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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. 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

  3. [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.

  4. 21 CFR 866.5630 - Beta-2-microglobulin immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... beta-2-microglobulin (a protein molecule) in serum, urine, and other body fluids. Measurement of beta-2-microglobulin aids in the diagnosis of active rheumatoid arthritis and kidney disease. (b) Classification....

  5. 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

  6. 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.

  7. 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)

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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 §...

  13. 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 §...

  14. 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 §...

  15. 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.

  16. 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.

  17. [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.

  18. [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.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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

  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. 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

  7. 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.

  8. 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

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

    DTIC Science & Technology

    2010-05-01

    Biochemistry 1973;12(24):4811-22. 5. Pedersen LO, Hansen AS, Olsen AC, Gerwien J, Nissen MH, Buus S. The interaction between beta 2- microglobulin (beta 2m...CHEMISTRY VOL. 285, NO. 11, pp. 7947–7956, March 12, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the... Devlin , H. L., and Mudryj, M. (2009) Cancer Lett. 274, 177–186 54. Brooke, G. N., and Bevan, C. L. (2009) Curr. Genomics 10, 18–25 55. Fuhrman, B

  10. 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.

  11. 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

  12. 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

  13. [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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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)

  2. 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.

  3. [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

  4. 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

  5. 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.

  6. 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

  7. 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

  8. [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)

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. Beta-2 Microglobulin Tumor Marker

    MedlinePlus

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  17. Activation of human platelets by antibodies to thymocytes and beta 2-microglobulin. I. Qualitative and quantitative aspects of the platelet aggregation induced by HATG and SA beta 2mG.

    PubMed Central

    Csákó, G; Suba, E A; Wistar, R

    1980-01-01

    The platelet effect of antibody preparations known to have immunosuppressive action was investigated by a turbidimetric method in vitro. Both horse anti-human thymocyte globulin (HATG) and sheep anti-human beta 2-microglobulin IgG (SA beta 2mG) caused the platelets to aggregate in all human platelet-rich plasmas (PRP) tested. The aggregation was usually irreversible and characterized by a sigmoid curve. The action is specific for the antibodies in HATG and SA beta 2mG because control preparations (horse normal IgG and IgG fractions of sheep normal, anti-dog IgG and anti-human IgA sera) were ineffective; further, heating (30 min at 56 degrees C) and BaSO4 or Al(OH)3 adsorption of HATG and SA beta 2mG did not alter their aggregating capability. When HATG and SA beta 2mG were added together to PRP, they induced aggregation in a simple additive manner. High antibody doses tended to decrease the extent of aggregation. The effect of platelet count on aggregation varied with both the dose level ('low' or 'high') and type (HATG or SA beta 2mG) of the inducer antibody. Using fixed submaximal doses, four main aggregation patterns could be recognized among 60 PRP: (i) high responses to both HATG and SA beta 2mG; (ii) high to HATG, low to SA beta 2mG; (iii) low to HATG, high to SA beta 2mG; and (iv) low to both. The results provide guidelines for quantitative aggregation studies with platelet antibodies and suggest that HATG and SA beta 2mG act through distinct platelet membrane components, the receptor for the latter being the best characterized protein of the mammalian cell membrane. PMID:6156042

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  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. 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.

  4. Beta-2 Microglobulin Kidney Disease Test

    MedlinePlus

    PLEASE NOTE: Your web browser does not have JavaScript enabled. Unless you enable Javascript , your ability to navigate and access the features of this website will be limited. ... Proceeds from website advertising help sustain Lab Tests Online. AACC is a not-for-profit organization and does not endorse non-AACC products and services. Advertising & Sponsorship: ...

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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.

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

    DTIC Science & Technology

    2009-03-14

    Odero-Marah V, Lue HW, Nomura T, Wang R, Chu G, Huang WC, and Chung LWK. (2008). Epithelial to Mesenchymal Transition (EMT) in Human Prostate...Cancer: Lessons Learned from ARCaP Model. Clinical and Experimental Metastasis. 25 (6):601-10, 2008. 4. Odero-Marah V, Wang R, Chu G, Xu J, Shi C...6198-206. 3. Zhau HE, Odero-Marah V, Lue HW, Nomura T, Wang R, Chu G, Huang WC, and Chung LWK. (2008). Epithelial to Mesenchymal Transition (EMT

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. Identification of Two Candidate Tumor Suppressor Genes on Chromosome 17p13.3: Assessment of Their Roles in Breast and Ovarian Carcinogenesis

    DTIC Science & Technology

    1999-07-01

    reported in primitive neuroectodermal tumors, carcinoma of the cervix uteri, medulloblastoma , osteosarcoma, astrocytoma (22), and acute myeloid leukemia and... thyroid carcinoma cells) 321 2 Ribosomal protein L8W’ 60S ribosomal protein L8 NR(human) 337 2 Beta-2-microglobulin Beta-2-microglobulin NRprecursor

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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, Bram; van Straalen, Nico M; Roelofs, Dick

    2017-03-18

    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 analyzed 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 co-regulated 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 eudaphic (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. This article is protected by copyright. All rights reserved.

