Superior PFS in CLL

    PubMed Central

    Thompson, Philip A.; O’Brien, Susan M.; Xiao, Lianchun; Wang, Xuemei; Burger, Jan A.; Jain, Nitin; Ferrajoli, Alessandra; Estrov, Zeev; Keating, Michael J.; Wierda, William G.

    2016-01-01

    High pre-treatment β2-microglobulin (B2M) level is associated with inferior survival outcomes. However, the prognostic and predictive significance of changes in B2M during treatment have not been reported. We analyzed 83 patients treated with ibrutinib-based regimens (66 relapsed/refractory) and 198 treatment-naïve (TN) patients treated with combined fludarabine, cyclophosphamide and rituximab (FCR) to characterize change in B2M and their relationship to clinical outcomes. B2M rapidly fell during treatment with ibrutinib; in multivariable analysis (MVA), patients who received FCR [OR 0.40 (0.18–0.90), p=0.027] were less likely to normalize B2M at 6 months than patients treated with ibrutinib. On univariable analysis, normalization of B2M was associated with superior progression-free survival (PFS) from the 6-month landmark in patients treated with ibrutinib-based regimens and FCR. On MVA, failure to normalize B2M at 6 months of treatment was associated with inferior PFS [HR 16.9 (1.3–220.0), p=0.031] for ibrutinib-treated patients, after adjusting for the effects of baseline B2M, stage, fludarabine-refractory disease and del(17p). In contrast, in FCR-treated patients, bone marrow MRD-negative status was the only variable significantly associated with superior PFS [HR 0.28 (0.12–0.67), p=0.004]. Normalization of B2M at 6 months in ibrutinib-treated patients thus was a useful predictor of subsequent PFS and may assist clinical decision-making. PMID:26588193

  17. Many de novo donor-specific antibodies recognize β2 -microglobulin-free, but not intact HLA heterodimers.

    PubMed

    Michel, K; Santella, R; Steers, J; Sahajpal, A; Downey, F X; Thohan, V; Oaks, M

    2016-05-01

    Solid-phase single antigen bead (SAB) assays are standard of care for detection and identification of donor-specific antibody (DSA) in patients who receive solid organ transplantation (SOT). While several studies have documented the reproducibility and sensitivity of SAB testing for DSA, there are little data available concerning its specificity. This study describes the identification of antibodies to β(2) -microglobulin-free human leukocyte antigen (β(2) -m-fHLA) heavy chains on SAB arrays and provides a reassessment of the clinical relevance of DSA testing by this platform. Post-transplant sera from 55 patients who were positive for de novo donor-specific antibodies on a SAB solid-phase immunoassay were tested under denaturing conditions in order to identify antibodies reactive with β(2) -m-fHLA or native HLA (nHLA). Antibodies to β(2) -m-fHLA were present in nearly half of patients being monitored in the post-transplant period. The frequency of antibodies to β(2) -m-fHLA was similar among DSA and HLA antigens that were irrelevant to the transplant (non-DSA). Among the seven patients with clinical or pathologic antibody-mediated rejection (AMR), none had antibodies to β(2) -m-fHLA exclusively; thus, the clinical relevance of β(2) -m-fHLA is unclear. Our data suggests that SAB testing produces false positive reactions due to the presence of β(2) -m-fHLA and these can lead to inappropriate assignment of unacceptable antigens during transplant listing and possibly inaccurate identification of DSA in the post-transplant period.

  18. Comparison of the aggregation of homologous β2-microglobulin variants reveals protein solubility as a key determinant of amyloid formation.

    PubMed

    Pashley, Clare L; Hewitt, Eric W; Radford, Sheena E

    2016-02-13

    The mouse and human β2-microglobulin protein orthologs are 70% identical in sequence and share 88% sequence similarity. These proteins are predicted by various algorithms to have similar aggregation and amyloid propensities. However, whilst human β2m (hβ2m) forms amyloid-like fibrils in denaturing conditions (e.g. pH2.5) in the absence of NaCl, mouse β2m (mβ2m) requires the addition of 0.3M NaCl to cause fibrillation. Here, the factors which give rise to this difference in amyloid propensity are investigated. We utilise structural and mutational analyses, fibril growth kinetics and solubility measurements under a range of pH and salt conditions, to determine why these two proteins have different amyloid propensities. The results show that, although other factors influence the fibril growth kinetics, a striking difference in the solubility of the proteins is a key determinant of the different amyloidogenicity of hβ2m and mβ2m. The relationship between protein solubility and lag time of amyloid formation is not captured by current aggregation or amyloid prediction algorithms, indicating a need to better understand the role of solubility on the lag time of amyloid formation. The results demonstrate the key contribution of protein solubility in determining amyloid propensity and lag time of amyloid formation, highlighting how small differences in protein sequence can have dramatic effects on amyloid formation. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  19. Predicting residual kidney function in hemodialysis patients using serum β-trace protein and β2-microglobulin.

    PubMed

    Wong, Jonathan; Sridharan, Sivakumar; Berdeprado, Jocelyn; Vilar, Enric; Viljoen, Adie; Wellsted, David; Farrington, Ken

    2016-05-01

    Residual kidney function (RKF) contributes significant solute clearance in hemodialysis patients. Kidney Diseases Outcomes Quality Initiative (KDOQI) guidelines suggest that hemodialysis dose can be safely reduced in those with residual urea clearance (KRU) of 2 ml/min/1.73 m(2) or more. However, serial measurement of RKF is cumbersome and requires regular interdialytic urine collections. Simpler methods for assessing RKF are needed. β-trace protein (βTP) and β2-microglobulin (β2M) have been proposed as alternative markers of RKF. We derived predictive equations to estimate glomerular filtration rate (GFR) and KRU based on serum βTP and β2M from 191 hemodialysis patients based on standard measurements of KRU and GFR (mean of urea and creatinine clearances) using interdialytic urine collections. These modeled equations were tested in a separate validation cohort of 40 patients. A prediction equation for GFR that includes both βTP and β2M provided a better estimate than either alone and contained the terms 1/βTP, 1/β2M, 1/serum creatinine, and a factor for gender. The equation for KRU contained the terms 1/βTP, 1/β2M, and a factor for ethnicity. Mean bias between predicted and measured GFR was 0.63 ml/min and 0.50 ml/min for KRU. There was substantial agreement between predicted and measured KRU at a cut-off level of 2 ml/min/1.73 m(2). Thus, equations involving βTP and β2M provide reasonable estimates of RKF and could potentially be used to identify those with KRU of 2 ml/min/1.73 m(2) or more to follow the KDOQI incremental hemodialysis algorithm.

  20. The linkage arrangement of four rabbit beta-like globin genes.

    PubMed

    Lacy, E; Hardison, R C; Quon, D; Maniatis, T

    1979-12-01

    Four different regions of rabbit beta-like globin gene sequences designated beta 1, beta 2, beta 3 and beta 4 were identified in a set of clones isolated from a bacteriophage lambda library of chromosomal DNA fragments (Maniatis et al., 1978). Restriction mapping and blot hybridization (Southern, 1975) studies indicate that a subset of these clones containing beta 1 and beta 2 hybridizes to an adult beta-globin cDNA clone (Maniatis et al., 1976) more efficiently than to a human gamma-globin cDNA clone (Wilson et al., 1978), while another subset containing beta 3 and beta 4 displays the converse hybridization specificity. beta 1 was identified as the adult beta-globin gene, while beta 2, beta 3 and beta 4 have not been identified with any known rabbit globin polypeptides. Cross-hybridization and transcriptional orientation experiments indicate that the set of beta-like gene clones contains overlapping restriction fragments encompassing 44 kb of rabbit chromosomal DNA. In addition, all four genes have the same transcriptional orientation and are arranged in the order 5'-beta 4-beta 3-beta 2-beta 1-3'.

  1. Correction of human. beta. sup S -globin gene by gene targeting

    SciTech Connect

    Shesely, E.G.; Hyungsuk Kim; Shehee, W.R.; Smithies, O. ); Papayannopoulou, T. ); Popovich, B.W. )

    1991-05-15

    As a step toward using gene targeting for gene therapy, the authors have corrected a human {beta}{sup S}-globin gene to the normal {beta}{sup A} allele by homologous recombination in the mouse-human hybrid cell line BSM. BSM is derived from a mouse erythroleukemia cell line and carries a single human chromosome 11 with the {beta}{sup S}-globin allele. A {beta}{sup A}-globin targeting construct containing a unique oligomer and a neomycin-resistance gene was electroporated into the BSM cells, which were then placed under G418 selection. Then 126 resulting pools containing a total {approx}29,000 G418-resistant clones were screened by PCR for the presence of a targeted recombinant: 3 positive pools were identified. A targeted clone was isolated by replating one of the positive pools into smaller pools and rescreening by PCR, followed by dilution cloning. Southern blot analysis demonstrated that the isolated clone had been targeted as planned. The correction of the {beta}{sup S} allele to {beta}{sup A} was confirmed both by allele-specific PCR and by allele-specific antibodies. Expression studies comparing the uninduced and induced RNA levels in unmodified BSM cells and in the targeted clone showed no significant alteration in the ability of the targeted clone to undergo induction, despite the potentially disrupting presence of a transcriptionally active neomycin gene 5{prime} to the human {beta}{sup A}-globin gene. Thus gene targeting can correct a {beta}{sup S} allele to {beta}{sup A}, and the use of a selectable helper gene need not significantly interfere with the induction of the corrected gene.

  2. Genomic organization of the mouse T-cell receptor beta-chain gene family.

    PubMed Central

    Lai, E; Barth, R K; Hood, L

    1987-01-01

    We have combined three different methods, deletion mapping of T-cell lines, field-inversion gel electrophoresis, and the restriction mapping of a cosmid clone, to construct a physical map of the murine T-cell receptor beta-chain gene family. We have mapped 19 variable (V beta) gene segments and the two clusters of diversity (D beta) and joining (J beta) gene segments and constant (C beta) genes. These members of the beta-chain gene family span approximately equal to 450 kilobases of DNA, excluding one potential gap in the DNA fragment alignments. Images PMID:3035555

  3. The genomic structure of the gene encoding the human transforming growth factor {beta} type II receptor (TGF-{beta} RII)

    SciTech Connect

    Takenoshita, Seiichi; Hagiwara, Koichi; Nagashima, Makoto; Gemma, Akihiko

    1996-09-01

    The genomic structure of the human transforming growth factor-{beta} type II receptor gene (TGF-{beta} RII) was determined by two PCR-based methods, the {open_quotes}long distance sequencer{close_quotes} method and the {open_quotes}promoter finder{close_quotes} method. Genomic fragments containing exons and adjacent introns were amplified by PCR, and the nucleotide sequences were determined by direct sequencing and subcloning sequencing. The TGF-{beta} RII protein is encoded by 567 codons in 7 exons. This is the first report about the genomic structure of a gene that belongs to the serine/threonine kinase type II receptor subfamily. Knowledge of the genomic structure of the TGF-{beta} RII gene will facilitate investigation of the TGF-{beta} RII gene will facilitate investigation of the TGF-{beta} signaling pathway in normal human cells and of the aberrations occurring during carcinogenesis. 18 refs., 2 figs., 1 tab.

  4. Beta globin gene cluster haplotypes of the beta thalassemia mutations observed in Denizli province of Turkey.

    PubMed

    Bahadır, Anzel; Öztürk, Onur; Atalay, Ayfer; Atalay, Erol Ömer

    2009-09-05

    Our aim is to identify the beta globin gene cluster haplotypes for the beta thalassemia mutations in Turkey at regional basis. Beta thalassemia mutations included in this study were IVS-I-110 (G>A), FSC 8/9 (+G), IVS-II-1 (G>A), IVS-I-5 (G>C), IVS-I-1 (G>A), IVS-I-6 (T>C) and FSC 8 (-AA). We studied 22 unrelated patients with β-thalassemia major and 72 unrelated healthy subjects from our Department's DNA bank. Haplotype analysis was done by polymerase chain reaction (PCR)-based restriction enzyme digestion for the beta globin gene cluster of the following polymorphic restriction sites: Hinc II 5' to ε, Hind III 5' to Gγ, Hind III in the IVS-II 5' to Aγ, Hinc II in pseudo β, Hinc II 3' to pseudo β, Ava II in β, Hinf I 3' to β. Associated haplotypes for the normal control samples (72 individuals, 144 chromosomes) were determined by Arlequin 3.1 software with unknown gametic phase. According to the results obtained, the most frequent beta globin gene cluster haplotypes in the normal population are (+----++), (+----+-), (-+-++++), (+-----+) with the frequencies of 28.6 %, 17.2 %, 9.8 % and 8.3 % respectively. IVS-I-110 mutation is linked with the haplotypes (+----++) and (+-----+). Observed haplotypes are (+----++) for FSC 8/9 (+G), (-+-+++-) for IVS-II-1 (G>A), (-+-++-+ and -+-++++) for IVS-I-5 (G>C), (+----+- and +------) for IVS-I-1 (G>A), (-++---+) for IVS-I-6 (T>C) and (+-----+) for FSC 8 (-AA). In conclusion, our region shows the Mediterranean character for the beta thalassemia mutations. According to the obtained results, IVS-I-110 (G>A) mutation linked with haplotype VII (+-----+), IVS-I-5 (G>C) mutation with haplotype IV (-+-++-+), codon 8/9 (+G) linked with haplotype I (+----++) were shown for the first time in Turkish population. The linkage of haplotype (+------) with the IVS-I-1 (G>A) mutation is reported for the first time in the published literature. In Denizli province of Turkey, beta globin gene cluster haplotypes of the normal population are

  5. Characterization of the mouse lymphotoxin-beta gene.

    PubMed

    Lawton, P; Nelson, J; Tizard, R; Browning, J L

    1995-01-01

    Lymphotoxin-beta (LT-beta) is a member of the TNF family of ligands which when expressed with lymphotoxin-alpha (LT-alpha, i.e., the original LT or TNF-beta) forms a heteromeric complex with LT-alpha on the cell surface. The mouse gene structure was determined by both cDNA cloning and analysis of a genomic DNA fragment encompassing the TNF/LT locus in the H-2 region of chromosome 17. The mouse and human genomic structures were found to be similar in terms of location in the class III region of the MHC; however, the mouse gene lacks one intron found in most members of the family. Both the cDNA and the genomic sequences revealed an altered splice donor in the conventional intron 2 position, rendering it nonfunctional. The altered gene retains an open reading frame such that an additional 66 amino acids are inserted into the stalk region connecting the transmembrane domain with the receptor binding domain encoded by exon 4 in this type II membrane protein. Northern analysis showed that this gene is expressed predominantly in lymphoid organs. The outlining of the complete mouse TNF locus will further studies of the relationship between these genes and immune function.

  6. Genomic effects of IFN-beta in multiple sclerosis patients.

    PubMed

    Weinstock-Guttman, Bianca; Badgett, Darlene; Patrick, Kara; Hartrich, Laura; Santos, Roseane; Hall, Dennis; Baier, Monika; Feichter, Joan; Ramanathan, Murali

    2003-09-01

    The purpose of this report was to characterize the dynamics of the gene expression cascades induced by an IFN-beta-1a treatment regimen in multiple sclerosis patients and to examine the molecular mechanisms potentially capable of causing heterogeneity in response to therapy. In this open-label pharmacodynamic study design, peripheral blood was obtained from eight relapsing-remitting multiple sclerosis patients just before and at 1, 2, 4, 8, 24, 48, 120, and 168 h after i.m. injection of 30 micro g of IFN-beta-1a. The total RNA was isolated from monocyte-depleted PBL and analyzed using cDNA microarrays containing probes for >4000 known genes. IFN-beta-1a treatment resulted in selective, time-dependent effects on multiple genes. The mRNAs for genes implicated in the anti-viral response, e.g., double-stranded RNA-dependent protein kinase, myxovirus resistance proteins 1 and 2, and guanylate binding proteins 1 and 2 were rapidly induced within 1-4 h of IFN-beta treatment. The mRNAs for several genes involved in IFN-beta signaling, such as IFN-alpha/beta receptor-2 and Stat1, were also increased. The mRNAs for lymphocyte activation markers, such as IFN-induced transmembrane protein 1 (9-27), IFN-induced transmembrane protein 2 (1-8D), beta(2)-microglobulin, and CD69, were also increased in a time-dependent manner. The findings demonstrate that IFN-beta treatment induces specific and time-dependent changes in multiple mRNAs in lymphocytes of multiple sclerosis patients that could provide a framework for rapid monitoring of the response to therapy.

  7. Transcriptional activation of cloned human beta-globin genes by viral immediate-early gene products.

    PubMed

    Green, M R; Treisman, R; Maniatis, T

    1983-11-01

    When the human beta-globin gene is transfected into Hela cells, no beta-globin RNA is detected unless the gene is linked to a viral transcription enhancer. In this paper we show that trans-acting adenovirus and herpesvirus (pseudorabies) transcriptional regulatory proteins can circumvent this enhancer requirement for detectable beta-globin transcription in transient expression assays. The viral gene products can be provided by constitutively expressed, integrated viral genes in established cell lines, by viral infection of permissive cells, or by transfection of cells with bacterial plasmids carrying the viral immediate-early genes. These results demonstrate the utility of transient expression assays for studying regulatory mechanisms involving trans-acting factors. Analysis of beta-globin promoter mutants indicates that between 75 and 128 bp of sequence 5' to the mRNA cap site is required for enhancer-dependent transcription in Hela cells. In contrast, beta-globin transcription in the presence of viral immediate-early gene products requires only 36 bp of 5'-flanking sequence, which includes the TATA box. Thus both cis and trans-acting viral factors activate beta-globin gene transcription in transient expression experiments, but the mechanisms by which they act appear to be fundamentally different.

  8. Urine β2-microglobulin is associated with clinical disease activity and renal involvement in female patients with systemic lupus erythematosus.

    PubMed

    Choe, J-Y; Park, S-H; Kim, S-K

    2014-12-01

    We investigated the association of serum and urine β2-microglobulin (β2MG) with renal involvement and clinical disease activity in systemic lupus erythematosus (SLE). Sixty-four female patients with SLE were enrolled. We assessed SLE disease activity (SLEDAI)-2K and measured serum and urine β2MG levels, as well as complement (C3 and C4) and anti-dsDNA levels. According to the SLEDAI scores, two groups were categorized: low (0-5 of SLEDAI) and high (6-19 of SLEDAI) disease activity groups. The presence of renal involvement was determined by renal SLEDAI score. Statistical analysis was performed using Spearman's correlation analysis, Mann-Whitney U test, multivariate regression analysis, and logistic regression analysis. Urine β2MG levels were significantly different between low and high SLEDAI groups (p = 0.001), but not for serum β2MG levels (p = 0.579). Patients with renal involvement showed higher urine β2MG levels compared to those without renal involvement (p < 0.001), but again there was not a difference in serum β2MG levels (p = 0.228). Urine β2MG was closely associated with SLEDAI (r = 0.363, p = 0.003), renal SLEDAI (r = 0.479, p < 0.001), urine protein/Cr (r = 0.416, p = 0.001), and ESR (r = 0.347, p = 0.006), but not serum β2MG (r = 0.245, p = 0.051). Urine β2MG level was identified as a surrogate for renal involvement (p = 0.009, OR = 1.017, 95% CI 1.004-1.030) and overall disease activity (p = 0.009, OR = 1.020, 95% CI 1.005-1.036). We demonstrated that urine β2MG levels are associated with renal involvement and overall clinical disease activity in SLE. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  9. The role of EKLF in human beta-globin gene competition.

    PubMed

    Wijgerde, M; Gribnau, J; Trimborn, T; Nuez, B; Philipsen, S; Grosveld, F; Fraser, P

    1996-11-15

    We have investigated the role of erythroid Kruppel-like factor (EKLF) in expression of the human beta-globin genes in compound EKLF knockout/human beta-locus transgenic mice. EKLF affects only the adult mouse beta-globin genes in homozygous knockout mice; heterozygous mice are unaffected. Here we show that EKLF knockout mice express the human epsilon and gamma-globin genes normally in embryonic red cells. However, fetal liver erythropoiesis, which is marked by a period of gamma- and beta-gene competition in which the genes are alternately transcribed, exhibits an altered ratio of gamma- to beta-gene transcription. EKLF heterozygous fetal livers display a decrease in the number of transcriptionally active beta genes with a reciprocal increase in the number of transcriptionally active gamma genes. beta-Gene transcription is absent in homozygous knockout fetuses with coincident changes in chromatin structure at the beta promoter. There is a further increase in the number of transcriptionally active gamma genes and accompanying gamma gene promoter chromatin alterations. These results indicate that EKLF plays a major role in gamma- and beta-gene competition and suggest that EKLF is important in stabilizing the interaction between the Locus Control Region and the beta-globin gene. In addition, these findings provide further evidence that developmental modulation of globin gene expression within individual cells is accomplished by altering the frequency and/or duration of transcriptional periods of a gene rather than changing the rate of transcription.

  10. Distribution of beta-glucosidase and beta-glucuronidase activity and of beta-glucuronidase gene gus in human colonic bacteria.

    PubMed

    Dabek, Marta; McCrae, Sheila I; Stevens, Valerie J; Duncan, Sylvia H; Louis, Petra

    2008-12-01

    beta-Glycosidase activities present in the human colonic microbiota act on glycosidic plant secondary compounds and xenobiotics entering the colon, with potential health implications for the human host. Information on beta-glycosidases is currently limited to relatively few species of bacteria from the human colonic ecosystem. We therefore screened 40 different bacterial strains that are representative of dominant bacterial groups from human faeces for beta-glucosidase and beta-glucuronidase activity. More than half of the low G+C% Gram-positive firmicutes harboured beta-glucosidase activity, while beta-glucuronidase activity was only found in some firmicutes within clostridial clusters XIVa and IV. Most of the Bifidobacterium spp. and Bacteroides thetaiotaomicron carried beta-glucosidase activity. A beta-glucuronidase gene belonging to family 2 glycosyl hydrolases was detected in 10 of the 40 isolates based on degenerate PCR. These included all nine isolates that gave positive assays for beta-glucuronidase activity, suggesting that the degenerate PCR could provide a useful assay for the capacity to produce beta-glucuronidase in the gut community. beta-Glucuronidase activity was induced by growth on d-glucuronic acid, or by addition of 4-nitrophenol-glucuronide, in Roseburia hominis A2-183, while beta-glucosidase activity was induced by 4-nitrophenol-glucopyranoside. Inducibility varied between strains.

  11. Capillary electrophoresis analysis of different variants of the amyloidogenic protein β2 -microglobulin as a simple tool for misfolding and stability studies.

    PubMed

    Bertoletti, Laura; Bisceglia, Federica; Colombo, Raffaella; Giorgetti, Sofia; Raimondi, Sara; Mangione, P Patrizia; De Lorenzi, Ersilia

    2015-10-01

    Free solution capillary electrophoresis with UV detection is here used to retrieve information on the conformational changes of wild-type β2 -microglobulin and a series of naturally and artificially created variants known to have different stability and amyloidogenic potential. Under nondenaturing conditions, the resolution of at least two folding conformers at equilibrium is obtained and a third species is detected for the less stable isoforms. Partial denaturation by using chaotropic agents such as acetonitrile or trifluoroethanol reveals that the separated peaks are at equilibrium, as the presence of less structured species is either enhanced or induced at the expenses of the native form. Reproducible CE data allow to obtain an interesting semiquantitative correlation between the peak areas observed and the protein stability. Thermal unfolding over the range 25-42°C is induced inside the capillary for the two pathogenic proteins (wtβ2 -microglobulin and D76N variant): the large differences observed upon small temperature variation draw attention on the robustness of analytical methods when dealing with proteins prone to misfolding and aggregation.

  12. Cloning of alpha-beta fusion gene from Clostridium perfringens and its expression.

    PubMed

    Bai, Jia-Ning; Zhang, Yan; Zhao, Bao-Hua

    2006-02-28

    To study the cloning of alpha-beta fusion gene from Clostridium perfringens and the immunogenicity of alpha-beta fusion expression. Cloning was accomplished after PCR amplification from strains NCTC64609 and C58-1 of the protective antigen genes of alpha-toxin and beta-toxin. The fragment of the gene was cloned using plasmid pZCPAB. This fragment coded for the gene with the stable expression of alpha-beta fusion gene binding. In order to verify the exact location of the alpha-beta fusion gene, domain plasmids were constructed. The two genes were fused into expression vector pBV221. The expressed alpha-beta fusion protein was identified by ELISA, SDS-PAGE, Western blotting and neutralization assay. The protective alpha-toxin gene (cpa906) and the beta-toxin gene (cpb930) were obtained. The recombinant plasmid pZCPAB carrying alpha-beta fusion gene was constructed and transformed into BL21(DE3). The recombinant strain BL21(DE3)(pZCPAB) was obtained. After the recombinant strain BL21(DE3)(pZCPAB) was induced by 42 degC, its expressed product was about 22.14% of total cellular protein at SDS-PAGE and thin-layer gel scanning analysis. Neutralization assay indicated that the antibody induced by immunization with alpha-beta fusion protein could neutralize the toxicity of alpha-toxin and beta-toxin. The obtained alpha-toxin and beta-toxin genes are correct. The recombinant strain BL21(DE3)(pZCPAB) could produce alpha-beta fusion protein. This protein can be used for immunization and is immunogenic. The antibody induced by immunization with alpha-beta fusion protein could neutralize the toxicity of alpha-toxin and beta-toxin.

  13. Design of retrovirus vectors for transfer and expression of the human. beta. -globin gene

    SciTech Connect

    Miller, A.D.; Bender, M.A.; Harris, E.A.S.; Kaleko, M.; Gelinas, R.E.

    1988-11-01

    Regulated expression of the human ..beta..-globin gene has been demonstrated in cultured murine erythroleukemia cells and in mice after retrovirus-mediated gene transfer. However, the low titer of recombinant viruses described to date results in relatively inefficient gene transfer, which limits their usefulness for animal studies and for potential gene therapy in humans for diseases involving defective ..beta..-globin genes. The authors found regions that interfered with virus production within intron 2 of the ..beta..-globin gene and on both sides of the gene. The flanking regions could be removed, but intron 2 was required for ..beta..-globin expression. Inclusion of ..beta..-globin introns necessitates an antisense orientation of the gene within the retrovirus vector. However, they found no effect of the antisense ..beta..-globin transcription on virus production. A region downstream of the ..beta..-globin gene that stimulates expression of the gene in transgenic mice was included in the viruses without detrimental effects on virus titer. Virus titers of over 10/sup 6/ CFU/ml were obtained with the final vector design, which retained the ability to direct regulated expression of human ..beta..-globin in murine erythroleukemia cells. The vector also allowed transfer and expression of the human ..beta..-globin gene in hematopoietic cells (CFU-S cells) in mice.

  14. [Fibrinogen beta chain gene mutation contributes to one congenital afibrinogenemia].

    PubMed

    Xu, Xiu-cai; Zhou, Rong-fu; Wu, Jing-sheng; Fang, Yi; Wang, Xue-feng; Zhai, Zhi-min; Wang, Hong-li

    2005-03-01

    To identify the fibrinogen (Fg) gene mutations in a Chinese pedigree of congenital afibrinogenemia. The plasma Fg activity and protein of the proband and his family members were detected. Genomic DNA was isolated from the peripheral blood mononuclear cells. All the exons and exon-intron boundaries of fibrinogen gene were amplified by PCR and sequenced thereafter. Two mutations, 7972 del G in FGB and T2543A in FGG, were found in the proband. FGG2543 is a polymorphism site, which lead to the polymorphism of gamma144 I/K. The G deletion at base 7972 of FGB contributes to the frameshift mutation after amino acid 419, resulting in the truncated beta chain without the terminal 27 amino acids. The latter may contributes to the pathogenetic mechanisms in Chinese congenital afibrinogenemia patients. The G deletion at base 7972 of FGB is identified for the first time.

  15. Characterization of two divergent beta-tubulin genes from Colletotrichum graminicola.

    PubMed

    Panaccione, D G; Hanau, R M

    1990-02-14

    We have cloned and sequenced two beta-tubulin genes, TUB1 and TUB2, from the phytopathogenic fungus, Colletotrichum graminicola. The nucleotide sequences of the coding regions of the two genes are only 72.8% homologous. This divergence is reflected in the deduced amino acid (aa) sequences which differ at 94 aa residues. Comparison with the aa sequences of other fungal beta-tubulins indicates that the C. graminicola TUB2 gene encodes a conserved isotype, whereas the C. graminicola TUB1 product is highly divergent. Both genes contain six identically placed introns and the position of each intron is conserved in other fungal beta-tubulin genes. Also typical of other fungal beta-tubulin genes, there is a pronounced bias in codon usage in the C. graminicola TUB2 gene; there is a lesser codon bias in TUB1 from C. graminicola. Both C. graminicola beta-tubulin genes are transcribed and yield similar sized messages.

  16. Lactogenic hormonal induction of long distance interactions between beta-casein gene regulatory elements

    USDA-ARS?s Scientific Manuscript database

    Lactogenic hormone regulation of beta-casein gene expression in mammary epithelial cells provides, an excellent model in which to study the mechanisms by which steroid and peptide hormone signaling control gene expression. Prolactin- and glucocorticoid-mediated induction of beta-casein gene express...

  17. Genomic evidence for independent origins of beta-like globin genes in monotremes and therian mammals.

    PubMed

    Opazo, Juan C; Hoffmann, Federico G; Storz, Jay F

    2008-02-05

    Phylogenetic reconstructions of the beta-globin gene family in vertebrates have revealed that developmentally regulated systems of hemoglobin synthesis have been reinvented multiple times in independent lineages. For example, the functional differentiation of embryonic and adult beta-like globin genes occurred independently in birds and mammals. In both taxa, the embryonic beta-globin gene is exclusively expressed in primitive erythroid cells derived from the yolk sac. However, the "epsilon-globin" gene in birds is not orthologous to the epsilon-globin gene in mammals, because they are independently derived from lineage-specific duplications of a proto beta-globin gene. Here, we report evidence that the early and late expressed beta-like globin genes in monotremes and therian mammals (marsupials and placental mammals) are the products of independent duplications of a proto beta-globin gene in each of these two lineages. Results of our analysis of genomic sequence data from a large number of vertebrate taxa, including sequence from the recently completed platypus genome, reveal that the epsilon- and beta-globin genes of therian mammals arose via duplication of a proto beta-globin gene after the therian/monotreme split. Our analysis of genomic sequence from the platypus also revealed the presence of a duplicate pair of beta-like globin genes that originated via duplication of a proto beta-globin gene in the monotreme lineage. This discovery provides evidence that, in different lineages of mammals, descendent copies of the same proto beta-globin gene may have been independently neofunctionalized to perform physiological tasks associated with oxygen uptake and storage during embryonic development.

  18. In vivo analysis of the murine beta-myosin heavy chain gene promoter.

    PubMed

    Rindt, H; Gulick, J; Knotts, S; Neumann, J; Robbins, J

    1993-03-05

    The 5' upstream region of the murine beta-myosin heavy chain (MHC) gene has been isolated and tested for its ability to drive gene expression in transgenic mice. Three classes of transgenic mice were generated. The constructs contained approximately 5000, 2500, and 600 base pairs of beta-MHC upstream sequence fused to the chloramphenicol acetyltransferase gene and were termed beta 5, beta 2.5, and beta .6, respectively. Muscle-specific expression was observed with all three constructs. However, only the beta 5 lines directed high levels of muscle-specific transgene expression in both pre- and postbirth mice. Expression driven by the two shorter constructs was two to three orders of magnitude lower when the chloramphenicol acetyltransferase specific activities were compared. These data suggest that a distal-positive element directs high levels of gene expression in the ventricle and in slow skeletal muscles. Analyses of transgene expression during heart maturation revealed that some of the beta 5 lines were not able to respond in an appropriate manner to developmental transcriptional cues. Unlike the endogenous beta-MHC gene, which is down regulated in the ventricles around the time of birth, reporter gene expression in the majority of the lines generated was not shut off in the ventricles of the adult animals. These data indicate that high levels of muscle-specific beta-MHC gene expression are dependent upon the combinatorial interactions of a number of sequence elements that are distributed over a large region of the gene's upstream sequence.

  19. Lack of neighborhood effects from a transcriptionally active phosphoglycerate kinase-neo cassette located between the murine beta-major and beta-minor globin genes.

    PubMed

    Kaufman, R M; Lu, Z H; Behl, R; Holt, J M; Ackers, G K; Ley, T J

    2001-07-01

    For the treatment of beta-globin gene defects, a homologous recombination-mediated gene correction approach would provide advantages over random integration-based gene therapy strategies. However, "neighborhood effects" from retained selectable marker genes in the targeted locus are among the key issues that must be taken into consideration for any attempt to use this strategy for gene correction. An Ala-to-Ile mutation was created in the beta6 position of the mouse beta-major globin gene (beta(6I)) as a step toward the development of a murine model system that could serve as a platform for therapeutic gene correction studies. The marked beta-major gene can be tracked at the level of DNA, RNA, and protein, allowing investigation of the impact of a retained phosphoglycerate kinase (PGK)-neo cassette located between the mutant beta-major and beta-minor globin genes on expression of these 2 neighboring genes. Although the PGK-neo cassette was expressed at high levels in adult erythroid cells, the abundance of the beta(6I) mRNA was indistinguishable from that of the wild-type counterpart in bone marrow cells. Similarly, the output from the beta-minor globin gene was also normal. Therefore, in this specific location, the retained, transcriptionally active PGK-neo cassette does not disrupt the regulated expression of the adult beta-globin genes. (Blood. 2001;98:65-73)

  20. Total beta-globin gene deletion has high frequency in Filipinos

    SciTech Connect

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

    1994-09-01

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

  1. Intestinal lactase as an autologous beta-galactosidase reporter gene for in vivo gene expression studies.

    PubMed

    Salehi, Siamak; Eckley, Lorna; Sawyer, Greta J; Zhang, Xiaohong; Dong, Xuebin; Freund, Jean-Noel; Fabre, John W

    2009-01-01

    Intestinal lactase has potential as an autologous beta-galactosidase reporter gene for long-term gene expression studies in vivo, using chromogenic, luminescent, and fluorogenic substrates developed for Escherichia coli beta-galactosidase. In normal rat tissues, reactivity with a chromogenic fucopyranoside (X-Fuc, the preferred substrate of lactase) was present only at the lumenal surface of small intestine epithelial cells. Full-length lactase (domains I-IV), mature lactase (domains III and IV), and a cytosolic form of mature lactase (domains III and IV, without the signal sequence or transmembrane region) were evaluated. Transfection of HuH-7 cells in vitro, and hydrodynamic gene delivery to the liver in vivo, resulted in excellent gene expression. The full-length and mature (homodimeric, membrane-bound) forms reacted strongly with X-Fuc but not with the corresponding galactopyranoside (X-Gal). However, the presumptively monomeric cytosolic lactase unexpectedly reacted equally well with both substrates. The fluorogenic substrate fluorescein-di-beta-D-galactopyranoside was cleaved by cytosolic lactase, but not by full-length or mature lactase. Full-length lactase, when expressed ectopically in hepatocytes in vivo, localized exclusively to the bile canalicular membrane. Intestinal lactase is highly homologous in mice, rats, and humans and has considerable potential for evaluating long-term gene expression in experimental animals and the clinic.

  2. Dopamine beta-hydroxylase gene modulates individuals’ empathic ability

    PubMed Central

    Gong, Pingyuan; Liu, Jinting; Li, She

    2014-01-01

    Dopamine beta-hydroxylase (DBH), an enzyme that converts dopamine to norepinephrine, has broad influences on social functions. In this study, we examined to what extent two polymorphisms (−1021C/T and a 19 bp insertion/deletion) in DBH gene modulate individuals’ empathic perception and response, which were measured, respectively, by reading the mind in the eyes test and the empathic concern subscale of interpersonal reactivity index. Results showed that polymorphism at −1021C/T, but not the 19 bp insertion/deletion, accounts for 2.3% variance of empathic perception and 1.4% variance of empathic response. Individuals with the CC genotype, which is associated with higher DBH activity, manifested greater empathic ability than those with CT/TT genotypes. These findings demonstrate the importance of DBH −1021C/T as a genetic basis of empathy and in predicting individual differences in social and affective processing. PMID:23988761

  3. [Nucleotide sequence of genes for alpha- and beta-subunits of luciferase from Photobacterium leiognathi].

    PubMed

    Illarionov, B A; Protopopova, M V; Karginov, V A; Mertvetsov, N P; Gitel'zon, I I

    1988-03-01

    Nucleotide sequence of the Photobacterium leiognathi DNA containing genes of alpha and beta subunits of luciferase has been determined. We also deduced amino acid sequence and molecular mass of luciferase and localized luciferase genes in the sequenced DNA fragment.

  4. Chromosome mapping of the human arrestin (SAG), {beta}-arrestin 2 (ARRB2), and {beta}-adrenergic receptor kinase 2 (ADRBK2) genes

    SciTech Connect

    Calabrese, G.; Sallese, M.; Stornaiuolo, A.

    1994-09-01

    Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, {beta}-arrestin. Both {beta}ARK and {beta}-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, {beta}ARK1, {beta}ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/{beta}-arrestin gene family includes arrestin (also known as S-antigen), {beta}-arrestin 1, and {beta}-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), {beta}arrestin 2 (ARRB2), and {beta}ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment of the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for {beta}-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for {beta}ARK2 (ADRBK2) to chromosome 22q11. 17 refs., 1 fig.

  5. The spectrum of beta-globin gene mutations in children with beta-thalassaemia major from Kota Kinabalu, Sabah, Malaysia.

    PubMed

    Thong, M K; Soo, T L

    2005-07-01

    Beta-thalassaemia major is one of the commonest genetic disorders in South East Asia. The strategy for the community control of beta-thalassaemia major requires the characterisation of the spectrum of beta-globin gene mutations in any multi-ethnic population. There is only a single report of mutation analyses of the beta-globin gene in an isolated Kadazandusun community in Kota Belud, Sabah, Malaysia, which showed the presence of a common 45 kb deletion. To confirm the observation that this large deletion is the commonest beta-globin gene mutation among the Kadazandusun and other indigenous populations in Sabah, Malaysia, we performed polymerase chain reaction (PCR) analysis of the beta-globin gene in ten children with beta-thalassaemia major attending the Thalassaemia Centre, Queen Elizabeth Hospital, the major paediatric referral centre in Kota Kinabalu, Sabah. The 45 kb deletion was confirmed to be the commonest mutation found in the Kadazandusun, Bajau and Murut populations, whereby it was detected in 19 out of the 20 (95 percent) alleles analysed. The other mutation was due to an IVS-1 position 1 G > T mutation. This finding confirmed the deletion in the homozygous state was associated with a severe phenotype. The reason for the predominance of this mutation in Kota Kinabalu is most likely to be due to founder effects and possibly intermarriages between the various ethnic groups. Prenatal diagnosis using PCR for this common mutation is feasible in this community. Medical workers and scientists at molecular diagnostic centres serving large South East Asian populations should incorporate a diagnostic strategy for this deletion in the appropriate population. Future studies on these indigenous ethnic groups in other areas and other groups in Sabah are required.

  6. Molecular analysis of the beta-globin gene cluster in the Niokholo Mandenka population reveals a recent origin of the beta(S) Senegal mutation.

    PubMed

    Currat, Mathias; Trabuchet, Guy; Rees, David; Perrin, Pascale; Harding, Rosalind M; Clegg, John B; Langaney, André; Excoffier, Laurent

    2002-01-01

    A large and ethnically well-defined Mandenka sample from eastern Senegal was analyzed for the polymorphism of the beta-globin gene cluster on chromosome 11. Five RFLP sites of the 5' region were investigated in 193 individuals revealing the presence of 10 different haplotypes. The frequency of the sickle-cell anemia causing mutation (beta(S)) in the Mandenka estimated from this sample is 11.7%. This mutation was found strictly associated with the single Senegal haplotype. Approximately 600 bp of the upstream region of the beta-globin gene were sequenced for a subset of 94 chromosomes, showing the presence of four transversions, five transitions, and a composite microsatellite polymorphism. The sequence of 22 beta(S) chromosomes was also identical to the previously defined Senegal haplotype, suggesting that this mutation is very recent. Monte Carlo simulations (allowing for a specific balancing selection model, a logistic growth of the population, and variable initial frequencies of the Senegal haplotype) were used to estimate the age of the beta(S) mutation. Resulting maximum-likelihood estimates are 45-70 generations (1,350-2,100 years) for very different demographic scenarios. Smallest confidence intervals (25-690 generations) are obtained under the hypothesis that the Mandenka population is large (N(e) >5,000) and stationary or that it has undergone a rapid demographic expansion to a current size of >5,000 reproducing individuals, which is quite likely in view of the great diversity found on beta(A) chromosomes.

  7. Expression of 3beta-HSD and P5betaR, genes respectively coding for Delta5-3beta-hydroxysteroid dehydrogenase and progesterone 5beta-reductase, in leaves and cell cultures of Digitalis lanata EHRH.

    PubMed

    Ernst, Mona; de Padua, Rodrigo Maia; Herl, Vanessa; Müller-Uri, Frieder; Kreis, Wolfgang

    2010-06-01

    Plants of the genus Digitalis produce 5 beta-cardenolides that are used in the therapy of cardiac insufficiency in humans. 3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) and progesterone 5 beta-reductase (P5 betaR) are both supposed to be important enzymes in the biosynthesis of these natural products. Activity and gene expression were demonstrated for both enzymes in cardenolide-accumulating leaves of Digitalis lanata but also in cardenolide-free permanent cell suspension cultures initiated from D. lanata leaf tissue. Enzyme activities were determined and quantified by HPLC and GC-MS methods. Expression of the respective genes, namely AY585867.1 (P5betaR gene) and DQ466890.1 (3beta-HSD gene), was made evident by real-time polymerase chain reaction (qPCR) analysis. We demonstrate for the first time that the P5betaR gene, encoding an enzyme described as a key enzyme in cardenolide biosynthesis, is also expressed in cardenolide-free tissues of cardenolide-containing plants.

  8. Ultrasound-mediated interferon {beta} gene transfection inhibits growth of malignant melanoma

    SciTech Connect

    Yamaguchi, Kazuki; Feril, Loreto B.; Tachibana, Katsuro; Takahashi, Akira; Matsuo, Miki; Endo, Hitomi; Harada, Yoshimi; Nakayama, Juichiro

    2011-07-22

    Highlights: {yields} Successful ultrasound-mediated transfection of melanoma (C32) cells with IFN-{beta} genes both in vitro and in vivo. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited proliferation of melanoma cells in vitro. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited melanoma tumor growth in vivo. -- Abstract: We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon {beta} (IFN-{beta}) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-{beta} in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-{beta} genes mixed with microbubbles. Successful sonotransfection with IFN-{beta} gene in vitro was confirmed by ELISA, which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-{beta} gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.

  9. Gene correction in the evolution of the T cell receptor beta chain

    PubMed Central

    1986-01-01

    Mutational mechanisms operating at the T cell receptor beta chain locus have been examined by comparison of the CT beta 1 and CT beta 2 gene sequences from Mus pahari, believed to be the oldest living species in the genus Mus, with those of inbred mice. Results indicate that a gene correction event independent of that suggested to have occurred in inbred mice has homogenized the M. pahari CT beta exon 1 sequences, minimizing diversity in this region of the molecule. These observations suggest that correction events such as gene conversion may occur frequently, even in pauci-gene families with as few as two members, and therefore play a significant role in gene diversification or homogenization of small as well as large gene families. PMID:3783089

  10. Hemoglobinopathies in the Dogon Country: presence of beta S, beta C, and delta A' genes.

    PubMed

    Ducrocq, R; Bennani, M; Bellis, G; Baby, M; Traore, K; Nagel, R L; Krishnamoorthy, R; Chaventre, A

    1994-07-01

    The population of the Dogon, located in Mali, is divided in an endogamic Noble class and two endogamic servant castes (Tanners and Blacksmiths). We find that the polymorphic frequencies of beta c, beta S, and, unexpectedly, a mutation of the delta-chain (delta A'), are geographically (valley vs. plateau) as well as social status dependent.