  10. 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.

  11. 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

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

    DTIC Science & Technology

    2009-05-31

    expression and survival through a lipogenic pathway in prostate cancer Wen-Chin Huang, Guodong Zhu , Haiyen E. Zhau and Leland W. K. Chung Emory...Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Requests for reprints: Wen-Chin Huang or Leland W.K. Chung , Molecular Urology...Konaka H, Sodek J, Zhau HE, Chung LW. Human Osteocalcin and Bone Sialoprotein Mediating Osteomimicry of Prostate Cancer Cells: Role of cAMP

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

    DTIC Science & Technology

    2011-05-01

    OUTCOMES: The third year of this DoD geant from May 1, 2010 to Apr 30, 2011: We collaborated with Dr. Leland Chung (Cedars-Sinai Medical Center) and...Sodek J, Zhau HE, Chung LW. Human Osteocalcin and Bone Sialoprotein Mediating Osteomimicry of Prostate Cancer Cells: Role of cAMP-Dependent Protein...Kinase A Signaling Pathway. Cancer Res 2005;65(6):2303-13. 10. Huang WC, Zhau HE, Chung LW. Androgen receptor survival signaling is blocked by anti

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

    DTIC Science & Technology

    2012-01-01

    Because there is currently no effective therapy for the management of hormone-refractory prostate cancer, we undertook the investi- gation of the...intratumorally with the h2M antibody as evaluated by their body weights and physical appearance (data not shown). This observation is concurred by the early...vivo . These results could have significant clinical implications. For example, the h2M antibody could be superior to other antiandrogenic therapies

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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

    2017-03-01

    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.

  1. 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.

  2. 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

  3. 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'.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. beta-adrenergic receptor gene polymorphisms and beta-blocker treatment outcomes in hypertension.

    PubMed

    Pacanowski, M A; Gong, Y; Cooper-Dehoff, R M; Schork, N J; Shriver, M D; Langaee, T Y; Pepine, C J; Johnson, J A

    2008-12-01

    Numerous studies have demonstrated that beta(1)- and beta(2)-adrenergic receptor gene (ADRB1 and ADRB2) variants influence cardiovascular risk and beta-blocker responses in hypertension and heart failure. We evaluated the relationship between ADRB1 and ADRB2 haplotypes, cardiovascular risk (death, nonfatal myocardial infarction (MI), and nonfatal stroke), and atenolol-based vs. verapamil sustained-release (SR)-based antihypertensive therapy in 5,895 coronary artery disease (CAD) patients. After an average of 2.8 years, death rates were higher in patients carrying the ADRB1 Ser49-Arg389 haplotype (hazard ratio (HR) 3.66, 95% confidence interval (95% CI) 1.68-7.99). This mortality risk was significant in patients randomly assigned to verapamil SR (HR 8.58, 95% CI 2.06-35.8) but not atenolol (HR 2.31, 95% CI 0.82-6.55), suggesting a protective role for the beta-blocker. ADRB2 haplotype associations were divergent within the treatment groups but did not remain significant after adjustment for multiple comparisons. ADRB1 haplotype variation is associated with mortality risk, and beta-blockers may be preferred in subgroups of patients defined by ADRB1 or ADRB2 polymorphisms.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. [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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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)

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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.

  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. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  4. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. ...

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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.

  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. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. A self-excising beta-recombinase/six cassette for repetitive gene deletion and homokaryon purification in Neurospora crassa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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.

  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. 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.

  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. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. Insect and wound induced GUS gene expression from a Beta vulgaris proteinase inhibitor gene promoter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  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. 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.

  13. 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

  14. 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

  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. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. [Investigation of beta-lactamase genes and clonal relationship among the extended-spectrum beta-lactamase producing nosocomial Escherichia coli isolates].

    PubMed

    Görgeç, Sündüz; Kuzucu, Çiğdem; Otlu, Barış; Yetkin, Funda; Ersoy, Yasemin

    2015-01-01

    Extended-spectrum beta-lactamase (ESBL) producing microorganisms currently cause a major problem. Among theseCTX-M beta-lactamase producing Escherichia coli has also disseminated worldwide as an important cause of both nosocomial and community-acquired infections. The aims of this study were to determine the prevalence of the beta-lactamase genes, antibiotic susceptibilities and clonal relationships of ESBL-producing nosocomial E.coli isolates. A total of 76 ESBL-producing E.coli strains isolated from urine (n= 26), blood (n= 25) and wound (n= 25) specimens of hospitalized patients identified as nosocomial infection agents according to the CDC criteria between June 2010-June 2011 were included in the study. Antibiotic susceptibilities of the isolates were detected by Kirby-Bauer disc diffusion method according to CLSI recommendations. ESBL production was tested by double disc diffusion method, and cefotaxime/cefotaxime-clavulanic acid E-test strips (AB Biodisk, Sweden) were used for indeterminate results. Presence of TEM, SHV, CTX-M, OXA-2 group, 0XA-10 group, PER, VEB and GES beta-lactamase genes were investigated by polymerase chain reaction (PCR) using specific primers. Pulsed-field gel electrophoresis (PFGE) method was used for the detection of clonal relationships among the strains. Most of the ESBL-producing E.coli strains were isolated from samples of inpatients in intensive care (35%), internal medicine (16%) and general surgery (13%) units. All of the 76 strains were found susceptible to imipenem, meropenem and amikacin; however all were resistant to cefotaxime and ceftriaxone. The susceptibility rates of the isolates to cefoxitin, ertapenem, cefoperazone/sulbactam, piperacillin-tazobactam, gentamicin, ciprofloxacin, cefepime, amoxicillin-clavulanic acid, aztreonam and ceftazidime were 96%, 83%, 63%, 61%, 50%, 41%, 25%, 21%, 20% and 18%, respectively. Among E.coli isolates, the frequency of CTX-M, TEM, OXA-2 group, PER, SHV and OXA-10 group beta

  2. Insertion of the beta Geo promoter trap into the Fem1c gene of ROSA3 mice.

    PubMed

    Schlamp, Cassandra L; Thliveris, Andrew T; Li, Yan; Kohl, Louis P; Knop, Claudia; Dietz, Joel A; Larsen, Inna V; Imesch, Pascal; Pinto, Lawrence H; Nickells, Robert W

    2004-05-01

    ROSA3 mice were developed by retroviral insertion of the beta Geo gene trap vector. Adult ROSA3 mice exhibit widespread expression of the trap gene in epithelial cells found in most organs. In the central nervous system the highest expression of beta Geo is found in CA1 pyramidal cells of the hippocampus, Purkinje cells of the cerebellum, and ganglion cells of the retina. Characterization of the genomic insertion site for beta Geo in ROSA3 mice shows that the trap vector is located in the first intron of Fem1c, a gene homologous to the sex-determining gene fem-1 of Caenorhabditis elegans. Transcription of the Rosa3 allele (R3) yields a spliced message that includes the first exon of Fem1c and the beta Geo coding region. Although normal processing of the Fem1c transcript is disrupted in homozygous Rosa3 (Fem1c(R3/R3)) mice, some tissues show low levels of a partially processed transcript containing exons 2 and 3. Since the entire coding region of Fem1c is located in these two exons, Fem1c(R3/R3) mice may still be able to express a putative FEM1C protein. To this extent, Fem1c(R3/R3) mice show no adverse effects in their sexual development or fertility or in the attenuation of neuronal cell death, another function that has been attributed to both fem-1 and a second mouse homolog, Fem1b. Examination of beta Geo expression in ganglion cells after exposure to damaging stimuli indicates that protein levels are rapidly depleted prior to cell death, making the beta Geo reporter gene a potentially useful marker to study early molecular events in damaged neurons.

  3. Gene fusions of signal sequences with a modified beta-glucuronidase gene results in retention of the beta-glucuronidase protein in the secretory pathway/plasma membrane.