  11. Cloning and sequencing of the gene encoding thermophilic beta-amylase of Clostridium thermosulfurogenes.

    PubMed Central

    Kitamoto, N; Yamagata, H; Kato, T; Tsukagoshi, N; Udaka, S

    1988-01-01

    A gene coding for thermophilic beta-amylase of Clostridium thermosulfurogenes was cloned into Bacillus subtilis, and its nucleotide sequence was determined. The nucleotide sequence suggested that the thermophilic beta-amylase is translated from monocistronic mRNA as a secretory precursor with a signal peptide of 32 amino acid residues. The deduced amino acid sequence of the mature beta-amylase contained 519 residues with a molecular weight of 57,167. The amino acid sequence of the C. thermosulfurogenes beta-amylase showed 54, 32, and 32% homology with those of the Bacillus polymyxa, soybean, and barley beta-amylases, respectively. Twelve well-conserved regions were found among the amino acid sequences of the four beta-amylases. To elucidate the mechanism rendering the C. thermosulfurogenes beta-amylase thermophilic, its amino acid sequence was compared with that of the B. polymyxa beta-amylase. The C. thermosulfurogenes beta-amyulase contained more Cys residues and fewer hydrophilic amino acid residues than the B. polymyxa beta-amylase did. Several regions were found in the amino acid sequence of the C. thermosulfurogenes beta-amylase, where the hydrophobicity was remarkably high as compared with that of the corresponding regions of the B. polymyxa beta-amylase. PMID:2461360

  12. Targeted disruption of the mouse beta1-adrenergic receptor gene: developmental and cardiovascular effects.

    PubMed Central

    Rohrer, D K; Desai, K H; Jasper, J R; Stevens, M E; Regula, D P; Barsh, G S; Bernstein, D; Kobilka, B K

    1996-01-01

    At least three distinct beta-adrenergic receptor (beta-AR) subtypes exist in mammals. These receptors modulate a wide variety of processes, from development and behavior, to cardiac function, metabolism, and smooth muscle tone. To understand the roles that individual beta-AR subtypes play in these processes, we have used the technique of gene targeting to create homozygous beta 1-AR null mutants (beta 1-AR -/-) in mice. The majority of beta 1-AR -/- mice die prenatally, and the penetrance of lethality shows strain dependence. Beta l-AR -/- mice that do survive to adulthood appear normal, but lack the chronotropic and inotropic responses seen in wild-type mice when beta-AR agonists such as isoproterenol are administered. Moreover, this lack of responsiveness is accompanied by markedly reduced stimulation of adenylate cyclase in cardiac membranes from beta 1-AR -/- mice. These findings occur despite persistent cardiac beta 2-AR expression, demonstrating the importance of beta 1-ARs for proper mouse development and cardiac function, while highlighting functional differences between beta-AR subtypes. Images Fig. 1 Fig. 3 PMID:8693001

  13. Dopamine beta-hydroxylase gene modulates individuals' empathic ability.

    PubMed

    Gong, Pingyuan; Liu, Jinting; Li, She; Zhou, Xiaolin

    2014-09-01

    Dopamine beta-hydroxylase (DBH), an enzyme that converts dopamine to norepinephrine, has broad influences on social functions. In this study, we examined to what extent two polymorphisms (-1021C/T and a 19 bp insertion/deletion) in DBH gene modulate individuals' empathic perception and response, which were measured, respectively, by reading the mind in the eyes test and the empathic concern subscale of interpersonal reactivity index. Results showed that polymorphism at -1021C/T, but not the 19 bp insertion/deletion, accounts for 2.3% variance of empathic perception and 1.4% variance of empathic response. Individuals with the CC genotype, which is associated with higher DBH activity, manifested greater empathic ability than those with CT/TT genotypes. These findings demonstrate the importance of DBH -1021C/T as a genetic basis of empathy and in predicting individual differences in social and affective processing. © The Author (2013). Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

  14. Inhibition of spermidine synthase gene expression by transforming growth factor-beta 1 in hepatoma cells.

    PubMed Central

    Nishikawa, Y; Kar, S; Wiest, L; Pegg, A E; Carr, B I

    1997-01-01

    We screened genes responsive to transforming growth factor-beta (TGF-beta 1) protein in a human hepatoma cell line (Hep3B) using a PCR-mediated differential display technique, in order to investigate the mechanisms involved in TGF-beta-induced growth suppression. We found a gene that was down-regulated by TGF-beta 1 to be completely identical in an approx. 620 bp segment to the gene for the enzyme spermidine synthase, which mediates the conversion of putrescine into spermidine. Both spermidine synthase mRNA expression and its enzyme activity were decreased after TGF-beta 1 treatment of Hep3B cells. The inhibition of spermidine synthase gene expression by TGF-beta 1 protein was also observed in other hepatoma cell lines. The expression of genes for other biosynthetic enzymes in polyamine metabolism (ornithine decarboxylase and S-adenosylmethionine decarboxylase) was also inhibited to the same extent as for spermidine synthase, while the gene expression of spermidine/spermine N1-acetyltransferase, a catabolic enzyme, was relatively resistant to TGF-beta 1. Spermine levels in Hep3B cells were decreased by TGF-beta 1 treatment, although the levels of spermidine and putrescine were unchanged, probably due to compensation by remaining spermidine/spermine N1-acetyltransferase activity. Exogenously added spermidine or spermine, but not putrescine, partially antagonized the growth-inhibitor effects of TGF-beta 1 on Hep3B cells. Our data suggest that down-regulation of gene expression of the enzymes involved in polyamine metabolism, including spermidine synthase, may be associated with the mechanism of TGF-beta-induced growth suppression. PMID:9020892

  15. Tumor-produced, active Interleukin-1 {beta} regulates gene expression in carcinoma-associated fibroblasts

    SciTech Connect

    Dudas, Jozsef; Fullar, Alexandra; Bitsche, Mario; Schartinger, Volker; Kovalszky, Ilona; Sprinzl, Georg Mathias; Riechelmann, Herbert

    2011-09-10

    Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial-mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1{beta} (IL1-{beta}) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-{beta} expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-{beta} processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated with IL1-{beta}. IL1-{beta} signaling was investigated by western blot and immunocytochemistry. IL1-{beta}-regulated genes were analyzed by real-time qPCR. SCC-25 cells produced 16 kD active IL1-{beta}, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NF{kappa}B{alpha}. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-{beta} reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-{beta}-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-{beta} in the tumor cells leads to IL1-{beta}-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression. -- Graphical abstract: SCC-25 cells produce active, processed IL1-{beta}. PDL fibroblasts possess receptor for IL1-{beta}, and its expression is increased 4.56-times in the

  16. A novel association of DQ alpha and DQ beta genes in the DRw10 haplotype. Determination of a DQw1 specificity by the DQ beta-chain.

    PubMed

    Merryman, P; Silver, J; Gregersen, P K; Solomon, G; Winchester, R

    1989-09-15

    The association of the class II genes of the DRw10 haplotype from a cell line, NASC, initiated from a member of a well characterized family, was analyzed by sequencing cDNA clones corresponding to DR beta I, DQ alpha, and DQ beta genes. An identical haplotype was also identified in the Raji cell line. In addition to typing as DRw10 and DQw1 with HLA typing sera both, the NASC and Raji cell lines were shown to react strongly with the monoclonal antibodies 109d6 (specific for DRw10 beta 1 and DRw53 beta 2 gene products) and Genox 3.5.3 (specific for DQw1) and exhibited the restriction fragment length polymorphism indicative of a DRw10, DQw1 haplotype. The DR beta 1 gene corresponding to the DRw10 specificity was found to have a first domain sequence different from all other DR beta I genes. Sequence analysis of the 3'-untranslated region of this DR beta-chain gene showed a significant divergence from the 3' untranslated region of the DRw53 family of haplotypes and a lesser divergence from that of the DRw52 and DR1/DR2 families. The sequence of the DQ beta genes corresponding to the DQw1 specificity in the DRw10 haplotype was found to be identical to the DQ beta gene from a DR1, DQw1 haplotype. Surprisingly, however, the DQ alpha gene did not resemble other DQw1-like DQ alpha genes, but was identical in sequence to the DQ alpha gene found in DR4 haplotypes. The novel association of DQ alpha and DQ beta genes in the DRw10 haplotype revealed in these studies may result from a double recombinational event. More consequentially, these studies strongly suggest that the DQw1 specificity recognized by Genox 3.5.3 is determined by the DQ beta chain and is not affected by the DQ alpha-chain.

  17. The beta-globin gene in Sardinian delta beta 0-thalassemia carries a C----T nonsense mutation at codon 39.

    PubMed

    Guida, S; Giglioni, B; Comi, P; Ottolenghi, S; Camaschella, C; Saglio, G

    1984-04-01

    Sardinian delta beta 0-thalassemia is an inherited syndrome characterized by the inactivity of the beta-globin gene and the persistent activity of the fetal gamma-globin genes, particularly the A gamma-globin gene. Previous mapping studies with restriction enzymes failed to show any abnormality in the non-alpha globin gene cluster. We have now examined the possibility that this syndrome might result from a single rather than two different defects. Restriction enzyme polymorphisms linked to the delta beta 0-thalassemic non-alpha globin fragments were defined providing the basis for cloning the delta beta 0-thalassemic beta-globin gene from the DNA of a heterozygous patient. This gene appears to carry a C----T single mutation causing the appearance of a stop codon at amino acid position 39 of the beta-globin gene. This mutation was previously reported in beta 0-thalassemic patients, in linkage with different haplotypes. We conclude that Sardinian delta beta 0-thalassemia is the result of two separate mutations, the former one (unknown) responsible for persistent expression of gamma-globin genes, the latter for beta 0-thalassemia.

  18. Molecular characterization of an atypical beta-thalassemia caused by a large deletion in the 5' beta-globin gene region.

    PubMed Central

    Popovich, B W; Rosenblatt, D S; Kendall, A G; Nishioka, Y

    1986-01-01

    We describe a Canadian family of Czechoslovakian descent that came to our attention because of an HbA2 percentage approximately twice that of an average case of heterozygous beta-thalassemia. This unique phenotype suggested to us the possibility of a novel genetic mechanism being responsible for their beta-thalassemia. To investigate this possibility, we mapped, cloned, and sequenced the mutant beta-globin allele. This molecular analysis demonstrated the presence of a unique 4,237 base pair (bp) deletion extending from 3.3 kilobases (kb) 5' of the beta-globin mRNA cap site to approximately the middle of beta IVS-2. This truncated beta-globin gene further extends the heterogeneity of mutations known to cause beta-thalassemia and delineates new sequences involved in nonhomologous recombination events in the beta-globin gene region. Images Fig. 2 Fig. 4 Fig. 5 Fig. 6 PMID:3799598

  19. Preprocedure and Postprocedure Predictive Values of Serum β2-Microglobulin for Contrast-Induced Nephropathy in Patients Undergoing Coronary Computed Tomography Angiography: A Comparison With Creatinine-Based Parameters and Cystatin C.

    PubMed

    Li, Suhua; Zheng, Zhenda; Tang, Xixiang; Peng, Long; Luo, Yanting; Dong, Ruimin; Zhao, Yunyue; Liu, Jinlai

    2015-01-01

    This study aimed to investigate the values of serum β2-microglobulin to predict contrast-induced nephropathy (CIN) before and early after coronary computed tomography angiography (CCTA), comparing with creatinine-based parameters and cystatin C. A total of 424 patients were enrolled. Serum β2-microglobulin, cystatin C, and creatinine were measured at 0, 24, and 48 hours of CCTA. Contrast-induced nephropathy was defined as an elevation of serum creatinine level by 25% or higher or 0.5 mg/dL or greater from baseline within 48 hours. The estimated glomerular filtration rate (eGFR) was calculated by the Modification of Diet in Renal Disease study equation. Receiver operating characteristic curves and multivariate logistic regression analysis were used to detect the efficiency of biomarkers in predicting CIN. Fifty-two subjects (12.26%) developed CIN. Before CCTA, CIN was predicted by both baseline β2-microglobulin (area under the receiver operating characteristic curve [AUC], 0.791; P < 0.001) and cystatin C (AUC, 0.781; P < 0.001), whereas creatinine and eGFR were not predictive. After CCTA, CIN was predicted by both the absolute post-CCTA levels of β2-microglobulin, cystatin C, creatinine, and eGFR (AUC, 0.842 vs 0.961 vs 0.691 vs 0.688 at 24 hours, P < 0.001; and 0.937 vs 1.000 vs 0.908 vs 0.898 at 48 hours, P < 0.001) and their relative changes (Δ) to baseline (AUC, 0.677 vs 0.846 vs 0.850 vs 0.844 at 24 hours, P < 0.001; and 0.731 vs 0.968 vs 0.984 vs 0.966 at 48 hours, P < 0.001). Multivariate regression analysis confirmed that baseline β2-microglobulin (odds ratio, 2.137; 95% confidence interval, 1.805-3.109; P < 0.001) and cystatin C (odds ratio, 1.873; 95% confidence interval, 1.667-2.341; P = 0.003) were independent predictors for CIN. Serum β2-microglobulin, with values superior to creatinine-based parameters and similar with cystatin C, was a useful biomarker for the prediction of CIN at pre-CCTA and early post-CCTA.

  20. Pretransplant β2-Microglobulin Is Associated with the Risk of Acute Graft-versus-Host-Disease after Allogeneic Hematopoietic Cell Transplant.

    PubMed

    Costa-Lima, Carolina; Miranda, Eliana Cristina Martins; Colella, Marcos Paulo; Aranha, Francisco Jose Penteado; de Souza, Carmino Antonio; Vigorito, Afonso Celso; De Paula, Erich Vinicius

    2016-07-01

    The risk of acute graft-versus-host disease (aGVHD) can be reliably estimated by the hematopoietic cell transplantation-specific comorbidity index (HCT-CI), which can be further refined by the incorporation of pre-hematopoietic cell transplantation (HCT) serum levels of inflammatory biomarkers such as ferritin and albumin. β2-Microglobulin (β2-m) is a key component of the MHC class I complex, which is independently associated with mortality and frailty in the general population. We took advantage of our institutional protocol that includes measurement of pre-HCT β2-m serum levels in the most patients to investigate whether pre-transplant β2-m levels were associated with the risk of aGVHD. One hundred three consecutive patients submitted to allogeneic HCT, of which 26 developed grades II to IV aGVHD, were included in the analysis. β2-m was significantly associated with age and HCT-CI. Higher levels of β2-m were observed in patients who developed aGVHD (P = .008). In the multivariate Cox regression model, β2-m and HCT-CI remained independently associated with the risk of developing aGVHD. In conclusion, the association between β2-m and the occurrence of aGVHD suggests that the measurement of this protein before HCT might represent an additional element for risk stratification of aGVHD.

  1. Malaria protection in β2-microglobulin-deficient mice lacking major histocompatibility complex class I antigens: essential role of innate immunity, including γδT cells

    PubMed Central

    Taniguchi, Tomoyo; Tachikawa, Saoko; Kanda, Yasuhiro; Kawamura, Toshihiko; Tomiyama-Miyaji, Chikako; Li, Changchun; Watanabe, Hisami; Sekikawa, Hiroho; Abo, Toru

    2007-01-01

    It is still controversial whether malaria protection is mediated by conventional immunity associated with T and B cells or by innate immunity associated with extrathymic T cells and autoantibody-producing B cells. Given this situation, it is important to examine the mechanism of malaria protection in β2-microglobulin-deficient (β2m(–/–)) mice. These mice lack major histocompatibility complex class I and CD1d antigens, which results in the absence of CD8+ T cells and natural killer T (NKT) cells. When C57BL/6 and β2m(–/–) mice were injected with parasitized (Plasmodium yoelii 17XNL) erythrocytes, both survived from the infection and showed a similar level of parasitaemia. The major expanding T cells were NK1.1– αβΤ-cell receptorint cells in both mice. The difference was a compensatory expansion of NK and γδT cells in β2m(–/–) mice, and an elimination experiment showed that these lymphocytes were critical for protection in these mice. These results suggest that malaria protection might be events of the innate immunity associated with multiple subsets with autoreactivity. CD8+ T and NKT cells may be partially related to this protection. PMID:17916163

  2. The monomer-seed interaction mechanism in the formation of the β2-microglobulin amyloid fibril clarified by solution NMR techniques.

    PubMed

    Yanagi, Kotaro; Sakurai, Kazumasa; Yoshimura, Yuichi; Konuma, Tsuyoshi; Lee, Young-Ho; Sugase, Kenji; Ikegami, Takahisa; Naiki, Hironobu; Goto, Yuji

    2012-09-21

    Amyloid fibrils are proteinous aggregates associated with various diseases, including Alzheimer's disease, type II diabetes, and dialysis-related amyloidosis. It is generally thought that, during the progression of these diseases, a precursor peptide or protein assumes a partially denatured structure, which interacts with the fibril seed to change into the final amyloid form. β2-Microglobulin (β2m), associated with dialysis-related amyloidosis, is known to form amyloid fibrils at low pH via a partially structured state. However, the molecular mechanism by which the conformation of β2m changes from the precursor to the final fibril structure is poorly understood. We performed various NMR experiments to characterize acid-denatured β2m. The analysis of the transverse relaxation rates revealed that acid-denatured β2m undergoes a structural exchange with an extensively unfolded form. The results of transferred cross-saturation experiments indicated that residues with a residual structure in the acid-denatured state are associated with the interaction with the fibril seed. Our experimental data suggest the partially structured state to be "activated" to become extensively unfolded, in which state the hydrophobic residues are exposed and associate with the seed. Our results provide general information about the extension of amyloid fibrils.

  3. Host Susceptibility to Brucella abortus Infection Is More Pronounced in IFN-γ knockout than IL-12/β2-Microglobulin Double-Deficient Mice

    PubMed Central

    Brandão, Ana Paula M. S.; Oliveira, Fernanda S.; Carvalho, Natalia B.; Vieira, Leda Q.; Azevedo, Vasco; Macedo, Gilson C.; Oliveira, Sergio C.

    2012-01-01

    Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. IFN-γ, IL-12, and CD8+ T lymphocytes are important components of host immune responses against B. abortus. Herein, IFN-γ and IL-12/β2-microglobulin (β2-m) knockout mice were used to determine whether CD8+ T cells and IL-12-dependent IFN-γ deficiency would be more critical to control B. abortus infection compared to the lack of endogenous IFN-γ. At 1 week after infection, IFN-γ KO and IL-12/β2-m KO mice showed increased numbers of bacterial load in spleens; however, at 3 weeks postinfection (p.i.), only IFN-γ KO succumbed to Brucella. All IFN-γ KO had died at 16 days p.i. whereas death within the IL-12/β2-m KO group was delayed and occurred at 32 days until 47 days postinfection. Susceptibility of IL-12/β2-m KO animals to Brucella was associated to undetectable levels of IFN-γ in mouse splenocytes and inability of these cells to lyse Brucella-infected macrophages. However, the lack of endogenous IFN-γ was found to be more important to control brucellosis than CD8+ T cells and IL-12-dependent IFN-γ deficiencies. PMID:22194770

  4. Stacked Sets of Parallel, In-register β-Strands of β2-Microglobulin in Amyloid Fibrils Revealed by Site-directed Spin Labeling and Chemical Labeling*

    PubMed Central

    Ladner, Carol L.; Chen, Min; Smith, David P.; Platt, Geoffrey W.; Radford, Sheena E.; Langen, Ralf

    2010-01-01

    β2-microglobulin (β2m) is a 99-residue protein with an immunoglobulin fold that forms β-sheet-rich amyloid fibrils in dialysis-related amyloidosis. Here the environment and accessibility of side chains within amyloid fibrils formed in vitro from β2m with a long straight morphology are probed by site-directed spin labeling and accessibility to modification with N-ethyl maleimide using 19 site-specific cysteine variants. Continuous wave electron paramagnetic resonance spectroscopy of these fibrils reveals a core predominantly organized in a parallel, in-register arrangement, by contrast with other β2m aggregates. A continuous array of parallel, in-register β-strands involving most of the polypeptide sequence is inconsistent with the cryoelectron microscopy structure, which reveals an architecture based on subunit repeats. To reconcile these data, the number of spins in close proximity required to give rise to spin exchange was determined. Systematic studies of a model protein system indicated that juxtaposition of four spin labels is sufficient to generate exchange narrowing. Combined with information about side-chain mobility and accessibility, we propose that the amyloid fibrils of β2m consist of about six β2m monomers organized in stacks with a parallel, in-register array. The results suggest an organization more complex than the accordion-like β-sandwich structure commonly proposed for amyloid fibrils. PMID:20335170

  5. β2-Microglobulin elicits itch-related responses in mice through the direct activation of primary afferent neurons expressing transient receptor potential vanilloid 1.

    PubMed

    Andoh, Tsugunobu; Maki, Takahito; Li, Sikai; Uta, Daisuke

    2017-09-05

    Uremic pruritus is an unpleasant symptom in patients undergoing hemodialysis, and the underlying mechanisms remain unclear. β2-Microglobulin (β2-MG) is well-known as an MHC class I molecule and its level is increased in the plasma of patients undergoing hemodialysis. In this study, we investigated whether β2-MG was a pruritogen in mice. Intradermal injections of β2-MG into the rostral back induced scratching in a dose-dependent manner. Intradermal injection of β2-MG into the cheek also elicited scratching, but not wiping. β2-MG-induced scratching was inhibited by the μ-opioid receptor antagonist naltrexone hydrochloride. β2-MG-induced scratching was not inhibited by antagonists of itch-related receptors (e.g., H1 histamine receptor (terfenadine), TP thromboxane receptor (DCHCH), BLT1 leukotriene B4 receptor (CMHVA), and proteinase-activated receptor 2 (FSLLRY-NH2)). However, β2-MG-induced scratching was attenuated in mice desensitized by repeated application of capsaicin and also by a selective transient receptor potential vanilloid 1 (TRPV1) antagonist (BCTC). In addition, β2-MG induced phosphorylation of extracellular signal-regulated kinase (a marker of activated neurons) in primary culture of dorsal root ganglion neurons that expressed TRPV1. These results suggest that β2-MG is a pruritogen and elicits itch-related responses, at least in part, through TRPV1-expressing primary sensory neurons. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Structure, Stability, and Aggregation of β-2 Microglobulin Mutants: Insights from a Fourier Transform Infrared Study in Solution and in the Crystalline State

    PubMed Central

    Ami, Diletta; Ricagno, Stefano; Bolognesi, Martino; Bellotti, Vittorio; Doglia, Silvia Maria; Natalello, Antonino

    2012-01-01

    β-2 microglobulin (β2m) is an amyloidogenic protein involved in dialysis-related amyloidosis. We report here the study of the structural properties of the protein in solution and in the form of single crystals by Fourier transform infrared (FTIR) spectroscopy and microspectroscopy. The investigation has been extended to four β2m mutants previously characterized by x-ray crystallography: Asp53Pro, Asp59Pro, Trp60Gly, and Trp60Val. These variants displayed very similar three-dimensional structures but different thermal stability and aggregation propensity, investigated here by FTIR spectroscopy. For each variant, appreciable spectral differences were found between the protein in solution and in single crystals, consisting in a downshift of the main β-sheet band and in better resolved turn and loop bands, indicative of reduced protein secondary structure dynamics in the crystalline state. Notably, the well-resolved spectra of the β2m crystalline variants enabled us to identify structural differences induced by the single amino acid mutations. Such differences encompass turn and loop structures that might affect the stability and aggregation propensity of the investigated β2m variants. This study highlights the potential of FTIR microspectroscopy to acquire useful structural information on protein crystals, complementary to the crystallographic analyses. PMID:22500768

  7. Structure and expression of the mouse beta-hexosaminidase genes, Hexa and Hexb.

    PubMed

    Yamanaka, S; Johnson, O N; Norflus, F; Boles, D J; Proia, R L

    1994-06-01

    Two genes, HEXA and HEXB, encode the alpha- and beta-subunits, respectively, of human beta-hexosaminidase. In the mouse, the corresponding genes are termed Hexa and Hexb. The subunits dimerize to yield three isozymes, beta-hexosaminidase A (alpha beta), B (beta beta), and S (alpha alpha), that have the capacity to degrade a variety of substrates containing beta-linked N-acetylglucosamine and N-acetylgalactosamine residues. Mutations in the HEXA or HEXB gene resulting in a beta-hexosaminidase deficiency cause Tay-Sachs or Sandhoff disease, respectively. As a prelude to the creation of mouse models of these lysosomal storage diseases, we have characterized the molecular biology of the mouse beta-hexosaminidase system. Protein sequences derived from the cloned Hexa and Hexb cDNAs were 55% identical to each other and were also very similar to the cognate human sequences: 84% sequence identity with human HEXA and 75% with HEXB. The mouse hexosaminidase subunits, when expressed in HeLa cells from the cDNAs, displayed specificity toward synthetic substrates similar to the human subunits. The Hexa and Hexb genes were 25 and 22 kb in length, respectively. Each gene was divided into 14 exons, with the positions of introns precisely matching those of the corresponding human genes. The 5' flanking regions of the mouse genes demonstrated promoter activity as ascertained by their ability to drive chloramphenicol acetyltransferase gene expression in transfected NIH 3T3 cells. The sequences of these regulatory regions were G+C-rich in the 200 bp upstream of the respective initiator ATGs. Several putative promoter elements were present, including Sp1, AP2, CAAT, and TATA motifs.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Beta-lactam antibiotic biosynthetic genes have been conserved in clusters in prokaryotes and eukaryotes.

    PubMed Central

    Smith, D J; Burnham, M K; Bull, J H; Hodgson, J E; Ward, J M; Browne, P; Brown, J; Barton, B; Earl, A J; Turner, G

    1990-01-01

    A cosmid clone containing closely linked beta-lactam antibiotic biosynthetic genes was isolated from a gene library of Flavobacterium sp. SC 12,154. The location within the cluster of the DNA thought to contain the gene for delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), the first step in the beta-lactam antibiotic biosynthetic pathway, was identified by a novel method. This DNA facilitated the isolation, by cross-hybridization, of the corresponding DNA from Streptomyces clavuligerus ATCC 27064, Penicillium chrysogenum Oli13 and Aspergillus nidulans R153. Evidence was obtained which confirmed that the cross-hybridizing sequences contained the ACVS gene. In each case the ACVS gene was found to be closely linked to other beta-lactam biosynthetic genes and constituted part of a gene cluster. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2107074

  9. Digitalis purpurea P5 beta R2, encoding steroid 5 beta-reductase, is a novel defense-related gene involved in cardenolide biosynthesis.

    PubMed

    Pérez-Bermúdez, Pedro; García, Aurelio A Moya; Tuñón, Iñaki; Gavidia, Isabel

    2010-02-01

    The stereospecific 5 beta-reduction of progesterone is a required step for cardiac glycoside biosynthesis in foxglove plants. Recently, we have isolated the gene P5 beta R, and here we investigate the function and regulation of P5 beta R2, a new progesterone 5 beta-reductase gene from Digitalis purpurea. P5 beta R2 cDNA was isolated from a D. purpurea cDNA library and further characterized at the biochemical, structural and physiological levels. Like P5 beta R, P5 beta R2 catalyzes the 5 beta-reduction of the Delta(4) double bond of several steroids and is present in all plant organs. Under stress conditions or on treatment with chemical elicitors, P5 beta R expression does not vary, whereas P5 beta R2 is highly responsive. P5 beta R2 expression is regulated by ethylene and hydrogen peroxide. The correlation between P5 beta R2 expression and cardenolide formation demonstrates the key role of this gene in cardenolide biosynthesis, and therefore in the chemical defense of foxglove plants.

  10. Molecular cloning and characterization of mutant and wild-type human. beta. -actin genes

    SciTech Connect

    Leavitt, J.; Gunning, P.; Porreca, P.; Ng, S.Y.; Lin, C.H.; Kedes, L.

    1984-10-01

    There are more than 20 ..beta..-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional ..beta..-actin genes, they used the new method of B. Seed for selecting genomic clones by homologous recombination. A derivative of the ..pi..VX miniplasmid, ..pi..AN7..beta..1, was constructed by insertion of the 600-base-pair 3' untranslated region of the ..beta..-actin mRNA expressed in human fibroblasts. Five clones containing ..beta..-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete ..beta..-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then use to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant ..beta..-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived closed verified the identity of the ..beta..-actin gene expressed in human fibroblasts.

  11. Genomic structure of the human beta-PIX gene and its alteration in gastric cancer.

    PubMed

    Li, Zhong you; Wang, You jie; Song, Jian ping; Kataoka, Hideki; Yoshii, Shigeto; Gao, Chang ming; Wang, Ya ping; Zhou, Jian nong; Ota, Satoshi; Tanaka, Masamitsu; Sugimura, Haruhiko

    2002-03-28

    beta-PIX, a newly identified p21-activated kinase (PAK)-interacting exchange factors (PIX), encodes a guanine nucleotide exchange factor for Rho guanosine triphosphatases. Characterization of beta-PIX gene was performed using the BAC Library method. The beta-PIX gene has 17 exons and an A/T polymorphism at the 32nd base upstream of the intron/exon junction of exon 7. The frequencies of genotypes A/T, A/A and T/T were 23.6% (13/55), 72.7% (40/55) and 3.6% (2/55), respectively; these frequencies are in Hardy-Weinberg equilibrium. Two out of 14 informative tumors (14.3%) were shown to have lost their heterozygosity at this locus, but no mutations in the remaining alleles were detected. In addition, we examined the gene-expression profile in another set of 30 gastric samples, but no significant over-expression of either the beta-PIX gene or the alpha-PIX gene was found. Though the beta-PIX gene has been speculated to potentially have tumor-related biological characteristics, the findings of the present study suggest that the involvement of beta-PIX gene in human gastric carcinogenesis is minimal.

  12. No evidence of the human chorionic gonadotropin-beta gene 5 betaV79M polymorphism in Mexican women.

    PubMed

    Dominguez-Lopez, Pablo; Diaz-Cueto, Laura; Ulloa-Aguirre, Alfredo; Lopez-Valle, Miguel Angel; Arechavaleta-Velasco, Fabian

    2008-01-01

    Human chorionic gonadotropin (hCG) is a placental hormone essential for the maintenance of pregnancy. Previous studies have shown a G to A transition in exon 3 of the hCGbeta gene 5, which changes the naturally occurring valine to methionine in codon 79. The frequency of this transition varies among different ethnic groups, being high in USA women, and less common, or absent, in various European populations. The purpose of the present study was to determine the frequency of the betaV79M allelic variant of the beta-subunit of hCG in a Mexican population, and to compare this frequency with those found in other ethnic groups. Placental DNA from 161 pregnant Mexican women was genotyped for the betaV79M by polymerase chain reaction (PCR)-restriction fragments length polymorphism analysis. No polymorphic betaV79M alleles were identified in the population studied. The allele and genotypic frequencies of betaV79M polymorphism in Mexican Mestizo women were significantly different from those reported for the US population, but not from five different European populations. In contrast to what has been found in women from the USA, it seems that the hCGbeta V79M polymorphism is absent or extremely rare in Mexican Mestizo women.

  13. On the road to gene therapy for beta-thalassemia and sickle cell anemia.

    PubMed

    Bank, Arthur

    2008-01-01

    Human globin gene therapy is a potential cure for sickle cell disease and beta-thalassemia (Cooley anemia). A clinical trial of this treatment is currently under way in Paris using lentiglobin vectors.

  14. Identification of Androgen Receptor and Beta-Catenin Target Genes in Prostate and Prostate Cancer

    DTIC Science & Technology

    2013-10-01

    Transdisciplinary Research in Epigenetics and Cancer Journal Clubs and Transdisciplinary Science Meetings, biweekly and monthly 3. To gain expertise...Target Genes in Prostate and Prostate Cancer PRINCIPAL INVESTIGATOR: Laura Lamb CONTRACTING ORGANIZATION: Washington University...TITLE AND SUBTITLE Identification of Androgen Receptor and Beta-Catenin Target Genes in Prostate and Prostate Cancer 5a. CONTRACT NUMBER Genes in

  15. APOE and A-betaPP Gene Variation in Cortical and Cerebrovascular Amyloid-beta Pathology and Alzheimer’s Disease: A Population-Based Analysis

    PubMed Central

    Peuralinna, Terhi; Tanskanen, Maarit; Mäkelä, Mira; Polvikoski, Tuomo; Paetau, Anders; Kalimo, Hannu; Sulkava, Raimo; Hardy, John; Lai, Shiao-Lin; Arepalli, Sampath; Hernandez, Dena; Traynor, Bryan J.; Singleton, Andrew; Tienari, Pentti J.; Myllykangas, Liisa

    2012-01-01

    Cortical and cerebrovascular amyloid-beta (A-beta) deposition is a hallmark of Alzheimer’s disease (AD), but also occurs in elderly people not affected by dementia. The apolipoprotein E (APOE) epsilon4 is a major genetic modulator of A-beta deposition and AD risk. Variants of the amyloid-beta protein precursor (A-betaPP) gene have been reported to contribute to AD and cerebral amyloid angiopathy (CAA). We analyzed the role of APOE and A-beta PP variants in cortical and cerebrovascular A-beta deposition, and neuropathologically verified AD (based on modified NIA-RI criteria) in a population-based autopsy sample of Finns aged ≥85 years (Vantaa85 + Study; n = 282). Our updated analysis of APOE showed strong associations of the epsilon4 allele with cortical (p = 4.91×10−17) and cerebrovascular (p = 9.87×10−11) A-beta deposition as well as with NIA-RI AD (p = 1.62×10−8). We also analyzed 60 single nucleotide polymorphisms (SNPs) at the A-betaPP locus. In single SNP or haplotype analyses there were no statistically significant A-betaPP locus associations with cortical or cerebrovascular A-beta deposition or with NIA-RI AD. We sequenced the promoter of the A-betaPP gene in 40 subjects with very high A-beta deposition, but none of these subjects had any of the previously reported or novel AD-associated mutations. These results suggest that cortical and cerebrovascular A-beta depositions are useful quantitative traits for genetic studies, as highlighted by the strong associations with the APOE epsilon4 variant. Promoter mutations or common allelic variation in the A-betaPP gene do not have a major contribution to cortical or cerebrovascular A-beta deposition, or very late-onset AD in this Finnish population based study. PMID:21654062

  16. Molecular cloning and characterization of the human beta-like globin gene cluster.

    PubMed

    Fritsch, E F; Lawn, R M; Maniatis, T

    1980-04-01

    The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.

  17. Allelic forms of the alpha- and beta-chain genes encoding DQw1-positive heterodimers.

    PubMed

    Turco, E; Care, A; Compagnone-Post, P; Robinson, C; Cascino, I; Trucco, M

    1987-01-01

    On chromosome 6, in the HLA region, the DQ subregion is located immediately centromeric to the DR subregion. Even though only three serological specificities to date have been officially recognized (DQw1, DQw2, and DQw3), it seems likely that the phenotypical polymorphism expressed by DQ molecules is much more complex. There are reasons to believe that fixed alpha-beta combinations exist, each of them associated with a different DR allele. DQw1 is a determinant present on DQ molecules that are found associated with DR1-, DR2-, and DRw6-positive haplotypes. By restriction fragment length polymorphism analysis, we recognized three allelic DQ-alpha and three allelic DQ-beta patterns associated with DQw1. In addition, one of these alpha/beta pairs associated with DR1, two with DR2, and a fourth with DRw6. We have obtained evidence using nucleotide sequencing that there are as many allelic forms of DQ-alpha and DQ-beta genes as there are different molecular DQ-alpha and DQ-beta patterns. The DQ-alpha and DQ-beta chains of DQw1-positive molecules each are encoded by at least three distinctly different allelic genes, and particular alpha/beta gene combinations are associated with the same DR alleles as their corresponding molecular alpha/beta pairs.

  18. Cloning and expression in Pichia pastoris of a blue mussel (Mytilus edulis) beta-mannanase gene.

    PubMed

    Xu, Bingze; Sellos, Daniel; Janson, Jan-Christer

    2002-03-01

    Using PCR, cloning and sequencing techniques, a 1.1-kb complementary DNA fragment encoding for a beta-mannanase (mannan endo-1,4-beta-mannosidase, EC 3.2.1.78) has been identified in the digestive gland of blue mussel, Mytilus edulis. The cDNA sequence shows significant sequence identity to several beta-mannanases in glycoside hydrolase family 5. The beta-mannanase gene has been isolated and sequenced from gill tissue of blue mussel and contains five introns. The beta-mannanase has been expressed extracellularly in Pichia pastoris using the Saccharomyces cerevisiae alpha-factor signal sequence. The beta-mannanase was produced in a 14-L fermenter with an expression level of 900 mg.L-1. The expression level is strongly affected by the induction temperature. A two-step purification procedure, composed of a combination of immobilized metal ion affinity chromatography and ion exchange chromatography, is required to give a pure beta-mannanase. However, due to post-translational modifications, structural varieties regarding molecular mass and isoelectric point were obtained. The specific activity of the purified recombinant M. edulis beta-mannanase was close to that of the wild-type enzyme. Also pH and temperature optima were the same as for the native protein. In conclusion, P. pastoris is regarded as a suitable host strain for the production of blue mussel beta-mannanase. This is the first time a mollusc beta-mannanase has been characterized at the DNA level.

  19. A Drosophila gene encoding a protein resembling the human beta-amyloid protein precursor.

    PubMed Central

    Rosen, D R; Martin-Morris, L; Luo, L Q; White, K

    1989-01-01

    We have isolated genomic and cDNA clones for a Drosophila gene resembling the human beta-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human beta-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development. Images PMID:2494667

  20. A Drosophila gene encoding a protein resembling the human. beta. -amyloid protein precursor

    SciTech Connect

    Rosen, D.R.; Martin-Morris, L.; Luo, L.; White, K. )

    1989-04-01

    The authors have isolated genomic and cDNA clones for a Drosophila gene resembling the human {beta}-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human {beta}-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development.

  1. The exon-intron organization of the human erythroid [beta]-spectrin gene

    SciTech Connect

    Amin, K.M.; Forget, B.G. ); Scarpa, A.L.; Curtis, P.J. ); Winkelmann, J.C. )

    1993-10-01

    The human erythrocyte [beta]-spectrin gene DNA has been cloned from overlapping human genomic phage and cosmid recombinants. The entire erythroid [beta]-spectrin mRNA is encoded by 32 exons that range in size from 49 to 871 bases. The exon/intron junctions have been identified and the exons mapped. There is no correlation between intron positions and the repeat units of 106 amino acids within domain II of the [beta]-spectrin gene. The scatter of the introns over the 17 repeats argues against the 106-amino-acid unit representing a minigene that underwent repeated duplication resulting in the present [beta]-spectrin gene. In fact, the two largest exons, exon 14 (871 bp) and 16 (757 bp), extend over 4 and 3 repeat units of 106 amino acids, respectively, while repeat [beta]10 is encoded by 4 exons. No single position of an intron in the [beta]-spectrin gene is conserved between any of the 17 [beta]-spectrin and 22 [alpha]-spectrin repeat units. The nucleotide sequences of the exon/intron boundaries conform to the consensus splice site sequences except for exon 20, whose 5[prime] donor splice-site sequence begins with GC. The [beta]-spectrin isoform present in the human brain, the skeletal muscle, and the cardiac muscle is an alternatively spliced product of the erythroid [beta]-spectrin gene. This splice site is located within the coding sequences of exon 32 and its utilization in nonerythroid tissues leads to the use of 4 additional downstream exons with a size range of 44 to 530 bp. 55 refs., 3 figs., 3 tabs.

  2. Comparative Studies of Vertebrate Beta Integrin Genes and Proteins: Ancient Genes in Vertebrate Evolution

    PubMed Central

    Holmes, Roger S.; Rout, Ujjwal K.

    2011-01-01

    Intregins are heterodimeric α- and β-subunit containing membrane receptor proteins which serve various cell adhesion roles in tissue repair, hemostasis, immune response, embryogenesis and metastasis. At least 18 α- (ITA or ITGA) and 8 β-integrin subunits (ITB or ITGB) are encoded on mammalian genomes. Comparative ITB amino acid sequences and protein structures and ITB gene locations were examined using data from several vertebrate genome projects. Vertebrate ITB genes usually contained 13–16 coding exons and encoded protein subunits with ∼800 amino acids, whereas vertebrate ITB4 genes contained 36-39 coding exons and encoded larger proteins with ∼1800 amino acids. The ITB sequences exhibited several conserved domains including signal peptide, extracellular β-integrin, β-tail domain and integrin β-cytoplasmic domains. Sequence alignments of the integrin β-cytoplasmic domains revealed highly conserved regions possibly for performing essential functions and its maintenance during vertebrate evolution. With the exception of the human ITB8 sequence, the other ITB sequences shared a predicted 19 residue α-helix for this region. Potential sites for regulating human ITB gene expression were identified which included CpG islands, transcription factor binding sites and microRNA binding sites within the 3′-UTR of human ITB genes. Phylogenetic analyses examined the relationships of vertebrate beta-integrin genes which were consistent with four major groups: 1: ITB1, ITB2, ITB7; 2: ITB3, ITB5, ITB6; 3: ITB4; and 4: ITB8 and a common evolutionary origin from an ancestral gene, prior to the appearance of fish during vertebrate evolution. The phylogenetic analyses revealed that ITB4 is the most likely primordial form of the vertebrate β integrin subunit encoding genes, that is the only β subunit expressed as a constituent of the sole integrin receptor ‘α6β4’ in the hemidesmosomes of unicellular organisms. PMID:24970121

  3. Physical mapping of the human T-cell antigen receptor (TCR) {beta}-chain gene complex

    SciTech Connect

    Yashim, Y.; So, A.K.

    1994-09-01

    The genetic variation of the TCR loci and their contribution to autoimmune diseases is poorly defined, in direct contrast to the clear examples of disease association with the Class I and II alleles of the major histocompatibility complex. We have therefore started to determine the gene organization and polymorphism of the TCR {beta} locus. Yeast artificial chromosomes (YACs) were used to construct a physical map of the germline human TCR {beta}-chain gene complex. Variable gene (V{beta}) sequences for the 25 known V{beta} subfamilies were amplified by PCR and were used as probes to screen a YAC library. Five positive YACs were identified. YACs designated B3, E11 and H11 of sizes 820, 400 and 600 kbp, respectively, were analyzed for their V{beta} content by pulse-field gel electrophoresis (PFGE). YAC B3 was found to contain all 25 V{beta} subfamilies, E11 for 14 and H11 for 7. B3 was also positive for the constant region genes. Restriction enzyme mapping of B3 located V{beta} and C{beta} gene regions to four Sfi I fragments of 280, 110, 90 and 125 kbp, and was in accordance with published data. The data thus showed that YAC B3 encoded a complete and unrearranged TCR {beta}-gene locus. The map was further resolved by locating restriction sites for Sal I and Bssll II on B3. Fluorescent in situ hybridization to human metaphase chromosomes localized B3 to chromosome 7q35. However, two additional signals were obtained: one attributable to V{beta} orphon cluster on chromosome 9q21; the second to the long arm of chromosome 2. PCR amplification of a chromosome 2 somatic cell hybrid using primers for all 25 V{beta} gene families revealed the signal was not attributable to a second orphon cluster. It is suggested that B3 is a chimeric YAC with an intact TCR {beta} locus flanked by chromosome 2 sequences. The determination of the TCR genomic organization will help extend studies of the role T-cells play in autoimmune diseases.

  4. Human beta-globin gene expression in transgenic mice is enhanced by a distant DNase I hypersensitive site.

    PubMed Central

    Curtin, P T; Liu, D P; Liu, W; Chang, J C; Kan, Y W

    1989-01-01

    Several lines of evidence suggest that erythroid-specific DNase I hypersensitive sites (HS) located far upstream of the human beta-globin gene are important in regulating beta-globin gene expression. We used the polymerase chain reaction technique to amplify and clone an 882-base-pair DNA fragment spanning one of these HS, designated HSII, which is located 54 kilobases upstream of the beta-globin gene. The cloned HSII fragment was linked to a human beta-globin gene in either the genomic (HSII-beta) or antigenomic (HSII-beta) orientation. These two constructs and a beta-globin gene alone (beta) were injected into fertilized mouse eggs, and expression was analyzed in liver and brain from day-16 transgenic fetuses. Five of 7 beta-transgenic fetuses expressed human beta-globin mRNA, but the level of expression per gene copy was low, ranging from 0.93 to 22.4% of mouse alpha-globin mRNA (average 9.9%). In contrast, 11 of 12 HSII-beta transgenic fetuses expressed beta-globin mRNA at levels per gene copy ranging from 31.3 to 336.6% of mouse alpha-globin mRNA (average 139.5%). Only three fetuses containing intact copies of the HSII-beta construct were produced. Two of three expressed human beta-globin mRNA at levels per gene copy of 179.2 and 387.1%. Expression of human beta-globin mRNA was tissue-specific in all three types of transgenic fetuses. These studies demonstrate that a small DNA fragment containing a single erythroid-specific HS can stimulate high-level human beta-globin gene expression in transgenic mice. Images PMID:2780563

  5. Cloning and sequencing of the beta-glucosidase gene from Acetobacter xylinum ATCC 23769.

    PubMed

    Tajima, K; Nakajima, K; Yamashita, H; Shiba, T; Munekata, M; Takai, M

    2001-12-31

    The beta-glucosidase gene (bglxA) was cloned from the genomic DNA of Acetobacter xylinum ATCC 23769 and its nucleotide sequence (2200 bp) was determined. This bglxA gene was present downstream of the cellulose synthase operon and coded for a polypeptide of molecular mass 79 kDa. The overexpression of the beta-glucosidase in A. xylinum caused a tenfold increase in activity compared to the wild-type strain. In addition, the action pattern of the enzyme was identified as G3ase activity. The deduced amino acid sequence of the bglxA gene showed 72.3%, 49.6%, and 45.1% identity with the beta-glucosidases from A. xylinum subsp. sucrofermentans, Cellvibrio gilvus, and Mycobacterium tuberculosis, respectively. Based on amino acid sequence similarities, the beta-glucosidase (BglxA) was assigned to family 3 of the glycosyl hydrolases.

  6. Understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in crossbred bulls

    NASA Astrophysics Data System (ADS)

    Deb, Rajib; Sajjanar, Basavaraj; Singh, Umesh; Alex, Rani; Raja, T. V.; Alyethodi, Rafeeque R.; Kumar, Sushil; Sengar, Gyanendra; Sharma, Sheetal; Singh, Rani; Prakash, B.

    2015-12-01

    Na+/K+-ATPase is an integral membrane protein composed of a large catalytic subunit (alpha), a smaller glycoprotein subunit (beta), and gamma subunit. The beta subunit is essential for ion recognition as well as maintenance of the membrane integrity. Present study was aimed to analyze the expression pattern of ATPase beta subunit genes (ATPase B1, ATPase B2, and ATPase B3) among the crossbred bulls under different ambient temperatures (20-44 °C). The present study was also aimed to look into the relationship of HSP70 with the ATPase beta family genes. Our results demonstrated that among beta family genes, transcript abundance of ATPase B1 and ATPase B2 is significantly ( P < 0.05) higher during the thermal stress. Pearson correlation coefficient analysis revealed that the expression of ATPase Β1, ATPase B2, and ATPase B3 is highly correlated ( P < 0.01) with HSP70, representing that the change in the expression pattern of these genes is positive and synergistic. These may provide a foundation for understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in cattle.

  7. Growth inhibition of human pancreatic cancer cells by human interferon-beta gene combined with gemcitabine.

    PubMed

    Endou, Masato; Mizuno, Masaaki; Nagata, Takuya; Tsukada, Kazuhiro; Nakahara, Norimoto; Tsuno, Takaya; Osawa, Hirokatsu; Kuno, Tomohiko; Fujita, Mitsugu; Hatano, Manabu; Yoshida, Jun

    2005-02-01

    We examined the anti-tumor effect of cationic multilamellar liposome containing human IFN-beta (huIFN-beta) gene against cultured human pancreatic cancer cells. We also evaluated the combined effect of huIFN-beta gene entrapped in liposomes and gemcitabine. Furthermore, we examined the anti-tumor mechanisms of the therapy, with emphasis on the Ras-related signal pathway. Three human pancreatic cancer cell lines (AsPc-1, MIAPaCa-2, and PANC-1) were used in this study. The growth inhibition together with the therapy were evaluated by WST-1 assay; the production of huIFN-beta protein was measured by ELISA; the cell cycle and apoptosis were analyzed using a FACScan flow cytometer; the protein levels of Son of sevenless (SOS-1) and Ras-GAP were measured by Western blotting; and the activation of Ras-GTP was evaluated by the immunoprecipitation method. As a result, we found that huIFN-beta gene entrapped in liposomes demonstrated a strong anti-tumor effect against human pancreatic cancer cells. The treatment that combined huIFN-beta gene entrapped in liposomes and gemcitabine was more effective than each treatment alone. Although gemcitabine remarkably reduced the level of SOS-1, the above combined therapy reduced the level of SOS-1 even more significantly. Both huIFN-beta gene entrapped in liposomes and the com-bination of huIFN-beta gene entrapped in liposomes and gemcitabine increased the level of Ras-GAP, and decreased the activity of Ras-GTP. These results suggest that this combination therapy can induce strong anti-tumor activity against human pancreatic cancer cells through the regulation of the Ras-related signal pathway.