    PubMed

    Yan, X; Gonzales, R A; Wagner, G J

    1997-11-01

    Signal sequences and endoplasmic reticulum (ER) retention signals are known to play central roles in targeting and translocation in the secretory pathway, but molecular aspects about their involvement are poorly understood. We tested the effectiveness of deduced signal sequences from various genes (hydroxyproline-rich glycoprotein [HRGP] from Phaseolus vulgaris; Serpin from Manduca sexta) to direct a modified beta-glucuronidase (GUS) protein into the secretory pathway in transgenic tobacco (Nicotiana tabacum L.). The reporter protein was not secreted to the cell wall/extracellular space as monitored using extracellular fluid analysis (low- or high-ionic-strength conditions) but occurred in membranes with a density of 1.16 to 1.20 g/mL. Membrane-bound GUS equilibrated with the plasma membrane (PM) and the ER on linear sucrose gradients with or without ethylenediaminetetraacetic acid, suggesting that GUS associates with the ER and the PM. Confocal microscopy of fixed cultured cells prepared from GUS control and HRGP signal peptide (SP)-GUS-expressing plants suggested only cytosolic localization in GUS-expressing plants but substantial peripheral localization in HRGP SP-GUS plants, which is consistent with GUS being associated with the PM. Aqueous two-phase partitioning of microsomal membranes from HRGP SP-GUS and Serpin SP-GUS transgenic leaves also indicated that GUS activity was enriched in the ER and the PM. These observations, together with hydrophobic moment plot analysis, suggest that properties of the SP-GUS protein result in its retention in the secretory pathway and PM.

  4. Gene fusions of signal sequences with a modified beta-glucuronidase gene results in retention of the beta-glucuronidase protein in the secretory pathway/plasma membrane.

    PubMed Central

    Yan, X; Gonzales, R A; Wagner, G J

    1997-01-01

    Signal sequences and endoplasmic reticulum (ER) retention signals are known to play central roles in targeting and translocation in the secretory pathway, but molecular aspects about their involvement are poorly understood. We tested the effectiveness of deduced signal sequences from various genes (hydroxyproline-rich glycoprotein [HRGP] from Phaseolus vulgaris; Serpin from Manduca sexta) to direct a modified beta-glucuronidase (GUS) protein into the secretory pathway in transgenic tobacco (Nicotiana tabacum L.). The reporter protein was not secreted to the cell wall/extracellular space as monitored using extracellular fluid analysis (low- or high-ionic-strength conditions) but occurred in membranes with a density of 1.16 to 1.20 g/mL. Membrane-bound GUS equilibrated with the plasma membrane (PM) and the ER on linear sucrose gradients with or without ethylenediaminetetraacetic acid, suggesting that GUS associates with the ER and the PM. Confocal microscopy of fixed cultured cells prepared from GUS control and HRGP signal peptide (SP)-GUS-expressing plants suggested only cytosolic localization in GUS-expressing plants but substantial peripheral localization in HRGP SP-GUS plants, which is consistent with GUS being associated with the PM. Aqueous two-phase partitioning of microsomal membranes from HRGP SP-GUS and Serpin SP-GUS transgenic leaves also indicated that GUS activity was enriched in the ER and the PM. These observations, together with hydrophobic moment plot analysis, suggest that properties of the SP-GUS protein result in its retention in the secretory pathway and PM. PMID:9390428

  5. Glycosaminoglycan storage in cultured neonatal murine mucopolysaccharidosis type VII neuroglial cells and correction by beta-glucuronidase gene transfer.

    PubMed

    Taylor, R M; Wolfe, J H

    1997-05-01

    The inherited deficiency of beta-glucuronidase activity causes the lysosomal storage disorder mucopolysaccharidosis (MPS) type VII (Sly disease). The sequential catabolism of glycosaminoglycans in lysosomes is blocked, and undegraded substrates accumulate in cells of many tissues, including neurons and glia in the brain. To evaluate the deficient metabolic pathway, primary cultures of mixed brain cells were established from newborn MPS VII mice. beta-Glucuronidase levels and glycosaminoglycan accumulation were studied in normal, carrier, and MPS VII cells. Retroviral vector-mediated transfer of a normal beta-glucuronidase cDNA corrected the enzymatic deficiency in MPS VII cells and restored glycosaminoglycan catabolism to normal. High levels of beta-glucuronidase expression were sustained in vector-corrected nondividing glial cell cultures for >2 months. These studies provide an in vitro model for evaluating somatic gene transfer in neural cells affected in mucopolysaccharidoses.

  6. Distinct functions for thyroid hormone receptors alpha and beta in brain development indicated by differential expression of receptor genes.