  8. Beta-thalassemia genes in French-Canadians: haplotype and mutation analysis of Portneuf chromosomes.

    PubMed Central

    Kaplan, F; Kokotsis, G; DeBraekeleer, M; Morgan, K; Scriver, C R

    1990-01-01

    beta-Thalassemia minor occurs at approximately 1% frequency in French-Canadians--in families residing in Portneuf County (population approximately 40,000) of Quebec province. We found eight different RFLP haplotypes at the beta-globin gene cluster in 37 normal persons and in 12 beta-thalassemia heterozygotes from six families. beta-Thalassemia genes in these families associated with two haplotypes only: Mediterranean I and Mediterranean II. There were two different beta-thalassemia mutations segregating in the Portneuf population: an RNA processing mutation (beta(+)IVS-1,nt110) on haplotype I (five families) and a point mutation leading to chain termination (beta(0) nonsense codon 39) on haplotype II (one family). The distribution of 5' haplotypes on normal beta A Portneuf chromosomes compared with other European populations was most similar to that in British subjects (data for French subjects have not yet been reported). Genealogical reconstructions traced the ancestry of carrier couples to settlers emigrating from several different regions of France to New France in the 17th century. These findings indicate genetic diversity of a greater degree among French-Canadians than recognized heretofore. Images Figure 4 PMID:1967205

  9. Beta-thalassemia genes in French-Canadians: haplotype and mutation analysis of Portneuf chromosomes.

    PubMed

    Kaplan, F; Kokotsis, G; DeBraekeleer, M; Morgan, K; Scriver, C R

    1990-01-01

    beta-Thalassemia minor occurs at approximately 1% frequency in French-Canadians--in families residing in Portneuf County (population approximately 40,000) of Quebec province. We found eight different RFLP haplotypes at the beta-globin gene cluster in 37 normal persons and in 12 beta-thalassemia heterozygotes from six families. beta-Thalassemia genes in these families associated with two haplotypes only: Mediterranean I and Mediterranean II. There were two different beta-thalassemia mutations segregating in the Portneuf population: an RNA processing mutation (beta(+)IVS-1,nt110) on haplotype I (five families) and a point mutation leading to chain termination (beta(0) nonsense codon 39) on haplotype II (one family). The distribution of 5' haplotypes on normal beta A Portneuf chromosomes compared with other European populations was most similar to that in British subjects (data for French subjects have not yet been reported). Genealogical reconstructions traced the ancestry of carrier couples to settlers emigrating from several different regions of France to New France in the 17th century. These findings indicate genetic diversity of a greater degree among French-Canadians than recognized heretofore.

  10. Arrested rearrangement of TCR V[beta] genes in thymocytes from children with x-linked severe combined immunodeficiency disease

    SciTech Connect

    Sleasman, J.W.; Harville, T.O.; White, G.B.; Barrett, D.J. ); George, J.F. ); Goodenow, M.M. Univ. of Alabama, Birmingham, AL )

    1994-07-01

    Human X-linked severe combined immunodeficiency disease (SCID) is an immunodeficiency disorder in which T cell development is arrested in the thymic cortex. B lymphocytes in children with X-linked SCID seem to differentiate normally. X-linked SCID is associated with a mutation in the gene that encodes the IL-2R [gamma]-chain. Because TCR-[beta] gene recombination is a pivotal initial event in T lymphocyte onteogeny within the thymus, the authors hypothesized that a failure to express normal IL-2R[gamma] could lead to impaired TCR-[beta] gene recombination in early thymic development. PCR was used to determine the status of TCR-[beta] gene-segment rearrangements in thymic DNA that had been obtained from children with X-linked SCID. The initial step in TCR-[beta] gene rearrangement, that of D[beta] to J[beta] recombination, was readily detected in all thymus samples from children with X-linked SCID; in contrast, V[beta] to DJ[beta] gene rearrangements were undetectable in the same samples. Both D[beta] to J[beta] and V[beta] to DJ[beta] TCR genes were rearranged in the thymic tissues obtained from immunologically normal children. The authors conclude that TCR[beta]-chain gene rearrangement is arrested in children with X-linked SCID. The results suggest a causative relationship between the failure of TCR [beta]-chain gene arrangements to proceed beyond DJ[beta] rearrangements and the production of a nonfunctional IL-2R [gamma]-chain. 45 refs., 3 figs.

  11. The human ATP synthase beta subunit gene: sequence analysis, chromosome assignment, and differential expression.

    PubMed

    Neckelmann, N; Warner, C K; Chung, A; Kudoh, J; Minoshima, S; Fukuyama, R; Maekawa, M; Shimizu, Y; Shimizu, N; Liu, J D

    1989-11-01

    In humans, the functional F0F1-ATP synthase beta subunit gene is located on chromosome 12 in the p13----qter region. Other partially homologous sequences have been detected on chromosomes 2 and 17. The bona fide beta subunit gene has 10 exons encoding a leader peptide of 49 amino acids and a mature protein of 480 amino acids. Thirteen Alu family DNA repeats are found upstream from the gene and in four introns. The gene has four "CCAAT" sequences upstream and in close proximity to the transcriptional initiation site. A 13-bp motif is found in the 5' nontranscribed region of both the beta subunit gene and an ADP/ATP translocator gene that is expressed in high levels in cardiac and skeletal muscle. Analysis of the beta subunit mRNA levels reveals marked differences among tissues. The highest levels are found in heart, lower levels in skeletal muscle, and the lowest levels in liver and kidney. These findings suggest that the tissue-specific levels of ATP synthase beta subunit mRNA may be generated through transcriptional control.

  12. Expression and nucleotide sequence of the Clostridium acetobutylicum beta-galactosidase gene cloned in Escherichia coli.

    PubMed Central

    Hancock, K R; Rockman, E; Young, C A; Pearce, L; Maddox, I S; Scott, D B

    1991-01-01

    A gene library for Clostridium acetobutylicum NCIB 2951 was constructed in the broad-host-range cosmid pLAFR1, and cosmids containing the beta-galactosidase gene were isolated by direct selection for enzyme activity on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) plates after conjugal transfer of the library to a lac deletion derivative of Escherichia coli. Analysis of various pSUP202 subclones of the lac cosmids on X-Gal plates localized the beta-galactosidase gene to a 5.1-kb EcoRI fragment. Expression of the Clostridium beta-galactosidase gene in E. coli was not subject to glucose repression. By using transposon Tn5 mutagenesis, two gene loci, cbgA (locus I) and cbgR (locus II), were identified as necessary for beta-galactosidase expression in E. coli. DNA sequence analysis of the entire 5.1-kb fragment identified open reading frames of 2,691 and 303 bp, corresponding to locus I and locus II, respectively, and in addition a third truncated open reading frame of 825 bp. The predicted gene product of locus I, CbgA (molecular size, 105 kDa), showed extensive amino acid sequence homology with E. coli LacZ, E. coli EbgA, and Klebsiella pneumoniae LacZ and was in agreement with the size of a polypeptide synthesized in maxicells containing the cloned 5.1-kb fragment. The predicted gene product of locus II, CbgR (molecular size, 11 kDa) shares no significant homology with any other sequence in the current DNA and protein sequence data bases, but Tn5 insertions in this gene prevent the synthesis of CbgA. Complementation experiments indicate that the gene product of cbgR is required in cis with cbgA for expression of beta-galactosidase in E. coli. Images PMID:1850729

  13. Structure of the gene for human. beta. /sub 2/-adrenergic receptor: expression and promoter characterization

    SciTech Connect

    Emorine, L.J.; Marullo, S.; Delavier-Klutchko, C.; Kaveri, S.V.; Durieu-Trautmann, O.; Strosberg, A.D.

    1987-10-01

    The genomic gene coding for the human ..beta../sub 2/-adrenergic receptor (..beta../sub 2/AR) from A431 epidermoid cells has been isolated. Transfection of the gene into eukaryotic cells restores a fully active receptor/GTP-binding protein/adenylate cyclase complex with ..beta../sub 2/AR properties. Southern blot analyses with ..beta../sub 2/AR-specific probes show that a single ..beta../sub 2/AR gene is common to various human tissues and that its flanking sequences are highly conserved among humans and between man and rabbit, mouse, and hamster. Functional significance of these regions is supported by the presence of a promoter region (including mRNA cap sites, two TATA boxes, a CAAT box, and three G + C-rich regions that resemble binding sites for transcription factor Sp1) 200-300 base pairs 5' to the translation initiation codon. In the 3' flanking region, sequences homologous to glucocorticoid-response elements might be responsible for the increased expression of the ..beta../sub 2/AR gene observed after treatment of the transfected cells with hydrocortisone. In addition, 5' to the promoter region, an open reading frame encodes a 251-residue polypeptide that displays striking homologies with protein kinases and other nucleotide-binding proteins.

  14. Regulatory Divergence among Beta-Keratin Genes during Bird Evolution.

    PubMed

    Bhattacharjee, Maloyjo Joyraj; Yu, Chun-Ping; Lin, Jinn-Jy; Ng, Chen Siang; Wang, Tzi-Yuan; Lin, Hsin-Hung; Li, Wen-Hsiung

    2016-11-01

    Feathers, which are mainly composed of α- and β-keratins, are highly diversified, largely owing to duplication and diversification of β-keratin genes during bird evolution. However, little is known about the regulatory changes that contributed to the expressional diversification of β-keratin genes. To address this issue, we studied transcriptomes from five different parts of chicken contour and flight feathers. From these transcriptomes we inferred β-keratin enriched co-expression modules of genes and predicted transcription factors (TFs) of β-keratin genes. In total, we predicted 262 TF-target gene relationships in which 56 TFs regulate 91 β-keratin genes; we validated 14 of them by in vitro tests. A dual criterion of TF enrichment and "TF-target gene" expression correlation identified 26 TFs as the major regulators of β-keratin genes. According to our predictions, the ancestral scale and claw β-keratin genes have common and unique regulators, whereas most feather β-keratin genes show chromosome-wise regulation, distinct from scale and claw β-keratin genes. Thus, after expansion from the β-keratin gene on Chr7 to other chromosomes, which still shares a TF with scale and claw β-keratin genes, most feather β-keratin genes have recruited distinct or chromosome-specific regulators. Moreover, our data showed correlated gene expression profiles, positive or negative, between predicted TFs and their target genes over the five studied feather regions. Therefore, regulatory divergences among feather β-keratin genes have contributed to structural differences among different parts of feathers. Our study sheds light on how feather β-keratin genes have diverged in regulation from scale and claw β-keratin genes and among themselves. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Genes and mechanisms involved in beta-amyloid generation and Alzheimer's disease.

    PubMed

    Steiner, H; Capell, A; Leimer, U; Haass, C

    1999-01-01

    Alzheimer's disease is characterized by the invariable accumulation of senile plaques that are predominantly composed of amyloid beta-peptide (Abeta). Abeta is generated by proteolytic processing of the beta-amyloid precursor protein (betaAPP) involving the combined action of beta- and gamma-secretase. Cleavage within the Abeta domain by alpha-secretase prevents Abeta generation. In some very rare cases of familial AD (FAD), mutations have been identified within the betaAPP gene. These mutations are located close to or at the cleavage sites of the secretases and pathologically effect betaAPP processing by increasing Abeta production, specifically its highly amyloidogenic 42 amino acid variant (Abeta42). Most of the mutations associated with FAD have been identified in the two presenilin (PS) genes, particularly the PS1 gene. Like the mutations identified within the betaAPP gene, mutations in PS1 and PS2 cause the increased generation of Abeta42. PS1 has been shown to be functionally involved in Notch signaling, a key process in cellular differentation, and in betaAPP processing. A gene knock out of PS1 in mice leads to an embryonic lethal phenotype similar to that of mice lacking Notch. In addition, absence of PS1 results in reduced gamma-secretase cleavage and leads to an accumulation of betaAPP C-terminal fragments and decreased amounts of Abeta. Recent work may suggest that PS1 could be the gamma-secretase itself, exhibiting the properties of a novel aspartyl protease. Mutagenesis of either of two highly conserved intramembraneous aspartate residues of PS1 leads to reduced Abeta production as observed in the PS1 knockout. A corresponding mutation in PS2 interfered with betaAPP processing and Notch signaling suggesting a functional redundancy of both presenilins. In this issue, some of the recent work on the molecular mechanisms involved in Alzheimer's disease (AD) as well as novel diagnostic approaches and risk factors for AD will be discussed. In the first

  16. Isolation and characterization of BetaM protein encoded by ATP1B4 - a unique member of the Na,K-ATPase {beta}-subunit gene family

    SciTech Connect

    Pestov, Nikolay B.; Zhao, Hao; Basrur, Venkatesha; Modyanov, Nikolai N.

    2011-09-09

    Highlights: {yields} Structural properties of BetaM and Na,K-ATPase {beta}-subunits are sharply different. {yields} BetaM protein is concentrated in nuclear membrane of skeletal myocytes. {yields} BetaM does not associate with a Na,K-ATPase {alpha}-subunit in skeletal muscle. {yields} Polypeptide chain of the native BetaM is highly sensitive to endogenous proteases. {yields} BetaM in neonatal muscle is a product of alternative splice mRNA variant B. -- Abstract: ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a {beta}-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase {beta}-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was

  17. Beta-globin gene cluster haplotypes and alpha-thalassemia in sickle cell disease patients from Trinidad.

    PubMed

    Jones-Lecointe, Altheia; Smith, Erskine; Romana, Marc; Gilbert, Marie-Georges; Charles, Waveney P; Saint-Martin, Christian; Kéclard, Lisiane

    2008-01-01

    In this study, we have determined the frequency of beta(S) haplotypes in 163 sickle cell disease patients from Trinidad. The alpha(3.7) globin gene deletion status was also studied with an observed gene frequency of 0.17. Among the 283 beta(S) chromosomes analyzed, the Benin haplotype was the most prevalent (61.8%) followed by Bantu (17.3%), Senegal (8.5%), Cameroon (3.5%), and Arab-Indian (3.2%), while 5.7% of them were atypical. This beta(S) haplotypes distribution differed from those previously described in other Caribbean islands (Jamaica, Guadeloupe, and Cuba), in agreement with the known involvement of the major colonial powers (Spain, France, and Great Britain) in the slave trade in Trinidad and documented an Indian origin of the beta(S) gene.

  18. Identification of stable normalization genes for quantitative real-time PCR in porcine articular cartilage

    PubMed Central

    2012-01-01

    Background Expression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results. The purpose of this study was to identify the most stable housekeeping genes in porcine articular cartilage subjected to a mechanical injury from a panel of 10 candidate genes. Results Ten candidate housekeeping genes were evaluated in three different treatment groups of mechanically impacted porcine articular cartilage. The genes evaluated were: beta actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hydroxymethylbilane synthase, hypoxanthine phosphoribosyl transferase, peptidylprolyl isomerase A (cyclophilin A), ribosomal protein L4, succinate dehydrogenase flavoprotein subunit A, TATA box binding protein, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein—zeta polypeptide. The stability of the genes was measured using geNorm, BestKeeper, and NormFinder software. The four most stable genes measured via geNorm were (most to least stable) succinate dehydrogenase flavoprotein, subunit A, peptidylprolyl isomerase A, glyceraldehyde-3-phosphate dehydrogenase, beta actin; the four most stable genes measured via BestKeeper were glyceraldehyde-3-phosphate dehydrogenase, peptidylprolyl isomerase A, beta actin, succinate dehydrogenase flavoprotein, subunit A; and the four most stable genes measured via NormFinder were peptidylprolyl isomerase A, succinate dehydrogenase flavoprotein, subunit A, glyceraldehyde-3-phosphate dehydrogenase, beta actin. Conclusions BestKeeper, geNorm, and NormFinder all generated similar results for the most stable genes in porcine articular cartilage. The use of these appropriate reference genes will facilitate accurate gene expression studies of porcine articular cartilage and suggest appropriate housekeeping genes for articular cartilage studies in other species. PMID:23146128

  19. Differential gene expression in response to transforming growth factor-beta1 by fetal and postnatal dermal fibroblasts.

    PubMed

    Rolfe, Kerstin J; Irvine, Laurie M; Grobbelaar, Addie O; Linge, Claire

    2007-01-01

    The multipotent growth factor transforming growth factor (TGF)-beta1 is consistently linked with fibrosis and scarring. The perfect (scarless) healing of cutaneous wounds in early gestational age fetuses is proposed to be due to this tissue's predominance of the TGF-beta3 isoform over the profibrotic TGF-beta1 and 2. Nevertheless, TGF-beta1 is present during wound healing in the early fetus and recently we demonstrated that relevant intracellular signaling pathways are activated (albeit transiently) on TGF-beta1 stimulation. This study aimed to determine whether TGF-beta1 has different effects on gene transcription in human fetal (<14 weeks) vs. human postnatal dermal fibroblasts, using real-time polymerase chain reaction. The regulation pattern of a number of TGF-beta response genes differed dramatically between the two cell sources. The typical autocrine loop of TGF-beta1 autoinduction did not occur in fetal fibroblasts and genes that are normally up-regulated, connective tissue growth factor and collagen type I were actually down-regulated. Furthermore, other response genes responded in a delayed fashion (TGF-beta3) compared with that seen in the more developmentally mature postnatal fibroblasts. Finally, genes unaltered by TGF-beta stimulation in postnatal cells, TGF-beta2 and collagen III, were up-regulated in fetal cells. These developmentally related differences in fibroblast response to TGF-beta1 may influence wound-healing outcome, i.e., perfect regeneration or fibrosis.

  20. Altered transcription of genes coding for class I histocompatibility antigens in murine tumor cells

    PubMed Central

    1983-01-01

    Three murine tumors induced by Moloney murine leukemia virus (M-MLV) which exhibited loss of some or all H-2 class I antigens at the cell surface were analyzed at the DNA and RNA level with molecular probes specific of H-2 heavy chains and beta 2-microglobulin sequences. No observable difference could be detected at the DNA level between the tumors and the parent animals. However, a decrease in H-2 mRNA was observed, especially in phenotypically H-2 negative tumor, BM5R, where H-2 transcripts were at least 30-fold less abundant. These results show that an H-2-negative character may result from a general alteration in the transcription of H-2 genes, which could reflect some kind of regulatory process. PMID:6311935

  1. Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence.

    PubMed

    Gholami, Khadijeh; Loh, Su Yi; Salleh, Naguib; Lam, Sau Kuen; Hoe, See Ziau

    2017-01-01

    Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies.

  2. Missense mutation of the {beta}-cardiac myosin heavy-chain gene in hypertrophic cardiomyopathy

    SciTech Connect

    Arai, Shoichi; Matsuoka, Rumiko; Hirayama, Kenji; Sakurai, Hisanao

    1995-09-11

    Hypertrophic cardiomyopathy occurs as an autosomal dominant familial disorder or as a sporadic disease without familial involvement. We describe a missense mutation of the {beta}-cardiac myosin heavy chain (MHC) gene, a G to T transversion (741 Gly{r_arrow}Trp) identified by direct sequencing of exon 20 in four individuals affected with familial hypertrophic cardiomyopathy. Three individuals with sporadic hypertrophic cardiomyopathy, whose parents are clinically and genetically unaffected, had sequence variations of exon 34 of the {alpha}-cardiac MHC gene (a C to T transversion, 1658 Asp{r_arrow}Asp, resulting in FokI site polymorphism), of intron 33 of the {alpha}-cardiac MHC gene (a G to A and an A to T transversion), and also of intron 14 of the {beta}-cardiac MHC gene (a C to T transversion in a patient with Noonan syndrome). Including our case, 30 missense mutations of the {beta}-cardiac MHC gene in 49 families have been reported thus far worldwide. Almost all are located in the region of the gene coding for the globular head of the molecule, and only one mutation was found in both Caucasian and Japanese families. Missense mutations of the {Beta}-cardiac MHC gene in hypertrophic cardiomyopathy may therefore differ according to race. 29 refs., 6 figs., 3 tabs.

  3. Methionine adenosyltransferase II beta subunit gene expression provides a proliferative advantage in human hepatoma.

    PubMed

    Martínez-Chantar, Maria L; García-Trevijano, Elena R; Latasa, M Ujue; Martín-Duce, Antonio; Fortes, Puri; Caballería, Juan; Avila, Matías A; Mato, José M

    2003-04-01

    Of the 2 genes (MAT1A, MAT2A) encoding methionine adenosyltransferase, the enzyme that synthesizes S-adenosylmethionine, MAT1A, is expressed in liver, whereas MAT2A is expressed in extrahepatic tissues. In liver, MAT2A expression associates with growth, dedifferentiation, and cancer. Here, we identified the beta subunit as a regulator of proliferation in human hepatoma cell lines. The beta subunit has been cloned and shown to lower the K(m) of methionine adenosyltransferase II alpha2 (the MAT2A product) for methionine and to render the enzyme more susceptible to S-adenosylmethionine inhibition. Methionine adenosyltransferase II alpha2 and beta subunit expression was analyzed in human and rat liver and hepatoma cell lines and their interaction studied in HuH7 cells. beta Subunit expression was up- and down-regulated in human hepatoma cell lines and the effect on DNA synthesis determined. We found that beta subunit is expressed in rat extrahepatic tissues but not in normal liver. In human liver, beta subunit expression associates with cirrhosis and hepatoma. beta Subunit is expressed in most (HepG2, PLC, and Hep3B) but not all (HuH7) hepatoma cell lines. Transfection of beta subunit reduced S-adenosylmethionine content and stimulated DNA synthesis in HuH7 cells, whereas down-regulation of beta subunit expression diminished DNA synthesis in HepG2. The interaction between methionine adenosyltransferase II alpha2 and beta subunit was demonstrated in HuH7 cells. Our findings indicate that beta subunit associates with cirrhosis and cancer providing a proliferative advantage in hepatoma cells through its interaction with methionine adenosyltransferase II alpha2 and down-regulation of S-adenosylmethionine levels.

  4. Cloning and characterization of the human beta2-glycoprotein I (beta2-GPI) gene promoter: roles of the atypical TATA box and hepatic nuclear factor-1alpha in regulating beta2-GPI promoter activity.

    PubMed Central

    Wang, Hsueh-Hsiao; Chiang, An-Na

    2004-01-01

    Beta2-glycoprotein I (beta2-GPI) is a plasma glycoprotein primarily synthesized in the liver. The interindividual variability of beta2-GPI expression in subjects with various metabolic syndromes and disease states suggests that it may have clinical importance. However, the regulation of beta2-GPI gene expression has not been clarified. To gain more insight into the control of beta2-GPI gene expression, we cloned the 4.1-kb 5'-flanking region and characterized the proximal promoter of the beta2- GPI gene in this study. Cis -acting elements required for beta2-GPI promoter activity were identified with transient transfection assays in the hepatoma cell lines HepG2 and Huh7 and in non-hepatic HeLa cells. Serial deletion analyses of the beta2-GPI 5'-flanking sequence revealed that the region from -197 to +7 had strong promoter activity in hepatoma cells but not in HeLa cells. Truncation and site-directed mutagenesis of putative cis -elements within this region showing an atypical TATA box and a HNF-1 (hepatic nuclear factor-1) element were both essential for the beta2-GPI promoter activity. Subsequent gel mobility shift assays confirmed the interaction of HNF-1alpha with the HNF-1 site residing downstream of the TATA box. Co-transfection of beta2-GPI promoter-luciferase vector with HNF-1alpha expression vector in Huh7 and HNF-1-deficient HeLa cells demonstrated the transactivation effect of HNF-1alpha on beta2-GPI promoter activity. In addition, overexpression of HNF-1alpha enhanced the endogenous beta2-GPI expression. These results suggest that the atypical TATA box and HNF-1 cis-element are critical for beta2-GPI transcription and HNF-1alpha may play an important role in cell-specific regulation of beta2-GPI gene expression. PMID:14984368

  5. The regulated expression of beta-globin genes introduced into mouse erythroleukemia cells.

    PubMed

    Chao, M V; Mellon, P; Charnay, P; Maniatis, T; Axel, R

    1983-02-01

    We have introduced a hybrid mouse-human beta-globin gene as well as the intact human beta-globin gene into murine erythroleukemia (MEL) cells and have demonstrated that these genes are appropriately regulated during differentiation of the MEL cell in culture. The addition of chemical inducers to cotransformed cells results in a 5 to 50 fold increase in the level of mRNA transcribed from the exogenous globin gene. S1 nuclease and primer extension analyses demonstrate that these mRNAs initiate and terminate correctly. Nuclear transcription experiments indicate that induction of hybrid mRNA results at least in part from the increase in the rate of globin gene transcription. Furthermore, the induction appears to be specific for globin genes within an erythroid cell. These results permit the study of expression of the globin gene during erythroid differentiation and suggest that the specific induction of the globin gene is an inherent property of DNA sequences within or flanking the beta-globin genes. Moreover, the fact that the human and hybrid globin genes are both inducible in MEL cells suggests that these regulatory sequences are conserved between mouse and human cells.

  6. Promoters for the human beta-hexosaminidase genes, HEXA and HEXB.

    PubMed

    Norflus, F; Yamanaka, S; Proia, R L

    1996-02-01

    Human lysosomal beta-hexosaminidases are encoded by two genes, HEXA and HEXB, specifying an alpha- and a beta-subunit, respectively. The subunits dimerize to form beta-hexosaminidase A (alpha beta), beta-hexosaminidase B (beta beta), and beta-hexosaminidase S (alpha alpha). This enzyme system has the capacity to degrade a variety of cellular substrates: oligosaccharides, glycosaminoglycans, and glycolipids containing beta-linked N-acetylglucosaminyl or N-galactosaminyl residues. Mutations in either the HEXA gene or HEXB gene lead to an accumulation of GM2 ganglioside in neurons, resulting in the severe neurodegenerative disorders termed the GM2 gangliosidoses. To identify the DNA elements responsible for hexosaminidase expression, we ligated the 5'-flanking sequences of both the human and mouse hexosaminidase genes to a chloramphenicol acetyltransferase (CAT) gene. The resulting plasmids were transfected into NIH-3T3 cells and CAT activity was determined as a measure of promoter strength. By 5' deletion analysis, it was found that essential sequences for HEXA expression resided within a 40-bp region between 100 bp and 60 bp upstream of the ATG initiation codon. This area contained two potential estrogen response element half-sites as well as potential binding sites for transcription factors NF-E1 and AP-2. Similarly, important HEXB promoter sequences were localized to a 60-bp region between 150 bp and 90 bp upstream of the ATG codon. By performing scanning mutagenesis on a 60-bp region within the 150-bp HEXB construct, we defined an essential promoter element of 12 bp that contained two potential AP-1 sites. The mouse Hexa and Hexb 5'-flanking sequences were found to contain regions similar in sequence, location, and activity to the essential promoter elements defined in the cognate human genes. No sequence similarity was found, however, between 5'-flanking regions of the HEXA and HEXB genes. These essential promoter elements represent potential sites for HEXA and

  7. Genomic organization and characterization of a three-gene rat adult beta-globin haplotype.

    PubMed

    Au, D M; Wong, W M; Tam, J W; Cheng, L Y; Lam, V M

    1995-11-20

    The isolation and detailed characterization of a three-beta-globin gene (GloB) haplotype in the Sprague-Dawley (S-D) rat is described. An enriched library, lambda SDHelib, was screened with a human GloB probe, humbg44, and from which a beta minor gene, Rathbbz, was isolated, sequenced and characterized. A S-D rat GloB-specific probe, Ratbgze12, derived from the Rathbbz gene, was then used to screen a S-D rat genomic library, lambda SDglib. The clone T1510 was isolated and identified to include the entire Rathbbz gene and part of another GloB gene, Rathbby, which was 5' upstream from Rathbbz. Chromosomal walking upstream using the riboprobe, rnaT71, led to the isolation of an overlapping clone, Ta49, which was shown to include two full-length GloB genes; the most 5' was Rathbbx followed by Rathbby. Sequence data suggests that Rathbbx is a beta major gene, whereas Rathbby is a hybrid gene of Rathbbx and Rathbbz. Genomic hybridization confirmed this particular three-gene haplotype in the S-D rat. This haplotype, a1, may be the prototype of the GloB cluster in rat.

  8. Identification and characterization of a novel repressor of beta-interferon gene expression.

    PubMed

    Keller, A D; Maniatis, T

    1991-05-01

    We have identified and characterized a novel repressor of human beta-interferon (beta-IFN) gene expression. This protein, designated PRDI-BF1, binds specifically to the PRDI element of the beta-IFN gene promoter and is distinct from previously reported proteins that bind to this sequence. PRDI-BF1 is an 88-kD protein containing five zinc-finger motifs. Cotransfection experiments in cultured mammalian cells revealed that PRDI-BF1 is a potent repressor of PRDI-dependent transcription. PRDI-BF1 blocks virus induction of the intact beta-IFN gene promoter and of synthetic promoters containing multiple PRDI sites. PRDI-BF1 can also block the SV40 enhancer when PRDI sites are located between the enhancer and the promoter. This repression is highly dependent on the location of the PRDI sites, however, indicating that PRDI-BF1 cannot act at a distance. On the basis of the properties of PRDI-BF1 and the observation that PRDI-BF1 mRNA accumulation is virus inducible, we propose that PRDI-BF1 may act as a postinduction repressor of the beta-IFN gene by displacing positive regulatory proteins from the PRDI site of the promoter.

  9. Identification and molecular characterization of four new large deletions in the beta-globin gene cluster.

    PubMed

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Couprie, Nicole; Francina, Alain

    2009-01-01

    Despite the fact that mutations in the human beta-globin gene cluster are essentially point mutations, a significant number of large deletions have also been described. We present here four new large deletions in the beta-globin gene cluster that have been identified on patients displaying an atypical hemoglobin phenotype (high HbF) at routine analysis. The first deletion, which spreads over 2.0 kb, removes the entire beta-globin gene, including its promoter, and is associated with a typical beta-thal minor phenotype. The three other deletions are larger (19.7 to 23.9 kb) and remove both the delta and beta-globin genes. Phenotypically, they look like an HPFH-deletion as they are associated with normal hematological parameters. The precise localization of their 5' and 3' breakpoints gives new insights about the differences between HPFH and (deltabeta)(0)-thalassemia at the molecular level. The importance of detection of these deletions in prenatal diagnosis and newborn screening of hemoglobinopathies is also discussed.

  10. Major histocompatibility complex gene product expression on pancreatic beta cells in acutely diabetic BB rats.

    PubMed Central

    Issa-Chergui, B.; Yale, J. F.; Vigeant, C.; Seemayer, T. A.

    1988-01-01

    Type I diabetes mellitus was induced in young, diabetes-prone BB rats by the passive transfer of concanavalin A-activated T lymphocytes from the spleens of acutely diabetic BB rats. The pancreas of the recipients was examined 1-2 days after the onset of glycosuria by immunocytochemistry by means of monoclonal antibodies for determining whether 1) Class I and/or II major histocompatibility gene complex (MHC) products were expressed on beta cells and 2) the mononuclear cell infiltrates were represented by T cells. Marked expression of Class I MHC gene products was evident on beta cells. In contrast, Class II MHC gene products were not identified on normal-appearing beta cells. Dendritic cells dispersed throughout the acinar and interstitial pancreas were markedly increased in number. The mononuclear cell infiltrate contained few cells (1-15%) recognized by a pan-T cell marker. Although it is possible that this passive transfer model might differ considerably from the spontaneously occurring diabetic state in the rat, this study suggests that 1) Class I, rather than Class II, MHC gene expression may be pivotal to beta-cell injury in diabetic rats, and 2) non-T cells may constitute an effector cell population central to beta-cell necrosis in Type I diabetes mellitus. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:3276208

  11. The chicken transforming growth factor-beta 3 gene: genomic structure, transcriptional analysis, and chromosomal location.

    PubMed

    Burt, D W; Dey, B R; Paton, I R; Morrice, D R; Law, A S

    1995-02-01

    In this paper, we report the isolation, characterization, and mapping of the chicken transforming growth factor-beta 3 (TGF-beta 3) gene. The gene contains seven exons and six introns spanning 16-kb of the chicken genome. A comparison of the 5'-flanking regions of human and chicken TGF-beta 3 genes reveals two regions of sequence conservation. The first contains ATF/CRE and TBP/TATA sequence motifs within an 87-bp region. The second is a 162-bp region with no known sequence motifs. Identification of transcription start sites using chicken RNA isolated from various embryonic and adult tissues reveals two sites of initiation, P1 and P2, which map to these two conserved regions. Comparison of 3'-flanking regions of chicken and mammalian TGF-beta 3 genes also revealed conserved sequences. The most significant homologies were found in the 3'-most end of the transcribed region. DNA sequence analysis of chicken TGF-beta 3 cDNAs isolated by 3'-RACE revealed multiple polyadenylation sites unusually distant from a poly(A) signal motif. A Msc I restriction fragment length polymorphism (RFLP) marker was used to map the TGFB3 locus to linkage group E7 on the East Lansing reference backcross. Linkage to the TH locus showed that the TGFB3 locus was physically located on chicken chromosome 5.

  12. Presence and diversity of the beta-lactamase gene in cat and dog staphylococci.

    PubMed

    Malik, Seidu; Christensen, Henrik; Peng, Haihong; Barton, Mary D

    2007-07-20

    Staphylococci are part of the normal microflora of humans and animals and some are potential pathogens that have become resistant to almost all known antibiotics. Despite the widespread reports of penicillin resistance in cat and dog staphylococci, the mechanism underlying penicillin resistance has not been examined. This study was aimed at investigating the molecular basis of resistance to penicillin in cat and dog staphylococcal isolates that showed phenotypic resistance to beta-lactam antibiotics. An 861 bp fragment of the structural blaZ gene which codes for beta-lactamase production in staphylococci was amplified by polymerase chain reaction (PCR) and the products were sequenced. Sequenced fragments were analysed by protein signature typing and sequences were compared to published blaZ sequences of human and bovine staphylococcal strains held in a public database. Four known protein signature types (1, 3, 5 and 6) and one new type (12) were identified in this study. When sequences were compared with published blaZ sequences, gene phylogenetic analysis revealed three major groups. The four variants of beta-lactamases types (A, B, C and D) belonged to each major group except for types A and D which were both in group II. These findings confirm that the blaZ gene is responsible for beta-lactamase production leading to subsequent resistance to beta-lactam antibiotics in feline and canine staphylococci and that the gene shows similar diversity and relatedness as found with blaZ sequences obtained from human and bovine staphylococci.

  13. A mutation of the beta-globin gene initiation codon, ATG-->AAG, found in a French Caucasian man.

    PubMed

    Lacan, Philippe; Aubry, Martine; Couprie, Nicole; Francina, Alain

    2005-01-01

    A new mutation of the beta-globin gene initiation codon, ATG-->AAG (Met-->Tyr), is reported in a man originating from the southeast of France. Typical hematological findings of beta-thalassemia (thal) trait were found. We emphasize the importance of characterizing uncommon beta-thal mutations for genetic counseling.

  14. Neofunctionalization of Chromoplast Specific Lycopene Beta Cyclase Gene (CYC-B) in Tomato Clade

    PubMed Central

    Mohan, Vijee; Pandey, Arun; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

    2016-01-01

    The ancestor of tomato underwent whole genome triplication ca. 71 Myr ago followed by widespread gene loss. However, few of the triplicated genes are retained in modern day tomato including lycopene beta cyclase that mediates conversion of lycopene to β-carotene. The fruit specific β-carotene formation is mediated by a chromoplast-specific paralog of lycopene beta cyclase (CYC-B) gene. Presently limited information is available about how the variations in CYC-B gene contributed to its neofunctionalization. CYC-B gene in tomato clade contained several SNPs and In-Dels in the coding sequence (33 haplotypes) and promoter region (44 haplotypes). The CYC-B gene coding sequence in tomato appeared to undergo purifying selection. The transit peptide sequence of CYC-B protein was predicted to have a stronger plastid targeting signal than its chloroplast specific paralog indicating a possible neofunctionalization. In promoter of two Bog (Beta old gold) mutants, a NUPT (nuclear plastid) DNA fragment of 256 bp, likely derived from a S. chilense accession, was present. In transient expression assay, this promoter was more efficient than the “Beta type” promoter. CARGATCONSENSUS box sequences are required for the binding of the MADS-box regulatory protein RIPENING INHIBITOR (RIN). The loss of CARGATCONSENSUS box sequence from CYC-B promoter in tomato may be related to attenuation of its efficiency to promote higher accumulation of β-carotene than lycopene during fruit ripening. PMID:27070417

  15. Sequence heterogeneity, multiplicity, and genomic organization of alpha- and beta-tubulin genes in sea urchins.

    PubMed Central

    Alexandraki, D; Ruderman, J V

    1981-01-01

    We analyzed the multiplicity, heterogeneity, and organization of the genes encoding the alpha and beta tubulins in the sea urchin Lytechinus pictus by using cloned complementary deoxyribonucleic acid (cDNA) and genomic tubulin sequences. cDNA clones were constructed by using immature spermatogenic testis polyadenylic acid-containing ribonucleic acid as a template. alpha- and beta-tubulin clones were identified by hybrid selection and in vitro translation of the corresponding messenger ribonucleic acids, followed by immunoprecipitation and two-dimensional gel electrophoresis of the translation products. The alpha cDNA clone contains a sequence that encodes the 48 C-terminal amino acids of alpha tubulin and 104 base pairs of the 3' nontranslated portion of the messenger ribonucleic acid. The beta cDNA insertion contains the coding sequence for the 100-C terminal amino acids of beta tubulin and 83 pairs of the 3' noncoding sequence. Hybrid selections performed at different criteria demonstrated the presence of several heterogeneous, closely related tubulin messenger ribonucleic acids, suggesting the existence of heterogeneous alpha- and beta-tubulin genes. Hybridization analyses indicated that there are at least 9 to 13 sequences for each of the two tubulin gene families per haploid genome. Hybridization of the cDNA probes to both total genomic DNA and cloned germline DNA fragments gave no evidence for close physical linkage of alpha-tubulin genes with beta-tubulin genes at the DNA level. In contrast, these experiments indicated that some genes within the same family are clustered. Images PMID:6287219

  16. Sequence heterogeneity, multiplicity, and genomic organization of. cap alpha. - and. beta. -tubulin genes in Sea Urchins

    SciTech Connect

    Alexandraki, D.; Ruderman, J.V.

    1981-12-01

    The authors analyzed the multiplicity, heterogeneity, and organization of the genes encoding the ..cap alpha.. and ..beta.. tubulins in the sea urchin Lytechinus pictus by using cloned complementary deoxyribonucleic acid (cDNA) and genomic tubulin sequences. cDNA clones were constructed by using immature spermatogenic testis polyadenylic acid-containing ribonucleic acid as a template. ..cap alpha.. and ..beta..-tubulin clones were identified by hybrid selection and in vitro translation of the corresponding messenger ribonucleic acids, followed by immunoprecipitation and two-dimensional gel electrophoresis of the translation products. The ..cap alpha.. cDNA clone contains a sequence that encodes the 48 C-terminal amino acids of ..cap alpha.. tubulin and 104 base pairs of the 3' nontranslated portion of the messenger ribonucleic acid. The ..beta.. cDNA insertion contains the coding sequence for the 100 C-terminal amino acids of ..beta.. tubulin and 83 base pairs of the 3' noncoding sequence. Hybrid selections performed at different criteria demonstrated the presence of several heterogeneous, closely related tubulin messenger ribonucleic acids, suggesting the existence of heterogeneous ..cap alpha..- and ..beta..-tubulin genes. Hybridization analyses indicated that there are at least 9 to 13 sequences for each of the two tubulin gene families per haploid genome. Hybridization of the cDNA probes to both total genomic DNA and cloned germline DNA fragments gave no evidence for close physical linkage of ..cap alpha..-tubulin genes with ..beta..-tubulin genes at the DNA level. In contrast, these experiments indicated that some genes within the same family are clustered.

  17. Retroviral transfer of a human beta-globin/delta-globin hybrid gene linked to beta locus control region hypersensitive site 2 aimed at the gene therapy of sickle cell disease.

    PubMed Central

    Takekoshi, K J; Oh, Y H; Westerman, K W; London, I M; Leboulch, P

    1995-01-01

    Human gamma-globin and delta-globin chains have been previously identified as strong inhibitors of the polymerization of hemoglobin S, in contrast to the beta-globin chain, which exerts only a moderate antisickling effect. However, gamma-globin and delta-globin are normally expressed at very low levels in adult erythroid cells, in contrast to beta-globin. We report the design of a beta-globin/delta-globin hybrid gene, beta/delta-sickle cell inhibitor 1 (beta/delta-SCI1) and its transduction by retrovirus-mediated gene transfer. The beta/delta-SCI1-encoding gene retains the overall structure of the human beta-globin gene, while incorporating specific amino acid residues from the delta chain previously found responsible for its enhanced antisickling properties. To achieve high expression levels of beta/delta-SCI1 in adult erythrocytes, the hybrid gene was placed under the transcriptional control of the human beta-globin promoter and the DNase I hypersensitive site 2 of the human beta locus control region. High-titer retroviruses were generated, and stable proviral transmission was achieved in infected cells. The mRNA expression levels of the beta/delta-SCI1 gene in infected, dimethyl sulfoxide-induced murine erythroleukemia cells approached 85% of the endogenous murine beta maj-globin mRNA, on a per gene basis, evidence that high gene expression levels were achieved in adult erythroid cells. Further evaluation of this strategy in transgenic animal models of sickle cell disease should assess its efficacy for the gene therapy of human patients. Images Fig. 4 Fig. 5 PMID:7708766

  18. Evolution and molecular characterization of a beta-globin gene from the Australian Echidna Tachyglossus aculeatus (Monotremata).

    PubMed

    Lee, M H; Shroff, R; Cooper, S J; Hope, R

    1999-07-01

    Coinciding with a period in evolution when monotremes, marsupials, and eutherians diverged from a common ancestor, a proto-beta-globin gene duplicated, producing the progenitors of mammalian embryonic and adult beta-like globin genes. To determine whether monotremes contain orthologues of these genes and to further investigate the evolutionary relationships of monotremes, marsupials, and eutherians, we have determined the complete DNA sequence of an echidna (Tachyglossus aculeatus) beta-like globin gene. Conceptual translation of the gene and sequence comparisons with eutherian and marsupial beta-like globin genes and echidna adult beta-globin indicate that the gene is adult expressed. Phylogenetic analyses do not clearly resolve the branching pattern of mammalian beta-like globin gene lineages and it is therefore uncertain whether monotremes have orthologues of the embryonic beta-like globin genes of marsupials and eutherians. Four models are proposed that provide a framework for interpreting further studies on the evolution of beta-like globin genes in the context of the evolution of monotremes, marsupials, and eutherians.

  19. The genomic fingerprinting of the coding region of the beta-tubulin gene in Leishmania identification.

    PubMed

    Luis, L; Ramírez, A; Aguilar, C M; Eresh, S; Barker, D C; Mendoza-León, A

    1998-06-01

    We have demonstrated the polymorphism of the beta-tubulin gene region in Leishmania and its value in the identification of the parasite. In this work we have shown that the coding region of the gene has sufficient variation to accurately discriminate these parasites at the subgenus level. Nevertheless, intrasubgenus diversity, for particular restriction enzymes, was found in New World Leishmania belonging to the Leishmania subgenus. For instance, differences were found between mexicana and amazonensis strains. A unique pattern at the species level was found in particular species of both subgenera, e.g. L. (L.) major strain P and L. (L.) tropica belonging to the Leishmania subgenus, and L. (V.) panamensis strain LS94 from the Viannia subgenus. Particular endonucleases are diagnostic in Leishmania species discrimination as in the case of PvuII for the mexicana and amazonensis. This variation evidenced in the beta-tubulin gene region of Leishmania also occurred in other Kinetoplastida e.g. Trypanosoma cruzi, Leptomonas spp. and Crithidia spp. Moreover, these organisms showed a different genomic fingerprinting for the beta-tubulin gene among them and also Leishmania. Thus, the polymorphism of the coding region of the beta-tubulin gene can be used as a molecular marker for the identification of Leishmania.

  20. Nitric oxide stimulates insulin gene transcription in pancreatic {beta}-cells

    SciTech Connect

    Campbell, S.C. . E-mail: s.c.campbell@ncl.ac.uk; Richardson, H.; Ferris, W.F.; Butler, C.S.; Macfarlane, W.M.

    2007-02-23

    Recent studies have identified a positive role for nitric oxide (NO) in the regulation of pancreatic {beta}-cell function. The aim of this study was to determine the effects of short-term exposure to NO on {beta}-cell gene expression and the activity of the transcription factor PDX-1. NO stimulated the activity of the insulin gene promoter in Min6 {beta}-cells and endogenous insulin mRNA levels in both Min6 and isolated islets of Langerhans. Addition of wortmannin prior to NO stimulation blocked the observed increases in insulin gene promoter activity. Although NO addition stimulated the phosphorylation of p38, inhibition by SB203580 did not block the effect of NO on the insulin gene promoter. NO addition also stimulated both the nuclear accumulation and the DNA binding activity of PDX-1. This study has shown that over 24 h, NO stimulates insulin gene expression, PI-3-kinase activity and the activity of the critical {beta}-cell transcription factor PDX-1.

  1. The maize auxotrophic mutant orange pericarp is defective in duplicate genes for tryptophan synthase beta.

    PubMed Central

    Wright, A D; Moehlenkamp, C A; Perrot, G H; Neuffer, M G; Cone, K C

    1992-01-01

    orange pericarp (orp) is a seedling lethal mutant of maize caused by mutations in the duplicate unlinked recessive loci orp1 and orp2. Mutant seedlings accumulate two tryptophan precursors, anthranilate and indole, suggesting a block in tryptophan biosynthesis. Results from feeding studies and enzyme assays indicate that the orp mutant is defective in tryptophan synthase beta activity. Thus, orp is one of only a few amino acid auxotrophic mutants to be characterized in plants. Two genes encoding tryptophan synthase beta were isolated from maize and sequenced. Both genes encode polypeptides with high homology to tryptophan synthase beta enzymes from other organisms. The cloned genes were mapped by restriction fragment length polymorphism analysis to approximately the same chromosomal locations as the genetically mapped factors orp1 and orp2. RNA analysis indicates that both genes are expressed in all tissues examined from normal plants. Together, the biochemical, genetic, and molecular data verify the identity of orp1 and orp2 as duplicate structural genes for the beta subunit of tryptophan synthase. PMID:1356534

  2. The maize auxotrophic mutant orange pericarp is defective in duplicate genes for tryptophan synthase beta.

    PubMed

    Wright, A D; Moehlenkamp, C A; Perrot, G H; Neuffer, M G; Cone, K C

    1992-06-01

    orange pericarp (orp) is a seedling lethal mutant of maize caused by mutations in the duplicate unlinked recessive loci orp1 and orp2. Mutant seedlings accumulate two tryptophan precursors, anthranilate and indole, suggesting a block in tryptophan biosynthesis. Results from feeding studies and enzyme assays indicate that the orp mutant is defective in tryptophan synthase beta activity. Thus, orp is one of only a few amino acid auxotrophic mutants to be characterized in plants. Two genes encoding tryptophan synthase beta were isolated from maize and sequenced. Both genes encode polypeptides with high homology to tryptophan synthase beta enzymes from other organisms. The cloned genes were mapped by restriction fragment length polymorphism analysis to approximately the same chromosomal locations as the genetically mapped factors orp1 and orp2. RNA analysis indicates that both genes are expressed in all tissues examined from normal plants. Together, the biochemical, genetic, and molecular data verify the identity of orp1 and orp2 as duplicate structural genes for the beta subunit of tryptophan synthase.

  3. Gene-gun DNA vaccination aggravates respiratory syncytial virus-induced pneumonitis.

    PubMed

    Bartholdy, Christina; Olszewska, Wieslawa; Stryhn, Anette; Thomsen, Allan Randrup; Openshaw, Peter J M

    2004-10-01

    A CD8+ T-cell memory response to respiratory syncytial virus (RSV) was generated by using a DNA vaccine construct encoding the dominant Kd-restricted epitope from the viral transcription anti-terminator protein M2 (M2(82-90)), linked covalently to human beta2-microglobulin (beta2m). Cutaneous gene-gun immunization of BALB/c mice with this construct induced an antigen-specific CD8+ T-cell memory. After intranasal RSV challenge, accelerated CD8+ T-cell responses were observed in pulmonary lymph nodes and virus clearance from the lungs was enhanced. The construct induced weaker CD8+ T-cell responses than those elicited with recombinant vaccinia virus expressing the complete RSV M2 protein, but stronger than those induced by a similar DNA construct without the beta2m gene. DNA vaccination led to enhanced pulmonary disease after RSV challenge, with increased weight loss and cell recruitment to the lung. Depletion of CD8+ T cells reduced, but did not abolish, enhancement of disease. Mice vaccinated with a construct encoding a class I-restricted lymphocytic choriomeningitis virus epitope and beta2m suffered more severe weight loss after RSV infection than unvaccinated RSV-infected mice, although RSV-specific CD8+ T-cell responses were not induced. Thus, in addition to specific CD8+ T cell-mediated immunopathology, gene-gun DNA vaccination causes non-specific enhancement of RSV disease without affecting virus clearance.