    PubMed Central

    Forrest, D; Hallböök, F; Persson, H; Vennström, B

    1991-01-01

    Thyroid hormones are essential for correct brain development, and since vertebrates express two thyroid hormone receptor genes (TR alpha and beta), we investigated TR gene expression during chick brain ontogenesis. In situ hybridization analyses showed that TR alpha mRNA was widely expressed from early embryonic stages, whereas TR beta was sharply induced after embryonic day 19 (E19), coinciding with the known hormone-sensitive period. Differential expression of TR mRNAs was striking in the cerebellum: TR beta mRNA was induced in white matter and granule cells after the migratory phase, suggesting a main TR beta function in late, hormone-dependent glial and neuronal maturation. In contrast, TR alpha mRNA was expressed in the earlier proliferating and migrating granule cells, and in the more mature granular and Purkinje cell layers after hatching, indicating a role for TR alpha in both immature and mature neural cells. Surprisingly, both TR genes were expressed in early cerebellar outgrowth at E9, before known hormone requirements, with TR beta mRNA restricted to the ventricular epithelium of the metencephalon and TR alpha expressed in migrating cells and the early granular layer. The results implicate TRs with distinct functions in the early embryonic brain as well as in the late phase of hormone requirement. Images PMID:1991448

  7. Gene structure and chromosomal localization of the human HSD11K gene encoding the kidney (type 2) isozyme of 11{beta}-hydroxysteroid dehydrogenase

    SciTech Connect

    Agarwal, A.K.; Rogerson, F.M.; Mune, T.; White, P.C.

    1995-09-01

    11{beta}-hydroxysteroid dehydrogenase (11{beta}HSD) converts glucocorticoids to inactive products and is thus thought to confer specificity for aldosterone on the type I mineralocorticoid receptor in the kidney. Recent studies indicate the presence of at least two isozymes of 11{beta}HSD. In vitro, the NAD{sup +}-dependent kidney (type 2) isozyme catalyzes 11{beta}-dehydrogenase but not reductase reactions, whereas the NADP{sup +}-dependent liver (type 1) isozyme catalyzes both reactions. We have now characterized the human gene encoding kidney 11{beta}HSD (HSD11K). A bacteriophage P1 clone was isolated after screening a human genomic library by hybridization with sheep HSD11K cDNA. The gene consists of 5 exons spread over 6 kb. The nucleotide binding domain lies in the first exon are GC-rich (80%), suggesting that the gene may be transcriptionally regulated by factors that recognize GC-rich sequences. Fluorescence in situ hybridization of metaphase chromosomes with a positive P1 clone localized the gene to chromosome 16q22. In contrast, the HSD11L (liver isozyme) gene is located on chromosome 1 and contains 6 exons; the coding sequences of these genes are only 21% identical. HSD11K is expressed at high levels in the placenta and kidney of midgestation human fetuses and at lower levels in lung and testes. Different transcriptional start sites are utilized in kidney and placenta. These data should be applicable to genetic analysis of the syndrome of apparent mineralocorticoid excess, which may represent a deficiency of 11{beta}HSD. 25 refs., 5 figs.

  8. The nagA gene of Penicillium chrysogenum encoding beta-N-acetylglucosaminidase.

    PubMed

    Díez, Bruno; Rodríguez-Sáiz, Marta; de la Fuente, Juan Luis; Moreno, Miguel Angel; Barredo, José Luis

    2005-01-15

    We purified the beta-N-acetylglucosaminidase from the filamentous fungus Penicillium chrysogenum and its N-terminal sequence was determined, showing the presence of a mixture of two proteins (P1 and P2). A genomic DNA fragment was cloned by using degenerated oligonucleotides from the Nt sequences. The nucleotide sequence showed the presence of an ORF (nagA gene) lacking introns, with a length of 1791 bp, and coding for a protein of 66.5 kDa showing similarity to acetylglucosaminidases. The NagA deduced protein includes P1 and P2 as incomplete forms of the mature protein, and contains putative features for protein maturation: an 18-amino acid signal peptide, a KEX2 processing site, and four glycosylation motifs. The sequence just after the signal peptide corresponds to P2 and that after the KEX2 site to P1. The nagA transcript has a size of about 2.1 kb and is present until the end of the fermentation process for penicillin production. NagA is one of the most largely represented proteins in P. chrysogenum, increasing along the fermentation process. The suitability of the nagA promoter (PnagA) for gene expression in fungi was demonstrated by expressing the bleomycin resistance gene (ble(R)) from Streptoalloteichus hindustanus in P. chrysogenum.