  4. Light controls phospholipase A2alpha and beta gene expression in Citrus sinensis.

    PubMed

    Liao, Hui-Ling; Burns, Jacqueline K

    2010-05-01

    The low-molecular weight secretory phospholipase A2alpha (CssPLA2alpha) and beta (CsPLA2beta) cloned in this study exhibited diurnal rhythmicity in leaf tissue of Citrus sinensis. Only CssPLA2alpha displayed distinct diurnal patterns in fruit tissues. CssPLA2alpha and CsPLA2beta diurnal expression exhibited periods of approximately 24 h; CssPLA2alpha amplitude averaged 990-fold in the leaf blades from field-grown trees, whereas CsPLA2beta amplitude averaged 6.4-fold. Diurnal oscillation of CssPLA2alpha and CsPLA2beta gene expression in the growth chamber experiments was markedly dampened 24 h after transfer to continuous light or dark conditions. CssPLA2alpha and CsPLA2beta expressions were redundantly mediated by blue, green, red and red/far-red light, but blue light was a major factor affecting CssPLA2alpha and CsPLA2beta expression. Total and low molecular weight CsPLA2 enzyme activity closely followed diurnal changes in CssPLA2alpha transcript expression in leaf blades of seedlings treated with low intensity blue light (24 micromol m(-2) s(-1)). Compared with CssPLA2alpha basal expression, CsPLA2beta expression was at least 10-fold higher. Diurnal fluctuation and light regulation of PLA2 gene expression and enzyme activity in citrus leaf and fruit tissues suggests that accompanying diurnal changes in lipophilic second messengers participate in the regulation of physiological processes associated with phospholipase A2 action.

  5. Transforming growth factor-beta receptor requirements for the induction of the endothelin-1 gene.

    PubMed

    Castañares, Cristina; Redondo-Horcajo, Mariano; Magan-Marchal, Noemi; Lamas, Santiago; Rodriguez-Pascual, Fernando

    2006-06-01

    Expression of the endothelin (ET)-1 gene is subject to complex regulation by numerous factors, among which the cytokine transforming growth factor-beta (TGF-beta) is one of the most important. TGF-beta action is based on the activation of the Smad signaling pathway. Smad proteins activate transcription of the gene by cooperation with activator protein-1 (AP-1) at specific sites on the ET-1 promoter. Smad signaling pathway is initiated by binding of the cytokine to a heteromeric complex of type I and type II receptors. Signal is then propagated to the nucleus by specific members of the Smad family. Most cell types contain a type I receptor known as ALK5. However, endothelial cells are unique because they coexpress an additional type I receptor named ALK1. These forms do not constitute redundant receptors with the same function, but they actually activate different Smad-mediated expression programs that lead to specific endothelial phenotypes. TGF-beta/ALK5/Smad3 pathway is associated to a mature endothelium because it leads to inhibition of cell migration/proliferation. Conversely, TGF-beta/ALK1/Smad5 activates both processes and is more related to the angiogenic state. We have analyzed the TGF-beta receptor subtype requirements for the activation of the ET-1 gene. For that purpose, we have overexpressed type I receptor and Smad isoforms in endothelial cells and analyzed the effect on ET-1 expression. Our experiments indicate that TGF-beta induces ET-1 expression preferentially through the activation of the ALK5/Smad3 pathway and, therefore, the expression of the vaso-constrictor may be associated to a quiescent and mature endothelial phenotype.

  6. Cell and Gene Therapy for the Beta-Thalassemias: Advances and Prospects.

    PubMed

    Mansilla-Soto, Jorge; Riviere, Isabelle; Boulad, Farid; Sadelain, Michel

    2016-04-01

    The beta-thalassemias are inherited anemias caused by mutations that severely reduce or abolish expression of the beta-globin gene. Like sickle cell disease, a related beta-globin gene disorder, they are ideal candidates for performing a genetic correction in patient hematopoietic stem cells (HSCs). The most advanced approach utilizes complex lentiviral vectors encoding the human β-globin gene, as first reported by May et al. in 2000. Considerable progress toward the clinical implementation of this approach has been made in the past five years, based on effective CD34+ cell mobilization and improved lentiviral vector manufacturing. Four trials have been initiated in the United States and Europe. Of 16 evaluable subjects, 6 have achieved transfusion independence. One of them developed a durable clonal expansion, which regressed after several years without transformation. Although globin lentiviral vectors have so far proven to be safe, this occurrence suggests that powerful insulators with robust enhancer-blocking activity will further enhance this approach. The combined discovery of Bcl11a-mediated γ-globin gene silencing and advances in gene editing are the foundations for another gene therapy approach, which aims to reactivate fetal hemoglobin (HbF) production. Its clinical translation will hinge on the safety and efficiency of gene targeting in true HSCs and the induction of sufficient levels of HbF to achieve transfusion independence. Altogether, the progress achieved over the past 15 years bodes well for finding a genetic cure for severe globin disorders in the next decade.

  7. A new compound heterozygous frameshift mutation in the type II 3{beta}-hydroxysteroid dehydrogenase 3{beta}-HSD gene causes salt-wasting 3{beta}-HSD deficiency congenital adrenal hyperplasia

    SciTech Connect

    Zhang, L.; Sakkal-Alkaddour, S.; Chang, Ying T.; Yang, Xiaojiang; Songya Pang

    1996-01-01

    We report a new compound heterozygous frameshift mutation in the type II 3{Beta}-hydroxysteroid dehydrogenase (3{beta}-HSD) gene in a Pakistanian female child with the salt-wasting form of 3{Beta}-HSD deficiency congenital adrenal hyperplasia. The etiology for her congenital adrenal hyperplasia was not defined. Although the family history suggested possible 3{beta}-HSd deficiency disorder, suppressed adrenal function caused by excess glucocorticoid therapy in this child at 7 yr of age did not allow hormonal diagnosis. To confirm 3{beta}-HSD deficiency, we sequenced the type II 3{beta}-HSD gene in the patient, her family, and the parents of her deceased paternal cousins. The type II 3{beta}-HSD gene region of a putative promotor, exons I, II, III, and IV, and exon-intron boundaries were amplified by PCR and sequenced in all subjects. The DNA sequence of the child revealed a single nucleotide deletion at codon 318 [ACA(Thr){r_arrow}AA] in exon IV in one allele, and two nucleotide deletions at codon 273 [AAA(Lys){r_arrow}A] in exon IV in the other allele. The remaining gene sequences were normal. The codon 318 mutation was found in one allele from the father, brother, and parents of the deceased paternal cousins. The codon 273 mutation was found in one allele of the mother and a sister. These findings confirmed inherited 3{beta}-HSD deficiency in the child caused by the compound heterozygous type II 3{beta}-HSD gene mutation. Both codons at codons 279 and 367, respectively, are predicted to result in an altered and truncated type II 3{beta}-HSD protein, thereby causing salt-wasting 3{beta}-HSD deficiency in the patient. 21 refs., 2 figs., 1 tab.

  8. Effect of Dactylogyrus catlaius (Jain 1961) infection in Labeo rohita (Hamilton 1822): innate immune responses and expression profile of some immune related genes.

    PubMed

    Dash, Pujarini; Kar, Banya; Mishra, Arpita; Sahoo, P K

    2014-03-01

    The monogenean ectoparasite, Dactylogyrus sp. is a major pathogen in freshwater aquaculture. The immune responses in parasitized fish were analyzed by quantitation of innate immune factors (natural agglutinin level, haemolysin titre, antiprotease, lysozyme and myeloperoxidase activities) in serum and immune-relevant gene expression in gill and anterior kidney. The antiprotease activity and natural agglutinin level were found to be significantly higher and lysozyme activity was significantly lower in parasitized fish. Most of the genes viz., beta2-microglobulin (beta2M), major histocompatibility complex I (MHCI), MHCII, tumor necrosis factor alpha (TNFalpha) and toll-like receptor 22 (TLR22) in gill samples were significantly down-regulated in the experimental group. In the anterior kidney, the expression of superoxide dismutase and interleukin 1beta (IL1beta) were significantly up-regulated whereas a significant down regulation of MHCII and TNFalpha was also observed. The down-regulation of most of the genes viz, MHCI, beta2M, MHCII, TLR22 and TNFalpha in infected gills indicated a well evolved mechanism in this parasite to escape the host immune response. The modulation of innate and adaptive immunity by this parasite can be further explored to understand host susceptibility.

  9. Structure of a Ruminococcus albus endo-1,4-beta-glucanase gene.

    PubMed Central

    Ohmiya, K; Kajino, T; Kato, A; Shimizu, S

    1989-01-01

    A chromosomal DNA fragment encoding an endo-1,4-beta-glucanase I (Eg I) gene from Ruminococcus albus cloned and expressed in Escherichia coli with pUC18 was fully sequenced by the dideoxy-chain termination method. The sequence contained a consensus promoter sequence and a structural amino acid sequence. The initial 43 amino acids of the protein were deduced to be a signal sequence, since they are missing in the mature protein (Eg I). High homology was found when the amino acid sequence of the Eg I was compared with that of endoglucanase E from Clostridium thermocellum. Codon usage of the gene was not biased. These results suggested that the properties of the Eg I gene from R. albus was specified from the known beta-glucanase genes of the other organisms. Images PMID:2687251

  10. Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals.

    PubMed

    Patel, Vidushi S; Cooper, Steven J B; Deakin, Janine E; Fulton, Bob; Graves, Tina; Warren, Wesley C; Wilson, Richard K; Graves, Jennifer A M

    2008-07-25

    Vertebrate alpha (alpha)- and beta (beta)-globin gene families exemplify the way in which genomes evolve to produce functional complexity. From tandem duplication of a single globin locus, the alpha- and beta-globin clusters expanded, and then were separated onto different chromosomes. The previous finding of a fossil beta-globin gene (omega) in the marsupial alpha-cluster, however, suggested that duplication of the alpha-beta cluster onto two chromosomes, followed by lineage-specific gene loss and duplication, produced paralogous alpha- and beta-globin clusters in birds and mammals. Here we analyse genomic data from an egg-laying monotreme mammal, the platypus (Ornithorhynchus anatinus), to explore haemoglobin evolution at the stem of the mammalian radiation. The platypus alpha-globin cluster (chromosome 21) contains embryonic and adult alpha- globin genes, a beta-like omega-globin gene, and the GBY globin gene with homology to cytoglobin, arranged as 5'-zeta-zeta'-alphaD-alpha3-alpha2-alpha1-omega-GBY-3'. The platypus beta-globin cluster (chromosome 2) contains single embryonic and adult globin genes arranged as 5'-epsilon-beta-3'. Surprisingly, all of these globin genes were expressed in some adult tissues. Comparison of flanking sequences revealed that all jawed vertebrate alpha-globin clusters are flanked by MPG-C16orf35 and LUC7L, whereas all bird and mammal beta-globin clusters are embedded in olfactory genes. Thus, the mammalian alpha- and beta-globin clusters are orthologous to the bird alpha- and beta-globin clusters respectively. We propose that alpha- and beta-globin clusters evolved from an ancient MPG-C16orf35-alpha-beta-GBY-LUC7L arrangement 410 million years ago. A copy of the original beta (represented by omega in marsupials and monotremes) was inserted into an array of olfactory genes before the amniote radiation (>315 million years ago), then duplicated and diverged to form orthologous clusters of beta-globin genes with different expression

  11. Pest protection conferred by A Beta vulgaris serine proteinase inhibitor gene

    USDA-ARS?s Scientific Manuscript database

    Proteinase inhibitors provide a means of engineering plant resistance to insect pests. A Beta vulgaris serine proteinase inhibitor gene (BvSTI) was fused to the constitutive CaMV35S promoter for over-expression in Nicotiana benthamiana plants to study its effect on lepidopteran insect pests. Indep...

  12. Using the NCBI Genome Databases to Compare the Genes for Human & Chimpanzee Beta Hemoglobin

    ERIC Educational Resources Information Center

    Offner, Susan

    2010-01-01

    The beta hemoglobin protein is identical in humans and chimpanzees. In this tutorial, students see that even though the proteins are identical, the genes that code for them are not. There are many more differences in the introns than in the exons, which indicates that coding regions of DNA are more highly conserved than non-coding regions.

  13. Direct cellobiose production from cellulose using sextuple beta-glucosidase gene deletion Neurospora crassa mutants

    USDA-ARS?s Scientific Manuscript database

    Direct cellobiose production from cellulose by a genetically modified fungus—Neurospora crassa, was explored in this study. A library of N. crassa sextuple beta-glucosidase (bgl) gene deletion strains was constructed. Various concentrations of cellobiose were detected in the culture broth of the N. ...

  14. Characterization and expression of the beta-N-acetylglucosaminidase gene family of Tribolium castaneum

    USDA-ARS?s Scientific Manuscript database

    Enzymes belonging to the Beta-N-acetylglucosaminidase (NAG) family cleave chitin oligosaccharides produced by the action of chitinases on chitin into the constituent N-acetylglucosamine monomer. Four genes encoding putative NAGs in the red flour beetle, Tribolium castaneum, namely TcNAG1, TcFDL, Tc...

  15. Using the NCBI Genome Databases to Compare the Genes for Human & Chimpanzee Beta Hemoglobin

    ERIC Educational Resources Information Center

    Offner, Susan

    2010-01-01

    The beta hemoglobin protein is identical in humans and chimpanzees. In this tutorial, students see that even though the proteins are identical, the genes that code for them are not. There are many more differences in the introns than in the exons, which indicates that coding regions of DNA are more highly conserved than non-coding regions.

  16. Nonblack patients with sickle cell disease have African. beta. sup s gene cluster haplotypes

    SciTech Connect

    Rogers, Z.R.; Powars, D.R.; Williams, W.D. ); Kinney, T.R. ); Schroeder, W.A. )

    1989-05-26

    Of 18 nonblack patients with sickle cell disease, 14 had sickle cell anemia, 2 had hemoglobin SC disease, and 2 had hemoglobin S-{beta}{sup o}-thalassemia. The {beta}{sup s} gene cluster haplotypes that were determined in 7 patients were of African origin and were identified as Central African Republic, Central African Republic minor II, Benin, and Senegal. The haplotype Central African Republic minor II was present on the {beta}{sup o}-thalassemia chromosome in 2 patients. None of 10 patients whose {alpha}-gene status was determined had {alpha}-thalassemia-2. These data strongly support the concept that the {beta}{sup s} gene on chromosome 11 of these individuals is of African origin and that the {alpha}-gene locus on chromosome 16 is of white or native American origin. The clinical severity of the disease in these nonblack patients is appropriate to their haplotype without {alpha}-thalassemia-2 and is comparable with that of black patients. All persons with congenital hemolytic anemia should be examined for the presence of sickle cell disease regardless of physical appearance or ethnic background.

  17. Retroviral-mediated transfer and expression of human. beta. -globin genes in cultured murine and human erythroid cells

    SciTech Connect

    Weber-Benarous, A.; Cone, R.D.; London, I.M.; Mulligan, R.C.

    1988-05-05

    The authors cloned human ..beta..-globin DNA sequences from a genomic library prepared from DNA isolated from the human leukemia cell line K562 and have used the retroviral vector pZip-NeoSV(X)1 to introduce a 3.0-kilobase segment encompassing the globin gene into mouse erythroleukemia cells. Whereas the endogenous K562 ..beta..-globin gene is repressed in K562 cells, when introduced into mouse erythroleukemia cells by retroviral-mediated gene transfer, the ..beta..-globin gene from K562 cells was transcribed and induced 5-20-fold after treatment of the cells with dimethyl sulfoxide. The transcripts were correctly initiated, and expression and regulation of the K562 gene were identical to the expression of a normal human ..beta..-globin gene transferred into mouse erythroleukemia cells in the same way. They have also introduced the normal human ..beta..-globin gene into K562 cells using the same retrovirus vector. SP6 analysis of the RNA isolated from the transduced cells showed that the normal ..beta..-globin gene was transcribed at a moderately high level, before or after treatment with hemin. Based on these data, they suggest that the lack of expression of the endogenous ..beta..-globin gene in K562 cells does not result from an alteration in the gene itself and may not result from a lack of factor(s) necessary for ..beta..-lobin gene transcription. Retroviral-mediated transfer of the human ..beta..-globin gene may, however, uniquely influence expression of the gene K562 cells.

  18. Origin and spread of beta-globin gene mutations in India, Africa, and Mediterranea: analysis of the 5' flanking and intragenic sequences of beta S and beta C genes.

    PubMed

    Trabuchet, G; Elion, J; Baudot, G; Pagnier, J; Bouhass, R; Nigon, V M; Labie, D; Krishnamoorthy, R

    1991-06-01

    Nucleotide polymorphisms of both the 5' flanking and intragenic regions of the human beta-globin gene were investigated by directly sequencing genomic DNA after amplification by the polymerase chain reaction in 47 subjects homozygous for the beta S or the beta C mutation. The sickle-cell mutation was found in the context of five different haplotypes defined by eight nucleotide substitutions and various structures of a region of the simple repeated sequence (AT) chi Ty. All subjects from the same geographic origin bear an identical chromosomal structure, defining the Senegal-, Bantu-, Benin-, Cameroon-, and Indian-type chromosomes. These results strengthen our previous conclusions about the multiple occurrence of the sickle-cell mutation. The Benin-type chromosome was also found among Algerian and Sicilian sickle-cell patients, whereas the Indian-type chromosome was observed in two geographically distant tribes, illustrating the spread of these sickle-cell genes. We also found that the intragenic sequence polymorphisms (frameworks) are not always in linkage disequilibrium with the BamH I polymorphism downstream from the beta-globin gene, as had been previously observed. Finally, we present a tentative phylogenetic tree of the different alleles at this locus. Some polymorphisms of this sequence might be contemporary with our last common ancestor, the great apes, that is, about 4-6 millions years old.

  19. [Alkaline-adapted beta-mannanase of Bacillus pumilus: gene heterologous expression and enzyme characterization].

    PubMed

    Tang, Jiajie; Guo, Su; Wang, Wei; Wei, Wei; Wei, Dongzhi

    2015-11-04

    We expressed a novel alkaline-adapted beta-mannanase gene and characterized the enzyme for potential industrial applications. We obtained a mannanase gene (named man(B)) from Bacillus pumilus Nsic2 and expressed the gene man(B) in Escherichia coli and Bacillus subtilis. Furthermore, we characterized the enzyme. The gene man(B) had an open reading frame of 1104 bp that encoded a polypeptide of 367-amino-acid beta-mannanase (Man(B)). The protein sequence showed the highest identity with the beta-mannanase from B. pumilus CCAM080065. We expressed the gene man(B) in E. coli BL21 (DE3) with the enzyme activity of 11021.3 U/mL. Compared with other mannanases, Man(B) showed higher stability under alkaline conditions and was stable at pH6.0 -9.0. The specific activity of purified Man(B) was 4191 ± 107 U/mg. The K(m) and V(max) values of purified Man(B) were 35.7 mg/mL and 14.9 μmol/(mL x min), respectively. Meanwhile, we achieved recombinant protein secretion expression in B. subtilis WB800N. We achieved heterologous expression of the gene man(B) and characterized its enzyme. The alkaline-adapted Man(B) showed potential value in industrial applications due to its pH stability.

  20. [Aspirin-PEI-beta-CyD as a novel non-viral vector for gene transfer].

    PubMed

    Wang, Zhong-Ren; Chen, Dan; Zhou, Jun; Tang, Gu-Ping

    2009-01-01

    To develop a novel non-viral gene delivery vector based on PEI-beta-CyD as backbone modified with aspirin, and to identify its physicochemical characters. 1, 1-carbonyldiimidazole (CDI) was used to bind aspirin onto PEI-beta-CyD to form PEI-beta-CyD-ASP. (1)H-NMR, FT-IR, UV and XRD were used to confirm the polymer structure. The ability of condensation was demonstrated by gel retardation assay. MTT assay was used to test the cell viability in B16, Hela and A293 cell lines. Transfection efficiency of the polymer was tested in B16 cells. The structure of PEI-beta-CyD-ASP was confirmed by (1)H-NMR, FT-IR, UV and XRD, which efficiently condensed plasmid DNA at the N/P ratio of 4. The copolymer showed low cytotoxicity and high transfection efficiency in B16 cells. The synthesized aspirin-PEI-beta-CyD might be a potential gene delivery vector.

  1. Isolation and functional characterization of a lycopene beta-cyclase gene that controls fruit colour of papaya (Carica papaya L.).

    PubMed

    Devitt, Luke C; Fanning, Kent; Dietzgen, Ralf G; Holton, Timothy A

    2010-01-01

    The colour of papaya fruit flesh is determined largely by the presence of carotenoid pigments. Red-fleshed papaya fruit contain lycopene, whilst this pigment is absent from yellow-fleshed fruit. The conversion of lycopene (red) to beta-carotene (yellow) is catalysed by lycopene beta-cyclase. This present study describes the cloning and functional characterization of two different genes encoding lycopene beta-cyclases (lcy-beta1 and lcy-beta2) from red (Tainung) and yellow (Hybrid 1B) papaya cultivars. A mutation in the lcy-beta2 gene, which inactivates enzyme activity, controls lycopene production in fruit and is responsible for the difference in carotenoid production between red and yellow-fleshed papaya fruit. The expression level of both lcy-beta1 and lcy-beta2 genes is similar and low in leaves, but lcy-beta2 expression increases markedly in ripe fruit. Isolation of the lcy-beta2 gene from papaya, that is preferentially expressed in fruit and is correlated with fruit colour, will facilitate marker-assisted breeding for fruit colour in papaya and should create possibilities for metabolic engineering of carotenoid production in papaya fruit to alter both colour and nutritional properties.

  2. Endomyocardial upregulation of beta1 adrenoreceptor gene expression and myocardial contractile reserve following cardiac resynchronization therapy.

    PubMed

    Vanderheyden, Marc; Mullens, Wilfried; Delrue, Leen; Goethals, Marc; Verstreken, Sofie; Wijns, William; de Bruyne, Bernard; Bartunek, Jozef

    2008-03-01

    Congestive heart failure (CHF) is associated with a blunted force-frequency relation (FFR) and myocardial contractile reserve (MCR) partially from a downregulation of beta1-adrenoreceptors (beta1-AR). We investigated whether acute and chronic cardiac resynchronization therapy (CRT) was capable of reversing the blunted FFR and MCR and if this was associated with upregulation of beta1-AR. Left ventricle dP/dtmax was invasively measured in 10 CHF patients (New York Heart Association class > or =3; ejection fraction <25%) during incremental dual chamber (DDD)-CRT pacing at 70, 90, 110, and 130 beats/min, with and without continuous infusion of intravenous dobutamine, immediately after CRT implantation (BL) and 4 months later (FU). In a subgroup of 5 patients, serial left ventricle beta1 and beta2-AR gene expression was measured using reverse transcriptase-polymerase chain reaction. Four months after the initiation of resynchronization therapy, DDD-CRT pacing results in a significant upward shift of the heart rate versus LV dP/dtmax relationship (P < .01) with force frequency amplification as evidenced by the steeper slope of the force frequency response (P = .04). Infusion of dobutamine recruits myocardial contractile reserve and increases the heart rate versus LV dP/dtmax relationship at BL and at FU (both P < .05). However, only at follow-up was an additional force frequency amplification noticed (P < .05) during dobutamine infusion. This observation was paralleled by a significant upregulation of beta1-AR gene expression (P = .02). Chronic CRT is associated with a partial restoration of the FFR and with a recruitment in myocardial contractile reserve, which is paralleled by upregulation of beta1-AR.

  3. Stimulator of IFN gene is critical for induction of IFN-beta during Chlamydia muridarum infection.

    PubMed

    Prantner, Daniel; Darville, Toni; Nagarajan, Uma M

    2010-03-01

    Type I IFN signaling has recently been shown to be detrimental to the host during infection with Chlamydia muridarum in both mouse lung and female genital tract. However, the pattern recognition receptor and the signaling pathways involved in chlamydial-induced IFN-beta are unclear. Previous studies have demonstrated no role for TLR4 and a partial role for MyD88 in chlamydial-induced IFN-beta. In this study, we demonstrate that mouse macrophages lacking TLR3, TRIF, TLR7, or TLR9 individually or both TLR4 and MyD88, still induce IFN-beta equivalent to wild type controls, leading to the hypothesis that TLR-independent cytosolic pathogen receptor pathways are crucial for this response. Silencing nucleotide-binding oligomerization domain 1 in HeLa cells partially decreased chlamydial-induced IFN-beta. Independently, small interfering RNA-mediated knockdown of the stimulator of IFN gene (STING) protein in HeLa cells and mouse oviduct epithelial cells significantly decreased IFN-beta mRNA expression, suggesting a critical role for STING in chlamydial-induced IFN-beta induction. Conversely, silencing of mitochondria-associated antiviral signaling proteins and the Rig-I-like receptors, RIG-I, and melanoma differentiation associated protein 5, had no effect. In addition, induction of IFN-beta depended on the downstream transcription IFN regulatory factor 3, and on activation of NF-kappaB and MAPK p38. Finally, STING, an endoplasmic reticulum-resident protein, was found to localize in close proximity to the chlamydial inclusion membrane during infection. These results indicate that C. muridarum induces IFN-beta via stimulation of nucleotide-binding oligomerization domain 1 pathway, and TLR- and Rig-I-like receptor-independent pathways that require STING, culminating in activation of IFN regulatory factor 3, NF-kappaB, and p38 MAPK.

  4. Gene expression of type 2 17 beta hydroxysteroid dehydrogenase in scalp hairs of hirsute women.

    PubMed

    Oliveira, Isabel O; Lhullier, Cintia; Brum, Ilma S; Spritzer, Poli Mara

    2003-09-01

    Androgens are the main hormonal regulators of human hair growth and they are related to clinical conditions such as hirsutism. The aim of this study was to analyze the gene expression of androgen receptor (AR) and type 2 17 beta hydroxysteroid dehydrogenase (17 beta-HSD) in keratinocytes of plucked scalp hairs from hirsute patients and normal subjects. We studied 58 women with hirsutism (31 with polycystic ovary syndrome (PCOS), 27 with idiopathic hirsutism (IH)); 15 control women; and 10 control men. Hirsutism was assessed by a modified Ferriman-Gallwey method. Hormonal status was assessed between days 2 and 10 of the menstrual cycle or on any day when the patients were amenorrheic. AR and type 2 17 beta-HSD mRNA levels were estimated by reverse transcription-polymerase chain reaction (RT-PCR). AR expression was similar in all groups. Type 2 17 beta-HSD gene expression in untreated hirsute patients was lower (2.1+/-0.10) than in normal women (3.1+/-0.17), and similar to men (1.8+/-0.22). Comparing hirsute patients, type 2 17 beta-HSD expression was higher in treated PCOS (3.0+/-0.34 versus 2.2+/-0.13) and IH patients (2.5+/-0.19 versus 2.0+/-0.15); hirsutism score was lower (P=0.003, PCOS; P=0.003, IH); and SHBG levels were higher (P=0.001, PCOS; P=0.024, IH) in treated patients. The free androgen index was lower in treated women (P=0.024 for the IH group). In conclusion, the lower expression of type 2 17 beta-HSD mRNA in scalp hairs of untreated hirsute patients suggests androgen metabolism disturbances with predominance of more potent androgens, as occurs in men. The enzyme's higher gene expression in treated hirsute patients could be an indirect evidence of restored enzyme activity and intracellular androgen metabolism.

  5. Sequences 5' of the basement membrane laminin beta 1 chain gene (LAMB1) direct the expression of beta-galactosidase during development of the mouse testis and ovary.

    PubMed

    Li, C; Gudas, L J

    1997-12-01

    The murine LAMB1 gene encoding laminin beta 1 is expressed in the developing male and female gonads and mesonephros. To identify the cis-acting elements regulating the expression of LAMB1, murine transgenic lines were generated by fusing regions of the LAMB1 gene to the Eschericia coli lacZ gene. The p3.9LAM beta gal construct contained approximately 4 kb of 5' flanking sequence and directed beta-galactosidase expression in many different organs including the kidney, mammary gland, and the male and female genital systems, the focus of this report. In male embryos, between gestational ages E 14.5 and birth beta-galactosidase was transiently expressed in the prospermatogonia cells of the testis and in the differentiating epithelial cells in the ductus deferens, ductus epididymis, and seminal vesicles. In female embryos, beta-galactosidase was not detected in the ovary until about 1 week after birth; at this time, beta-galactosidase was expressed by oocytes of primary and secondary follicles. In contrast, transgenic mice carrying the first 0.7 kb of LAMB1 fused to the lacZ gene expressed beta-galactosidase only in the prospermatogonia cells of the testis. Thus, the cis-acting element(s) necessary for the expression of the LAMB1 gene in prospermatogonia cells are located in the first 0.7 kb of LAMB1 5' flanking sequence; element(s) required for expression of the LAMB1 gene in oocytes and epithelial cells of the mesonephric ducts, mesonephric tubules, the ductus deferens, ductus epididymis, and seminal vesicles are located with 4 kb 5' of the transcription initiation site.

  6. In vivo regulation of the mouse beta myosin heavy chain gene.

    PubMed

    Knotts, S; Rindt, H; Neumann, J; Robbins, J

    1994-12-09

    The interactions of trans-acting factors with their respective cis-acting elements in the 5' upstream region of the beta myosin heavy chain gene (MyHC) regulate its tissue- and developmental stage-specific expression. The role of three conserved elements, an MCAT or TEF-1 binding site, a C-rich region, and a beta e3 region, in muscle-specific gene expression was analyzed in vivo. Each cis-acting site was ablated in the context of the beta MyHC promoter, fused to the chloramphenicol acetyltransferase reporter gene, and used to generate transgenic mice. In contrast to results obtained in vitro, the data demonstrate that mutating any one of these cis-acting elements does not affect the level or tissue specificity of transgene expression. Sequences upstream of -600 can functionally substitute for any one of these regulatory cassettes and are important both for high levels of expression as well as for controlled muscle specificity. Mutation of any two of the cis-acting elements also does not affect transgene expression. However, simultaneous mutation of the three sites significantly reduces expression, indicating that these conserved sequences do play an important role and that combinatorial interactions underlie the beta MyHC's regulation.

  7. Molecular characteristics of the alpha- and beta-tubulin genes of Nosema philosamiae.

    PubMed

    Zhu, Feng; Shen, Zhongyuan; Xu, Li; Guo, Xijie

    2013-11-01

    Microsporidia are intracellular parasites of insects and other higher eukaryotes. The microsporidian Nosema philosamiae Talukdar, 1961 was isolated from the eri silkworm, Philosamia cynthia ricini Grote. In the present study, alpha- and beta-tubulin genes from N. philosamiae were characterized. The identity analysis of nucleotide and amino acid sequences indicated high similarity with species of Nosema Nägeli, 1857 sensu lato (nucleotide sequences, > or = 96.0%; amino acid sequences, > or = 99.0%). However, the tubulin genes of N. philosamiae share low sequence similarity with that of N. ceranae Fries, Feng, da Silva, Slemenda et Pieniazek, 1996 (strain BRL01) and a Nosema/Vairimorpha species. Phylogenies based on alpha-, beta- and combined alpha- plus beta-tubulin gene sequences showed that N. philosamiae, along with the true Nosema species, forms a separate clade with a high bootstrap value, with N. ceranae BRL01 forming a clade of its own. The results indicated that the alpha- and beta-tubulin sequences may be useful as a diagnostic tool to discriminate the true Nosema group from the Nosema/Vairimorpha group.

  8. Molecular characterization of the gene for human interleukin-1[beta] converting enzyme (IL1BC)

    SciTech Connect

    Cerretti, D.P.; Hollingsworth, L.T.; Kozlosky, C.J.; Nelson, N. ); Valentine, M.B. ); Shapiro, D.N.; Morris, S.W. Univ. of Tennessee College of Medicine, Memphis, TN )

    1994-04-01

    Interleukin-1[beta] (IL-1[beta]) mediates a wide range of immune and inflammatory responses. The active cytokine is generated by proteolytic cleavage of an inactive precursor by a protease called the IL-1[beta] converting enzyme (ICE). A cDNA encoding this protease was recently isolated. A human genomic clone containing the ICE gene (IL1BC) was isolated using the cDNA as a probe. The gene consists of 10 exons spanning at least 10.6 kb. 5[prime]-anchored polymerase chain reaction indicated a single transcription start site [approximately]33 bp upstream of the initiator Met codon. The 5[prime]-flanking region does not have an apparent TATA box but may contain an initiator (Inr) promotor element. However, transcriptional activity could not be detected with a fusion gene containing the 5[prime]-flanking region linked to the bacterial chloramphenicol acetyltransferase gene (CAT) when transfected into the human acute monocytic leukemia cell line THP-1. Using the genomic IL1BC clone, the authors have confirmed the localization of the gene to chromosome 11 band q22.2-q22.3 by fluorescence in situ hybridization. 34 refs., 2 figs., 1 tab.

  9. Expression of the Escherichia coli beta-glucuronidase gene in Pseudocercosporella herpotrichoides.

    PubMed Central

    Bunkers, G J

    1991-01-01

    The plant-pathogenic fungus Pseudocercosporella herpotrichoides has been successfully transformed by using two different positive selection systems in combination with the Escherichia coli gusA gene. The selectable markers used in this study were the hygromycin B phosphotransferase gene (hph) from E. coli and the gene (bml) for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa. A lower transformation rate was obtained with the bml system than with the hph system. Conversely, cotransformation frequencies, as determined with medium plates containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, were higher with bml than with hph as the selectable marker. The hygromycin-resistant transformants were mitotically stable, and both the selectable gene and gusA were maintained through conidiation. The vector DNA was integrated into the genome, and the number and sites of insertion varied among transformants. Enzyme assays of mycelial extracts showed that beta-glucuronidase activity was highest in transformants with a high gusA copy number. Expression of gusA during growth of the fungus on plants was easily detectable and did not affect pathogenicity. These results form the basis for construction of a versatile and sensitive reporter gene system for P. herpotrichoides. Images PMID:1746951

  10. Specific transcription and RNA splicing defects in five cloned beta-thalassaemia genes.

    PubMed

    Treisman, R; Orkin, S H; Maniatis, T

    1983-04-14

    Transcriptional analysis of five different cloned beta-thalassaemia genes introduced into cultured mammalian cells revealed specific defects in transcription and RNA splicing. A single base change 87 base pairs to the 5' side of the mRNA cap site significantly lowers the level of transcription and therefore appears to represent a promoter mutation. Three genes contain different single base changes in the first intervening sequence (IVS) 5' splice site. One mutation, at IVS1 position 1, inactivates the splice site completely; the other two, at IVS1 positions 5 and 6, reduce its activity. Each mutation activates the same three cryptic splice sites. The fifth gene contains a single base change within IVS2 at position 745, which results in the formation of abnormal beta-globin RNA that contains an extra exon.

  11. Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector.

    PubMed Central

    Cone, R D; Weber-Benarous, A; Baorto, D; Mulligan, R C

    1987-01-01

    We introduced a human beta-globin gene into murine erythroleukemia (MEL) cells by infection with recombinant retroviruses containing the complete genomic globin sequence. The beta-globin gene was correctly regulated during differentiation, steady-state mRNA levels being induced 5- to 30-fold after treatment of the cells with the chemical inducer dimethyl sulfoxide. Studies using vectors which yield integrated proviruses lacking transcriptional enhancer sequences indicated that neither retroviral transcription nor the retroviral enhancer sequences themselves had any obvious effect on expression of the globin gene. Viral RNA expression also appeared inducible, being considerably depressed in uninduced MEL cells but approaching normal wild-type levels after dimethyl sulfoxide treatment. We provide data which suggest that the control point for both repression and subsequent activation of virus expression in MEL cells lies in the viral enhancer element. Images PMID:3029570

  12. Rapid Evolution of Beta-Keratin Genes Contribute to Phenotypic Differences That Distinguish Turtles and Birds from Other Reptiles

    PubMed Central

    Li, Yang I.; Kong, Lesheng; Ponting, Chris P.; Haerty, Wilfried

    2013-01-01

    Sequencing of vertebrate genomes permits changes in distinct protein families, including gene gains and losses, to be ascribed to lineage-specific phenotypes. A prominent example of this is the large-scale duplication of beta-keratin genes in the ancestors of birds, which was crucial to the subsequent evolution of their beaks, claws, and feathers. Evidence suggests that the shell of Pseudomys nelsoni contains at least 16 beta-keratins proteins, but it is unknown whether this is a complete set and whether their corresponding genes are orthologous to avian beak, claw, or feather beta-keratin genes. To address these issues and to better understand the evolution of the turtle shell at a molecular level, we surveyed the diversity of beta-keratin genes from the genome assemblies of three turtles, Chrysemys picta, Pelodiscus sinensis, and Chelonia mydas, which together represent over 160 Myr of chelonian evolution. For these three turtles, we found 200 beta-keratins, which indicate that, as for birds, a large expansion of beta-keratin genes in turtles occurred concomitantly with the evolution of a unique phenotype, namely, their plastron and carapace. Phylogenetic reconstruction of beta-keratin gene evolution suggests that separate waves of gene duplication within a single genomic location gave rise to scales, claws, and feathers in birds, and independently the scutes of the shell in turtles. PMID:23576313

  13. Rapid evolution of Beta-keratin genes contribute to phenotypic differences that distinguish turtles and birds from other reptiles.

    PubMed

    Li, Yang I; Kong, Lesheng; Ponting, Chris P; Haerty, Wilfried

    2013-01-01

    Sequencing of vertebrate genomes permits changes in distinct protein families, including gene gains and losses, to be ascribed to lineage-specific phenotypes. A prominent example of this is the large-scale duplication of beta-keratin genes in the ancestors of birds, which was crucial to the subsequent evolution of their beaks, claws, and feathers. Evidence suggests that the shell of Pseudomys nelsoni contains at least 16 beta-keratins proteins, but it is unknown whether this is a complete set and whether their corresponding genes are orthologous to avian beak, claw, or feather beta-keratin genes. To address these issues and to better understand the evolution of the turtle shell at a molecular level, we surveyed the diversity of beta-keratin genes from the genome assemblies of three turtles, Chrysemys picta, Pelodiscus sinensis, and Chelonia mydas, which together represent over 160 Myr of chelonian evolution. For these three turtles, we found 200 beta-keratins, which indicate that, as for birds, a large expansion of beta-keratin genes in turtles occurred concomitantly with the evolution of a unique phenotype, namely, their plastron and carapace. Phylogenetic reconstruction of beta-keratin gene evolution suggests that separate waves of gene duplication within a single genomic location gave rise to scales, claws, and feathers in birds, and independently the scutes of the shell in turtles.

  14. Expression of cytokine genes in human cardiac allografts: correlation of IL-6 and transforming growth factor-beta (TGF-beta) with histological rejection.

    PubMed Central

    Zhao, X M; Frist, W H; Yeoh, T K; Miller, G G

    1993-01-01

    Cytokines may play critical roles in allograft rejection. Currently, a clear pattern of cytokine production that correlates with rejection has not emerged. Our preliminary studies suggested a trend toward increased IL-6 and TGF-beta gene expression in cardiac allografts during rejection. We have extended these studies using reverse transcriptase/polymerase chain reaction (RT/PCR) to detect the expression of IL-6, TGF-beta, and T cell receptor beta chain constant region (TCR-beta) genes in 21 additional consecutive myocardial biopsies obtained from six heart transplant patients and from five pre-transplant donor hearts. Cytokine gene expression was compared with histological diagnosis of rejection. There was strong correlation between IL-6 as well as TGF-beta gene expression, and histological rejection (6/8 biopsies with versus 0/7 without rejection (P = 0.006) and 7/9 biopsies with versus 0/7 without rejection (P = 0.003) respectively). Neither IL-6 nor TGF-beta transcripts were detected in any pre-transplant donor heart. TCR-beta chain mRNA was found in all allograft biopsies regardless of the presence of rejection, but was absent in pre-transplant donor hearts. Our results indicate that expression of IL-6 and TGF-beta is highly correlated with allograft rejection and thus may play an important role in regulation of cardiac allograft rejection. T cell infiltration of allografted myocardium is invariably detected by PCR regardless of histological rejection. The long-term functional significance of these cells in transplanted hearts needs further investigation. Images Fig. 1 PMID:8370174

  15. Transfer of nonselectable genes into mouse teratocarcinoma cells and transcription of the transferred human. beta. -globin gene

    SciTech Connect

    Wagner, E.F.; Mintz, B.

    1982-02-01

    Teratocarcinoma (TCC) stem cells can function as vehicles for the introduction of specific recombinant genes into mice. Because most genes do not code for a selectable marker, the authors investigated the transformation efficiency of vectors with a linked selectable gene. In one series, TCC cells first selected for thymidine kinase deficiency were treated with DNA from the plasmid vector PtkH..beta..1 containing the human genomic ..beta..-globin gene and the thymidine kinase gene of herpes simplex virus. A high transformation frequency was obtained after selection in hypoxanthine-aminopterin-thymidine medium. Hybridization tests revealed that the majority of transformants had intact copies of the human gene among three to six total copies per cell. These were associated with cellular DNA sequences as judged from the presence of additional new restriction fragments and from stability of the sequences in tumors produced by injecting the cells subcutaneously. Total polyadenylate-containing RNA from cell cultures of two out of four transformants examined showed hybridization to the human gene probe: one RNA species resembled mature human ..beta..-globin mRNA transcripts; the others were of larger size. In differentiating tumors, various tissues, including hematopoietic cells of TCC provenance could be found. In a second model set of experiments, wild-type TCC cells were used to test a dominant-selection scheme with pSV-gpt vectors. Numerous transformants were isolated, and their transfected DNA was apparently stably integrated. Thus, any gene of choice can be transferred into TCC stem cells even without mutagenesis of the cells, and selected cell clones can be characterized. Cells of interest may then be introduced into early embryos to produce new mouse strains with predetermined genetic changes.

  16. Inter-MAR association contributes to transcriptionally active looping events in human beta-globin gene cluster.

    PubMed

    Wang, Li; Di, Li-Jun; Lv, Xiang; Zheng, Wei; Xue, Zheng; Guo, Zhi-Chen; Liu, De-Pei; Liang, Chi-Chuan

    2009-01-01

    Matrix attachment regions (MARs) are important in chromatin organization and gene regulation. Although it is known that there are a number of MAR elements in the beta-globin gene cluster, it is unclear that how these MAR elements are involved in regulating beta-globin genes expression. Here, we report the identification of a new MAR element at the LCR (locus control region) of human beta-globin gene cluster and the detection of the inter-MAR association within the beta-globin gene cluster. Also, we demonstrate that SATB1, a protein factor that has been implicated in the formation of network like higher order chromatin structures at some gene loci, takes part in beta-globin specific inter-MAR association through binding the specific MARs. Knocking down of SATB1 obviously reduces the binding of SATB1 to the MARs and diminishes the frequency of the inter-MAR association. As a result, the ACH establishment and the alpha-like globin genes and beta-like globin genes expressions are affected either. In summary, our results suggest that SATB1 is a regulatory factor of hemoglobin genes, especially the early differentiation genes at least through affecting the higher order chromatin structure.

  17. Dominant-negative mutation in the beta2 and beta6 proteasome subunit genes affect alternative cell fate decisions in the Drosophila sense organ lineage.

    PubMed

    Schweisguth, F

    1999-09-28

    In Drosophila, dominant-negative mutations in the beta2 and beta6 proteasome catalytic subunit genes have been identified as dominant temperature-sensitive (DTS) mutations. At restrictive temperature, beta2 and beta6 DTS mutations confer lethality at the pupal stage. I investigate here the role of proteasome activity in regulating cell fate decisions in the sense organ lineage at the early pupal stage. Temperature-shift experiments in beta2 and beta6 DTS mutant pupae occasionally resulted in external sense organs with two sockets and no shaft. This double-socket phenotype was strongly enhanced in conditions in which Notch signaling was up-regulated. Furthermore, conditional overexpression of the beta6 dominant-negative mutant subunit led to shaft-to-socket and to neuron-to-sheath cell fate transformations, which are both usually associated with increased Notch signaling activity. Finally, expression of the beta6 dominant-negative mutant subunit led to the stabilization of an ectopically expressed nuclear form of Notch in imaginal wing discs. This study demonstrates that mutations affecting two distinct proteasome catalytic subunits affect two alternative cell fate decisions and enhance Notch signaling activity in the sense organ lineage. These findings raise the possibility that the proteasome targets an active form of the Notch receptor for degradation in Drosophila.

  18. Nonrandom association of polymorphic restriction sites in the beta-globin gene cluster.

    PubMed

    Antonarakis, S E; Boehm, C D; Giardina, P J; Kazazian, H H

    1982-01-01

    By using probes for epsilon-, Psibeta(1)-, and beta-globin genes, we found four additional polymorphic restriction sites that have frequencies >0.1 in persons of Mediterranean area origin, Asian Indians, and American Blacks. Three of these (HincII sites) and the two previously described polymorphic HindIII sites [one in intervening sequence (IVS) II of each gamma-globin gene] are distributed over 32 kilobases (kb) of DNA located 5' to the delta-globin gene. This region of DNA comprises two-thirds of the beta-globin gene cluster. Since each of these five polymorphic sites can be present (+) or absent (-), in theory there exist 32 possible combinations of sites (haplotypes). However, in Italians, Greeks, Indians, and Turks, 3 of the 32 haplotypes, (+----), (-+-++), and (-++-+), account for 92% of 89 beta(A) chromosomes examined. The observed frequencies for these haplotypes are 0.64, 0.15, and 0.13 in the populations studied, in contrast to expected frequencies (based on the observed gene frequencies at each of the five sites) of 0.20, 0.006, and 0.005, respectively. In American Blacks, a fourth haplotype, (----+), which is rare in non-Black populations, has a frequency of 0.37 in contrast to its expected frequency of 0.05. These results suggest a nonrandom association of DNA sequences over 32 kb 5' to the delta-globin gene in all populations studied. Two other polymorphic sites 3' to the delta gene (the newly discovered Ava II site in IVS II of the beta-globin gene and the BamHI site 3' to it) are nonrandomly associated with each other but randomly distributed with respect to the above haplotypes. This suggests that randomization of sequences has occurred within 12 kb of DNA between these two nonrandomly associated sequence clusters. Nonrandom association of polymorphic restriction sites has practical consequences in that it limits the usefulness of these additional HincII sites for prenatal diagnosis of hemoglobinopathies by linkage analysis. These sites provide

  19. Structure, chromosome location, and expression of the human. gamma. -actin gene: Differential evolution, location, and expression of the cytoskeletal BETA- and. gamma. -actin genes

    SciTech Connect

    Erba, H.P.; Eddy, R.; Shows, T.; Kedes, L.; Gunning, P.

    1988-04-01

    The accumulation of the cytoskeletal ..beta..-and ..gamma..-actin mRNAs was determined in a variety of mouse tissues and organs. The ..beta..-iosform is always expressed in excess of the ..gamma..-isoform. However, the molar ratio of ..beta..- to ..gamma..-actin mRNA varies from 1.7 in kidney and testis to 12 in sarcomeric muscle to 114 in liver. The authors conclude that, whereas the cytoskeletal ..beta..- and ..gamma..-actins are truly coexpressed, their mRNA levels are subject to differential regulation between different cell types. The human ..gamma..-actin gene has been cloned and sequenced, and its chromosome location has been determined. The gene is located on human chromosome 17, unlike ..beta..-actin which is on chromosome 7. Thus, if these genes are also unlinked in the mouse, the coexpression of the ..beta..- and ..gamma..-actin genes in rodent tissues cannot be determined by gene linkage. Comparison of the human ..beta..- and ..gamma..-actin genes reveals that noncoding sequences in the 5'-flanking region and in intron III have been conserved since the duplication that gave rise to these two genes. In contrast, there are sequences in intron III and the 3'-untranslated region which are not present in the ..beta..-actin gene but are conserved between the human ..gamma..-actin and the Xenopus borealis type 1 actin genes. Such conserved noncoding sequences may contribute to the coexpression of ..beta..- and ..gamma..-actin or to the unique regulation and function of the ..gamma..-actin gene. Finally, the authors demonstrate that the human ..gamma..-actin gene is expressed after introduction into mouse L cells and C2 myoblasts and that, upon fusion of C2 cells to form myotubes, the human ..gamma..-actin gene is appropriately regulated.

  20. A novel transgenic mouse model produced from lentiviral germline integration for the study of beta-thalassemia gene therapy.

    PubMed

    Li, Wei; Xie, Shuyang; Guo, Xinbing; Gong, Xiuli; Wang, Shu; Lin, Dan; Zhang, Jingzhi; Ren, Zhaorui; Huang, Shuzhen; Zeng, Fanyi; Zeng, Yitao

    2008-03-01

    beta-thalassemia is one of the most common genetic diseases in the world and requires extensive therapy. Lentiviral-mediated gene therapy has been successfully exploited in the treatment of beta-thalassemia and showed promise in clinical application. Using a human beta-globin transgenic mouse line in a beta-thalassemia diseased model generated with a lentiviral-mediated approach, we investigate the stable therapeutic effect on a common thalassemia syndrome. Human beta-globin gene lentiviral vector was constr ucted, followed by subzonal microinjection into single-cell embryos of beta(IVS-2-654)-thalassemia mice to generate a transgenic line. Human beta-globin gene expression was examined with RT-PCR, Western-blotting and ELISA. The hematologic parameters and tissue pathology were investigated over time in founder mice and their off-spring. Transgenic mice with stable expression of the lentivirus carrying human beta-globin gene were obtained. A marked improvement in red blood cell indices and a dramatic reduction in red blood cell anisocytosis, poikilocytosis and target cells were observed. Nucleated cell proportion was greatly decreased in bone marrow, and splenomegaly with extramedullary hematopoiesis was ameliorated. Iron deposition in liver was also reduced. There was a two-fold increase in the survival rate of the beta(IVS-2-654) mice carrying human beta-globin transgene. Significantly, the germline integration of the lentiviral construct was obtained and stable hematologic phenotype correction was observed over the next two generations of the transgenic mice. The generation of human beta-globin transgenic mice in a beta(IVS-2-654)-thalassemia mouse mediated with lentiviral vectors provides a useful model and offers an attractive means to investigate the transgenic stable therapeutic effect in beta-thalassemia.

  1. beta-Glucuronidase is an optimal normalization control gene for molecular monitoring of chronic myelogenous leukemia.

    PubMed

    Lee, Joong Won; Chen, Qiaofang; Knowles, Daniel M; Cesarman, Ethel; Wang, Y Lynn

    2006-07-01

    Quantitative monitoring of breakpoint cluster region (BCR)-Abelson kinase (ABL) transcripts has become indispensable in the clinical care of patients with chronic myelogenous leukemia. Because quantity and quality of RNA in clinical samples are highly variable, a suitable internal normalization control is required for accurate BCR-ABL quantification. However, few studies have examined suitability of the control genes using criteria relevant to residual disease testing. In this study, we evaluated a number of control genes with the application of several novel criteria, including control gene performance on serial patient sample testing and in a residual disease model. We also examined expression of the control genes in BCR-ABL-positive K562 cells in response to Gleevec treatment. We found that beta-glucuronidase is the best control gene among those studied. Importantly, ABL, a widely used control gene, generates misleading BCR-ABL changes that potentially affect the clinical management of chronic myelogenous leukemia patients.

  2. Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei beta-mannanase gene containing a cellulose binding domain.

    PubMed Central

    Stålbrand, H; Saloheimo, A; Vehmaanperä, J; Henrissat, B; Penttilä, M

    1995-01-01

    beta-Mannanase (endo-1,4-beta-mannanase; mannan endo-1,4-beta-mannosidase; EC 3.2.1.78) catalyzes endo-wise hydrolysis of the backbone of mannan and heteromannans, including hemicellulose polysaccharides, which are among the major components of plant cell walls. The gene man1, which encodes beta-mannanase, of the filamentous fungus Trichoderma reesei was isolated from an expression library by using antiserum raised towards the earlier-purified beta-mannanase protein. The deduced beta-mannanase consists of 410 amino acids. On the basis of hydrophobic cluster analysis, the beta-mannanase was assigned to family 5 of glycosyl hydrolases (cellulase family A). The C terminus of the beta-mannanase has strong amino acid sequence similarity to the cellulose binding domains of fungal cellulases and is preceded by a serine-, threonine-, and proline-rich region. Consequently, the beta-mannanase is probably organized similarly to the T. reesei cellulases, having a catalytic core domain separated from the substrate-binding domain by an O-glycosylated linker. Active beta-mannanase was expressed and secreted by using the yeast Saccharomyces cerevisiae as the host. The results indicate that the man1 gene encodes the two beta-mannanases with different isoelectric points (pIs 4.6 and 5.4) purified earlier from T. reesei. PMID:7793911

  3. Comparison of the canine and human acid {beta}-galactosidase gene

    SciTech Connect

    Ahern-Rindell, A.J.; Kretz, K.A.; O`Brien, J.S.

    1996-05-17

    Several canine cDNA libraries were screened with human {beta}-galactosidase cDNA as probe. Seven positive clones were isolated and sequenced yielding a partial (2060 bp) canine {beta}-galactosidase cDNA with 86% identity to the human {beta}-galactosidase cDNA. Preliminary analysis of a canine genomic library indicated conservation of exon number and size. Analysis by Northern blotting disclosed a single mRNA of 2.4 kb in fibroblasts and liver from normal dogs and dogs affected with GM1 gangliosidosis. Although incomplete, these results indicate canine GM1 gangliosidosis is a suitable animal model of the human disease and should further efforts to devise a gene therapy strategy for its treatment. 20 refs., 2 figs., 1 tab.

  4. Regulation of the beta-hydroxyacyl ACP dehydratase gene of Picea mariana by alternative splicing.

    PubMed

    Tai, Helen H; Williams, Martin; Iyengar, Abhinav; Yeates, Jessica; Beardmore, Tannis

    2007-01-01

    The gene for beta-hydroxyacyl ACP dehydratase, a de novo fatty acid biosynthetic enzyme, was cloned from Picea mariana (black spruce) and consists of five exons and four introns. The first intron of the beta-hydroxyacyl ACP dehydratase mRNA is alternatively spliced. Retention of intron 1 in splice variants results in truncation of the beta-hydroxyacyl ACP dehydratase ORF at a premature termination codon. In addition, splicing of intron 1 was found to be associated with cold temperature. mRNAs retaining intron 1 increase with seed imbibition at 22 degrees C but not 4 degrees C, whereas, splicing of intron 1 increases in winter weeks with temperatures below freezing. These results provide evidence that alternative splicing may also contribute to regulation of lipid biosynthesis in Picea mariana.

  5. MHC evolution in three salmonid species: a comparison between class II alpha and beta genes.

    PubMed

    Gómez, Daniela; Conejeros, Pablo; Marshall, Sergio H; Consuegra, Sofia

    2010-08-01

    The genes of the major histocompatibility complex (MHC) are amongst the most variable in vertebrates and represent some of the best candidates to study processes of adaptive evolution. However, despite the number of studies available, most of the information on the structure and function of these genes come from studies in mammals and birds in which the MHC class I and II genes are tightly linked and class II alpha exhibits low variability in many cases. Teleost fishes are among the most primitive vertebrates with MHC and represent good organisms for the study of MHC evolution because their class I and class II loci are not physically linked, allowing for independent evolution of both classes of genes. We have compared the diversity and molecular mechanisms of evolution of classical MH class II alpha and class II beta loci in farm populations of three salmonid species: Oncorhynchus kisutch, Oncorhynchus mykiss and Salmo salar. We found single classical class II loci and high polymorphism at both class II alpha and beta genes in the three species. Mechanisms of evolution were common for both class II genes, with recombination and point mutation involved in generating diversity and positive selection acting on the peptide-binding residues. These results suggest that the maintenance of variability at the class IIalpha gene could be a mechanism to increase diversity in the MHC class II in salmonids in order to compensate for the expression of one single classical locus and to respond to a wider array of parasites.

  6. Expression of Caveolin-1 reduces cellular responses to TGF-{beta}1 through down-regulating the expression of TGF-{beta} type II receptor gene in NIH3T3 fibroblast cells

    SciTech Connect

    Lee, Eun Kyung; Lee, Youn Sook; Han, In-Oc; Park, Seok Hee . E-mail: parks@skku.edu

    2007-07-27

    Transcriptional repression of Transforming Growth Factor-{beta} type II receptor (T{beta}RII) gene has been proposed to be one of the major mechanisms leading to TGF-{beta} resistance. In this study, we demonstrate that expression of Caveolin-1 (Cav-1) gene in NIH3T3 fibroblast cells down-regulates the expression of T{beta}RII gene in the transcriptional level, eventually resulting in the decreased responses to TGF-{beta}. The reduced expression of T{beta}RII gene by Cav-1 appeared to be due to the changes of the sequence-specific DNA binding proteins to either Positive Regulatory Element 1 (PRE1) or PRE2 of the T{beta}RII promoter. In addition, Cav-1 expression inhibited TGF-{beta}-mediated cellular proliferation and Plasminogen Activator Inhibitor (PAI)-1 gene expression as well as TGF-{beta}-induced luciferase activity. Furthermore, the inhibition of endogeneous Cav-1 by small interfering RNA increased the expression of T{beta}RII gene. These findings strongly suggest that expression of Cav-1 leads to the decreased cellular responsiveness to TGF-{beta} through down-regulating T{beta}RII gene expression.

  7. Analysis and expression of the alpha-expansin and beta-expansin gene families in maize

    NASA Technical Reports Server (NTRS)

    Wu, Y.; Meeley, R. B.; Cosgrove, D. J.

    2001-01-01

    Expansins comprise a multigene family of proteins in maize (Zea mays). We isolated and characterized 13 different maize expansin cDNAs, five of which are alpha-expansins and eight of which are beta-expansins. This paper presents an analysis of these 13 expansins, as well as an expression analysis by northern blotting with materials from young and mature maize plants. Some expansins were expressed in restricted regions, such as the beta-expansins ExpB1 (specifically expressed in maize pollen) and ExpB4 (expressed principally in young husks). Other expansins such as alpha-expansin Exp1 and beta-expansin ExpB2 were expressed in several organs. The expression of yet a third group was not detected in the selected organs and tissues. An analysis of expansin sequences from the maize expressed sequence tag collection is also presented. Our results indicate that expansin genes may have general, overlapping expression in some instances, whereas in other cases the expression may be highly specific and limited to a single organ or cell type. In contrast to the situation in Arabidopsis, beta-expansins in maize seem to be more numerous and more highly expressed than are alpha-expansins. The results support the concept that beta-expansins multiplied and evolved special functions in the grasses.

  8. A pseudodeficiency allele (D152N) of the human beta-glucuronidase gene.

    PubMed

    Vervoort, R; Islam, M R; Sly, W; Chabas, A; Wevers, R; de Jong, J; Liebaers, I; Lissens, W

    1995-10-01

    We present evidence that a 480G-->A transition in the coding region of the beta-glucuronidase gene, which results in an aspartic-acid-to-asparagine substitution at amino acid position 152 (D152N), produces a pseudodeficiency allele (GUSBp) that leads to greatly reduced levels of beta-glucuronidase activity without apparent deleterious consequences. The 480G-->A mutation was found initially in the pseudodeficient mother of a child with mucopolysaccharidosis VII (MPSVII), but it was not on her disease-causing allele, which carried the L176F mutation. The 480G-->A change was also present in an unrelated individual with another MPSVII allele who had unusually low beta-glucuronidase activity, but whose clinical symptoms were probably unrelated to beta-glucuronidase deficiency. This individual also had an R357X mutation, probably on his second allele. We screened 100 unrelated normal individuals for the 480G-->A mutation with a PCR method and detected one carrier. Reduced beta-glucuronidase activity following transfection of COS cells with the D152N cDNA supported the causal relationship between the D152N allele and pseudodeficiency. The mutation reduced the fraction of expressed enzyme that was secreted. Pulse-chase experiments indicated that the reduced activity in COS cells was due to accelerated intracellular turnover of the D152N enzyme. They also suggested that a potential glycosylation site created by the mutation is utilized in approximately 50% of the enzyme expressed.

  9. Localisation of Neuregulin 1-{beta}3 to different sub-nuclear structures alters gene expression

    SciTech Connect

    Wang, Ming; Trim, Carol M.; Gullick, William J.

    2011-02-15

    Neuregulins are growth factors that signal via the ErbB3 and ErbB4 receptors. Here we show using immunohistochemistry that they are often expressed in the nucleus of a range of tumour types including soft tissue and breast. The Neuregulin 1 type I-{beta}3 (NRG1-{beta}3) isoform localises to two sub-nuclear compartments in animal cells, nucleoli and spliceosomes. We used NRG1-{beta}3 tagged with photoactivatable GFP and demonstrated that this re-localised from nucleoli to spliceosomes over 90 min. Tyrosine kinase activity was not required for retaining the NRG1-{beta}3 within the nucleus. Mutation of the lysines 14 and 16 or 15 and 16 together prevented nucleolar uptake while four positively charged residues were identified which were required for spliceosome uptake. Molecular modelling suggests that three of these may form a binding site. We showed using a kinome array that NRG1-{beta}3 and a mutant exclusively localising to spliceosomes increased phosphorylation and/or expression of the HER4 and HER2 receptors. Using a transcriptomic analysis the same two constructs induced expression of several messenger RNAs and we confirmed the increased expression at the protein level of the most highly induced, Heat Shock Protein 70B'. These results suggest that Neuregulin activates receptor signalling in spliceosomes leading to altered gene expression.

  10. Behavioral responses to odorants in drosophila require nervous system expression of the beta integrin gene myospheroid.

    PubMed

    Bhandari, Poonam; Gargano, Julia Warner; Goddeeris, Matthew M; Grotewiel, Michael S

    2006-09-01

    Integrins are cell adhesion molecules that mediate numerous developmental processes in addition to a variety of acute physiological events. Two reports implicate a Drosophila beta integrin, betaPS, in olfactory behavior. To further investigate the role of integrins in Drosophila olfaction, we used Gal4-driven expression of RNA interference (RNAi) transgenes to knock down expression of myospheroid (mys), the gene that encodes betaPS. Expression of mys-RNAi transgenes in the wing reduced betaPS immunostaining and produced morphological defects associated with loss-of-function mutations in mys, demonstrating that this strategy knocked down mys function. Expression of mys-RNAi transgenes in the antennae, antennal lobes, and mushroom bodies via two Gal4 lines, H24 and MT14, disrupted olfactory behavior but did not alter locomotor abilities or central nervous system structure. Olfactory behavior was normal in flies that expressed mys-RNAi transgenes via other Gal4 lines that specifically targeted the antennae, the projection neurons, the mushroom bodies, bitter and sweet gustatory neurons, or Pox neuro neurons. Our studies confirm that mys is important for the development or function of the Drosophila olfactory system. Additionally, our studies demonstrate that mys is required for normal behavioral responses to both aversive and attractive odorants. Our results are consistent with a model in which betaPS mediates events within the antennal lobes that influence odorant sensitivity.

  11. Molecular cloning and functional characterization of beta-N-acetylglucosaminidase genes from Sf9 cells.

    PubMed

    Aumiller, Jared J; Hollister, Jason R; Jarvis, Donald L

    2006-06-01

    Sf9, a cell line derived from the lepidopteran insect, Spodoptera frugiperda, is widely used as a host for recombinant glycoprotein expression and purification by baculovirus vectors. Previous studies have shown that this cell line has one or more beta-N-acetylglucosaminidase activities that may be involved in the degradation and/or processing of N-glycoprotein glycans. However, these enzymes and their functions remain poorly characterized. Therefore, the goal of this study was to isolate beta-N-acetylglucosaminidase genes from Sf9 cells, over-express the gene products, and characterize their enzymatic activities. A degenerate PCR approach yielded three Sf9 cDNAs, which appeared to encode two distinct beta-N-acetylglucosaminidases, according to bioinformatic analyses. Baculovirus-mediated expression of these two cDNA products induced membrane-associated beta-N-acetylglucosaminidase activities in Sf9 cells, which cleaved terminal N-acetylglucosamine residues from the alpha-3 and -6 branches of a biantennary N-glycan substrate with acidic pH optima and completely hydrolyzed chitotriose to its constituent N-acetylglucosamine monomers. GFP-tagged forms of both enzymes exhibited punctate cytoplasmic fluorescence, which did not overlap with either lysosomal or Golgi-specific dyes. Together, these results indicated that the two new Sf9 genes identified in this study encode broad-spectrum beta-N-acetylglucosaminidases that appear to have unusual intracellular distributions. Their relative lack of substrate specificity and acidic pH optima are consistent with a functional role for these enzymes in glycoprotein glycan and chitin degradation, but not with a role in N-glycoprotein glycan processing.

  12. Genetic structures at the origin of acquisition of the beta-lactamase bla KPC gene.

    PubMed

    Naas, Thierry; Cuzon, Gaelle; Villegas, Maria-Virginia; Lartigue, Marie-Frédérique; Quinn, John P; Nordmann, Patrice

    2008-04-01

    Genetic structures surrounding the carbapenem-hydrolyzing Ambler class A bla KPC gene were characterized in several KPC-positive Klebsiella pneumoniae and Pseudomonas aeruginosa strains isolated from the United States, Colombia, and Greece. The bla KPC genes were associated in all cases with transposon-related structures. In the K. pneumoniae YC isolate from the United States, the beta-lactamase bla KPC-2 gene was located on a novel Tn3-based transposon, Tn4401. Tn4401 was 10 kb in size, was delimited by two 39-bp imperfect inverted repeat sequences, and harbored, in addition to the beta-lactamase bla KPC-2 gene, a transposase gene, a resolvase gene, and two novel insertion sequences, ISKpn6 and ISKpn7. Tn4401 has been identified in all isolates. However, two isoforms of this transposon were found: Tn4401a was found in K. pneumoniae YC and K. pneumoniae GR from the United States and Greece, respectively, and differed by a 100-bp deletion, located just upstream of the bla KPC-2 gene, compared to the sequence of Tn4401b, which was found in the Colombian isolates. In all isolates tested, Tn4401 was flanked by a 5-bp target site duplication, the signature of a recent transposition event, and was inserted in different open reading frames located on plasmids that varied in size and nature. Tn4401 is likely at the origin of carbapenem-hydrolyzing beta-lactamase KPC mobilization to plasmids and its further insertion into various-sized plasmids identified in nonclonally related K. pneumoniae and P. aeruginosa isolates.

  13. Increased interleukin-1beta mRNA expression in skin biopsies of horses with Culicoides hypersensitivity following challenge with Culicoides nubeculosus extract.

    PubMed

    Kolm, Gabriela; Knapp, Elzbieta; Wagner, Regina; Klein, Dieter

    2006-09-15

    Interleukin-1beta (IL-1beta) is a primary cytokine of the skin that has a pivotal role in keratinocyte differentiation, epidermal wound healing and host defense. Pathological increase of cutaneous IL-1beta is associated with edema formation, epidermal hyperproliferation and atopic dermatitis in humans. However, in horses the role of cutaneous IL-1beta in edema formation and allergic skin disease has not been characterised so far. Particularly in Culicoides hypersensitivity (CHS), intradermal injection of Culicoides extract may be associated with enhanced transcription of local IL-1beta. To examine the mRNA expression of IL-1beta and its receptor antagonist IL-1RA in the skin of horses, biopsy specimens of horses affected and non-affected by CHS prior and following intradermal challenge with a commercial C. nubeculosus extract were examined. Our hypothesis was that cutaneous IL-1beta mRNA was significantly upregulated in horses with CHS in response to Culicoides allergen. Biopsies were taken from sites prior to and 4 h following intradermal challenge with C. nubeculosus extract. In order to obtain reliable data, real time PCR was performed and genes of interest were normalized using three different housekeeping genes, beta-actin, GAPDH, beta-2-microglobulin. No significant difference was detected in non-challenged cutaneous IL-1beta mRNA and IL-1RA mRNA levels between CHS affected and non-affected horses. Intradermal injection of C. nubeculosus extract resulted in local upregulation of IL-1beta mRNA both in horses with typical history, characteristic clinical signs for CHS and a positive intradermal skin test (IDT), and non-affected horses with a negative IDT. However, the difference in prior and post challenged site IL-1beta mRNA levels only reached statistical significance in the affected horses (p=0.01 versus 0.7). In contrast, IL-1RA mRNA levels did not demonstrate any modification following intradermal injection with C. nubeculosus in either group. In contrast

  14. Insertion/deletion-related polymorphisms in the human T cell receptor beta gene complex

    PubMed Central

    1989-01-01

    Insertion/deletion related polymorphisms (IDRP) involving stretches of 15-30 kb within the human TCR-beta gene complex were revealed by pulse- field gel electrophoresis. Two independent IDRP systems were detected by analysis of Sfi I- and Sal I-digested human DNA samples using probes for TCR C and V region gene segments. The allelic nature of these systems was verified in family studies, and mapping data allowed localization of one area of insertion/deletion among the V gene segments and the other near the C region genes. All but one of 50 individuals tested could be typed for the two allelic systems, and gene frequencies for the two allelic forms were 0.37/0.61 and 0.46/0.54, indicating that these polymorphisms are widespread. PMID:2571667

  15. Crop wild relative populations of Beta vulgaris allow direct mapping of agronomically important genes.

    PubMed

    Capistrano-Gossmann, Gina G; Ries, D; Holtgräwe, D; Minoche, A; Kraft, T; Frerichmann, S L M; Rosleff Soerensen, T; Dohm, J C; González, I; Schilhabel, M; Varrelmann, M; Tschoep, H; Uphoff, H; Schütze, K; Borchardt, D; Toerjek, O; Mechelke, W; Lein, J C; Schechert, A W; Frese, L; Himmelbauer, H; Weisshaar, B; Kopisch-Obuch, F J

    2017-06-06

    Rapid identification of agronomically important genes is of pivotal interest for crop breeding. One source of such genes are crop wild relative (CWR) populations. Here we used a CWR population of <200 wild beets (B. vulgaris ssp. maritima), sampled in their natural habitat, to identify the sugar beet (Beta vulgaris ssp. vulgaris) resistance gene Rz2 with a modified version of mapping-by-sequencing (MBS). For that, we generated a draft genome sequence of the wild beet. Our results show the importance of preserving CWR in situ and demonstrate the great potential of CWR for rapid discovery of causal genes relevant for crop improvement. The candidate gene for Rz2 was identified by MBS and subsequently corroborated via RNA interference (RNAi). Rz2 encodes a CC-NB-LRR protein. Access to the DNA sequence of Rz2 opens the path to improvement of resistance towards rhizomania not only by marker-assisted breeding but also by genome editing.

  16. Crop wild relative populations of Beta vulgaris allow direct mapping of agronomically important genes

    PubMed Central

    Capistrano-Gossmann, Gina G.; Ries, D.; Holtgräwe, D.; Minoche, A.; Kraft, T.; Frerichmann, S.L.M.; Rosleff Soerensen, T.; Dohm, J. C.; González, I.; Schilhabel, M.; Varrelmann, M.; Tschoep, H.; Uphoff, H.; Schütze, K.; Borchardt, D.; Toerjek, O.; Mechelke, W.; Lein, J. C.; Schechert, A. W.; Frese, L.; Himmelbauer, H.; Weisshaar, B.; Kopisch-Obuch, F. J.

    2017-01-01

    Rapid identification of agronomically important genes is of pivotal interest for crop breeding. One source of such genes are crop wild relative (CWR) populations. Here we used a CWR population of <200 wild beets (B. vulgaris ssp. maritima), sampled in their natural habitat, to identify the sugar beet (Beta vulgaris ssp. vulgaris) resistance gene Rz2 with a modified version of mapping-by-sequencing (MBS). For that, we generated a draft genome sequence of the wild beet. Our results show the importance of preserving CWR in situ and demonstrate the great potential of CWR for rapid discovery of causal genes relevant for crop improvement. The candidate gene for Rz2 was identified by MBS and subsequently corroborated via RNA interference (RNAi). Rz2 encodes a CC-NB-LRR protein. Access to the DNA sequence of Rz2 opens the path to improvement of resistance towards rhizomania not only by marker-assisted breeding but also by genome editing. PMID:28585529

  17. A single-base change at a splice site in a beta 0-thalassemic gene causes abnormal RNA splicing.

    PubMed

    Treisman, R; Proudfoot, N J; Shander, M; Maniatis, T

    1982-07-01

    We have studied the structure and transcription of a cloned human beta-globin gene from a fetus diagnosed for beta 0 thalassemia. The sequence of the beta 0 gene differs from that of a normal beta-globin gene at positions 1 and 74 of the second intervening sequence (IVS2). The position 1 change alters the GT dinucleotide conserved at 5' splice sites, while the position 74 change is a common sequence polymorphism. When the cloned beta 0 gene is introduced into HeLa cells by use of an SV40-derived plasmid vector, two abnormally spliced cytoplasmic beta-globin RNAs are detected. The predominant RNA differs from normal beta-globin mRNA by the insertion of the first 47 nucleotides of IVS2 between exons 2 and 3. The less abundant RNA comprises the normal first exon spliced directly to the third. Analysis of nuclear RNA suggests that the beta 0 transcript is inefficiently spliced and that the removal of the two intervening sequences is coupled.

  18. A self-excising beta-recombinase/six cassette for repetitive gene deletion and homokaryon purification in Neurospora crassa

    USDA-ARS?s Scientific Manuscript database

    In a previous study we developed a cassette employing a bacterial beta-recombinase acting on six recognition sequences (beta-rec/six), which allowed repetitive site-specific gene deletion and marker recycling in Neurospora crassa. However, only one positive selection marker was used in the cassette...

  19. Low autocrine interferon beta production as a gene therapy approach for AIDS: Infusion of interferon beta-engineered lymphocytes in macaques chronically infected with SIVmac251.

    PubMed

    Gay, Wilfried; Lauret, Evelyne; Boson, Bertrand; Larghero, Jérome; Matheux, Franck; Peyramaure, Sophie; Rousseau, Véronique; Dormont, Dominique; De Maeyer, Edward; Le Grand, Roger

    2004-09-25

    The aim of this study was to evaluate gene therapy for AIDS based on the transduction of circulating lymphocytes with a retroviral vector giving low levels of constitutive macaque interferon beta production in macaques chronically infected with a pathogenic isolate of SIVmac251. Two groups of three animals infected for more than one year with a pathogenic primary isolate of SIVmac251 were included in this study. The macaques received three infusions of their own lymphocytes transduced ex vivo with the construct encoding macaque IFN-beta (MaIFN-beta or with a vector carrying a version of the MaIFN-beta gene with a deletion preventing translation of the mRNA. Cellular or plasma viremia increased transiently following injection in most cases, regardless of the retroviral construct used. Transduced cells were detected only transiently after each infusion, among the peripheral blood mononuclear cells of all the animals, with copy numbers of 10 to 1000 per 106 peripheral mononuclear cells. Long-term follow-up indicated that the transitory presence of such a small number of cells producing such small amounts of MaIFN-beta did not prevent animals from the progressive decrease in CD4+ cell count typical of infection with simian immunodeficiency virus. These results reveal potential pitfalls for future developments of gene therapy strategies of HIV infection.

  20. Increased gene dosage plays a predominant role in the initial stages of evolution of duplicate TEM-1 beta lactamase genes.

    PubMed

    Dhar, Riddhiman; Bergmiller, Tobias; Wagner, Andreas

    2014-06-01

    Gene duplication is important in evolution, because it provides new raw material for evolutionary adaptations. Several existing hypotheses about the causes of duplicate retention and diversification differ in their emphasis on gene dosage, subfunctionalization, and neofunctionalization. Little experimental data exist on the relative importance of gene expression changes and changes in coding regions for the evolution of duplicate genes. Furthermore, we do not know how strongly the environment could affect this importance. To address these questions, we performed evolution experiments with the TEM-1 beta lactamase gene in Escherichia coli to study the initial stages of duplicate gene evolution in the laboratory. We mimicked tandem duplication by inserting two copies of the TEM-1 gene on the same plasmid. We then subjected these copies to repeated cycles of mutagenesis and selection in various environments that contained antibiotics in different combinations and concentrations. Our experiments showed that gene dosage is the most important factor in the initial stages of duplicate gene evolution, and overshadows the importance of point mutations in the coding region. © 2014 The Author(s). Evolution © 2014 The Society for the Study of Evolution.

  1. Identification of an immunodominant region on the I-A beta chain using site-directed mutagenesis and DNA-mediated gene transfer

    PubMed Central

    1988-01-01

    To identify which polymorphic residues determine the allospecific antibody binding sites on A beta polypeptides, mutant Ak beta genes were constructed encoding single or multiple amino acids of the d allele at 14 polymorphic positions in the beta 1 domain. Cell lines expressing these genes were analyzed by quantitative immunofluorescence using 16 mAbs reactive to Ak beta or Ad beta. Substitution of d allele residues at positions 63 and 65-67 in the Ak beta polypeptide resulted in the loss of binding of all Ak beta-reactive antibodies and the gain of binding of most Ad beta-reactive antibodies. Two Ad beta-reactive mAbs bound to the mutant Ak beta polypeptide containing d allele- characteristic residue at position 40. In contrast, substitution of the other polymorphic residues in the NH2-terminal and COOH-terminal regions of the beta 1 domain did not alter antibody binding. PMID:2450160

  2. The human and mouse sex-determining SRY genes repress the Rspol/beta-catenin signaling.

    PubMed

    Lau, Yun-Fai Chris; Li, Yunmin

    2009-04-01

    The sex-determining region Y (SRY) is the gene on the Y chromosome responsible for switching on male sex determination during mammalian embryogenesis. In its absence, ovaries develop in the embryo. Hence, ovarian determination and differentiation is considered to be a default, or passive, developmental pathway. Recently this classical paradigm of sex determination has been challenged with the discovery of the R-spondin 1 (RSPO1) as an active ovarian determinant. Mutations of RSPO1 cause a female-to-male sex reversal. RSPO1 synergizes with WNT4 in activating an ovarian development in the bipotential gonad via the canonical Wnt signaling. Early studies showed that SRY represses such Wnt signaling, but also generated discrepancies on whether only mouse Sry is capable of inhibiting such Wnt signaling and whether both human and mouse SRY proteins are able to interact with beta-catenin, the intracellular messenger responsible for executing the Wnt signals. Our studies show that both human SRY and mouse Sry are capable of repressing the Rspo1/Wnt/beta-catenin signaling. However, the repression activities vary among different SRY/Sry proteins and paradoxically related to the presence and/or size of an acidic/glutamine-rich domain. The HMG box of human SRY could bind directly to beta-catenin while the mouse Sry binds to beta-catenin via its HMG box and glutamine-rich domain. The results clarify some of the initial discrepancies, and raise the possibility that SRY interacts with beta-catenin in the nucleus and represses the transcriptional activation of the Rspo1/Wnt target genes involved in ovarian determination, thereby switching on testis determination.

  3. The 5alpha-reductase type 1, but not type 2, gene is expressed in anagen hairs plucked from the vertex area of the scalp of hirsute women and normal individuals.

    PubMed

    Oliveira, I O; Lhullier, C; Brum, I S; Spritzer, P M

    2003-10-01

    The aim of the present study was to determine the expression of the genes for type 1 (SDR5A1) and type 2 (SDR5A2) 5alpha-reductase isoenzymes in scalp hairs plucked from 33 hirsute patients (20 with polycystic ovary syndrome and 13 with idiopathic hirsutism) and compare it with that of 10 men and 15 normal women. SDR5A1 and SDR5A2 expression was estimated by RT-PCR using the gene of the ubiquitously expressed protein 2-microglobulin as an internal control. The results are expressed as arbitrary units in relation to beta2-microglobulin absorbance (mean SEM). SDR5A2 expression was not detected in any hair samples analyzed in this study. No differences were found in SDR5A1 mRNA levels between men and normal women (0.78+/-0.05 vs 0.74+/-0.06, respectively). SDR5A1 gene expression in the cells of hair plucked from the scalp of normal women (0.85+/-0.04) and of women with polycystic ovary syndrome (0.78+/-0.05) and idiopathic hirsutism (0.80+/-0.06) was also similar. These results indicate that SDR5A1 gene expression in the follicular keratinocytes from the vertex area of the scalp seems not to be related to the differences in hair growth observed between normal men and women and hirsute patients. Further studies are needed to investigate the expression of the 5alpha-reductase genes in other scalp follicular compartments such as dermal papillae, and also in hair follicles from other body sites, in order to elucidate the mechanism of androgen action on the hair growth process and related diseases.

  4. beta. amyloid gene duplication in Alzheimer's disease and karyotypically normal Down syndrome

    SciTech Connect

    Delabar, J.; Goldgaber, D.; Lamour, Y.; Nicole, A.; Huret, J.; De Groucy, J.; Brown, P.; Gajdusek, D.C.; Sinet, P.

    1987-03-13

    With the recently cloned complementary DNA probe, lambdaAm4 for the chromosome 21 gene encoding brain amyloid polypeptide (..beta.. amyloid protein) of Alzheimer's disease, leukocyte DNA from three patients with sporadic Alzheimer's disease and two patients with karyotypically normal Down syndrome was found to contain three copies of this bene. Because a small region of chromosome 21 containing the ets-2 gene is duplicated in patients with Alzheimer's disease, as well as in karyotypically normal Down syndrome, duplication of a subsection of the critical segment of chromosome 21 that is duplicated in Down syndrome may be the genetic defect in Alzeimer's disease.

  5. Targets for dioxin: Genes for plasminogen activator inhibitor-2 and interleukin-1. beta

    SciTech Connect

    Sutter, T.R.; Guzman, K.; Dold, K.M. ); Greenlee, W.F. )

    1991-10-18

    Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), a widespread environmental contaminant, may elicit its effects by altering gene expression in susceptible cells. Five TCDD-responsive complementary DNA clones were isolated from a human keratinocyte cell line. One of these clones encodes plasminogen activator inhibitor-2, a factor that influences growth and differentiation by regulating proteolysis of the extracellular matrix. Another encodes the cytokine interleukin-1{beta}. Thus, TCDD alters the expression of growth regulator genes and has effects similar to those of other tumor-promoting agents that affect both inflammation and differentiation.

  6. TGF-Beta Gene Polymorphisms in Food Allergic versus Non-Food Allergic Eosinophilic Esophagitis

    DTIC Science & Technology

    2014-12-01

    AD_________________ Award Number: W81XWH-11-1-0741 TITLE: Gene Food Allerg PRINCIPAL INVESTIGATOR: David Broide MB ChB CONTRACTING...TYPE Final 3. DATES COVERED (From - To) 15 Sep 2011 to 2014 4. TITLE AND SUBTITLE TGF-Beta Gene Polymorphisms in Food Allergic 5a. CONTRACT NUMBER...versus Non- Food Allergic Eosinophilic Esophagitis 5b. GRANT NUMBER W81XWH-11-1-0741 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) David Broide MB ChB

  7. Gene-gene interaction between PPAR gamma 2 and ADR beta 3 increases obesity risk in children and adolescents.

    PubMed

    Ochoa, M C; Marti, A; Azcona, C; Chueca, M; Oyarzábal, M; Pelach, R; Patiño, A; Moreno-Aliaga, M J; Martínez-González, M A; Martínez, J A

    2004-11-01

    Multiple genes are likely to be involved in obesity and these genes may interact with environmental factors to influence obesity risk. Our aim was to explore the synergistic contribution of the two polymorphisms: Pro12Ala of the PPAR gamma 2 gene and Trp64Arg of the ADR beta 3 gene to obesity risk in a Spanish children and adolescent population. We designed a sex- and age-matched case-control study. Participants were 185 obese and 185 control children (aged 5-18 y) from the Navarra region, recruited through Departments of Pediatrics (Hospital Virgen del Camino, Navarra University Clinic and several Primary Health Centers). The obesity criterion (case definition) was BMI above the 97th percentile according to Spanish BMI reference data for age and gender. Anthropometric parameters were measured by standard protocols. The genotype was assessed by PCR-RFLP after digestion with BstUI for PPAR gamma 2 mutation and BstNI for ADR beta 3 variants. Face-to-face interviews were conducted to assess the physical activity. Using a validated physical activity questionnaire, we computed an activity metabolic equivalent index (METs h/week), which represents the physical exercise during the week for each participant. Statistical analysis was performed by conditional logistic regression, taking into account the matching between cases and controls. Carriers of the polymorphism Pro12Ala of the PPAR gamma 2 gene had a significantly higher obesity risk than noncarriers (odds ratio (OR)=2.18, 95% CI=1.09-4.36) when we adjusted for sex, age and physical activity. Moreover, the risk of obesity was higher (OR=2.59, 95% CI=1.17-5.34) when family history of obesity was also taken into account in the model. The OR for obesity linked to both polymorphisms (PPAR gamma 2 and ADR beta 3) was 5.30 (95% CI=1.08-25.97) when we adjusted for sex, age and physical activity. After adjustment for family history of obesity, the OR for carriers of both polymorphisms was 19.5 (95% CI=2.43-146.8). A

  8. Use of microarray analysis to unveil transcription factor and gene networks contributing to Beta cell dysfunction and apoptosis.

    PubMed

    Eizirik, Decio L; Kutlu, Burak; Rasschaert, Joanne; Darville, Martine; Cardozo, Alessandra K

    2003-11-01

    The beta cell fate following immune-mediated damage depends on an intricate pattern of dozens of genes up- or downregulated in parallel and/or sequentially. We are utilizing microarray analysis to clarify the pattern of gene expression in primary rat beta cells exposed to the proapoptotic cytokines, IL-1beta and/or IFN-gamma. The picture emerging from these experiments is that beta cells are not passive bystanders of their own destruction. On the contrary, beta cells respond to damage by activating diverse networks of transcription factors and genes that may either lead to apoptosis or preserve viability. Of note, cytokine-exposed beta cells produce and release chemokines that may contribute to the homing and activation of T cells and macrophages during insulitis. Several of the effects of cytokines depend on the activation of the transcription factor, NF-kappaB. NF-kappaB blocking prevents cytokine-induced beta cell death, and characterization of NF-kappaB-dependent genes by microarray analysis indicated that this transcription factor controls diverse networks of transcription factors and effector genes that are relevant for maintenance of beta cell differentiated status, cytosolic and ER calcium homeostasis, attraction of mononuclear cells, and apoptosis. Identification of this and additional "transcription factor networks" is being pursued by cluster analysis of gene expression in insulin-producing cells exposed to cytokines for different time periods. Identification of complex gene patterns poses a formidable challenge, but is now technically feasible. These accumulating evidences may finally unveil the molecular mechanisms regulating the beta cell "decision" to undergo or not apoptosis in early T1DM.

  9. Increase in expression level of alpha- and beta-tubulin genes in Arabidopsis seedlings under hypergravity conditions

    NASA Astrophysics Data System (ADS)

    Saito, Y.; Soga, K.; Wakabayashi, K.; Hoson, T.

    Hypergravity, a gravitational force of more than 1 g, suppresses elongation growth of shoots of various plants. The analysis of the changes in gene expression by hypergravity treatment in Arabidopsis hypocotyls by the differential display method showed that a gene encoding alpha-tubulin, which is a component of microtubules, was up-regulated by hypergravity [Yoshioka et al. (2003) Adv. Space Res. 31: 2187-2193]. However, the detailed relation between hypergravity treatment and the changes in alpha-tubulin gene levels has not been determined yet. Microtubules are formed by the spontaneous polymerization of dimers consisting of one alpha- and one beta-tubulin protein molecule. Thus, the expression levels of beta-tubulin genes may also be affected by hypergravity. In the present study, we examined the dose-response and the time course relations of change in the expression of both alpha- and beta-tubulin genes in Arabidopsis hypocotyls grown under hypergravity conditions. Elongation growth of Arabidopsis hypocotyls was suppressed by increasing gravity. The expression levels of both alpha- and beta-tubulin genes were increased depending on the magnitude of gravity. At 300 g, expression levels of alpha- and beta-tubulin genes were about 400% and 350% of the 1 g control, respectively. The increases in expression of both tubulin genes were detected within a few hours, when the seedlings grown at 1 g were transferred to hypergravity conditions. The increase in alpha- and beta-tubulin genes preceded or occurred at the same time as growth suppression. These results suggest that Arabidopsis hypocotyls regulate the expression level of alpha- and beta-tubulin genes promptly in response to gravity stimuli. The increase in the amount of microtubules due to the activation of tubulin gene expression may be involved in the regulation by gravity signal of shoot growth.

  10. Influence of coding region polymorphism on the peripheral expression of a human TCR V[beta] gene

    SciTech Connect

    Vissinga, C.S.; Charmley, P.; Concannon, P. )

    1994-02-01

    A number of human TCR V[beta] gene segments are reported to be polymorphic, with alleles differing by one or a small number of amino acid substitutions. In the absence of detailed structural information regarding the interaction of specific positions in the TCR with Ag or MHC, the significance of such variation is difficult to assess. In this report the relative use of the two common alleles of the human V[beta]6.7 gene, 6.7a and 6.7b, which differ by two nonconservative amino acid substitutions, and the use of two common alleles of the V[beta]12.2 gene, which differ by only silent substitutions, were measured in PBL derived from individuals heterozygous for these alleles. Equal use of V[beta]12.2 alleles was observed, consistent with the inability of selection mechanisms to discriminate between the products of these alleles that are indistinguishable at the amino acid level. However, statistically significant skewing in the use of V[beta]6.7 alleles was observed in 15 of 16 individuals studied. Expression levels for each allele ranged from 16 to 84% of the total V[beta]6.7 signal in heterozygous individuals, with either the 6.7a or the 6.7b allele predominant in different individuals. Based on segregation studies in families, it seems unlikely that other unidentified polymorphism in the TCR[beta] locus, such as in the V[beta]6.7 promoter, was responsible for the differential allele expression. Family studies provided no evidence for an association between specific HLA haplotypes and V[beta]6.7 allele use. These results indicate that even modest allelic variation in human TCR V[beta] coding regions can have a significant impact on the expression of human V[beta] genes in the peripheral repertoire. 29 refs., 4 figs.

  11. Validation of commonly used reference genes for sleep-related gene expression studies

    PubMed Central

    Lee, Kil S; Alvarenga, Tathiana A; Guindalini, Camila; Andersen, Monica L; Castro, Rosa MRPS; Tufik, Sergio

    2009-01-01

    Background Sleep is a restorative process and is essential for maintenance of mental and physical health. In an attempt to understand the complexity of sleep, multidisciplinary strategies, including genetic approaches, have been applied to sleep research. Although quantitative real time PCR has been used in previous sleep-related gene expression studies, proper validation of reference genes is currently lacking. Thus, we examined the effect of total or paradoxical sleep deprivation (TSD or PSD) on the expression stability of the following frequently used reference genes in brain and blood: beta-actin (b-actin), beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and hypoxanthine guanine phosphoribosyl transferase (HPRT). Results Neither TSD nor PSD affected the expression stability of all tested genes in both tissues indicating that b-actin, B2M, GAPDH and HPRT are appropriate reference genes for the sleep-related gene expression studies. In order to further verify these results, the relative expression of brain derived neurotrophic factor (BDNF) and glycerol-3-phosphate dehydrogenase1 (GPD1) was evaluated in brain and blood, respectively. The normalization with each of four reference genes produced similar pattern of expression in control and sleep deprived rats, but subtle differences in the magnitude of expression fold change were observed which might affect the statistical significance. Conclusion This study demonstrated that sleep deprivation does not alter the expression stability of commonly used reference genes in brain and blood. Nonetheless, the use of multiple reference genes in quantitative RT-PCR is required for the accurate results. PMID:19445681

  12. Beta-Arrestin2 regulates RANKL and ephrins gene expression in response to bone remodeling in mice.

    PubMed

    Pierroz, Dominique D; Rufo, Anna; Bianchi, Estelle N; Glatt, Vaida; Capulli, Mattia; Rucci, Nadia; Cavat, Fanny; Rizzoli, René; Teti, Anna; Bouxsein, Mary L; Ferrari, Serge L

    2009-05-01

    PTH-stimulated intracellular signaling is regulated by the cytoplasmic adaptor molecule beta-arrestin. We reported that the response of cancellous bone to intermittent PTH is reduced in beta-arrestin2(-/-) mice and suggested that beta-arrestins could influence the bone mineral balance by controlling RANKL and osteoprotegerin (OPG) gene expression. Here, we study the role of beta-arrestin2 on the in vitro development and activity of bone marrow (BM) osteoclasts (OCs) and Ephrins ligand (Efn), and receptor (Eph) mRNA levels in bone in response to PTH and the changes of bone microarchitecture in wildtype (WT) and beta-arrestin2(-/-) mice in models of bone remodeling: a low calcium diet (LoCa) and ovariectomy (OVX). The number of PTH-stimulated OCs was higher in BM cultures from beta-arrestin2(-/-) compared with WT, because of a higher RANKL/OPG mRNA and protein ratio, without directly influencing osteoclast activity. In vivo, high PTH levels induced by LoCa led to greater changes in TRACP5b levels in beta-arrestin2(-/-) compared with WT. LoCa caused a loss of BMD and bone microarchitecture, which was most prominent in beta-arrestin2(-/-). PTH downregulated Efn and Eph genes in beta-arrestin2(-/-), but not WT. After OVX, vertebral trabecular bone volume fraction and trabecular number were lower in beta-arrestin2(-/-) compared with WT. Histomorphometry showed that OC number was higher in OVX-beta-arrestin2(-/-) compared with WT. These results indicate that beta-arrestin2 inhibits osteoclastogenesis in vitro, which resulted in decreased bone resorption in vivo by regulating RANKL/OPG production and ephrins mRNAs. As such, beta-arrestins should be considered an important mechanism for the control of bone remodeling in response to PTH and estrogen deprivation.