  9. Ectopic mitotic recombination in Drosophila probed with bacterial beta-galactosidase gene-based reporter transgenes.

    PubMed Central

    Bärtsch, S; Dücker, K; Würgler, F E; Sengstag, C

    1997-01-01

    Plasmids were constructed to investigate homologous mitotic recombination in Drosophila cells. Heteroalleles containing truncated but overlapping segments of the bacterial beta-galactosidase gene (lacZ) were positioned either on separate plasmids or as direct repeats on the same chromosome. Recombination reconstituted a functional lacZgene leading to expression of LacZ+activity detectable by histochemical staining. High extrachromosomal recombination (ECR) frequencies between unlinked heteroalleles were observed upon transient co-transfection into Drosophila melanogaster Schneider line 2 (S2) cells. Stably transfected cells containing the lacZ heteroalleles linked on a chromosome exhibited intrachromosomal recombination (ICR) frequencies two orders of magnitude lower than ECR frequencies. Recombination was inducible by exposing the cells to ethyl methanesulphonate or mitomycin C. Recombination products were characterized by multiplex PCR analysis and unequal sister chromatid recombination was found as the predominant mechanism reconstituting the lacZ gene. To investigate recombination in vivo imaginal disc cells from transgenic larvae carrying the reporter gene on the X chromosome were isolated and stained for LacZ+ activity. The presence of a few LacZ+ clones indicated that mitotic recombination events occurred at frequencies two orders of magnitude lower than the corresponding event in cultured cells and late during larval development. PMID:9380517

  10. Early peroxisome proliferator-activated receptor gamma regulated genes involved in expansion of pancreatic beta cell mass

    PubMed Central

    2011-01-01

    Background The progression towards type 2 diabetes depends on the allostatic response of pancreatic beta cells to synthesise and secrete enough insulin to compensate for insulin resistance. The endocrine pancreas is a plastic tissue able to expand or regress in response to the requirements imposed by physiological and pathophysiological states associated to insulin resistance such as pregnancy, obesity or ageing, but the mechanisms mediating beta cell mass expansion in these scenarios are not well defined. We have recently shown that ob/ob mice with genetic ablation of PPARγ2, a mouse model known as the POKO mouse failed to expand its beta cell mass. This phenotype contrasted with the appropriate expansion of the beta cell mass observed in their obese littermate ob/ob mice. Thus, comparison of these models islets particularly at early ages could provide some new insights on early PPARγ dependent transcriptional responses involved in the process of beta cell mass expansion Results Here we have investigated PPARγ dependent transcriptional responses occurring during the early stages of beta cell adaptation to insulin resistance in wild type, ob/ob, PPARγ2 KO and POKO mice. We have identified genes known to regulate both the rate of proliferation and the survival signals of beta cells. Moreover we have also identified new pathways induced in ob/ob islets that remained unchanged in POKO islets, suggesting an important role for PPARγ in maintenance/activation of mechanisms essential for the continued function of the beta cell. Conclusions Our data suggest that the expansion of beta cell mass observed in ob/ob islets is associated with the activation of an immune response that fails to occur in POKO islets. We have also indentified other PPARγ dependent differentially regulated pathways including cholesterol biosynthesis, apoptosis through TGF-β signaling and decreased oxidative phosphorylation. PMID:22208362

  11. Targeting of lacZ reporter gene expression with radioiodine-labelled phenylethyl-beta- d-thiogalactopyranoside.

    PubMed

    Lee, Kyung-Han; Byun, Sang Sung; Choi, Joon Hun; Paik, Jin-Young; Choe, Yearn Seong; Kim, Byung-Tae

    2004-03-01

    There has recently been increasing interest in the development of radioprobes that specifically target proteins transcribed from expression of reporter genes of interest. The purpose of this study was to develop a radioprobe that targets one of the most widely used reporter genes, the bacterial lacZ gene. We synthesised and purified radioiodine-labelled phenylethyl-beta- d-thiogalactopyranoside (PETG), a competitive inhibitor specific against Escherichia coli beta-galactosidase. We showed that [(125)I]iodo-PETG specifically binds to beta-galactosidase as verified by column chromatography and polyacrylamide gel electrophoresis after incubation of radiotracer with the protein. We also showed through enzyme kinetic studies that iodo-PETG retains inhibitory action against beta-galactosidase activity. COS-7 cells infected with a recombinant adenovirus expressing the lacZ gene had viral titre-dependent enhancements in [(125)I]iodo-PETG uptake ( r(2)=0.897; P=0.001), which reached up to 642.5%+/-16.7% of control levels ( P<0.00001). Moreover, the level of uptake was highly correlated to luminescent measurements of beta-galactosidase activity ( r(2)=0.878; P<0.0001). These results confirm that radioiodine-labelled PETG specifically targets beta-galactosidase and that its uptake rates faithfully reflect levels of expression of the lacZ reporter gene. Further investigations were performed in nude mice bearing human neuroblastoma tumours transferred with the lacZ gene. Compared with control tumours, lacZ-expressing tumours were slightly better visualised on [(123)I]iodo-PETG images and had a modest increase in tumour to muscle count ratio (2.6+/-0.2 vs 1.9+/-0.1, P<0.05). The present results provide proof-of-principle for the potential of radiolabelled inhibitors as promising radiotracers to monitor lacZ gene expression levels. Future modifications to improve cell permeability should enhance in vivo contrast levels and may allow the use of radiolabelled beta

  12. Regulation of nitrogen metabolism, starch utilisation and the beta-hbd-adh1 gene cluster in Clostridium acetobutylicum.