  13. Interaction of SOCS3 with NonO attenuates IL-1beta-dependent MUC8 gene expression.

    PubMed

    Song, Kyoung Seob; Kim, Kyubo; Chung, Kwang Chul; Seol, Jae Hong; Yoon, Joo-Heon

    2008-12-19

    The intracellular negatively regulatory mechanism which affects IL-1beta-induced MUC8 gene expression remains unclear. We found that SOCS3 overexpression suppressed IL-1beta-induced MUC8 gene expression in NCI-H292 cells, whereas silencing of SOCS3 restored IL-1beta-induced MUC8 gene expression. Sequentially activated ERK1/2, RSK1, and CREB by IL-1beta were not affected by SOCS3, indicating that SOCS3 has an independent mechanism of action. Using immunoprecipitaion and nano LC mass analysis, we found that SOCS3 bound NonO (non-POU-domain containing, octamer-binding domain protein) in the absence of IL-1beta, whereas IL-1beta treatment dissociated the direct binding of SOCS3 and NonO. A dominant-negative SOCS3 mutant (Y204F/Y221F) did not bind to NonO. Interestingly, SOCS3 overexpression dramatically suppressed MUC8 gene expression in cells transfected with wild-type or siRNA of NonO. Moreover, silencing of SOCS3 dramatically increased NonO-mediated MUC8 gene expression caused by IL-1beta compared to NonO overexpression alone, suggesting that SOCS3 acts as a suppressor by regulating the action of NonO.

  14. Species-dependent expression of the hyoscyamine 6 beta-hydroxylase gene in the pericycle.

    PubMed

    Kanegae, T; Kajiya, H; Amano, Y; Hashimoto, T; Yamada, Y

    1994-06-01

    The tropane alkaloid scopolamine is synthesized in the pericycle of branch roots in certain species of the Solanaceae. The enzyme responsible for the synthesis of scopolamine from hyoscyamine is hyoscyamine 6 beta-hydroxylase (H6H). The gene for H6H was isolated from Hyoscyamus niger. It has an exon/intron organization very similar to those for ethylene-forming enzymes, suggesting a common evolutionary origin. The 827-bp 5' flanking region of the H6H gene was fused to the beta-glucuronidase (GUS) reporter gene and transferred to three solanaceous species by Agrobacterium-mediated transformation systems: H. niger and belladonna (Atropa belladonna), which have high and low levels, respectively, of H6H mRNA in the root, and tobacco (Nicotiana tabacum), which has no endogenous H6H gene. Histochemical analysis showed that GUS expression occurred in the pericycle and at the root meristem of transgenic H. niger hairy roots, but only at the root meristem of transgenic H. niger hairy roots, but only at the root meristem of hairy roots and plants of transgenic tobacco. In transgenic hairy roots and regenerated plants of belladonna, the root meristem was stained with GUS activity, except for a few transformants in which the vascular cylinder was also stained. These studies indicate that the cell-specific expression of the H6H gene is controlled by some genetic regulation specific to scopolamine-producing plants.

  15. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

    SciTech Connect

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    2015-12-04

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  16. The effect of alfuzosin on renal resistive index, urinary electrolytes and β2 microglobulin levels and TGF β-1 levels of kidney tissue in rats with unilateral ureteropelvic junction obstruction.

    PubMed

    Kose, Faik; Turkyilmaz, Zafer; Sonmez, Kaan; Karabulut, Ramazan; Poyraz, Aylar; Gulbahar, Ozlem; Aral, Arzu; Damar, Cagri; Kaya, Cem; Can Basaklar, Abdullah

    2016-09-01

    In this study, it was aimed to determine the effects of alfuzosin on experimentally generated unilateral partial ureteropelvic junction obstruction (UPO) in rats. Thirty Long-Evans rats were randomly allocated into five groups. In control group (C), nothing was performed; in group Sham (S) only laparotomy was done; in Alfuzosin group (A) only alfuzosin was administered for two weeks (10 mg/kg/day p.o.) without any surgery; in UPO group, unilateral UP junction obstruction was produced; and in the Group UPT (ureteropelvic obstruction + treatment), alfuzosin was administered for two weeks (10 mg/kg/day p.o.) in addition to UPO production. Renal pelvic anteroposterior diameters were determined with ultrasonography (USG) and renal arterial resistivity indexes by color Doppler USG. Urine was collected both at the beginning and at the end of the experiment for 24 h in all the groups and at the end of the experiment, blood samples were obtained. Blood and urine electrolytes and TGF-β1, urine density, urine β2 microglobulin levels were determined. Renal tissue samples harvested from all of the rats were histopathologically evaluated. Results were determined using one-way ANOVA t-test; p < 0.05 was accepted as significant. Urine density in the UPT group was lower with respect to UPO group and blood electrolytes were preserved as close to normal (p < 0.05). In the UPT group, urine TGF-β1 and blood TGF-β1, blood β2 microglobulin levels and histopathologic damage scores were lower compared to the UPO group (p < 0.05). It is shown in this experimental unilateral partial UPO model that alfuzosin treatment prevents obstructive renal damage.

  17. Isolation of novel human cDNA (hGMF-gamma) homologous to Glia Maturation Factor-beta gene.

    PubMed

    Asai, K; Fujita, K; Yamamoto, M; Hotta, T; Morikawa, M; Kokubo, M; Moriyama, A; Kato, T

    1998-03-13

    A novel full-length human cDNA homologous to Glia Maturation Factor-beta (GMF-beta) gene was isolated. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein sequence of 142 amino acid residues. The deduced amino acid sequences of its putative product is highly homologous to human GMF-beta (82% identity) and named for GMF-gamma. Northern blot analysis indicated that a message of 0.9 kb long, but not 4.1 kb of GMF-beta, is predominantly expressed in human lung, heart, and placenta.

  18. Poly(beta-amino esters): procedures for synthesis and gene delivery.

    PubMed

    Green, Jordan J; Zugates, Gregory T; Langer, Robert; Anderson, Daniel G

    2009-01-01

    Non-viral gene delivery systems are promising as they avoid many problems of viral gene therapy by having increased design flexibility, high safety, large DNA cargo capacity, and ease of manufacture. Here, we describe the use of polymeric vectors, in particular biodegradable poly(beta-amino esters) (PBAEs), for non-viral gene delivery. These polymers are able to self-assemble with DNA and form positively charged gene delivery nanoparticles. Methods for synthesis of these polymers, particle self-assembly, and transfection using these particles are delineated. A standard protocol is presented as well as a high-throughput screening technique that can be used to more quickly optimize transfection parameters for efficient delivery.

  19. Lactogenic hormonal induction of long distance interactions between beta-casein gene regulatory elements.

    PubMed

    Kabotyanski, Elena B; Rijnkels, Monique; Freeman-Zadrowski, Courtneay; Buser, Adam C; Edwards, Dean P; Rosen, Jeffrey M

    2009-08-21

    Lactogenic hormone regulation of beta-casein gene expression in mammary epithelial cells provides an excellent model in which to study the mechanisms by which steroid and peptide hormone signaling control gene expression. Prolactin- and glucocorticoid-mediated induction of beta-casein gene expression involves two principal regulatory regions, a proximal promoter and a distal enhancer located in the mouse approximately -6 kb upstream of the transcription start site. Using a chromosome conformation capture assay and quantitative real time PCR, we demonstrate that a chromatin loop is created in conjunction with the recruitment of specific transcription factors and p300 in HC11 mammary epithelial cells. Stimulation with both prolactin and hydrocortisone is required for the induction of these long range interactions between the promoter and enhancer, and no DNA looping was observed in nontreated cells or cells treated with each of the hormones separately. The lactogenic hormone-induced interaction between the proximal promoter and distal enhancer was confirmed in hormone-treated primary three-dimensional mammary acini cultures. In addition, the developmental regulation of DNA looping between the beta-casein regulatory regions was observed in lactating but not in virgin mouse mammary glands. Furthermore, beta-casein mRNA induction and long range interactions between these regulatory regions were inhibited in a progestin-dependent manner following stimulation with prolactin and hydrocortisone in HC11 cells expressing human PR-B. Collectively, these data suggest that the communication between these regulatory regions with intervening DNA looping is a crucial step required to both create and maintain active chromatin domains and regulate transcription.

  20. Position-independent human beta-globin gene expression mediated by a recombinant adeno-associated virus vector carrying the chicken beta-globin insulator.

    PubMed

    Inoue, T; Yamaza, H; Sakai, Y; Mizuno, S; Ohno, M; Hamasaki, N; Fukumaki, Y

    1999-01-01

    The position-independent expression of transgenes in target cells is an essential subject for determining effective gene therapies. The chicken beta-globin insulator blocks the effects of regulatory sequences on transcriptional units at differential domains. We prepared a recombinant adeno-associated virus (rAAV) containing various combinations of the DNase I-hypersensitive site 2 (HS2), 3 (HS3), and 4 (HS4) core elements from the human beta-globin locus control region (LCR), the human beta-globin gene, and the herpes virus thymidine kinase promoter driven neomycin-resistant gene (neoR) (rHS432, rHS43, rHS42, rHS32, and rHS2), and also rAAV containing two copies of the 250-bp core sequence of the chicken beta-globin insulator on both sides of the rHS2 (rIns/HS2/2Ins). After isolating neomycin-resistant mouse erythroleukemia (MEL) cells infected with each rAAV, we analyzed the rAAV genome by Southern blots and polymerase chain reaction (PCR), using primers specific for HS core elements and the human beta-globin gene. All clones contained a single copy of the rAAV genome in the chromosome, however, some of them had a rearranged proviral genome. In five clones with a single unrearranged rAAV genome for each rAAV construct, we assayed the expression of the human b-globin gene relative to the endogenous mouse beta maj-globin gene, using quantitative reverse transcriptase (RT)-PCR. In clones infected with rHS432, the expression level of the human beta-globin gene ranged from 51.6% to 765.6% of that in the mouse beta maj-globin gene. Likewise, in rHS43, the expression level ranged from 36.7% to 259.0%; in rHS42, from 47.8% to 207.0%; in rHS32, from 47.9% to 105.4%; and in rHS2, from 6.1% to 172.1%, indicating a high variability of expression level in clones infected with recombinant virus lacking the insulator. In contrast, in clones infected with rIns/HS2/Ins, the range of expression of the human beta-globin gene ranged from 52.8% to 58.3% of that in the mouse beta maj

  1. Mutation in a structural gene for a beta-tubulin specific to testis in Drosophila melanogaster.

    PubMed Central

    Kemphues, K J; Raff, R A; Kaufman, T C; Raff, E C

    1979-01-01

    By two-dimensional gel electrophoresis of tubulins prepared from tissues of Drosophila melanogaster we have identified a beta-tubulin subunit that is present only in the testis. Furthermore, we have isolated, as a male sterile, a third chromosome dominant mutation [ms(3)KKD] in the structural gene for this beta-tubulin. Males heterozygous for this mutation produce no motile spermatozoa. Beginning with meiosis, all processes in spermatogenesis are abnormal to some extent. Many microtubules (including both cytoplasmic microtubules and doublet tubules of the axoneme) show aberrant structure in cross section, and the overall morphology of the developing spermatids is disorganized. Testes from these males were shown, by two-dimensional gel electrophoresis, to contain both the normal testis-specific beta-tubulin and an electrophoretic variant of this tubulin in equal amounts. Both wild-type and mutant testis-specific beta-tubulins were characterized by vinblastine sulfate precipitation, coassembly with purified Drosophila embryo tubulin, and peptide mapping. Images PMID:115008

  2. Beta-oxidation in hepatocyte cultures from mice with peroxisomal gene knockouts.

    PubMed

    Dirkx, Ruud; Meyhi, Els; Asselberghs, Stanny; Reddy, Janardan; Baes, Myriam; Van Veldhoven, Paul P

    2007-06-08

    Beta-oxidation of carboxylates takes place both in mitochondria and peroxisomes and in each pathway parallel enzymes exist for each conversion step. In order to better define the substrate specificities of these enzymes and in particular the elusive role of peroxisomal MFP-1, hepatocyte cultures from mice with peroxisomal gene knockouts were used to assess the consequences on substrate degradation. Hepatocytes from mice with liver selective elimination of peroxisomes displayed severely impaired oxidation of 2-methylhexadecanoic acid, the bile acid intermediate trihydroxycholestanoic acid (THCA), and tetradecanedioic acid. In contrast, mitochondrial beta-oxidation rates of palmitate were doubled, despite the severely affected inner mitochondrial membrane. As expected, beta-oxidation of the branched chain compounds 2-methylhexadecanoic acid and THCA was reduced in hepatocytes from mice with inactivation of MFP-2. More surprisingly, dicarboxylic fatty acid oxidation was impaired in MFP-1 but not in MFP-2 knockout hepatocytes, indicating that MFP-1 might play more than an obsolete role in peroxisomal beta-oxidation.

  3. Cloning of the {beta}3 chain gene (LAMB3) of human laminin 5, a candidate gene in junctional epidermolysis bullosa

    SciTech Connect

    Pulkkinen, L.; Christiano, A.M.; Uitto, J.

    1995-01-01

    Laminin 5 consists of three polypeptides, {alpha}3, {beta}3, and {gamma}2, encoded by the genes LAMA3, LAMB3, and LAMC2, respectively. In this study, we have elucidated the exon-intron organization of the human LAMB3 gene. Characterization of five overlapping {lambda} phage DNA clones revealed that the gene was approximately 29 kb in size. Subsequent sequence data revealed that the gene consisted of 23 exons that varied from 64 to 379 bp in size, accounting for the full-length cDNA with an open reading frame of 3516 hp encoding 1172 amino acids. Comparison of the LAMB3 gene structure with the previously characterized LAMB1 gene revealed that LAMB3 was considerably more compact. Knowledge of the exon-intron organization of the LAMB3 gene will facilitate elucidation of mutations in patients with the junctional forms of epidermolysis bullosa, some of which have been associated with mutations in the laminin 5 genes. 33 refs., 3 figs., 2 tabs.

  4. Reference gene for primary culture of prostate cancer cells.

    PubMed

    Souza, Aline Francielle Damo; Brum, Ilma Simoni; Neto, Brasil Silva; Berger, Milton; Branchini, Gisele

    2013-04-01

    Selection of reference genes to normalize mRNA levels between samples is critical for gene expression studies because their expression can vary depending on the tissues or cells used and the experimental conditions. We performed ten cell cultures from samples of prostate cancer. Cells were divided into three groups: control (with no transfection protocol), cells transfected with siRNA specific to knockdown the androgen receptor and cells transfected with inespecific siRNAs. After 24 h, mRNA was extracted and gene expression was analyzed by Real-time qPCR. Nine candidates to reference genes for gene expression studies in this model were analyzed (aminolevulinate, delta-, synthase 1 (ALAS1); beta-actin (ACTB); beta-2-microglobulin (B2M); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine phosphoribosyltransferase 1 (HPRT1); succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (SDHA); TATA box binding protein (TBP); ubiquitin C (UBC); tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ)). Expression stability was calculated NormFinder algorithm to find the most stable genes. NormFinder calculated SDHA as the most stable gene and the gene with the lowest intergroup and intragroup variation, and indicated GAPDH and SDHA as the best combination of two genes for the purpose of normalization. Androgen receptor mRNA expression was evaluated after normalization by each candidate gene and showed statistical difference in the transfected group compared to control group only when normalized by combination of GAPDH and SDHA. Based on the algorithm analysis, the combination of SDHA and GAPDH should be used to normalize target genes mRNA levels in primary culture of prostate cancer cells submitted to transfection with siRNAs.

  5. Expression of the human BETA-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells

    SciTech Connect

    Bender, M.A.; Miller, A.D.; Gelinas, R.E.

    1988-04-01

    Replication-defective amphotropic retrovirus vectors containing either the human ..beta..-globin gene with introns or an intronless ..beta..-globin minigene were constructed and used to study ..beta..-globin expression following gene transfer into hematopoietic cells. The ..beta..-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the ..beta..-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human ..beta..-globin RNA. Introduction of a virus containing the ..beta..-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human ..beta..-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human ..beta..-globin gene was 6 to 110% as active as the endogenous mouse ..beta../sup maj/-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced ..beta..-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy.

  6. Expression of the human beta-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells.

    PubMed Central

    Bender, M A; Miller, A D; Gelinas, R E

    1988-01-01

    Replication-defective amphotropic retrovirus vectors containing either the human beta-globin gene with introns or an intronless beta-globin minigene were constructed and used to study beta-globin expression following gene transfer into hematopoietic cells. The beta-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the beta-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human beta-globin RNA. Introduction of a virus containing the beta-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human beta-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human beta-globin gene was 6 to 110% as active as the endogenous mouse beta maj-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced beta-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy. Images PMID:3288863

  7. Systemic hyperosmolality improves beta-glucuronidase distribution and pathology in murine MPS VII brain following intraventricular gene transfer.

    PubMed

    Ghodsi, A; Stein, C; Derksen, T; Martins, I; Anderson, R D; Davidson, B L

    1999-11-01

    Mucopolysaccharidosis VII, a classical lysosomal storage disease, is caused by deficiency of the enzyme beta-glucuronidase. Central nervous system (CNS) manifestations are severe with accumulations of storage vacuoles in all cell types. Intraventricular gene transfer can lead to transduction of the ependyma, with production and secretion of beta-glucuronidase into the cerebral spinal fluid and underlying cortex resulting in reversal of disease pathology restricted to the periventricular areas. We tested if systemic hyperosmolality would increase the distribution of beta-glucuronidase in brain parenchyma after intraventricular virus injection. Mice were administered mannitol, intraperitoneally, 20 days after gene transfer and 1 day prior to sacrifice. Mannitol-induced systemic hyperosmolality caused a marked penetration of beta-glucuronidase into the brain parenchyma. If mannitol was administered at the time of the intraventricular injection of virus, there was penetration of vector across the ependymal cell layer, with infection of cells in the subependymal region. This also resulted in increased beta-glucuronidase activity throughout the brain. Sections of brains from beta-glucuronidase-deficient mice showed correction of cellular pathology in the subependymal region plus cortical structures away from the ventricular wall. These data indicate that virus-mediated gene transfer to the brain via the ventricles, coupled with systemic mannitol administration, can lead to extensive CNS distribution of beta-glucuronidase with concomitant correction of the storage defect. Our findings have positive therapeutic implications for the treatment of CNS disorders with gene transfer vectors and recombinant proteins.

  8. Evidence that Mls-2 antigens which delete V beta 3+ T cells are controlled by multiple genes.

    PubMed

    Pullen, A M; Marrack, P; Kappler, J W

    1989-05-01

    V beta 3+ T cells are eliminated in Mls-2a mice carrying some, but not all, H-2 types. Analysis of AKXD and BXD recombinant inbred strains showed that Mls-2a (formerly Mlsc) was not the product of a single gene and suggested that at least two non-H-2 genes control V beta 3 levels. Studies of the progeny of a B10.BR x (C3H/HeJ x B10.BR)F1 backcross confirmed the existence of two V beta 3+ T cell deleting genes: one unlinked and one linked to Ly-7, which we propose be called Mls-2 and Mls-3, respectively. Mls-2a induces partial deletion of V beta 3+ T cells with a bias toward deleting CD4+ cells. It stimulates V beta 3+ hybrids and may be linked to Mtv-13 on chromosome 4. A third non-H-2 gene is implicated in enhancing the presentation of Mls-2a. Mls-3a causes elimination of all V beta 3+ T cells in H-2k and H-2d mice but poorly stimulates V beta 3+ hybrids.

  9. Differential gene expression in well-regulated and dysregulated pancreatic beta-cell (MIN6) sublines.

    PubMed

    Lilla, Valérie; Webb, Gene; Rickenbach, Katharina; Maturana, Andres; Steiner, Donald F; Halban, Philippe A; Irminger, Jean-Claude

    2003-04-01

    To identify genes involved in regulated insulin secretion, we have established and characterized two sublines derived from the mouse pancreatic beta-cell line MIN6, designated B1 and C3. They have a similar insulin content, but differ in their secretory properties. B1 responded to glucose in a concentration- and cell confluence-dependent manner, whereas C3 did not. B1 cells were stimulated by phorbol 12-myristate 13-acetate, leucine, arginine, glibenclamide, isobutylmethylxanthine, and KCl, whereas C3 did not respond (leucine, arginine, and glibenclamide) or responded to a lesser extent (isobutylmethylxanthine, phorbol 12-myristate 13-acetate, and KCl). Although intracellular Ca(2+) rose in response to glucose in B1 but not C3 cells, KCl increased intracellular Ca(2+) in a similar manner in both sublines. GLUT-1, GLUT-2, Kir6.2, and SUR1 expression was not significantly different between B1 and C3 cells, whereas E-cadherin was more abundantly expressed in B1 cells. A more complete list of differentially expressed genes was established by suppression subtractive hybridization and high density (Affymetrix) oligonucleotide microarrays. Genes were clustered according to known or putative function. Those involved in metabolism, intracellular signaling, cytoarchitecture, and cell adhesion are of potential interest. These two sublines should be useful for identification of the genes and mechanisms involved in regulated insulin secretion of the pancreatic beta-cell.

  10. Chronic tonsillitis is not associated with beta defensin 1 gene polymorphisms in Turkish population.

    PubMed

    Arslan, Fatih; Babakurban, Seda Turkoglu; Erbek, Selim S; Sahin, Feride I; Terzi, Yunus Kasım

    2015-04-01

    Defensins are antimicrobial peptides expressed on mucosal surfaces. They function as part of the innate immune system. Palatine tonsils play important roles in innate immune system. However, our knowledge on the pathophysiology of chronic tonsils is limited. The aim of this study was to investigate the association between beta defensin 1 gene single nucleotide polymorphisms and chronic tonsillitis. Prospective, non-randomized, controlled clinical study. Tertiary referral center. Eighty six patients with chronic tonsillitis and eighty controls without history of chronic tonsillitis were enrolled in this study. Genotypes were determined by restriction fragment length polymorphism analyses after polymerase chain reaction. Genotype and allele frequencies of the -20G/A (rs11362), -44C/G (rs1800972) and -52G/A (rs1799946) single nucleotide polymorphisms were not statistically different between patients and control groups (p>0.05). In this study, we found that DEFB1 gene -20G/A, -44C/G and -52G/A single nucleotide polymorphisms were not associated with chronic tonsillitis. Studies, which analyse other polymorphism of the beta defensin 1 gene in large case series, should be conducted to understand the role of DEFB1 gene on chronic tonsillitis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  11. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells.

    PubMed

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes.

  12. A Putatively Functional Haplotype in the Gene Encoding Transforming Growth Factor Beta-1 as a Potential Biomarker for Radiosensitivity

    SciTech Connect

    Schirmer, Markus A.; Brockmoeller, Juergen; Rave-Fraenk, Margret; Virsik, Patricia; Wilken, Barbara; Kuehnle, Elna; Campean, Radu; Hoffmann, Arne O.; Mueller, Katarina; Goetze, Robert G.; Neumann, Michael; Janke, Joerg H.; Nasser, Fatima; Wolff, Hendrik A.; Ghadimi, B. Michael; Schmidberger, Heinz; Hess, Clemens F.; Christiansen, Hans; Hille, Andrea

    2011-03-01

    Purpose: To determine whether genetic variability in TGFB1 is related to circulating transforming growth factor-{beta}1 (TGF-{beta}1) plasma concentrations after radiotherapy and to radiosensitivity of lymphoid cells. Patients and Methods: Transforming growth factor-{beta}1 plasma concentrations (n = 79) were measured in patients 1 year after radiotherapy and chromosomal aberrations (n = 71) ex vivo before therapy start. Furthermore, TGF-{beta}1 secretion and apoptosis were measured in isolated peripheral blood mononuclear cells of 55 healthy volunteers. These phenotypes were analyzed in relation to five germline polymorphisms in the 5' region of the TGFB1 gene. Because of high linkage disequilibrium, these five polymorphisms reflect frequent genetic variation in this region. A presumed impact of TGF-{beta}1 on DNA damage or repair was measured as micronucleus formation in 30 lymphoblastoid cell lines. Results: We identified a hypofunctional genetic haplotype termed H3 tagging the 5' region of the TGFB1 gene encoding TGF-{beta}1. H3 was associated with lower TGF-{beta}1 plasma concentrations in patients (p = 0.01) and reduced TGF-{beta}1 secretion in irradiated peripheral blood mononuclear cells (p = 0.003). Furthermore, cells with H3 were less prone to induction of chromosomal aberrations (p = 0.001) and apoptosis (p = 0.003) by irradiation. The hypothesis that high TGF-{beta}1 could sensitize cells to DNA damage was further supported by increased micronuclei formation in 30 lymphoblastoid cell lines when preincubated with TGF-{beta}1 before irradiation (p = 0.04). Conclusions: On the basis of TGF-{beta}1 plasma levels and radiation sensitivity of lymphoid cells, this study revealed a putatively hypofunctional TGFB1 haplotype. The significance of this haplotype and the suggested link between TGF-{beta}1 function and DNA integrity should be further explored in other cell types, as well as other experimental and clinical conditions.

  13. Characterization of methionine adenosyltransferase 2beta gene expression in skeletal muscle and subcutaneous adipose tissue from obese and lean pigs.

    PubMed

    Fang, Qian; Yin, Jingdong; Li, Fengna; Zhang, Jinxiao; Watford, Malcolm

    2010-06-01

    Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine. Two genes (MAT1A and MAT2A) encode for the catalytic subunit of MAT, while a third gene (MAT2beta) encodes for a regulatory subunit (MAT II beta) that regulates the activity of the MAT2A-encoded isoenzyme and intracellular S-adenosylmethionine levels. Our previous work identified MAT2beta as a candidate gene for intramuscular fat (IMF) deposition in porcine skeletal muscle by microarray technology. Here, we cloned porcine MAT2beta cDNA and compared its expression pattern in subcutaneous adipose tissue and skeletal muscle from obese (Rongchang Breed) and lean (Pig Improvement Company, PIC) pigs (n = 6). The porcine MAT2beta cDNA was 1,800 bp long and encodes for 334 amino acids sharing high similarity with other species. MAT2beta is expressed at a higher level in liver and duodenum, followed by the stomach, fat and longissinus dorsi muscle. As expected, both subcutaneous fat content and IMF content were higher in obese than in lean pigs (both P < 0.01). MAT2beta mRNA abundance was lower in both subcutaneous adipose tissue and skeletal muscle in obese pigs compared with lean pigs (both P < 0.01). MAT II beta protein content was lower in skeletal muscle in obese than in lean pigs (P < 0.05), whereas the opposite was observed in subcutaneous adipose tissue (P < 0.01). These data demonstrated an obesity-related expression variation of the MAT II beta subunit in skeletal muscle and adipose tissue in pigs, and suggest a novel role for the MAT2beta gene in regulation of IMF deposition in skeletal muscle.

  14. Allelic polymorphism in IL-1 beta and IL-1 receptor antagonist (IL-1Ra) genes in inflammatory bowel disease.

    PubMed Central

    Bioque, G; Crusius, J B; Koutroubakis, I; Bouma, G; Kostense, P J; Meuwissen, S G; Peña, A S

    1995-01-01

    Recent reports have shown that allele 2 of the IL-1 receptor antagonist (IL-1Ra) gene is over-represented in ulcerative colitis (UC). Healthy individuals carrying allele 2 of this gene have increased production of IL-1Ra protein. Since the final outcome of the biological effects of IL-1 beta may depend on the relative proportion of these two cytokines, we have studied if a TaqI polymorphism in the IL-1 beta gene, which is relevant to IL-1 beta protein production, may be involved in the genetic susceptibility to UC and Crohn's disease (CD), in association with the established IL-1Ra gene polymorphism. Polymorphisms in the closely linked genes for IL-1 beta and IL-1Ra were typed in 100 unrelated Dutch patients with UC, 79 with CD, and 71 healthy controls. The polymorphic regions in exon 5 of the IL-1 beta gene and in intron 2 of the IL-1Ra gene, were studied by polymerase chain reaction (PCR)-based methods. The IL-1 beta allele frequencies in UC and CD patients did not differ from those in healthy controls. In order to study if the IL-1 beta gene polymorphism might participate synergistically with the IL-1Ra gene polymorphism in susceptibility to UC and CD, individuals were distributed into carriers and non-carriers of allele 2 of the genes encoding IL-1 beta and IL-1Ra, in each of the patient groups and controls. Results indicated a significant association of this pair of genes, estimated by the odds ratio (OR) after performing Fisher's exact test, in the UC group (P = 0.023, OR = 2.81), as well as in the CD group (P = 0.01, OR = 3.79). Thus, non-carriers of IL-1 beta allele 2 were more often present in the subgroup of patients carrying the IL-1Ra allele 2. By contrast, no association of these alleles was detected in the group of healthy controls (P = 1.00, OR = 0.92). These results suggest that the IL-1 beta/IL-1Ra allelic cluster may participate in defining the biological basis of predisposition to chronic inflammatory bowel diseases. PMID:7586694

  15. beta(S)-Globin gene cluster haplotypes in the West Bank of Palestine.

    PubMed

    Samarah, Fekri; Ayesh, Suhail; Athanasiou, Miranda; Christakis, John; Vavatsi, Norma

    2009-01-01

    Sickle cell disease is an inherited autosomal recessive disorder of the beta-globin chain. In Palestine it is accompanied by a low level of Hb F (mean 5.14%) and a severe clinical presentation. In this study, 59 Palestinian patients, homozygotes for Hb S were studied for their haplotype background. Eight polymorphic sites in the beta-globin gene cluster were examined. The Benin haplotype was predominant with a frequency of 88.1%, followed by a frequency of 5.1% for the Bantu haplotype. One chromosome was found to carry the Cameroon haplotype (0.85%). Three atypical haplotypes were also found (5.95%). Heterogeneity was observed in Hb F production, ranging between 1.5 and 17.0%, whereas the (G)gamma ratio was homogeneous among all haplotypes with a normal amount of about 41%. Our results are in agreement with previous reports of the Benin haplotype origin in the Mediterranean.

  16. Comprehensive identification of single nucleotide polymorphisms associated with beta-lactam resistance within pneumococcal mosaic genes.

    PubMed

    Chewapreecha, Claire; Marttinen, Pekka; Croucher, Nicholas J; Salter, Susannah J; Harris, Simon R; Mather, Alison E; Hanage, William P; Goldblatt, David; Nosten, Francois H; Turner, Claudia; Turner, Paul; Bentley, Stephen D; Parkhill, Julian

    2014-08-01

    Traditional genetic association studies are very difficult in bacteria, as the generally limited recombination leads to large linked haplotype blocks, confounding the identification of causative variants. Beta-lactam antibiotic resistance in Streptococcus pneumoniae arises readily as the bacteria can quickly incorporate DNA fragments encompassing variants that make the transformed strains resistant. However, the causative mutations themselves are embedded within larger recombined blocks, and previous studies have only analysed a limited number of isolates, leading to the description of "mosaic genes" as being responsible for resistance. By comparing a large number of genomes of beta-lactam susceptible and non-susceptible strains, the high frequency of recombination should break up these haplotype blocks and allow the use of genetic association approaches to identify individual causative variants. Here, we performed a genome-wide association study to identify single nucleotide polymorphisms (SNPs) and indels that could confer beta-lactam non-susceptibility using 3,085 Thai and 616 USA pneumococcal isolates as independent datasets for the variant discovery. The large sample sizes allowed us to narrow the source of beta-lactam non-susceptibility from long recombinant fragments down to much smaller loci comprised of discrete or linked SNPs. While some loci appear to be universal resistance determinants, contributing equally to non-susceptibility for at least two classes of beta-lactam antibiotics, some play a larger role in resistance to particular antibiotics. All of the identified loci have a highly non-uniform distribution in the populations. They are enriched not only in vaccine-targeted, but also non-vaccine-targeted lineages, which may raise clinical concerns. Identification of single nucleotide polymorphisms underlying resistance will be essential for future use of genome sequencing to predict antibiotic sensitivity in clinical microbiology.

  17. A pseudodeficiency allele (D152N) of the human {beta}-glucuronidase gene

    SciTech Connect

    Vervoort, R.; Liebaers, I.; Lissens, W.

    1995-10-01

    We present evidence that a 480G{r_arrow}A transition in the coding region of the {Beta}glucuronidase gene, which results in an aspartic-acid-to-asparagine substitution at amino acid position 152 (D152N), produces a pseudodeficiency allele (GUSBp) that leads to greatly reduced levels of {Beta}-glucuronidase activity without apparent deleterious consequences. The 48OG{r_arrow}A mutation was found initially in the pseudodeficient mother of a child with mucopolysaccharidosis VII (MPSVII), but it was not on her disease-causing allele, which carried the L176F mutation. The 480G{r_arrow}A change was also present in an unrelated individual with another MPSVII allele who had unusually low {Beta}-glucuronidase activity, but whose clinical symptoms were probably unrelated to {Beta}-glucuronidase deficiency. This individual also had an R357X mutation, probably on his second allele. We screened 100 unrelated normal individuals for the 480G{r_arrow}A mutation with a PCR method and detected one carrier. Reduced {Beta}-glucuronidase activity following transfection of COS cells with the D152N cDNA supported the causal relationship between the D152N allele and pseudodeficiency. The mutation reduced the fraction of expressed enzyme that was secreted. Pulse-chase experiments indicated that the reduced activity in COS cells was due to accelerated intracellular turnover of the D152N enzyme. They also suggested that a potential glycosylation site created by the mutation is utilized in {approximately}50% of the enzyme expressed. 25 refs., 3 figs., 3 tabs.

  18. Independent regulatory elements in the upstream region of the Drosophila beta 3 tubulin gene (beta Tub60D) guide expression in the dorsal vessel and the somatic muscles.

    PubMed

    Damm, C; Wolk, A; Buttgereit, D; Löher, K; Wagner, E; Lilly, B; Olson, E N; Hasenpusch-Theil, K; Renkawitz-Pohl, R

    1998-07-01

    The beta 3 tubulin gene (beta Tub60D) is a structural gene expressed during mesoderm development from the extended germ band stage onward. Expression within the individual mesodermal derivatives is guided by different control elements. The upstream regions allow expression in the dorsal vessel and the somatic mesoderm while enhancers localized in the first intron guide expression in the visceral mesoderm. Deletion analysis carried out in transgenic flies revealed independent regulatory elements for the dorsal vessel and the somatic mesoderm. For expression in the somatic mesoderm, a 279-bp region is absolutely essential. This region contains a binding site for the Drosophila myocyte-specific enhancer binding factor 2 (D-MEF2), a MADS-box transcription factor known to be essential for mesoderm development. Deletion or mutation of this D-MEF2 binding site strongly reduces transcription. This pattern is consistent with the strongly reduced expression of beta 3 tubulin in D-mef2 mutant embryos. This analysis furthermore reveals that the D-MEF2 binding site acts in concert with nearby cis regulatory elements. These data show that the upstream control region of the beta 3 tubulin gene is an early target of the D-MEF2 transcriptional activator.

  19. Renal tubular function in children with beta-thalassemia minor.

    PubMed

    Kalman, Süleyman; Atay, A Avni; Sakallioglu, Onur; Ozgürtaş, Taner; Gök, Faysal; Kurt, Ismail; Kürekçi, A Emin; Ozcan, Okan; Gökçay, Erdal

    2005-10-01

    beta-thalassemia minor is a common heterozygous haemoglobinopathy that is characterized by both microcytosis and hypochromia. It requires no treatment. It has been postulated that low-grade haemolysis, tubular iron deposition and toxins derived from erythrocytes might cause renal tubular damage in adult patients with beta-thalassemia minor. Our aim was to investigate the renal tubular functions in children with beta-thalassemia minor and to determine its possible harmful effects. The study was conducted on 32 children (14 female and 18 male) at the age of 5.8 +/- 3.1 years (range 2-14 years) with beta-thalassemia minor. The patients were classified as anaemic (haemoglobin (Hb) 11 g/dL) (Group 2, n = 18). A control group was formed with 18 healthy children whose ages and sexes match those in other groups (Group 3, n = 18). Fractional excretion of sodium (FE(Na), %), fractional excretion of magnesium (FE(Mg), %), fractional excretion of uric acid (FE(UA), %) and tubular phosphorus reabsorption (TPR,%) were calculated with standard formulas. Urinary calcium excretion (mg/kg per 24 h), zinc (Zn) (microg/dL), glucosuria (mg/dL), beta-2 microglobulin (mg/dL) and N-acetyl-beta-D-glycosaminidase (NAG, U/mmol creatinine) levels were measured through biochemical methods. There was no statistically significant difference among the three groups in terms of the results of FE(Na) (%), FE(Mg) (%), FE(UA) (%), TPR (%), calciuria (mg/kg per 24 h), NAG, urine Zn, proteinuria, glucosuria or urine beta- 2 microglobulin levels (P > 0.05). On the contrary of children with beta-thalassemia major, renal tubular dysfunction has not been determined in children with beta-thalassemia minor in the present study.

  20. Insect and wound induced GUS gene expression from a Beta vulgaris proteinase inhibitor gene promoter

    USDA-ARS?s Scientific Manuscript database

    Inducible gene promoters that are specifically activated by pathogen invasion or insect pest attack are needed for effective expression of resistance genes to control plant diseases. In the present study, a promoter from a serine proteinase inhibitor gene (BvSTI) shown to be up-regulated in resist...

  1. Haplotyping the human T-cell receptor. beta. -chain gene complex by use of restriction fragment length polymorphisms

    SciTech Connect

    Charmley, P.; Chao, A.; Gatti, R.A. ); Concannon, P. ); Hood, L. )

    1990-06-01

    The authors have studied the genetic segregation of human T-cell receptor {beta}-chain (TCR{beta}) genes on chromosome 7q in 40 CEPH (Centre d'Etude du Polymorphisme Humain) families by using restriction fragment length polymorphisms (RFLPs). They constructed haplotypes from eight RFLPs by using variable- and constant-region cDNA probes, which detect polymorphisms that span more than 600 kilobases of the TCR{beta} gene complex. Analysis of allele distributions between TCR{beta} genes revealed significant linkage disequilibrium between only 6 of the 28 different pairs of RFLPs. This linkage disequilibrium strongly influences the most efficient order to proceed for typing of these RFLPs in order to achieve maximum genetic informativeness, which in this study revealed a 97.3% level of heterozygosity within the TCR{beta} gene complex. The results should provide new insight into recent reports of disease associations with the TCR{beta} gene complex and should assist in designing future experiments to detect or confirm the existence of disease-susceptibility loci in this region of the human genome.

  2. Expression of the human. beta. -globin gene following retroviral-mediated transfer into multipotential hematopoietic progenitors of mice

    SciTech Connect

    Karlsson, S.; Bodine, D.M.; Perry, L.; Papayannopoulou, T.; Nienhuis, A.W. )

    1988-08-01

    Efficient transfer of the {beta}-globin gene into primitive hematopoietic progenitors was achieved with consistent and significant expression in the progeny of those cells. Retroviral vectors containing the intact genomic human {beta}-globin gene and the neomycin (G418)-resistance (neo{sup R}) gene were constructed. These gave titers of 10{sup 6} or more neo{sup R} colony-forming units/ml when packaged in {psi}2 cells. Mouse bone marrow cells were infected by coculture with producer cells and injected into lethally irradiated animals. Several parameters were varied to enhance infection frequency of colony-forming units, spleen (CFU-S); overall 41% of 116 foci studied contained an intact proviral genome. The human {beta}-globin gene was expressed in 31 of 35 CFU-S-derived spleen colonies that contained the intact vector genome at levels ranging from 1% to 5% of that of the mouse {beta}-globin genes. Infected bone marrow cells were also injected into genetically anemic W/W{sup v} recipients without prior irradiation. Human {beta}-globin chains were detected in circulating erythrocytes by immunofluorescent staining with a specific monoclonal antibody. All animals injected with donor cells that had been cultured in G418 (1 mg/ml) for 48 hr after retroviral infection had circulating erythrocytes containing human {beta}-globin chains between 3 and 8 weeks after transplantation.

  3. Segregation of cardiac and skeletal muscle-specific regulatory elements of the beta-myosin heavy chain gene.

    PubMed Central

    Rindt, H; Knotts, S; Robbins, J

    1995-01-01

    The beta-myosin heavy chain (beta-MyHC) gene is expressed in cardiac and slow skeletal muscles. To examine the regulatory sequences that are required for the gene's expression in the two compartments in vivo, we analyzed the expression pattern of a transgene consisting of the beta-MyHC gene 5' upstream region linked to the chloramphenicol acetyltransferase reporter gene. By using 5600 bp of 5' upstream region, the transgene was expressed at high levels in the slow skeletal muscles. Decreased levels of thyroid hormone led to the up-regulation of the transgene in both cardiac and skeletal muscles, mimicking the behavior of the endogenous beta-MyHC gene. After deleting the distal 5000 bp, the level of reporter gene expression was strongly reduced. However, decreased levels of thyroid hormone led to an 80-fold skeletal muscle-specific increase in transgene expression, even upon the ablation of a conserved cis-regulatory element termed MCAT, which under normal (euthyroid) conditions abolishes muscle-specific expression. In contrast, cardiac-specific induction was not detected with the deletion construct. These observations indicate that the cardiac and skeletal muscle regulatory elements can be functionally segregated on the beta-MyHC gene promoter. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7878016

  4. Segregation of cardiac and skeletal muscle-specific regulatory elements of the beta-myosin heavy chain gene.

    PubMed

    Rindt, H; Knotts, S; Robbins, J

    1995-02-28

    The beta-myosin heavy chain (beta-MyHC) gene is expressed in cardiac and slow skeletal muscles. To examine the regulatory sequences that are required for the gene's expression in the two compartments in vivo, we analyzed the expression pattern of a transgene consisting of the beta-MyHC gene 5' upstream region linked to the chloramphenicol acetyltransferase reporter gene. By using 5600 bp of 5' upstream region, the transgene was expressed at high levels in the slow skeletal muscles. Decreased levels of thyroid hormone led to the up-regulation of the transgene in both cardiac and skeletal muscles, mimicking the behavior of the endogenous beta-MyHC gene. After deleting the distal 5000 bp, the level of reporter gene expression was strongly reduced. However, decreased levels of thyroid hormone led to an 80-fold skeletal muscle-specific increase in transgene expression, even upon the ablation of a conserved cis-regulatory element termed MCAT, which under normal (euthyroid) conditions abolishes muscle-specific expression. In contrast, cardiac-specific induction was not detected with the deletion construct. These observations indicate that the cardiac and skeletal muscle regulatory elements can be functionally segregated on the beta-MyHC gene promoter.

  5. Effect of polymorphic variants of GH, Pit-1, and beta-LG genes on milk production of Holstein cows.

    PubMed

    Heidari, M; Azari, M A; Hasani, S; Khanahmadi, A; Zerehdaran, S

    2012-04-01

    Effect of polymorphic variants of growth hormone (GH), beta-lactoglobulin (beta-LG), and Pit-1 genes on milk yield was analyzed in a Holstein herd. Genotypes of the cows for these genes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Allele frequencies were 0.884 and 0.116 for L and V variants of GH, 0.170 and 0.830 for A and B variants of Pit-1, and 0.529 and 0.471 for A and B variants of beta-LG, respectively. GLM procedure of SAS software was used to test the effects of these genes on milk yield. Results indicated significant effects of these genes on milk yield (P < 0.05). Cows with LL genotype of GH produced more milk than cows with LVgenotype (P < 0.05). Also, for Pit-1 gene, animals with AB genotype produced more milk than BB genotype (P < 0.05). In the case of beta-LG gene, milk yield of animals with AA genotype was more than BB genotype (P < 0.01). Therefore, it might be concluded that homozygote genotypes of GH (LL) and beta-LG (AA) were superior compared to heterozygote genotypes, whereas, the heterozygote genotype of Pit-1 gene (AB) was desirable.

  6. A second gene for cerulean cataracts maps to the {beta} crystallin region on chromosome 22

    SciTech Connect

    Kramer, P.; Yount, J.; Lovrien, E.

    1996-08-01

    Cogenital cataracts are one of the most common major eye abnormalities and often lead to blindness in infants. At least a third of all cases are familial. Within this group, highly penetrant, autosomal dominant forms of congenital cataracts (ADCC) are most common. ADCC is a genetically heterogeneous group of disorders, in which at least eight different loci have been identified for nine clinically distinct forms. Among these, Armitage et al. mapped a gene for cerulean blue cataracts to chromosome 17q24. Bodker et al. described a large family with cerulean blue cataracts, in which the affected daughter of affected first cousins was presumed to be homozygous for the purported gene. We report linkage in this family to the region on chromosome 22q that includes two {beta} crystallin genes (CRYBB2, CRYBB3) and one pseudogene (CRYBB2P1). The affected female in question is homozygous at all markers. 25 refs., 1 fig., 1 tab.

  7. Thyroid hormone resistance: a novel mutation in thyroid hormone receptor beta (THRB) gene - case report.

    PubMed

    Işık, Emregül; Beck Peccoz, Paolo; Campi, Irene; Özön, Alev; Alikaşifoğlu, Ayfer; Gönç, Nazlı; Kandemir, Nurgün

    2013-01-01

    Thyroid hormone resistance (THR) is a dominantly inherited syndrome characterized by reduced sensitivity to thyroid hormones. It is usually caused by mutations in the thyroid hormone receptor beta (THRB) gene. In the present report, we describe the clinical and laboratory characteristics and genetic analysis of patients with a novel THRB gene mutation. The index patient had been misdiagnosed as hyperthyroidism and treated with antithyroid drugs since eight days of age. Thyroid hormone results showed that thyrotropin (thyroid-stimulating hormone, TSH) was never suppressed despite elevated thyroid hormone levels, and there was no symptom suggesting hyperthyroidism. A heterozygous mutation at codon 350 located in exon 9 of the THRB gene was detected in all the affected members of the family. It is important to consider thyroid hormone levels in association with TSH levels to prevent inappropriate treatment and the potential complications, such as clinical hypothyroidism or an increase in goiter size.

  8. Gene expression responses of European flounder (Platichthys flesus) to 17-beta estradiol.

    PubMed

    Williams, Tim D; Diab, Amer M; George, Stephen G; Sabine, Victoria; Chipman, James K

    2007-02-05

    Male European flounder (Platichthys flesus) were intraperitoneally injected with 10mg/kg 17-beta estradiol and tissues taken from individuals over a timecourse of 16 days. The GENIPOL P. flesus cDNA microarray was employed to detect hepatic gene expression differences between fish treated with estradiol and saline controls. Known biomarkers of estrogen exposure, choriogenin L and vitellogenins, showed sustained induction over the time-course. Among 175 identified clones showing sustained statistically significant induction or repression, those associated with the Gene Ontology terms mitochondria, amino acid synthesis, ubiquitination and apoptosis were included amongst those induced while those associated with immune function, electron transport, cell signalling and protein phosphorylation were repressed. Thus, we show the gene expression response of an environmentally relevant fish species to a high dose of an estrogenic endocrine disruptor and also report the sequencing of a further 2121 flounder ESTs.