    PubMed

    Woods, D R; Reid, S J

    1995-10-01

    The successful genetic manipulation of Clostridium acetobutylicum for the increased production of solvents will depend on an understanding of gene structure and regulation in the bacterium. The glutamine synthetase (glnA) gene is regulated by antisense RNA, transcribed from a downstream promoter, in the opposite direction to the glnA gene. An open reading frame (ORF) was detected downstream of the glnA gene, which has sequence homology to response regulators with anti-termination activity and may be involved in sensing nitrogen conditions. The expression of the linked beta-hbd, adh1 and fixB genes was investigated throughout the bacterial growth cycle by RNA hybridisation techniques. The adh1 gene was independently expressed as a 2.4-kb transcript which peaked at 12 h, immediately prior to the solventogenic phase. The beta-hbd and fixB genes were transcribed throughout the acidogenic and solventogenic phases. A regulator gene, regA, which complements a Bacillus subtilis ccpA mutant, has been identified and sequenced from C. acetobutylicum P262. The regA gene repressed the degradation of starch by an uncharacterised C. acetobutylicum gene, and may therefore play a role in the utilisation of carbohydrate substrates in this organism.

  13. Regulatory elements and structural features of Beta vulgaris polygalacturonase-inhibiting protein gene for fungal and pest control

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polygalacturonase-inhibiting proteins (PGIPs) are involved in plant defense. PGIPs are cell wall leucine-rich repeat (LRR) proteins that are known to inhibit pathogen and pest polygalacturonases (PGs) during the infection process. Several sugar beet (Beta vulgaris L.) PGIP genes (BvPGIP) were clon...

  14. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme of escherichia coli.

    PubMed Central

    Burgers, P M; Kornberg, A; Sakakibara, Y

    1981-01-01

    An Escherichia coli mutant, dnaN59, stops DNA synthesis promptly upon a shift to a high temperature; the wild-type dnaN gene carried in a transducing phage encodes a polypeptide of about 41,000 daltons [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553; Yuasa, S. & Sakakibara, Y. (1980) Mol. Gen. Genet. 180, 267-273]. We now find that the product of dnaN gene is the beta subunit of DNA polymerase III holoenzyme, the principal DNA synthetic multipolypeptide complex in E. coli. The conclusion is based on the following observations: (i) Extracts from dnaN59 cells were defective in phage phi X174 and G4 DNA synthesis after the mutant cells had been exposed to the increased temperature. (ii) The enzymatic defect was overcome by addition of purified beta subunit but not by other subunits of DNA polymerase III holoenzyme or by other replication proteins required for phi X174 DNA synthesis. (iii) Partially purified beta subunit from the dnaN mutant, unlike that from the wild type, was inactive in reconstituting the holoenzyme when mixed with the other purified subunits. (iv) Increased dosage of the dnaN gene provided by a plasmid carrying the gene raised cellular levels of the beta subunit 5- to 6-fold. PMID:6458041

  15. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme of escherichia coli.

    PubMed

    Burgers, P M; Kornberg, A; Sakakibara, Y

    1981-09-01

    An Escherichia coli mutant, dnaN59, stops DNA synthesis promptly upon a shift to a high temperature; the wild-type dnaN gene carried in a transducing phage encodes a polypeptide of about 41,000 daltons [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553; Yuasa, S. & Sakakibara, Y. (1980) Mol. Gen. Genet. 180, 267-273]. We now find that the product of dnaN gene is the beta subunit of DNA polymerase III holoenzyme, the principal DNA synthetic multipolypeptide complex in E. coli. The conclusion is based on the following observations: (i) Extracts from dnaN59 cells were defective in phage phi X174 and G4 DNA synthesis after the mutant cells had been exposed to the increased temperature. (ii) The enzymatic defect was overcome by addition of purified beta subunit but not by other subunits of DNA polymerase III holoenzyme or by other replication proteins required for phi X174 DNA synthesis. (iii) Partially purified beta subunit from the dnaN mutant, unlike that from the wild type, was inactive in reconstituting the holoenzyme when mixed with the other purified subunits. (iv) Increased dosage of the dnaN gene provided by a plasmid carrying the gene raised cellular levels of the beta subunit 5- to 6-fold.