  9. Transcriptome analysis of sugar beet root maggot (Tetanops myopaeformis) genes modulated by the Beta vulgaris host.

    PubMed

    Li, Haiyan; Smigocki, Ann C

    2016-10-03

    Sugar beet root maggot (SBRM, Tetanops myopaeformis von Röder) is a major but poorly understood insect pest of sugar beet (Beta vulgaris L.). The molecular mechanisms underlying plant defense responses are well documented, however, little information is available about complementary mechanisms for insect adaptive responses to overcome host resistance. To date, no studies have been published on SBRM gene expression profiling. Suppressive subtractive hybridization (SSH) generated more than 300 SBRM ESTs differentially expressed in the interaction of the pest with a moderately resistant (F1016) and a susceptible (F1010) sugar beet line. Blast2GO v. 3.2 search indicated that over 40% of the differentially expressed genes had known functions, primarily driven by fruit fly D. melanogaster genes. Expression patterns of 18 selected EST clones were confirmed by RT-PCR analysis. Gene Ontology (GO) analysis predicted a dominance of metabolic and catalytic genes involved in the interaction of SBRM with its host. SBRM genes functioning during development, regulation, cellular process, signaling and under stress conditions were annotated. SBRM genes that were common or unique in response to resistant or susceptible interactions with the host were identified and their possible roles in insect responses to the host are discussed.

  10. A physical map linking the five CD1 human thymocyte differentiation antigen genes.

    PubMed Central

    Yu, C Y; Milstein, C

    1989-01-01

    Human CD1 is a family of thymocyte differentiation antigens which consist of heavy chains with mol. wts between 43 and 49 kd binding to beta 2 microglobulin. They are distant relatives of the major histocompatibility complex (MHC) class I and II products. Five human CD1 genes have been described. Three (CD1A, -B and -C) code for the serologically defined CD1a, -b and -c antigens. The protein products of the other two genes, CD1D and CD1E, remain unknown. All CD1 genes are located on chromosome 1 and hence are independent of the MHC locus. In this paper, the tight linkage of the CD1 genes has been established by pulse field gel electrophoresis, cosmid cloning and walking techniques. The 190 kb of DNA linking all five CD1 genes has been spanned by 14 overlapping cosmids. The order of the genes in the CD1 complex is CD1D-CD1A-CD1C-CD1B-CD1E, and, with the exception of CD1B, they are arranged in the same transcriptional orientation. The genes are evenly spaced in the complex except for the distance between CD1D and CD1A, which is two to three times greater than the average. Images PMID:2583117

  11. Structure, expression, and molecular mapping of a divergent member of the class I HLA gene family

    SciTech Connect

    Srivastava, R.; Chorney, M.J.; Lawrance, S.K.; Pan, J.; Smith, Z.; Smith, C.L.; Weissman, S.M.

    1987-06-01

    A class I gene distinct from HLA-A, -B, or -C was identified in a cosmid clone and transfected into mouse L cells. The gene, placed adjacent to the polyoma enhancer, produced a full-length class I mRNA and high levels of a 43-kDa protein in the cytoplasm. The surface expression of the gene product required its association with human ..beta../sub 2/-microglobulin. The protein was recognized by a xenoantiserum raised against a mixture of human B- and T-cell lines. The product was also serologically reactive the HLA framework monoclonal antibodies. The complete nucleotide sequence of the gene was determined and a specific oligonucleotide probe was synthesized. This probe was used to identify a full-length mRNA transcript in a B-lymphoblastoid cell line (JY). The gene was mapped within a 190-kilobase Not I restriction fragment located in the telomeric portion of the human major histocompatibility complex. Distinct features of the gene include (i) the structure of the promoter, (ii) the position of the translation initiation site, (iii) a frameshift mutation at the carboxyl terminus, (iv) the insertion of an Alu repeat element in the eighth exon, (v) divergence in the derived amino acid sequence, and (vi) the lack of expression of the gene in some cells.

  12. Cloning and sequence of the gene encoding a cefotaxime-hydrolyzing class A beta-lactamase isolated from Escherichia coli.

    PubMed Central

    Ishii, Y; Ohno, A; Taguchi, H; Imajo, S; Ishiguro, M; Matsuzawa, H

    1995-01-01

    Escherichia coli TUH12191, which is resistant to piperacillin, cefazolin, cefotiam, ceftizoxime, cefuzonam, and aztreonam but is susceptible to cefoxitin, latamoxef, flomoxef, and imipenem, was isolated from the urine of a patient treated with beta-lactam antibiotics. The beta-lactamase (Toho-1) purified from the bacteria had a pI of 7.8, had a molecular weight of about 29,000, and hydrolyzed beta-lactam antibiotics such as penicillin G, ampicillin, oxacillin, carbenicillin, piperacillin, cephalothin, cefoxitin, cefotaxime, ceftazidime, and aztreonam. Toho-1 was markedly inhibited by beta-lactamase inhibitors such as clavulanic acid and tazobactam. Resistance to beta-lactams, streptomycin, spectinomycin, sulfamethoxazole, and trimethoprim was transferred by conjugational transfer from E. coli TUH12191 to E. coli ML4903, and the transferred plasmid was about 58 kbp, belonging to incompatibility group M. The cefotaxime resistance gene for Toho-1 was subcloned from the 58-kbp plasmid by transformation of E. coli MV1184. The sequence of the gene for Toho-1 was determined, and the open reading frame of the gene consisted of 873 or 876 bases (initial sequence, ATGATG). The nucleotide sequence of the gene (DDBJ accession number D37830) was found to be about 73% homologous to the sequence of the gene encoding a class A beta-lactamase produced by Klebsiella oxytoca E23004. According to the amino acid sequence deduced from the DNA sequence, the precursor consisted of 290 or 291 amino acid residues, which contained amino acid motifs common to class A beta-lactamases (70SXXK, 130SDN, and 234KTG). Toho-1 was about 83% homologous to the beta-lactamase mediated by the chromosome of K. oxytoca D488 and the beta-lactamase mediated by the plasmid of E. coli MEN-1. Therefore, the newly isolated beta-lactamase Toho-1 produced by E. coli TUH12191 is similar to beta-lactamases produced by K. oxytoca D488, K. oxytoca E23004, and E. coli MEN-1 rather than to mutants of TEM or SHV enzymes

  13. Expression of the leukemia-associated CBF{beta}/SMMHC chimeric gene causes transformation of 3T3 cells

    SciTech Connect

    Hajra, A.; Liu, P.; Collins, E.S.

    1994-09-01

    A pericentric inversion of chromosome 16 (inv(16)(p13;q22)) is consistently seen in acute myeloid leukemia of the M4Eo subtype. This inversion fuses almost the entire coding region of the gene encoding of the {beta} subunit of the heterodimeric transcription factor CBF/PEBP2 to the region of the MYH11 gene encoding the rod domain for the smooth muscle myosin heavy chain (SMMHC). To investigate the biological properties of the CBF{beta}/SMMHC fusion protein, we have generated 3T3 cell lines that stably express the CBF{beta}/SMMHC chimeric cDNA or the normal, nonchimeric CBF{beta} and SMMHC cDNAs. 3T3 cells expressing CBF{beta}/SMMHC acquire a transformed phenotype, as indicated by altered cell morphology, formation of foci, and growth in soft agar. Cells constitutively overexpressing the normal CBF{beta} cDNA or the rod region of SMMHC remain nontransformed. Western blot analysis using antibodies to CBF{beta} and the SMMHC rod demonstrates that stably transfected cells express the appropriate chimeric or normal protein. Electrophoretic mobility shift assays reveal that cells transformed by the chimeric cDNA do not have a CBF-DNA complex of the expected mobility, but instead contain a large complex with CBF DNA-binding activity that fails to migrate out of the gel wells. In order to define the regions of CBF{beta}/SMMHC necessary for 3T3 transformation, we have stably transfected cells with mutant CBF{beta}/SMMHC cDNAs containing various deletions of the coding region. Analysis of these cell lines indicates that the transformation property of CBF{beta}/SMMHC requires regions of CBF{beta} known to be necessary for association with the DNA-binding CBF{alpha} subunit, and also requires an intact SMMHC carboxyl terminus, which is necessary for formation of the coiled coil domain of the myosin rod.

  14. Mutations in the c-erbA beta 1 gene: do they underlie euthyroid fibromyalgia?

    PubMed

    Lowe, J C; Cullum, M E; Graf, L H; Yellin, J

    1997-02-01

    Fibromyalgia, a chronic condition of widespread pain, stiffness, and fatigue, has proven unresponsive to drugs, the use of which is based on the 'serotonin-deficiency hypothesis'. An alternative hypothesis-failed transcription regulation by thyroid hormone-can explain the serotonin deficiency and other objective findings and symptoms of euthyroid fibromyalgia. Virtually every feature of fibromyalgia corresponds to signs or symptoms associated with failed transcription regulation by thyroid hormone. In hypothyroid fibromyalgia, failed transcription regulation would result from thyroid-hormone deficiency. In euthyroid fibromyalgia, failed transcription regulation may result from low-affinity thyroid hormone receptors coded by a mutated c-erbA beta 1 gene, yielding partial peripheral resistance to thyroid hormone. The hypothesis of this paper is that, in euthyroid fibromyalgia, a mutant c-erbA beta 1 gene (or alternately, the c-erbA alpha 1 gene) results in low-affinity thyroid-hormone receptors that prevent normal thyroid hormone regulation of transcription. As in hypothyroidism, this would cause a shift toward alpha-adrenergic dominance and increases in both cyclic adenosine 3'-5'-phosphate phosphodiesterase and inhibitory Gi proteins. The result would be tissue-specific hypothyroid-like symptoms despite normal circulating thyroid-hormone levels.

  15. Beta-lactamase-producing Pseudomonas aeruginosa: Phenotypic characteristics and molecular identification of virulence genes.

    PubMed

    Ullah, Waheed; Qasim, Muhammad; Rahman, Hazir; Jie, Yan; Muhammad, Noor

    2017-03-01

    Pseudomonas aeruginosa causes common infections in immunocompromised and cystic fibrosis patients. However, drug resistance capability and release of virulence factors play a key role in bacterial pathogenicity. Beta-lactamase-producing clinical isolates of P. aeruginosa were screened for biofilm formation and pigment production. Subsequently, all the isolates were subjected to the detection of six virulence genes (OprI, OprL, LasB, PlcH, ExoS, and ToxA). Among beta-lactamase-producing isolates (n=54), about 85.18% (n=46) were biofilm producers. Pigment production was observed in 92.59% (n=50) isolates. Clinical samples were subsequently screened for detection of virulence factors. Among them, 40.74% (n=22) isolates were found to be OprI positive, while 29.62% (n=16) were OprL producers. In the case of LasB and PlcH, 24% (n=13) and 18.5% (n=10) isolates produced these virulence genes, respectively. Among the isolates, 37.03% (n=20) and 33.33% (n=18) expressed virulence factors ExoS and ToxA, respectively. Furthermore, 42.59% (n=23) isolates coproduced more than one type of virulence factors. In the current study, pigment display, biofilm formation, and virulence genes were detected in P. aeruginosa clinical isolates. Such factors could be crucial in the development of drug resistance. Copyright © 2016. Published by Elsevier Taiwan LLC.

  16. Developmental regulation of {beta}-hexosaminidase {alpha}- and {beta}-subunit gene expression in the rat reproductive system

    SciTech Connect

    Trasler, J.M.; Wakamatsu, N.; Gravel, R.A.; Benoit, G.

    1994-09-01

    {beta}-Hexosaminidase is an essential lysosomal enzyme whose absence in man results in a group of disorders, the G{sub M2} gangliosidoses. Enzyme activity for {beta}-hexosaminidase is many fold higher in the epididymis than in other tissues, is present in sperm and is postulated to be required for mammalian fertilization. To better understand how {beta}-hexosaminidase is regulated in the reproductive system, we quantitated the mRNA expression of the {alpha}- and {beta}-subunits (Hex {alpha} and Hex {beta}) of the enzyme in the developing rat testis and epididymis. Hex {alpha} mRNA was differentially expressed and abundant in adult rat testis and epididymis, 13- and 2-fold brain levels, respectively. In contrast, Hex {beta} mRNA levels in the testis and epididymis were .3- and 5-fold brain levels. Within the epididymis both Hex {alpha} and Hex {beta} mRNA concentrations were highest in the corpus, 1.5-fold and 9-fold initial segment values, respectively. During testis development from 7-91 days of age, testis levels of Hex {alpha} mRNA increased 10-fold and coincided with the appearance of spermatocytes and spermatids in the epithelium. In isolated male germ cells, Hex {alpha} expression was most abundant in haploid round spermatids. Hex {alpha} mRNA was undetectable after hypophysectomy and returned to normal after testosterone administration and the return of advanced germ cells to the testis. Hex {beta} mRNA was expressed at constant low levels throughout testis development. In the caput-corpus and cauda regions of the epididymis Hex {alpha} mRNA levels increased 2-fold between 14 and 91 days; during the same developmental period epididymal Hex {beta} mRNA levels increased dramatically, by 10-20 fold. In summary, Hex {alpha} and Hex {beta} mRNAs are differentially and developmentally expressed at high levels in the rat testis and epididymis and augur for an important role for {beta}-hexosaminidase in normal male reproductive function.

  17. Double gene deletion reveals the lack of cooperation between PPAR{alpha} and PPAR{beta} in skeletal muscle

    SciTech Connect

    Bedu, E.; Desplanches, D.; Pequignot, J.; Bordier, B.; Desvergne, B. . E-mail: beatrice.desvergne@unil.ch

    2007-06-15

    The peroxisome proliferator-activated receptors (PPARs) are involved in the regulation of most of the pathways linked to lipid metabolism. PPAR{alpha} and PPAR{beta} isotypes are known to regulate muscle fatty acid oxidation and a reciprocal compensation of their function has been proposed. Herein, we investigated muscle contractile and metabolic phenotypes in PPAR{alpha}-/-, PPAR{beta}-/-, and double PPAR{alpha}-/- {beta}-/- mice. Heart and soleus muscle analyses show that the deletion of PPAR{alpha} induces a decrease of the HAD activity ({beta}-oxidation) while soleus contractile phenotype remains unchanged. A PPAR{beta} deletion alone has no effect. However, these mild phenotypes are not due to a reciprocal compensation of PPAR{beta} and PPAR{alpha} functions since double gene deletion PPAR{alpha}-PPAR{beta} mostly reproduces the null PPAR{alpha}-mediated reduced {beta}-oxidation, in addition to a shift from fast to slow fibers. In conclusion, PPAR{beta} is not required for maintaining skeletal muscle metabolic activity and does not compensate the lack of PPAR{alpha} in PPAR{alpha} null mice.

  18. Structure of a rice beta-glucanase gene regulated by ethylene, cytokinin, wounding, salicylic acid and fungal elicitors.

    PubMed

    Simmons, C R; Litts, J C; Huang, N; Rodriguez, R L

    1992-01-01

    A rice beta-glucanase gene was sequenced and its expression analyzed at the level of mRNA accumulation. This gene (Gns1) is expressed at relatively low levels in germinating seeds, shoots, leaves, panicles and callus, but it is expressed at higher levels in roots. Expression in the roots appears to be constitutive. Shoots express Gns1 at much higher levels when treated with ethylene, cytokinin, salicylic acid, and fungal elicitors derived from the pathogen Sclerotium oryzae or from the non-pathogen Saccharomyces cereviseae. Shoots also express Gns1 at higher levels in response to wounding. Expression in the shoots is not significantly affected by auxin, gibberellic acid or abscisic acid. The beta-glucanase shows 82% amino acid similarity to the barley 1,3;1,4-beta-D-glucanases, and from hybridization studies it is the beta-glucanase gene in the rice genome closest to the barley 1,3;1,4-beta-glucanase EI gene. The mature peptide has a calculated molecular mass of 32 kDa. The gene has a large 3145 bp intron in the codon for the 25th amino acid of the signal peptide. The gene exhibits a very strong codon bias of 99% G + C in the third position of the codon in the mature peptide coding region, but only 61% G + C in the signal peptide region.

  19. Identification of appropriate reference genes for human mesenchymal stem cell analysis by quantitative real-time PCR.

    PubMed

    Li, Xiuying; Yang, Qiwei; Bai, Jinping; Xuan, Yali; Wang, Yimin

    2015-01-01

    Normalization to a reference gene is the method of choice for quantitative reverse transcription-PCR (RT-qPCR) analysis. The stability of reference genes is critical for accurate experimental results and conclusions. We have evaluated the expression stability of eight commonly used reference genes found in four different human mesenchymal stem cells (MSC). Using geNorm, NormFinder and BestKeeper algorithms, we show that beta-2-microglobulin and peptidyl-prolylisomerase A were the optimal reference genes for normalizing RT-qPCR data obtained from MSC, whereas the TATA box binding protein was not suitable due to its extensive variability in expression. Our findings emphasize the significance of validating reference genes for qPCR analyses. We offer a short list of reference genes to use for normalization and recommend some commercially-available software programs as a rapid approach to validate reference genes. We also demonstrate that the two reference genes, β-actin and glyceraldehyde-3-phosphate dehydrogenase, are frequently used are not always successful in many cases.

  20. Selection and evaluation of new reference genes for RT-qPCR analysis in Epinephelus akaara based on transcriptome data

    PubMed Central

    Wang, Huan; Zhang, Xiang; Liu, Qiaohong; Liu, Xiaochun; Ding, Shaoxiong

    2017-01-01

    Groupers are an economically important fish species in world fishery markets. Because many studies using RT-qPCR have addressed gene expression in groupers, appropriate reference genes are required to obtain reliable and accurate results. In this study, the most suitable reference genes were identified from eleven candidate genes of one of the most valuable species, Epinephelus akaara, in a range of different experimental conditions. Using the software packages geNorm, NormFinder, BestKeeper and refFinder, three traditionally used reference genes, β-actin (β-ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-2-microglobulin (B2M), were identified as not suitable for E. akaara gene expression studies, whereas two newly identified reference genes, conserved oligomeric Golgi complex subunit 5 (Cog5) and brefeldin a-inhibited guanine nucleotide-exchange protein 1 (ARFGEF1), could be universally applied under all the tested conditions. These data provide the foundation for more precise results in RT-qPCR studies of gene expression in E. akaara and other Epinephelus species. PMID:28182746

  1. Selection and evaluation of new reference genes for RT-qPCR analysis in Epinephelus akaara based on transcriptome data.

    PubMed

    Wang, Huan; Zhang, Xiang; Liu, Qiaohong; Liu, Xiaochun; Ding, Shaoxiong

    2017-01-01

    Groupers are an economically important fish species in world fishery markets. Because many studies using RT-qPCR have addressed gene expression in groupers, appropriate reference genes are required to obtain reliable and accurate results. In this study, the most suitable reference genes were identified from eleven candidate genes of one of the most valuable species, Epinephelus akaara, in a range of different experimental conditions. Using the software packages geNorm, NormFinder, BestKeeper and refFinder, three traditionally used reference genes, β-actin (β-ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-2-microglobulin (B2M), were identified as not suitable for E. akaara gene expression studies, whereas two newly identified reference genes, conserved oligomeric Golgi complex subunit 5 (Cog5) and brefeldin a-inhibited guanine nucleotide-exchange protein 1 (ARFGEF1), could be universally applied under all the tested conditions. These data provide the foundation for more precise results in RT-qPCR studies of gene expression in E. akaara and other Epinephelus species.

  2. Low beta-glucuronidase enzyme activity and mutations in the human beta-glucuronidase gene in mild mucopolysaccharidosis type VII, pseudodeficiency and a heterozygote.

    PubMed

    Vervoort, R; Gitzelmann, R; Bosshard, N; Maire, I; Liebaers, I; Lissens, W

    1998-01-01

    Deficiency of beta-glucuronidase is the cause of the human lysosomal storage disorder mucopolysaccharidosis type VII (MPS VII). The wide interfamilial variation in the presentation of this disorder complicates clinical diagnosis. Since greatly reduced beta-glucuronidase enzyme activity may also be found in healthy individuals (pseudodeficiency), diagnosis based on the biochemical phenotype is also difficult. This is illustrated by the patients studied here, who had extremely mild symptoms confined to the spine, or tachycardia, or upper respiratory infection, and who had low beta-glucuronidase activity, and excessive granulation of granulocytes and monocytes on routine blood smears. Low enzyme activity was caused by mutations in the beta-glucuronidase gene in all cases. One patient was homozygous for the previously described D152N allele. Family information and 35SO4-uptake studies clearly demonstrated that he was pseudodeficient, with symptoms unrelated to his low beta-glucuronidase activity. Two patients of another family were compound heterozygotes for a C38G and a Y626H allele, and were probably extremely mild MPS VII patients. The low beta-glucuronidase activity in another mild MPS VII patient was due to reduced biosynthesis of stable mRNA from one allele, and a W446X mutation on the second. Extremely low beta-glucuronidase enzyme activity was also found in the serum of a carrier of a 1801deltaT allele, possibly as a consequence of a dominant-negative effect. A combination of investigations is necessary in order to differentiate between mild disease and pseudodeficiency in individuals with enzyme activities close to the threshold.

  3. Characterization of horse (Equus caballus) T-cell receptor beta chain genes

    SciTech Connect

    Schrenzel, M.D.; Watson, J.L.; Ferrick, D.A.

    1994-12-31

    Genes encoding the horse (Equus caballus) T-cell receptor beta chain (TCRB) were cloned and characterized. Of 33 cDNA clones isolated from the mesenteric lymph node, 30 had functionally rearranged gene segments, and three contained germline sequences. Sixteen unique variable segments (TCRBV), 14 joining genes (TCRBJ), and two constant region genes (TCRBC) were identified. Horse TCRBV were grouped into nine families based on similarity to human sequences. TCRBV2 and TCRBV12 were the most commonly represented horse families. Analysis of predicted protein structure revealed the presence of conserved regions similar to those seen in TCRB of other species. A decanucleotide promoter sequence homologous to those found in humans and mice was located in the 5{prime} untranslated region of one horse gene. Germline sequences included the 5{prime} region of the TCRBD2 gene with flanking heptamer/nonamer recombination signals and portions of the TCRBJ2-C2 intro. Southern blot hybridizations demonstrated restriction fragment length polymorphisms at the TCRBC locus among different horse breeds.

  4. Beta s gene in Central Iran is in linkage disequilibrium with the Indian-Arab haplotype.

    PubMed

    Rahgozar, S; Poorfathollah, A A; Moafi, A R; Old, J M

    2000-11-01

    Sickle cell anemia is not considered to be a significant disease in Iran, although the sickle cell trait is estimated to have a high incidence in the Southern provinces. Since 1977, when the presence of a mild sickle cell anemia was reported in this country, there have been no further investigations published giving precise data on the incidence and origins of the sickle cell mutation in Iran. We report here the finding of patients with the sickle cell trait, sickle cell anemia, and sickle-beta thalassemia in Central Iran. A survey of 300 individuals from a village in Southeast Esfahan revealed an incidence of the sickle cell trait of 8.33%. "Cascade screening" enabled 96 relatives in four surrounding villages to be tested, and the at-risk couples were offered counseling as a "sickle cell control program." The hematological indices and HbF levels of the affected patients were determined. The HbF levels were unusually high, ranging from 18% to 41.4%, and SS patients with the highest levels were asymptomatic. Linkage analysis revealed the betaS gene haplotype in this population to be the Indian-Arab haplotype.

  5. Cloning of the elk TSH beta-subunit cDNA and seasonal expression of the pituitary glycoprotein hormone genes.

    PubMed

    Clark, Rena J; Furlan, Michael A; Chedrese, P Jorge

    2005-06-01

    We report the elk (Cervus elaphus) thyroid stimulating hormone (TSH) beta-subunit cDNA cloning, nucleotide and deduced amino acid sequences. The TSH beta-subunit cDNA was obtained by RT-PCR of polyadenylated pituitary RNA. The deduced elk TSH beta-subunit peptide chain shares between 93 to 99% sequence similarities with the reported TSH beta-subunit of a sub-set of related species. The TSH beta-subunit gene is expressed in the elk pituitary gland as a mature transcript of approximately 600 bases, which corresponds to the size of the mRNA expressed in the sheep pituitary gland. Seasonal expression of the pituitary gonadotropin genes was investigated by Northern blot analyses. Samples of elk pituitary glands collected during the breeding season showed elevated steady state levels of common alpha-subunit and FSH and LH beta-subunit gene expression, consistent with the seasonal reproductive cycling of this species. Samples collected before the breeding season demonstrated decreased expression of the gonadotropin genes. TSH, which is not directly tied to reproduction, had similar levels of expression, regardless of the animal's reproductive status.

  6. Human neural stem cells transduced with IFN-beta and cytosine deaminase genes intensify bystander effect in experimental glioma.

    PubMed

    Ito, S; Natsume, A; Shimato, S; Ohno, M; Kato, T; Chansakul, P; Wakabayashi, T; Kim, S U

    2010-05-01

    Previously, we have shown that the genetically modified human neural stem cells (NSCs) show remarkable migratory and tumor-tropic capability to track down brain tumor cells and deliver therapeutic agents with significant therapeutic benefit. Human NSCs that were retrovirally transduced with cytosine deaminase (CD) gene showed remarkable 'bystander killer effect' on the glioma cells after application of the prodrug, 5-fluorocytosine (5-FC). Interferon-beta (IFN-beta) is known for its antiproliferative effects in a variety of cancers. In our pilot clinical trial in glioma, the IFN-beta gene has shown potent antitumor activity in patients with malignant glioma. In the present study, we sought to examine whether human NSCs genetically modified to express both CD and IFN-beta genes intensified antitumor effect on experimental glioma. In vitro studies showed that CD/IFN-beta-expressing NSCs exerted a remarkable bystander effect on human glioma cells after the application of 5-FC, as compared with parental NSCs and CD-expressing NSCs. In animal models with human glioma orthotopic xenograft, intravenously infused CD/IFN-beta-expressing NSCs produced striking antitumor effect after administration of the prodrug 5-FC. Furthermore, the same gene therapy regimen prolonged survival periods significantly in the experimental animals. The results of the present study indicate that the multimodal NSC-based treatment strategy might have therapeutic potential against gliomas.

  7. Mice doubly deficient in the midkine and pleiotrophin genes exhibit deficits in the expression of beta-tectorin gene and in auditory response.

    PubMed

    Zou, Peng; Muramatsu, Hisako; Sone, Michihiko; Hayashi, Hideo; Nakashima, Tsutomu; Muramatsu, Takashi

    2006-07-01

    alpha-Tectorin and beta-tectorin are major noncollagenous proteins of the tectorial membrane, which plays a crucial role in the reception of sonic signals in the cochlea. Midkine and pleiotrophin are closely related proteins that serve as growth factors and cytokines. In mice doubly deficient in the midkine gene and pleiotrophin gene, expression of beta-tectorin mRNA was nearly abolished in the cochlea on day 1 and 7 after birth. Expression of alpha-tectorin mRNA was unaffected in the double knockout mice, and expression of beta-tectorin mRNA was not altered in mice deficient in only the midkine or pleiotrophin gene. In newborn wild-type mice, both midkine and pleiotrophin were expressed in the greater epithelial ridge of the cochlea, in which beta-tectorin mRNA was strongly expressed. These results indicate that either midkine or pleiotrophin is required for significant expression of beta-tectorin. In agreement with the view that beta-tectorin is essential for normal auditory function, mice doubly deficient in both midkine and pleiotrophin genes exhibited very severe auditory deficits. We observed that mice deficient in either midkine or pleiotrophin gene were also impaired in their auditory response, but the level of the deficit was generally low or moderate. The present finding illustrates the importance of growth factor expression in the cochlea for auditory function.

  8. Effect of interleukin-1beta gene functional polymorphism on dorsolateral prefrontal cortex activity in schizophrenic patients.

    PubMed

    Papiol, Sergi; Molina, Vicente; Rosa, Araceli; Sanz, Javier; Palomo, Tomás; Fañanás, Lourdes

    2007-12-05

    Hypoactivity of the dorsolateral prefrontal cortex (DLPFC) during cognitive tasks is among the most consistent findings in schizophrenia. The biological factors contributing to this hypofrontality are only partially known. Previous reports have shown the influence of genes mapped to IL-1 cluster (i) in the risk to develop schizophrenia and (ii) on brain morphological abnormalities in these patients. Moreover, Interleukin-1beta (IL-1beta), encoded by IL-1B gene (IL-1 cluster, chromosome 2q13) has a key role in dopaminergic differentiation and dendrite growth in developing cortical neurons. The authors explored the role of a genetic functional polymorphism at IL-1B gene in relation to DLPFC activity. DLPFC (left and right) metabolic activity was measured in a sample of 19 DSM-IV diagnosed schizophrenic patients of Spanish origin using a procedure based on MRI/PET image fusion. During PET studies, subjects performed a contingent Continuous Performance Test aiming to activate DLPFC. Functional promoter polymorphism -511 C/T (rs16944) of IL-1B gene was genotyped in these patients. Those patients who were allele 2 (-511 T) carriers showed a lower metabolic activity in the left DLPFC with respect to patients homozygous for allele 1 (-511 C) (U = 16, z = -2.32, P = 0.02). Our results suggest that hypofrontality reported in some schizophrenic patients might be explained, at least in part, by this functional polymorphism at IL-1B gene. Genetic variants with influence on brain functionality may account for the neurocognitive heterogeneity observed in schizophrenic patients.

  9. Human T-cell receptor v{beta} gene polymorphism and multiple sclerosis

    SciTech Connect

    Wei, S.; Charmley, P.; Birchfield, R.I.; Concannon, P.

    1995-04-01

    Population-based genetic associations have been reported between RFLPs detected with probes corresponding to the genes encoding the {beta} chain of the T-cell receptor for antigen (RCRB) and a variety of autoimmune disorders. In the case of multiple sclerosis (MS), these studies have localized a putative disease-associated gene to a region of {approximately}110 kb in length, located within the TCRB locus. In the current study, all 14 known TCRBV (variable region) genes within the region of localization were mapped and identified. The nucleotide sequences of these genes were determined in a panel of six MS patients and six healthy controls, who were human-leukocyte antigen and TCRB-RFLP haplotype matched. Nine of the 14 TCRBV genes studied showed evidence of polymorphism. PCR-based assays for each of these polymorphic genes were developed, and allele and genotype frequencies were determined in a panel of DNA samples from 48 MS patients and 60 control individuals. No significant differences in allele, genotype, or phenotype frequencies were observed between the MS patients and controls for any of the 14 TCRBV-gene polymorphisms studied. In light of the extensive linkage disequilibrium across the region studied, the saturating numbers of polymorphisms examined, and the direct sequence analysis of all BV genes in the region, these results suggest that it is unlikely that germ-line polymorphism in the TCRBV locus makes a major contribution to MS susceptibility. The TCRBV coding region-specific markers generated in these studies, as well as the approach of testing for associations with specific functionally relevant polymorphic sites within individual BV genes, should be useful in the evaluation of the many reported disease associations involving the human TCRB region. 22 refs., 1 fig., 3 tabs.

  10. Detection of genes mediating beta-lactamase production in isolates of enterobacteria recovered from wild pets in Saudi Arabia

    PubMed Central

    Hassan, Sabry A.; Shobrak, Mohammed Y.

    2015-01-01

    Aim: To determine the genetic basis and types of beta-lactamase encountered among enterobacterial isolates of wild pets from the animal exhibit. Materials and Methods: A total of 17 beta-lactamase-producing enterobacteria recovered from fecal samples of wild pet animals were analyzed for a selected beta-lactamase gene by polymerase chain reaction. Results: Molecular analysis identified one or more β-lactamase-encoding genes in 14 enterobacterial isolates as a single or gene combination. The most frequent extended-spectrum β-lactamases types were TEM and CTX-M, and the most common AmpC enzymes were CMY-2 and DHA types. Conclusions: The study is the first in Saudi Arabia, have established the presence of β-lactamase-encoding genes in the fecal isolates of wild pets. PMID:27047051

  11. Increased gene expression of Alzheimer disease beta-amyloid precursor protein in senescent cultured fibroblasts.

    PubMed

    Adler, M J; Coronel, C; Shelton, E; Seegmiller, J E; Dewji, N N

    1991-01-01

    The pathological hallmark of Alzheimer disease is the accumulation of neurofibrillary tangles and neuritic plaques in the brains of patients. Plaque cores contain a 4- to 5-kDa amyloid beta-protein fragment which is also found in the cerebral blood vessels of affected individuals. Since amyloid deposition in the brain increases with age even in normal people, we sought to establish whether the disease state bears a direct relationship with normal aging processes. As a model for biological aging, the process of cellular senescence in vitro was used. mRNA levels of beta-amyloid precursor protein associated with Alzheimer disease were compared in human fibroblasts in culture at early passage and when the same fibroblasts were grown to senescence after more than 52 population doublings. A dramatic increase in mRNA was observed in senescent IMR-90 fibroblasts compared with early-passage cells. Hybridization of mRNA from senescent and early proliferating fibroblasts with oligonucleotide probes specific for the three alternatively spliced transcripts of the gene gave similar results, indicating an increase during senescence of all three forms. A similar, though more modest, increase in message levels was also observed in early-passage fibroblasts made quiescent by serum deprivation; with repletion of serum, however, the expression returned to previous low levels. ELISAs were performed on cell extracts from senescent, early proliferating, and quiescent fibroblasts, and quiescent fibroblasts repleted with serum for over 48 hr, using polyclonal antibodies to a synthetic peptide of the beta-amyloid precursor. The results confirmed that the differences in mRNA expression were partially reflected at the protein level. Regulated expression of beta-amyloid precursor protein may be an important determinant of growth and metabolic responses to serum and growth factors under physiological as well as pathological conditions.

  12. 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.

  13. Adenovirus-mediated gene transfer and expression of human beta-glucuronidase gene in the liver, spleen, and central nervous system in mucopolysaccharidosis type VII mice.

    PubMed

    Ohashi, T; Watabe, K; Uehara, K; Sly, W S; Vogler, C; Eto, Y

    1997-02-18

    Mucopolysaccharidosis type VII (Sly syndrome) is a lysosomal storage disease caused by inherited deficiency of the lysosomal enzyme beta-glucuronidase. A murine model of this disorder has been well characterized and used to study a number of forms of experimental therapies, including gene therapy. We produced recombinant adenovirus that expresses human beta-glucuronidase and administered this recombinant adenovirus to beta-glucuronidase-deficient mice intravenously. The beta-glucuronidase activities in liver and spleen were elevated to 40% and 20%, respectively, of the heterozygote enzymatic level at day 16. Expression persisted for at least 35 days. Pathological abnormalities of these tissues were also improved, and the elevated levels of urinary glycosaminoglycans were reduced in treated mice. However, the beta-glucuronidase activity in kidney and brain was not significantly increased. After administration of the recombinant adenovirus directly into the lateral ventricles of mutant mice, the beta-glucuronidase activity in crude brain homogenates increased to 30% of heterozygote activity. Histochemical demonstration of beta-glucuronidase activity in brain revealed that the enzymatic activity was mainly in ependymal cells and choroid. However, in some regions, the adenovirus-mediated gene expression was also evident in brain parenchyma associated with vessels and in the meninges. These results suggest that adenovirus-mediated gene delivery might improve the central nervous system pathology of mucopolysaccharidosis in addition to correcting visceral pathology.

  14. Epistatic interaction between beta2-adrenergic receptor and neuropeptide Y genes influences LDL-cholesterol in hypertension.

    PubMed

    Tomaszewski, Maciej; Charchar, Fadi J; Lacka, Beata; Pesonen, Ullamari; Wang, William Y S; Zukowska-Szczechowska, Ewa; Grzeszczak, Wladyslaw; Dominiczak, Anna F

    2004-11-01

    Beta2-adrenergic receptor gene and neuropeptide Y gene may potentially influence lipid metabolism and overall energy balance. Therefore, we examined associations of these genes with lipid fractions and obesity-related phenotypes in hypertensive subjects. A total of 638 white individuals from 212 Polish families with clustering of essential hypertension were phenotyped for cardiovascular risk determinants. Each subject was genotyped for functional polymorphisms of beta2-adrenergic receptor gene (Arg16Gly and Gln27Glu) and neuropeptide Y (Leu7Pro). Of 3 common haplotypes of beta2-adrenergic receptor gene, Arg16Gln27 was overtransmitted to offspring with elevated levels of total cholesterol (Z=2.2; P=0.026) and LDL-cholesterol (Z=3.2; P=0.002). Individually, Leu7Pro was not associated with any of the metabolic phenotypes in family-based tests or case-control analyses. However, in the presence of Arg allele of Arg16Gly and Gln allele of Gln27Glu, homozygosity for Leu variant of the Leu7Pro polymorphism was associated with 2.1-increased odds ratio (confidence interval, 1.10 to 3.81; P=0.024) of elevated LDL in hypertensive subjects, independent of age, gender, body mass index, adjusted blood pressures, antihypertensive therapy, and use of nonselective beta-blockers and diuretics. Consistently, there was a significant multilocus association among variants of Arg16Gly, Gln27Glu, and Leu7Pro in hypertensive probands with elevated LDL (cases; P=0.028) but not in hypertensive subjects with normal LDL (controls). This study revealed an association of LDL-cholesterol with beta2-adrenergic receptor gene haplotype and provided evidence for epistatic interaction between beta2-adrenergic receptor gene and neuropeptide Y gene in determination of LDL-cholesterol in patients with essential hypertension.

  15. Atypical haplotypes linked to the beta S gene in Africa are likely to be the product of recombination.

    PubMed

    Srinivas, R; Dunda, O; Krishnamoorthy, R; Fabry, M E; Georges, A; Labie, D; Nagel, R L

    1988-09-01

    We report here the haplotypes of 10 MstII-defined SS patients and a S/beta o thalassemia from the Central African Republic, exhibiting 7 different atypical haplotypes that are different from the typical Bantu haplotype that characterize over 93% of the beta s bearing chromosomes in that region of Africa. Of the seven atypical haplotypes, six can be easily interpreted as the result of recombination around the "hot spot" 5' of the beta gene, between a typical Bantu haplotype and other haplotypes available in the normal population. Except for one case that requires further study, this result demonstrates that the main mutational event leading to sickle hemoglobin in Bantu-speaking Africa was the mutation of the beta gene in a Bantu haplotype background.

  16. Repression of the c-fms gene in fibroblast cells by c-Myc-MM-1-TIF1beta complex.

    PubMed

    Satou, Akiko; Hagio, Yuko; Taira, Takahiro; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2004-08-13

    MM-1 has been reported to repress the E-box-dependent transcription activity of c-Myc by recruiting histone deacetylase 1 complex via TIF1beta/KAP1. In this study, to identify target genes for c-Myc-MM-1-TIF1beta, we established rat-1 cells harboring the dominant-negative form of TIF1beta to abrogate the pathway from TIF1beta to MM-1-c-Myc. This cell line, in which transcription activity of c-Myc was activated, was found to be tumorigenic. By DNA-microarray analysis of this cell line, expression and promoter activity of the c-fms oncogene were found to be upregulated. Of the two promoters, pE1 and pE2, in the c-fms gene, pE1 promoter activity was found to be activated in an E-box-dependent manner.

  17. In vivo topological analysis of Ste2, a yeast plasma membrane protein, by using beta-lactamase gene fusions.

    PubMed Central

    Cartwright, C P; Tipper, D J

    1991-01-01

    Gene fusions were constructed between Ste2, the receptor for the Saccharomyces cerevisiae alpha-factor, and beta la, the secreted form of beta-lactamase encoded by the bla gene of pBR322. The Ste2 and beta la components were linked by a processing fragment (P) from the yeast killer preprotoxin containing a C-terminal lysine-arginine site for cleavage by the Golgi-associated Kex2 protease. Ste2 is predicted to have a rhodopsinlike topology, with an external N terminus and seven transmembrane segments. Fusions to three of the four Ste2 domains predicted to be external resulted in beta la secretion from yeast cells. A fusion at a site just preceding the first transmembrane segment was an exception; the product was cell associated, indicating that the first 44 residues of Ste2 are insufficient to direct secretion of beta la; translocation of this domain presumably requires the downstream transmembrane segment. Expression of fusions located in two domains predicted to be cytoplasmic failed to result in beta la secretion. Following insertion of the preprotoxin signal peptide (S) between the Ste2 and P components of these cytoplasmic fusions, secretion of beta la activity occurred, which is consistent with inversion of the orientation of the beta la reporter. Conversely, insertion of S between Ste2 and P in an external fusion sharply reduced beta la secretion. Complementary information about both cytoplasmic and external domains of Ste2 was therefore provided, and most aspects of the predicted topology were confirmed. The steady-state levels of beta la detected were low, presumably because of efficient degradation of the fusions in the secretory pathway; levels, however, were easily detectable. This method should be valuable in the analysis of in vivo topologies of both homologous and foreign plasma membrane proteins expressed in yeast cells. Images PMID:2017168

  18. Genomic organization of the human {beta}-catenin gene (CTNNB1)

    SciTech Connect

    Nollet, F.; Berx, G.; Molemans, F.; Roy, F. van

    1996-03-05

    The cytoplasmic {beta}-catenin protein is implicated in signal transduction and associates with both the cell-cell adhesion protein E-cadherin and the tumor suppressor gene product APC. We determined the primary structure of the human {beta}-catenin gene (CTNNB1) by analysis cDNA and genomic clones. The size of the complete gene was determined to be 23.2 kb. Restriction mapping and partial sequence analysis revealed 16 exons. All splice donor and acceptor sites were conformable to the GT/AG rule. The exon size ranged from 61 to 790 bp. Half of the introns were smaller than 550 bp, with the smallest being 84 pb and the longest being 6700 bp. The intron-exon boundaries did not coincide either with conserved sites in the 12 armadillo repeat sequences of {beta}-catenin or with intron-exon boundaries in the armadillo gene of Drosophila. A major site for transcription initiation was identified as an A residue 214 nucleotides upstream of the ATG initiation codon. The resulting transcript is 3362 nucleotides long. Compared to the previously published mRNA sequence, additional residues were identified, 16 at the 5{prime} end and 766 at the 3{prime} end of the mRNA. An alternative splice acceptor site within exon 16 reduced the 3{prime} UTR sequence by 159 bp. Polymerase chain reaction on cDNA from 14 human cell lines demonstrated the general occurrence of both splice variants. The 5{prime}-flanking region is highly GC-rich and lacks a CCAAT box, but contains a TATA box and potential binding sites for several transcription factors, such as NFkB, SP1, AP2, and EGR1. Both a 437-bp fragment and a 6-kb fragment, containing about 4.7 kb of the 5{prime}-flanking region in addition to the noncoding exon 1 and 1 kb of intron 1, showed clear promoter activity when these fragments were linked to a secreted alkaline phosphatase reporter gene and transfected into a mouse epithelial cell line. 53 refs., 5 figs., 2 tabs.

  19. Comparative evolution of the mitochondrial cytochrome b gene and nuclear beta-fibrinogen intron 7 in woodpeckers.

    PubMed

    Prychitko, T M; Moore, W S

    2000-07-01

    Most molecular phylogenetic studies of vertebrates have been based on DNA sequences of mitochondrial-encoded genes. MtDNA evolves rapidly and is thus particularly useful for resolving relationships among recently evolved groups. However, it has the disadvantage that all of the mitochondrial genes are inherited as a single linkage group so that only one independent gene tree can be inferred regardless of the number of genes sequenced. Introns of nuclear genes are attractive candidates for independent sources of rapidly evolving DNA: they are pervasive, most of their nucleotides appear to be unconstrained by selection, and PCR primers can be designed for sequences in adjacent exons where nucleotide sequences are conserved. We sequenced intron 7 of the beta-fibrinogen gene (beta-fibint7) for a diversity of woodpeckers and compared the phylogenetic signal and nucleotide substitution properties of this DNA sequence with that of mitochondrial-encoded cytochrome b (cyt b) from a previous study. A few indels (insertions and deletions) were found in the beta-fibint7 sequences, but alignment was not difficult, and the indels were phylogentically informative. The beta-fibint7 and cyt b gene trees were nearly identical to each other but differed in significant ways from the traditional woodpecker classification. Cyt b evolves 2.8 times as fast as beta-fibint7 (14. 0 times as fast at third codon positions). Despite its relatively slow substitution rate, the phylogenetic signal in beta-fibint7 is comparable to that in cyt b for woodpeckers, because beta-fibint7 has less base composition bias and more uniform nucleotide substitution probabilities. As a consequence, compared with cyt b, beta-fibint7 nucleotide sites are expected to enter more distinct character states over the course of evolution and have fewer multiple substitutions and lower levels of homoplasy. Moreover, in contrast to cyt b, in which nearly two thirds of nucleotide sites rarely vary among closely related taxa

  20. Polymorphism of the ADRB2 gene and response to inhaled beta- agonists in children with asthma: a meta-analysis.

    PubMed

    Finkelstein, Yaron; Bournissen, Facundo Garcia; Hutson, Janine R; Shannon, Michael

    2009-11-01

    About 9% of children have asthma, corresponding to almost 6.8 million children in the USA and 1.1 million in the UK. Asthma exacerbations are the leading cause of pediatric emergency room visits and impose a large burden on the individual, family, and society. There is mounting evidence that therapeutic failure of inhaled beta-agonists is associated with polymorphisms of the beta(2)-adrenergic receptor gene (ADRB2); specifically, mutations leading to amino acid changes at positions 16 and 27, which alter down-regulation of the beta(2)-adrenergic receptor (beta(2)AR), induce resistance to the smooth-muscle relaxing effect of beta(2)-adrenergic agonists. We conducted a meta-analysis to examine the association between ADRB2 polymorphisms and the response to inhaled beta(2)-adrenergic agonists in children with asthma. We included all published studies until November 2008, in which asthmatic children underwent testing for acute bronchodilator response, defined as > or = 15% improvement in forced expiratory volume in 1 second (FEV(1)) and single nucleotide polymorphism (SNP) genotyping for positions 16 and/or 27 of the beta(2)AR. Individual and summary odds ratios were calculated using a random effects model. We identified three case-control or family-based studies involving 960 asthmatic children (692 children with negative beta(2)-bronchodilator response, defined as < 15% improvement in FEV(1) and 268 children with positive bronchodilator response). We found a significant association between favorable therapeutic response to inhaled beta(2)-adrenergic agonists in asthmatic children and the Arg/Arg phenotype at position 16 of the beta(2)AR [OR = 1.77; 95% CI (1.01; 3.1); p = 0.029], compared with the Arg/Gly or Gly/Gly phenotypes. The beneficial effect of Arg at position 16 of the beta(2)AR was most pronounced in African-American asthmatic children [OR = 3.54; 95% CI (1.37, 9.13)]. There was no association between clinical response to beta(2)-agonists and polymorphism

  1. Hematopoietic stem cell mobilization strategies for gene therapy of beta thalassemia and sickle cell disease.

    PubMed

    Yannaki, Evangelia; Stamatoyannopoulos, George

    2010-08-01

    Effective gene therapy for hemoglobinopathies will require high numbers of autologous gene-engineered hematopoetic stem cells to be reintroduced into the patients. Stem cell mobilization using G-CSF is the most convenient and effective approach to achieve this goal, but it can have severe side effects in sickle cell anemia and be potentially harmful in the case of severe thalassemia. Hence, the optimal way of collection of hematopoetic stem cells from patients with thalassemia and sickle cell disease needs to be determined. In this paper, we review the possible risks of G-CSF mobilization in hemoglobinopathies and we outline the approaches used in an on-going clinical trial in which pretreatment with hydroxyurea is used to reduce potential risks of G-CSF administration to patients with severe beta thalassemia.

  2. Characterization of nematode resistance genes in the section Procumbentes genus Beta: response to two populations of Heterodera schachtii.

    PubMed

    Klinke, A; Müller, J; Wricke, G

    1996-10-01

    Three species of the section Procumbentes genus Beta, nine monosomic additions, and five translocation lines were tested for resistance to two Heterodera schachtii populations. Nematode population 129-v (129-virulent) was selected for virulence to resistance gene(s) transferred from chromosome 1 of Beta procumbens to the diploid resistant sugar beet KWS-NR1. This population is considered to be a pathotype. The unselected sib population 129-av (129-avirulent) was reared continuously on fodder rape, Brassica napus cv Velox. Monosomic additions with chromosome 1 from the three species of the section Procumbentes were susceptible to population 129-v, regardless of the origin of the alien chromosome. Translocations with a gene(s) for resistance from chromosome 7 of B. procumbens and B. webbiana were also susceptible to the pathotype. However, a monosomic addition with chromosome 7 of B. webbiana was resistant to population 129-v. The three wild beets of the section Procumbentes, Beta procumbens, Beta webbiana and Beta patellaris, also were highly resistant to the two populations. The results indicate the existence of just two different major genes for resistance to H. schachtii in the entire Procumbentes section.

  3. Worldwide diversity of Klebsiella pneumoniae that produce beta-lactamase blaKPC-2 gene.

    PubMed

    Cuzon, Gaelle; Naas, Thierry; Truong, HaVy; Villegas, Maria Virginia; Wisell, Karin T; Carmeli, Yehuda; Gales, Ana C; Venezia, Shiri Navon; Quinn, John P; Nordmann, Patrice

    2010-09-01

    Klebsiella pneumoniaeisolates that produce carbapenemases (KPCs) are rapidly disseminating worldwide. To determine their genetic background, we investigated 16 blaKPC-2-harboring K. pneumoniae isolates from 5 countries. The isolates were multidrug resistant, possessed the blaKPC-2 gene, and differed by additional Beta-lactamase content. They harbored a naturally chromosome-encoded bla gene (blaSHV-1 [12.5%], blaSHV-11 [68.7%], or blaOKP-AVB [18.8%]) and several acquired and plasmid-encoded genes (blaTEM-1 [81.3%], blaCTX-M-2 [31.3%], blaCTX-M-12 [12.5%], blaCTX-M-15 [18.7%], and blaOXA-9 [37.5%]). The blaKPC-2 gene was always associated with 1 of the Tn4401 isoforms (a, b, or c). Tn4401 was inserted on different-sized plasmids that belonged to different incompatibility groups. Several blaKPC-containing K. pneumoniae clones were found: 9 different pulsotypes with 1 major (sequence type 258) and 7 minor distinct allelic profiles. Different clones harboring different plasmids but having identical genetic structure, Tn4401, could be at the origin of the worldwide spread of this emerging resistance gene.

  4. Pituitary transcription factor Prop-1 stimulates porcine follicle-stimulating hormone beta subunit gene expression.

    PubMed

    Aikawa, Satoko; Kato, Takako; Susa, Takao; Tomizawa, Kyoko; Ogawa, Satoshi; Kato, Yukio

    2004-11-12

    Molecular cloning of the transcription factor that modulates the expression of porcine follicle-stimulating hormone beta subunit (FSHbeta) gene was performed by the yeast one-hybrid cloning system using the -852/-746 upstream region (Fd2) as a bait sequence. We eventually cloned a pituitary transcription factor, Prop-1, which has been identified as an upstream transcription factor of Pit-1 gene. Binding ability of Prop-1 to the bait sequence was confirmed using recombinant Prop-1, and the binding property was investigated by DNase I footprinting, revealing that Prop-1 certainly bound to the large AT-rich region throughout the Fd2. Co-transfection of Prop-1 expression vector together with a reporter gene fused with Fd2 in CHO cells demonstrated an attractive stimulation of reporter gene expression. Immunohistochemistry of adult porcine pituitary confirmed the colocalization of the Prop-1 and FSHbeta subunit. This study is the first to report that Prop-1 participates in the regulation of FSHbeta gene. The present finding will provide new insights into the development of pituitary cell lineage and combined pituitary hormone deficiency (CPHD), since why the defect of Prop-1 causes CPHD including gonadotropins (FSH and LH) has yet to be clarified.

  5. Hormonal regulation of platypus Beta-lactoglobulin and monotreme lactation protein genes.

    PubMed

    Enjapoori, Ashwantha Kumar; Lefèvre, Christophe M; Nicholas, Kevin R; Sharp, Julie A

    2017-02-01

    Endocrine regulation of milk protein gene expression in marsupials and eutherians is well studied. However, the evolution of this complex regulation that began with monotremes is unknown. Monotremes represent the oldest lineage of extant mammals and the endocrine regulation of lactation in these mammals has not been investigated. Here we characterised the proximal promoter and hormonal regulation of two platypus milk protein genes, Beta-lactoglobulin (BLG), a whey protein and monotreme lactation protein (MLP), a monotreme specific milk protein, using in vitro reporter assays and a bovine mammary epithelial cell line (BME-UV1). Insulin and dexamethasone alone provided partial induction of MLP, while the combination of insulin, dexamethasone and prolactin was required for maximal induction. Partial induction of BLG was achieved by insulin, dexamethasone and prolactin alone, with maximal induction using all three hormones. Platypus MLP and BLG core promoter regions comprised transcription factor binding sites (e.g. STAT5, NF-1 and C/EBPα) that were conserved in marsupial and eutherian lineages that regulate caseins and whey protein gene expression. Our analysis suggests that insulin, dexamethasone and/or prolactin alone can regulate the platypus MLP and BLG gene expression, unlike those of therian lineage. The induction of platypus milk protein genes by lactogenic hormones suggests they originated before the divergence of marsupial and eutherians.

  6. Enantioselective Degradation Mechanism of Beta-Cypermethrin in Soil From the Perspective of Functional Genes.

    PubMed

    Yang, Zhong-Hua; Ji, Guo-Dong

    2015-12-01

    The behavior and mechanisms of the enantioselective degradation of beta-cypermethrin were studied in soil. The four isomers were degraded at different rates, and the enantiomer fractions of alpha-cypermethrin and theta-cypermethrin exceeded 0.5. Moreover, 3-phenoxybenzoic acid, phenol, and protocatechuic acid were detected; based on the presence of these metabolites, we predicted the degradation pathway and identified the functional genes that are related to this degradation process. We established quantitative relationships between the data on degradation kinetics and functional genes; we found that the quantitative relationships between different enantiomers differed even under the same conditions, and the genes pobA and pytH played key roles in limiting the degradation rate. Data obtained using path analysis revealed that the same gene had different direct and indirect effects on the degradation of different isomers. A mechanism was successfully proposed to explain the selective degradation of chiral compounds based on the perspective of functional genes. © 2015 Wiley Periodicals, Inc.

  7. Nucleotide sequence analysis of beta tubulin gene in a wide range of dermatophytes.

    PubMed

    Rezaei-Matehkolaei, Ali; Mirhendi, Hossein; Makimura, Koichi; de Hoog, G Sybren; Satoh, Kazuo; Najafzadeh, Mohammad Javad; Shidfar, Mohammad Reza

    2014-10-01

    We investigated the resolving power of the beta tubulin protein-coding gene (BT2) for systematic study of dermatophyte fungi. Initially, 144 standard and clinical strains belonging to 26 species in the genera Trichophyton, Microsporum, and Epidermophyton were identified by internal transcribe spacer (ITS) sequencing. Subsequently, BT2 was partially amplified in all strains, and sequence analysis performed after construction of a BT2 database that showed length ranged from approximately 723 (T. ajelloi) to 808 nucleotides (M. persicolor) in different species. Intraspecific sequence variation was found in some species, but T. tonsurans, T. equinum, T. concentricum, T. verrucosum, T. rubrum, T. violaceum, T. eriotrephon, E. floccosum, M. canis, M. ferrugineum, and M. audouinii were invariant. The sequences were found to be relatively conserved among different strains of the same species. The species with the closest resemblance were Arthroderma benhamiae and T. concentricum and T. tonsurans and T. equinum with 100% and 99.8% identity, respectively; the most distant species were M. persicolor and M. amazonicum. The dendrogram obtained from BT2 topology was almost compatible with the species concept based on ITS sequencing, and similar clades and species were distinguished in the BT2 tree. Here, beta tubulin was characterized in a wide range of dermatophytes in order to assess intra- and interspecies variation and resolution and was found to be a taxonomically valuable gene.

  8. Several homozygous mutations in the gene for 11{beta}-hydroxysteroid dehydrogenase type 2 in patients with apparent mineralocorticoid excess

    SciTech Connect

    Wilson, R.C.; Harbison, M.D.; Hanauske-Abel, H.M.; Licholai, T.

    1995-10-01

    Four deleterious mutations are described in the gene for HSD11B2, which encodes the type 2 isoenzyme of 11{beta}-hydroxysteroid dehydrogenase (11{beta}HSD2). In seven families with one or more members affected by apparent mineralocortiocoid excess, this disorder is shown to be the result of a deficiency in 11{beta}HSD2. Surprisingly, the patients are all homozygous for their mutation. This results from consanguinity in two families and possibly from endogamy or a founder effect in four of the other five families. The absence of compound heterozygotes remains to be investigated. 25 refs., 3 figs., 2 tabs.

  9. Recessive mutations in the gene encoding the beta-subunit of rod phosphodiesterase in patients with retinitis pigmentosa.

    PubMed

    McLaughlin, M E; Sandberg, M A; Berson, E L; Dryja, T P

    1993-06-01

    We have found four mutations in the human gene encoding the beta-subunit of rod cGMP phosphodiesterase (PDE beta) that cosegregate with autosomal recessive retinitis pigmentosa, a degenerative disease of photoreceptors. In one family two affected siblings both carry allelic nonsense mutations at codons 298 and 531. Affected individuals have abnormal rod and cone electroretinograms. PDE beta is the second member of the phototransduction cascade besides rhodopsin that is absent or altered as a cause of retinitis pigmentosa, suggesting that other members of this pathway may be defective in other forms of this disease.

  10. Beta*, a UV-inducible smaller form of the beta subunit sliding clamp of DNA polymerase III of Escherichia coli. I. Gene expression and regulation.

    PubMed

    Paz-Elizur, T; Skaliter, R; Blumenstein, S; Livneh, Z

    1996-02-02

    The 40.6-kDa beta subunit of DNA polymerase III of Escherichia coli is a sliding DNA clamp responsible for tethering the polymerase to DNA and endowing it with high processivity (Stukenberg, P. T., Studwell-Vaughan, P. S., and O'Donnell, M. (1991) J. Biol. Chem. 266, 11328-11334). UV irradiation of E. coli induces a smaller 26-kDa form of the beta subunit, termed beta*, that, when overproduced from a plasmid, increases UV resistance of E. coli (Skaliter, R., Paz-Elizur, T., and Livneh, Z. (1996) J. Biol. Chem. 271, 2478-2481). Here we show that this protein is synthesized from a UV-inducible internal gene, termed dnaN*, that is located in-frame inside the coding region of dnaN, encoding the beta subunit. The initiation codon and the Shine-Dalgarno sequence of dnaN* were identified by site-directed mutagenesis. The dnaN* transcript was shown to be induced upon treatment with nalidixic acid, and transcriptional dnaN*-cat gene fusions were UV inducible, suggesting induction of dnaN* at the transcriptional level. Analysis of translational dnaN*-lacZ gene fusions revealed that UV induction was abolished in strains carrying the recA56, lexA3, or delta rpoH mutations, indicating involvement of both SOS and heat shock stress responses in the induction process. Expression of dnaN* represents a strategy of producing several proteins with related functional domains from a single gene.

  11. A striking similarity in the organization of the E-selectin and beta interferon gene promoters.

    PubMed Central

    Whitley, M Z; Thanos, D; Read, M A; Maniatis, T; Collins, T

    1994-01-01

    Transcription of the endothelial leukocyte adhesion molecule 1 (E-selectin or ELAM-1) gene is induced by the inflammatory cytokines interleukin-1 beta and tumor necrosis factor alpha (TNF-alpha). In this report, we identify four positive regulatory domains (PDI to PDIV) in the E-selectin promoter that are required for maximal levels of TNF-alpha induction in endothelial cells. In vitro DNA binding studies reveal that two of the domains contain novel adjacent binding sites for the transcription factor NF-kappa B (PDIII and PDIV), a third corresponds to a recently described CRE/ATF site (PDII), and a fourth is a consensus NF-kappa B site (PDI). Mutations that decrease the binding of NF-kappa B to any one of the NF-kappa B binding sites in vitro abolished cytokine-induced E-selectin gene expression in vivo. Previous studies demonstrated a similar correlation between ATF binding to PDII and E-selectin gene expression. Here we show that the high-mobility-group protein I(Y) [HMG I(Y)] also binds specifically to the E-selectin promoter and thereby enhances the binding of both ATF-2 and NF-kappa B to the E-selectin promoter in vitro. Moreover, mutations that interfere with HMG I(Y) binding decrease the level of cytokine-induced E-selectin expression. The organization of the TNF-alpha-inducible element of the E-selectin promoter is remarkably similar to that of the virus-inducible promoter of the human beta interferon gene in that both promoters require NF-kappa B, ATF-2, and HMG I(Y). We propose that HMG I(Y) functions as a key architectural component in the assembly of inducible transcription activation complexes on both promoters. Images PMID:7523851

  12. Activation of nicotinamide N-methyltransferase gene promoter by hepatocyte nuclear factor-1beta in human papillary thyroid cancer cells.

    PubMed

    Xu, Jimin; Capezzone, Marco; Xu, Xiao; Hershman, Jerome M

    2005-02-01

    We previously demonstrated that the human nicotinamide N-methytransferase (NNMT) gene was highly expressed in many papillary thyroid cancers and cell lines. The expression in other papillary and follicular cancers or cell lines and normal thyroid cells was low or undetectable. To gain an understanding of the molecular mechanism of this cell-specific expression, the NNMT promoter was cloned and studied by luciferase reporter gene assay. The promoter construct was expressed highly in papillary cancer cell lines, including those with higher (e.g. BHP 2-7) and lower (e.g. BHP 14-9) NNMT gene expression, and expressed weakly in follicular thyroid cancer cell lines. Further study with 5'-deletion promoter construct suggested that the NNMT promoter was regulated differently in BHP 2-7 and BHP 14-9 cells. In BHP 2-7 cells, promoter activity was dependent on an upstream sequence. In BHP 14-9 cells, sequence in the basal promoter region contributed notably to the overall promoter activity. RT-PCR or Western blot analysis indicated that hepatocyte nuclear factor-1beta (HNF-1beta) was expressed in only papillary cancer cell lines with high NNMT gene expression. HNF-1beta was not expressed or expressed very weakly in other papillary, follicular, and Hurthle cancer cell lines and primary cultures of normal thyroid cells and benign thyroid conditions. A HNF-1 binding site was identified in the NNMT basal promoter region. Mutations in this site decreased NNMT promoter activity in the HNF-1beta-positive BHP 2-7 cells, but not in the HNF-1beta-negative BHP 14-9 cells. HNF-1beta bound to the HNF-1 site specifically as a homodimer as determined by gel retardation assays with HNF-1beta-specific antibody. Cotransfection of a HNF-1beta expression plasmid increased NNMT promoter activity significantly in both HNF-1beta-positive and -negative thyroid cancer cell lines and Hep G2 liver cancer cells. Furthermore, transient expression of HNF-1beta in BHP 14-9 cells increased endogenous NNMT

  13. The rs1800471 Polymorphism of TGFB1 Gene, Serum TGF-Beta1 Level and Chronic Kidney Disease Progression.

    PubMed

    Kiliś-Pstrusińska, K; Mastalerz-Migas, A; Zwolińska, D; Grzeszczak, W; Zachwieja, K; Zachwieja, J; Madziarska, K; Hyla Klekot, L

    2015-01-01

    The aim of the study was to investigate whether rs1800471 polymorphism in TGFB1 gene is associated with the development and progression of non-diabetic chronic kidney disease. Moreover, we examined the serum TGF-beta1 concentration and its association with that polymorphism and progression of the disease. We applied two different methodological approaches. Firstly, a family based study was carried out, comprised of 109 patients with non-diabetic chronic kidney disease and their 218 healthy parents, using the transmission/disequilibrium test. The rs1800471 polymorphism and serum TGF-beta1 level were determined in all subjects. Serum TGF-beta1 concentration was also measured in 40 healthy controls. Secondly, we performed a case-control orientated study to determine whether rs1800471 polymorphism and other factors influence the progression of renal impairment. We found no relationships between rs1800471 polymorphism allele transfer and the incidence or progression of non-diabetic chronic kidney disease. We found, however, that the serum TGF-beta1 was significantly higher in patients than in controls. In conclusion, rs1800471 polymorphism in TGFB1 gene does not have an impact on the development and progression of non-diabetic chronic kidney disease caused by primary glomerulopathy and chronic interstitial nephritis. The increased serum TGF-beta1 concentration in such patients suggests its role in the pathomechanism of the disease. Circulating TGF-beta1 level is determined in a multifactorial way, not by rs1800471 polymorphism in TGFB1 gene.

  14. Gene organization and plasticity of the beta-lactam genes in different filamentous fungi.

    PubMed

    Gutiérrez, S; Fierro, F; Casqueiro, J; Martín, J F

    1999-01-01

    The genes pcbAB, pcbC and penDE encoding enzymes that catalyze the three steps of the penicillin biosynthesis have been cloned from Penicillium chrysogenum and Aspergillus nidulans. They are located in a cluster in Penicillium chrysogenum, Penicillium notatum, Aspergillus nidulans and Penicillium nalgiovense. The three genes are clustered in chromosome I (10.4 Mb) of P. chrysogenum, in chromosome II of P. notatum (9.6 Mb) and in chromosome VI (3.0 Mb) of A. nidulans. The cluster of the penicillin biosynthetic genes is amplified in strains with high level of antibiotic production. About five to six copies of the cluster are present in the AS-P-78 strain and 11 to 14 copies in the E1 strain (an industrial isolate), whereas only one copy is present in the wild type (NRRL 1951) strain and in the low producer Wis 54-1255 strain. The amplified region in strains AS-P-78 and E1 is arranged in tandem repeats of 106.5 or 57.6-kb units, respectively. In Acremonium chrysogenum the genes involved in cephalosporin biosynthesis are separated in at least two clusters. The pcbAB and pcbC genes are linked in the so-called 'early cluster' of genes involved in the cephalosporin biosynthesis. The 'late cluster', which includes the cefEF and cefG genes, is involved in the last steps of cephalosporin biosynthesis. The 'early cluster' was located in chromosome VII (4.6 Mb) in the C10 strain and the 'late cluster' in chromosome I (2.2 Mb). Both clusters are present in a single copy in the A. chrysogenum genome, in the wild-type and in the high cephalosporin-producing C10 strains.

  15. Conservation and divergence of autonomous pathway genes in the flowering regulatory network of Beta vulgaris.

    PubMed

    Abou-Elwafa, Salah F; Büttner, Bianca; Chia, Tansy; Schulze-Buxloh, Gretel; Hohmann, Uwe; Mutasa-Göttgens, Effie; Jung, Christian; Müller, Andreas E

    2011-06-01

    The transition from vegetative growth to reproductive development is a complex process that requires an integrated response to multiple environmental cues and endogenous signals. In Arabidopsis thaliana, which has a facultative requirement for vernalization and long days, the genes of the autonomous pathway function as floral promoters by repressing the central repressor and vernalization-regulatory gene FLC. Environmental regulation by seasonal changes in daylength is under control of the photoperiod pathway and its key gene CO. The root and leaf crop species Beta vulgaris in the caryophyllid clade of core eudicots, which is only very distantly related to Arabidopsis, is an obligate long-day plant and includes forms with or without vernalization requirement. FLC and CO homologues with related functions in beet have been identified, but the presence of autonomous pathway genes which function in parallel to the vernalization and photoperiod pathways has not yet been reported. Here, this begins to be addressed by the identification and genetic mapping of full-length homologues of the RNA-regulatory gene FLK and the chromatin-regulatory genes FVE, LD, and LDL1. When overexpressed in A. thaliana, BvFLK accelerates bolting in the Col-0 background and fully complements the late-bolting phenotype of an flk mutant through repression of FLC. In contrast, complementation analysis of BvFVE1 and the presence of a putative paralogue in beet suggest evolutionary divergence of FVE homologues. It is further shown that BvFVE1, unlike FVE in Arabidopsis, is under circadian clock control. Together, the data provide first evidence for evolutionary conservation of components of the autonomous pathway in B. vulgaris, while also suggesting divergence or subfunctionalization of one gene. The results are likely to be of broader relevance because B. vulgaris expands the spectrum of evolutionarily diverse species which are subject to differential developmental and/or environmental regulation

  16. Conservation and divergence of autonomous pathway genes in the flowering regulatory network of Beta vulgaris

    PubMed Central

    Abou-Elwafa, Salah F.; Büttner, Bianca; Chia, Tansy; Schulze-Buxloh, Gretel; Hohmann, Uwe; Mutasa-Göttgens, Effie; Jung, Christian; Müller, Andreas E.

    2011-01-01

    The transition from vegetative growth to reproductive development is a complex process that requires an integrated response to multiple environmental cues and endogenous signals. In Arabidopsis thaliana, which has a facultative requirement for vernalization and long days, the genes of the autonomous pathway function as floral promoters by repressing the central repressor and vernalization-regulatory gene FLC. Environmental regulation by seasonal changes in daylength is under control of the photoperiod pathway and its key gene CO. The root and leaf crop species Beta vulgaris in the caryophyllid clade of core eudicots, which is only very distantly related to Arabidopsis, is an obligate long-day plant and includes forms with or without vernalization requirement. FLC and CO homologues with related functions in beet have been identified, but the presence of autonomous pathway genes which function in parallel to the vernalization and photoperiod pathways has not yet been reported. Here, this begins to be addressed by the identification and genetic mapping of full-length homologues of the RNA-regulatory gene FLK and the chromatin-regulatory genes FVE, LD, and LDL1. When overexpressed in A. thaliana, BvFLK accelerates bolting in the Col-0 background and fully complements the late-bolting phenotype of an flk mutant through repression of FLC. In contrast, complementation analysis of BvFVE1 and the presence of a putative paralogue in beet suggest evolutionary divergence of FVE homologues. It is further shown that BvFVE1, unlike FVE in Arabidopsis, is under circadian clock control. Together, the data provide first evidence for evolutionary conservation of components of the autonomous pathway in B. vulgaris, while also suggesting divergence or subfunctionalization of one gene. The results are likely to be of broader relevance because B. vulgaris expands the spectrum of evolutionarily diverse species which are subject to differential developmental and/or environmental regulation

  17. Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus).

    PubMed

    Yang, Chang Geng; Wang, Xian Li; Tian, Juan; Liu, Wei; Wu, Fan; Jiang, Ming; Wen, Hua

    2013-09-15

    Quantitative real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) has been used frequently to study gene expression related to fish immunology. In such studies, a stable reference gene should be selected to correct the expression of the target gene. In this study, seven candidate reference genes (glyceraldehyde-3-phosphate dehydrogenase (GADPH), ubiquitin-conjugating enzyme (UBCE), 18S ribosomal RNA (18S rRNA), beta-2-microglobulin (B2M), elongation factor 1 alpha (EF1A), tubulin alpha chain-like (TUBA) and beta actin (ACTB)), were selected to analyze their stability and normalization in seven tissues (liver, spleen, kidney, brain, heart, muscle and intestine) of Nile tilapia (Oreochromis niloticus) challenged with Streptococcus agalactiae or Streptococcus iniae, respectively. The results showed that all the candidate reference genes exhibited tissue-dependent transcriptional variations. With PBS injection as a control, UBCE was the most stable and suitable single reference gene in the intestine, liver, brain, kidney, and spleen after S. iniae infection, and in the liver, kidney, and spleen after S. agalactiae infection. EF1A was the most suitable in heart and muscle after S. iniae or S. agalactiae infection. GADPH was the most suitable gene in intestine and brain after S. agalactiae infection. In normal conditions, UBCE and 18S rRNA were the most stably expressed genes across the various tissues. These results showed that for RT-qPCR analysis of tilapia, selecting two or more reference genes may be more suitable for cross-tissue analysis of gene expression.

  18. Alteration of gene expression by alcohol exposure at early neurulation

    PubMed Central

    2011-01-01

    Background We have previously demonstrated that alcohol exposure at early neurulation induces growth retardation, neural tube abnormalities, and alteration of DNA methylation. To explore the global gene expression changes which may underline these developmental defects, microarray analyses were performed in a whole embryo mouse culture model that allows control over alcohol and embryonic variables. Result Alcohol caused teratogenesis in brain, heart, forelimb, and optic vesicle; a subset of the embryos also showed cranial neural tube defects. In microarray analysis (accession number GSM9545), adopting hypothesis-driven Gene Set Enrichment Analysis (GSEA) informatics and intersection analysis of two independent experiments, we found that there was a collective reduction in expression of neural specification genes (neurogenin, Sox5, Bhlhe22), neural growth factor genes [Igf1, Efemp1, Klf10 (Tieg), and Edil3], and alteration of genes involved in cell growth, apoptosis, histone variants, eye and heart development. There was also a reduction of retinol binding protein 1 (Rbp1), and de novo expression of aldehyde dehydrogenase 1B1 (Aldh1B1). Remarkably, four key hematopoiesis genes (glycophorin A, adducin 2, beta-2 microglobulin, and ceruloplasmin) were absent after alcohol treatment, and histone variant genes were reduced. The down-regulation of the neurospecification and the neurotrophic genes were further confirmed by quantitative RT-PCR. Furthermore, the gene expression profile demonstrated distinct subgroups which corresponded with two distinct alcohol-related neural tube phenotypes: an open (ALC-NTO) and a closed neural tube (ALC-NTC). Further, the epidermal growth factor signaling pathway and histone variants were specifically altered in ALC-NTO, and a greater number of neurotrophic/growth factor genes were down-regulated in the ALC-NTO than in the ALC-NTC embryos. Conclusion This study revealed a set of genes vulnerable to alcohol exposure and genes that were

  19. Cloning and nucleotide sequence of the gene coding for enzymatically active fragments of the Bacillus polymyxa beta-amylase.

    PubMed Central

    Kawazu, T; Nakanishi, Y; Uozumi, N; Sasaki, T; Yamagata, H; Tsukagoshi, N; Udaka, S

    1987-01-01

    The gene encoding beta-amylase was cloned from Bacillus polymyxa 72 into Escherichia coli HB101 by inserting HindIII-generated DNA fragments into the HindIII site of pBR322. The 4.8-kilobase insert was shown to direct the synthesis of beta-amylase. A 1.8-kilobase AccI-AccI fragment of the donor strain DNA was sufficient for the beta-amylase synthesis. Homologous DNA was found by Southern blot analysis to be present only in B. polymyxa 72 and not in other bacteria such as E. coli or B. subtilis. B. polymyxa, as well as E. coli harboring the cloned DNA, was found to produce enzymatically active fragments of beta-amylases (70,000, 56,000, or 58,000, and 42,000 daltons), which were detected in situ by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Nucleotide sequence analysis of the cloned 3.1-kilobase DNA revealed that it contains one open reading frame of 2,808 nucleotides without a translational stop codon. The deduced amino acid sequence for these 2,808 nucleotides encoding a secretory precursor of the beta-amylase protein is 936 amino acids including a signal peptide of 33 or 35 residues at its amino-terminal end. The existence of a beta-amylase of larger than 100,000 daltons, which was predicted on the basis of the results of nucleotide sequence analysis of the gene, was confirmed by examining culture supernatants after various cultivation periods. It existed only transiently during cultivation, but the multiform beta-amylases described above existed for a long time. The large beta-amylase (approximately 160,000 daltons) existed for longer in the presence of a protease inhibitor such as chymostatin, suggesting that proteolytic cleavage is the cause of the formation of multiform beta-amylases. Images PMID:2435707

  20. Cloning and nucleotide sequence of the gene coding for enzymatically active fragments of the Bacillus polymyxa beta-amylase.

    PubMed

    Kawazu, T; Nakanishi, Y; Uozumi, N; Sasaki, T; Yamagata, H; Tsukagoshi, N; Udaka, S

    1987-04-01

    The gene encoding beta-amylase was cloned from Bacillus polymyxa 72 into Escherichia coli HB101 by inserting HindIII-generated DNA fragments into the HindIII site of pBR322. The 4.8-kilobase insert was shown to direct the synthesis of beta-amylase. A 1.8-kilobase AccI-AccI fragment of the donor strain DNA was sufficient for the beta-amylase synthesis. Homologous DNA was found by Southern blot analysis to be present only in B. polymyxa 72 and not in other bacteria such as E. coli or B. subtilis. B. polymyxa, as well as E. coli harboring the cloned DNA, was found to produce enzymatically active fragments of beta-amylases (70,000, 56,000, or 58,000, and 42,000 daltons), which were detected in situ by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Nucleotide sequence analysis of the cloned 3.1-kilobase DNA revealed that it contains one open reading frame of 2,808 nucleotides without a translational stop codon. The deduced amino acid sequence for these 2,808 nucleotides encoding a secretory precursor of the beta-amylase protein is 936 amino acids including a signal peptide of 33 or 35 residues at its amino-terminal end. The existence of a beta-amylase of larger than 100,000 daltons, which was predicted on the basis of the results of nucleotide sequence analysis of the gene, was confirmed by examining culture supernatants after various cultivation periods. It existed only transiently during cultivation, but the multiform beta-amylases described above existed for a long time. The large beta-amylase (approximately 160,000 daltons) existed for longer in the presence of a protease inhibitor such as chymostatin, suggesting that proteolytic cleavage is the cause of the formation of multiform beta-amylases.

  1. [Influence of interleukin-1 beta gene polymorphism and childhood maltreatment on antidepressant treatment].

    PubMed

    Chen, Ying; Zhang, Zhijun; Xu, Zhi; Pu, Mengjia; Geng, Leiyu

    2015-12-01

    To explore the influence of interleukin-1 beta (IL1B) gene polymorphism and childhood maltreatment on antidepressant treatment. Two hundred and four patients with major depressive disorder (MDD) have received treatment with single antidepressant drugs and were followed up for 8 weeks. Hamilton depression scale-17 (HAMD-17) was used to evaluate the severity of depressive symptoms and therapeutic effect. Childhood maltreatment was assessed using Childhood Trauma Questionnaire, a 28-item Short Form (CTQ-SF). Single nucleotide polymorphism (SNP) of the IL1B gene was determined using a SNaPshot method. Correlation of rs16944 gene polymorphism with response to treatment was analyzed using Unphased 3.0.13 software. The main and interactive effects of SNP and childhood maltreatment on the antidepressant treatment were analyzed using Logistic regression analysis. No significant difference of gender, age, year of education, family history, episode time, and antidepressant agents was detected between the remitters and non-remitters. Association analysis has found that the SNP rs16944 in the IL1B AA genotype carriers antidepressant response was poorer (χ2=3.931, P=0.047). No significant difference was detected in the CTQ scores between the two groups. Genetic and environmental interaction analysis has demonstrated a significant correlation between rs16944 AA genotype and childhood maltreatment and poorer response to antidepressant treatment. The SNP rs16944 in the IL1B gene and its interaction with childhood maltreatment may influence the effect of antidepressant treatment for patients with MDD.

  2. Interleukin 10 and transforming growth factor beta 1 gene polymorphisms in juvenile idiopathic arthritis.

    PubMed

    Harsini, S; Ziaee, V; Maddah, M; Rezaei, A; Sadr, M; Zoghi, S; Moradinejad, M H; Tahghighi, F; Aghighi, Y; Rezaei, N

    2016-01-01

    The aim of this study is to identify the associations between interleukin 10 (IL-10) and transforming growth factor beta 1 (TGF-β1) gene polymorphisms and individual susceptibility to juvenile idiopathic arthritis (JIA) in a group of Iranian patients. Cytokine genes, including IL-10 and TGF-β1, are known to play important roles in the pathogenesis of JIA. Using polymerase chain reaction with sequence-specific primers method, the frequency of alleles, genotypes and haplotypes of IL-10 (positions -1082, -819, -592) and TGF-β1 (codon 10, codon 25) single-nucleotide polymorphisms (SNPs) were investigated in 55 patients with JIA as a case group and compared with 140 healthy unrelated controls. The G allele was significantly less frequent at TGF-β1 codon 25 in patients with JIA than in the controls (p < 0.01). The frequency of CT genotype at TGF-β1 codon 10 was found to be higher in healthy individuals in comparison with that in patients group (p = 0.04). We observed no differences in the frequency of alleles, genotypes and haplotypes of IL-10 gene between the groups of patients and controls. Considering the low frequency of existence of TGF-β1 G allele at codon 25 as well as TGF-β1 CT genotype at codon 10 in patients with JIA, it seems that these cytokine gene polymorphisms could play role as the protective factors against JIA.

  3. Two peptides derived from trout IL-1beta have different stimulatory effects on immune gene expression after intraperitoneal administration.

    PubMed

    Hong, Suhee; Secombes, Chris J

    2009-07-01

    The aim of this study was to examine the biological activities of two IL-1beta derivatives on immune gene expression (i.e. IL-1beta, TNF-alpha, IL-8, MX, lysozyme) in fish using RT-PCR analysis, as a means to establish whether such peptides have value as immunostimulants in vivo. Two functional domains (P1 and P3) of the trout IL-1beta molecule were produced as synthetic peptides and tested for biological effects following intraperitoneal administration into rainbow trout (Oncorhynchus mykiss). P1 and P3 showed different regulatory effects on the examined genes. P1 did not stimulate proinflammatory gene expression but induced rapid expression of the antiviral gene MX. In contrast, P3 showed more widespread stimulatory effects, and increased expression of the proinflammatory genes IL-1beta and IL-8, as well as the antibacterial lysozyme gene. Such data confirm that it is possible to produce bioactive peptide derivatives of cytokine molecules, and in addition that it is possible to engineer the peptides for different stimulatory repertoires, that may have value in enhancing particular types of immune response to enhance disease resistance in fish.

  4. Candidate Gene Study of TRAIL and TRAIL Receptors: Association with Response to Interferon Beta Therapy in Multiple Sclerosis Patients

    PubMed Central

    Órpez-Zafra, Teresa; Pinto-Medel, María Jesús; Oliver-Martos, Begoña; Ortega-Pinazo, Jesús; Arnáiz, Carlos; Guijarro-Castro, Cristina; Varadé, Jezabel; Álvarez-Lafuente, Roberto; Urcelay, Elena; Sánchez-Jiménez, Francisca

    2013-01-01

    TRAIL and TRAIL Receptor genes have been implicated in Multiple Sclerosis pathology as well as in the response to IFN beta therapy. The objective of our study was to evaluate the association of these genes in relation to the age at disease onset (AAO) and to the clinical response upon IFN beta treatment in Spanish MS patients. We carried out a candidate gene study of TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 genes. A total of 54 SNPs were analysed in 509 MS patients under IFN beta treatment, and an additional cohort of 226 MS patients was used to validate the results. Associations of rs1047275 in TRAILR-2 and rs7011559 in TRAILR-4 genes with AAO under an additive model did not withstand Bonferroni correction. In contrast, patients with the TRAILR-1 rs20576-CC genotype showed a better clinical response to IFN beta therapy compared with patients carrying the A-allele (recessive model: p = 8.88×10−4, pc = 0.048, OR = 0.30). This SNP resulted in a non synonymous substitution of Glutamic acid to Alanine in position 228 (E228A), a change previously associated with susceptibility to different cancer types and risk of metastases, suggesting a lack of functionality of TRAILR-1. In order to unravel how this amino acid change in TRAILR-1 would affect to death signal, we performed a molecular modelling with both alleles. Neither TRAIL binding sites in the receptor nor the expression levels of TRAILR-1 in peripheral blood mononuclear cell subsets (monocytes, CD4+ and CD8+ T cells) were modified, suggesting that this SNP may be altering the death signal by some other mechanism. These findings show a role for TRAILR-1 gene variations in the clinical outcome of IFN beta therapy that might have relevance as a biomarker to predict the response to IFN beta in MS. PMID:23658636

  5. Transfer and expression of three cloned human non-HLA-A,B,C class I major histocompatibility complex genes in mutant lymphoblastoid cells.

    PubMed Central

    Shimizu, Y; Geraghty, D E; Koller, B H; Orr, H T; DeMars, R

    1988-01-01

    The HLA-A, -B, and -C class I human histocompatibility antigens and the genes that encode them have been isolated and characterized. Apparently complete class I non-HLA-A, B, C genes have been identified on HindIII-generated 5.4-kilobase (kb), 6.0-kb, and 6.2-kb DNA fragments derived from lymphoblastoid cell line (LCL) 721. We studied the expressibility of these genes by subcloning them into the nonintegrating pHeBo vector and transferring the chimeric plasmids into mutant LCL 721.221. This mutant was derived from LCL 721 by means of immunoselections following gamma-ray mutagenesis that eliminated expressions of the HLA-A, -B, and -C alpha chains. The HLA-A, B, C-null phenotype of mutant 721.221 made it possible to monitor the expression of class I genes transferred into it by assaying cell surface binding of monoclonal antibodies BBM.1 and W6/32, which recognize beta 2-microglobulin and HLA class I alpha-chain epitopes, respectively. Increased binding of BBM.1 and W6/32 was clearly observed in transferents containing the class I gene of the 6.0-kb DNA fragment but not in transferents containing the class I genes of the 5.4- and 6.2-kb DNA fragments. However, one-dimensional gel electrophoresis of BBM.1 and W6/32 immunoprecipitates made with [35S]methionine-labeled cell lysates showed that transfer of each non-HLA-A, B, C class I gene into 721.221 resulted in the appearance of an alpha chain that coprecipitated with beta 2-microglobulin. The three previously unreported alpha chains differed from each other in size and were smaller than HLA-A, -B, and -C alpha chains. These observations clearly show that these three cloned, nonallelic, non-HLA-A, B, C class I genes encode alpha chains that can be expressed in human cells. Images PMID:3257565

  6. DNA methylation of retrotransposons, DNA transposons and genes in sugar beet (Beta vulgaris L.).

    PubMed

    Zakrzewski, Falk; Schmidt, Martin; Van Lijsebettens, Mieke; Schmidt, Thomas

    2017-03-03

    The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses activity of transposable elements (TEs), affects gene expression, and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of the worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%) in particular, satellite DNA, retrotransposons, and DNA transposons. Genome-wide cytosine methylation in the sugar beet genome was studied in leaves and leaf-derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H=A, C, and T), and CHH sites, whereas the TE pattern differed, depending on the classes 1 (retrotransposons) and 2 (DNA transposons), respectively. Along genes and TEs, the CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing toward a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome-wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared to leaves. This article is protected by copyright. All rights reserved.

  7. Genus Beta Human Papillomavirus E6 Proteins Vary in Their Effects on the Transactivation of p53 Target Genes

    PubMed Central

    White, Elizabeth A.; Walther, Johanna; Javanbakht, Hassan

    2014-01-01

    ABSTRACT The genus beta human papillomaviruses (beta HPVs) cause cutaneous lesions and are thought to be involved in the initiation of some nonmelanoma skin cancers (NMSCs), particularly in patients with the genetic disorder epidermodysplasia verruciformis (EV). We have previously reported that at least two of the genus beta HPV E6 proteins bind to and/or increase the steady-state levels of p53 in squamous epithelial cells. This is in contrast to a well-characterized ability of the E6 proteins of cancer-associated HPVs of genus alpha HPV, which inactivate p53 by targeting its ubiquitin-mediated proteolysis. In this study, we have investigated the ability of genus beta E6 proteins from eight different HPV types to block the transactivation of p53 target genes following DNA damage. We find that the E6 proteins from diverse beta HPV species and types vary in their capacity to block the induction of MDM2, p21, and proapoptotic genes after genotoxic stress. We conclude that some genus beta HPV E6 proteins inhibit at least some p53 target genes, although perhaps not by the same mechanism or to the same degree as the high-risk genus alpha HPV E6 proteins. IMPORTANCE This study addresses the ability of various human papillomavirus E6 proteins to block the activation of p53-responsive cellular genes following DNA damage in human keratinocytes, the normal host cell for HPVs. The E6 proteins encoded by the high-risk, cancer-associated HPV types of genus alpha HPV have a well-established activity to target p53 degradation and thereby inhibit the response to DNA damage. In this study, we have investigated the ability of genus beta HPV E6 proteins from eight different HPV types to block the ability of p53 to transactivate downstream genes following DNA damage. We find that some, but not all, genus beta HPV E6 proteins can block the transactivation of some p53 target genes. This differential response to DNA damage furthers the understanding of cutaneous HPV biology and may help

  8. Genus beta human papillomavirus E6 proteins vary in their effects on the transactivation of p53 target genes.

    PubMed

    White, Elizabeth A; Walther, Johanna; Javanbakht, Hassan; Howley, Peter M

    2014-08-01

    The genus beta human papillomaviruses (beta HPVs) cause cutaneous lesions and are thought to be involved in the initiation of some nonmelanoma skin cancers (NMSCs), particularly in patients with the genetic disorder epidermodysplasia verruciformis (EV). We have previously reported that at least two of the genus beta HPV E6 proteins bind to and/or increase the steady-state levels of p53 in squamous epithelial cells. This is in contrast to a well-characterized ability of the E6 proteins of cancer-associated HPVs of genus alpha HPV, which inactivate p53 by targeting its ubiquitin-mediated proteolysis. In this study, we have investigated the ability of genus beta E6 proteins from eight different HPV types to block the transactivation of p53 target genes following DNA damage. We find that the E6 proteins from diverse beta HPV species and types vary in their capacity to block the induction of MDM2, p21, and proapoptotic genes after genotoxic stress. We conclude that some genus beta HPV E6 proteins inhibit at least some p53 target genes, although perhaps not by the same mechanism or to the same degree as the high-risk genus alpha HPV E6 proteins. This study addresses the ability of various human papillomavirus E6 proteins to block the activation of p53-responsive cellular genes following DNA damage in human keratinocytes, the normal host cell for HPVs. The E6 proteins encoded by the high-risk, cancer-associated HPV types of genus alpha HPV have a well-established activity to target p53 degradation and thereby inhibit the response to DNA damage. In this study, we have investigated the ability of genus beta HPV E6 proteins from eight different HPV types to block the ability of p53 to transactivate downstream genes following DNA damage. We find that some, but not all, genus beta HPV E6 proteins can block the transactivation of some p53 target genes. This differential response to DNA damage furthers the understanding of cutaneous HPV biology and may help to explain the

  9. A gene encoding the major beta tubulin of the mitotic spindle in Physarum polycephalum plasmodia

    SciTech Connect

    Burland, T.G.; Paul, E.C.A.; Oetliker, M.; Dove, W.F.

    1988-03-01

    The multinucleate plasmodium of Physarum polycephalum is unusual among eucaryotic cells in that it uses tubulins only in mitotic-spindle microtubules; cytoskeletal, flagellar, and centriolar microtubules are absent in this cell type. The authors identified a ..beta..-tubulin cDNA clone, ..beta..105, which is shown to correspond to the transcript of the betC ..beta..-tubulin locus and to encode ..beta..2 tubulin, the ..beta.. tubulin expressed specifically in the plasmodium and used exclusively in the mitotic spindle. Physarum amoebae utilize tubulins in the cytoskeleton, centrioles, and flagella, in addition to the mitotic spindle. Sequence analysis shows that ..beta..2 tubulin is only 83% identical to the two ..beta.. tubulins expressed in amoebae. This compares with 70 to 83% identity between Physarum ..beta..2 tubulin and the ..beta.. tubulins of yeasts, fungi, alga, trypanosome, fruit fly, chicken, and mouse. On the other hand, Physarum ..beta..2 tubulin is no more similar to, for example, Aspergillus ..beta.. tubulins than it is to those of Drosophila melanogaster or mammals. Several eucaryotes express at least one widely diverged ..beta.. tubulin as well as one or more ..beta.. tubulins that conform more closely to a consensus ..beta..-tubulin sequence. The authors suggest that ..beta..-tubulins diverge more when their expression pattern is restricted, especially when this restriction results in their use in fewer functions. This divergence among ..beta.. tubulins could have resulted through neutral drift. For example, exclusive use of Physarum ..beta..2 tubulin in the spindle may have allowed more amino acid substitutions than would be functionally tolerable in the ..beta.. tubulins that are utilized in multiple microtubular organelles. Alternatively, restricted use of ..beta.. tubulins may allow positive selection to operate more freely to refine ..beta..-tubulin function.

  10. GeneSpeed Beta Cell: An Online Genomics Data Repository and Analysis Resource Tailored for the Islet Cell Biologist

    PubMed Central

    Quayum, Nayeem; Kutchma, Alecksandr; Sarkar, Suparna A.; Juhl, Kirstine; Gradwohl, Gerard; Mellitzer, Georg; Hutton, John C.; Jensen, Jan

    2008-01-01

    Objective. We here describe the development of a freely available online database resource, GeneSpeed Beta Cell, which has been created for the pancreatic islet and pancreatic developmental biology investigator community. Research Design and Methods. We have developed GeneSpeed Beta Cell as a separate component of the GeneSpeed database, providing a genomics-type data repository of pancreas and islet-relevant datasets interlinked with the domain-oriented GeneSpeed database. Results. GeneSpeed Beta Cell allows the query of multiple published and unpublished select genomics datasets in a simultaneous fashion (multiexperiment viewing) and is capable of defining intersection results from precomputed analysis of such datasets (multidimensional querying). Combined with the protein-domain categorization/assembly toolbox provided by the GeneSpeed database, the user is able to define spatial expression constraints of select gene lists in a relatively rigid fashion within the pancreatic expression space. We provide several demonstration case studies of relevance to islet cell biology and development of the pancreas that provide novel insight into islet biology. Conclusions. The combination of an exhaustive domain-based compilation of the transcriptome with gene array data of interest to the islet biologist affords novel methods for multidimensional querying between individual datasets in a rapid fashion, presently not available elsewhere. PMID:18795106

  11. Ethanol production from cellobiose by Zymobacter palmae carrying the Ruminocuccus albus beta-glucosidase gene.

    PubMed

    Yanase, Hideshi; Yamamoto, Keiko; Sato, Dai; Okamoto, Kenji

    2005-07-21

    Its metabolic characteristics suggest Zymobacter palmae gen. nov., sp. nov. could serve as a useful new ethanol-fermenting bacterium, but its biotechnological exploitation would require certain genetic improvements. We therefore established a method for transforming Z. palmae using the broad-host vector plasmids pRK290, pMFY31 and pMFY40 as a source of transforming DNA. Using electroporation, the frequency of transformation was 10(5) to 10(6) transformants/mug of DNA. To confer the ability to ferment cellobiose, which is a hydrolysis product from cellulosic materials treated enzymatically or with acid, the beta-glucosidase gene from Ruminococcus albus was introduced into Z. palmae, where its expression was driven by its endogenous promoter. About 56% of the enzyme expressed was localized on the cell-surface or in the periplasm. The recombinant Z. palmae could ferment 2% cellobiose to ethanol, producing 95% of the theoretical yield with no