  16. Inter-ethnic polymorphism of the beta-globin gene locus control region (LCR) in sickle-cell anemia patients.

    PubMed

    Périchon, B; Ragusa, A; Lapouméroulie, C; Romand, A; Moi, P; Ikuta, T; Labie, D; Elion, J; Krishnamoorthy, R

    1993-06-01

    Sequence polymorphisms within the 5'HS2 segment of human locus control region is described among sickle cell anemia patients. Distinct polymorphic patterns of a simple sequence repeat are observed in strong linkage disequilibrium with each of the five major beta s haplotypes. Potential functional relevance of this polymorphic region in globin gene expression is discussed.

  17. Identification of a Novel Single Nucleotide Polymorphism in Porcine Beta-Defensin-1 Gene.

    PubMed

    Pruthviraj, D R; Usha, A P; Venkatachalapathy, R T

    2016-03-01

    Porcine beta-defensin-1 (PBD-1) gene plays an important role in the innate immunity of pigs. The peptide encoded by this gene is an antimicrobial peptide that has direct activity against a wide range of microbes. This peptide is involved in the co-creation of an antimicrobial barrier in the oral cavity of pigs. The objective of the present study was to detect polymorphisms, if any, in exon-1 and exon-2 regions of PBD-1 gene in Large White Yorkshire (LWY) and native Ankamali pigs of Kerala, India. Blood samples were collected from 100 pigs and genomic DNA was isolated using phenol chloroform method. The quantity of DNA was assessed in a spectrophotometer and quality by gel electrophoresis. Exon-1 and exon-2 regions of PBD-1 gene were amplified by polymerase chain reaction (PCR) and the products were subjected to single strand conformation polymorphism (SSCP) analysis. Subsequent silver staining of the polyacrylamide gels revealed three unique SSCP banding patterns in each of the two exons. The presence of single nucleotide polymorphisms (SNPs) was confirmed by nucleotide sequencing of the PCR products. A novel SNP was found in the 5'-UTR region of exon-1 and a SNP was detected in the mature peptide coding region of exon-2. In exon-1, the pooled population frequencies of GG, GT, and TT genotypes were 0.67, 0.30, and 0.03, respectively. GG genotype was predominant in both the breeds whereas TT genotype was not detected in LWY breed. Similarly, in exon-2, the pooled population frequencies of AA, AG, and GG genotypes were 0.50, 0.27, and 0.23, respectively. AA genotype was predominant in LWY pigs whereas GG genotype was predominant in native pigs. These results suggest that there exists a considerable genetic variation at PBD-1 locus and further association studies may help in development of a PCR based genotyping test to select pigs with better immunity.

  18. TGF beta secreted by B16 melanoma antagonizes cancer gene immunotherapy bystander effect.

    PubMed

    Penafuerte, Claudia; Galipeau, Jacques

    2008-08-01

    Tumor-targeted delivery of immune stimulatory genes, such as pro-inflammatory cytokines and suicide genes, has shown to cure mouse models of cancer. Total tumor eradication was also found to occur despite subtotal tumor engineering; a phenomenon coined the "bystander effect". The bystander effect in immune competent animals arises mostly from recruitment of a cancer lytic cell-mediated immune response to local and distant tumor cells which escaped gene modification. We have previously described a Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Interleukin 2 (IL2) fusokine (aka GIFT2) which serves as a potent anticancer cytokine and it here served as a means to understand the mechanistic underpinnings to the immune bystander effect in an immune competent model of B16 melanoma. As expected, we observed that GIFT2 secreted by genetically engineered B16 tumor cells induces a bystander effect on non modified B16 cells, when admixed in a 1:1 ratio. However, despite keeping the 1:1 ratio constant, the immune bystander effect was completely lost as the total B16 cell number was increased from 10(4) to 10(6) which correlated with a sharp reduction in the number of tumor-infiltrating NK cells. We found that B16 secrete biologically active TGFbeta which in turn inhibited GIFT2 dependent immune cell proliferation in vitro and downregulated IL-2R beta expression and IFN gamma secretion by NK cells. In vivo blockade of B16 originating TGFbeta significantly improved the immune bystander effect arising from GIFT2. We propose that cancer gene immunotherapy of pre-established tumors will be enhanced by blockade of tumor-derived TGFbeta.

  19. Cystatin B-deficient mice have increased expression of apoptosis and glial activation genes

    SciTech Connect

    Lieuallen, Kimberly; Pennacchio, Len A.; Park, Morgan; Myers, Richard M.; Lennon, Gregory G.

    2001-07-05

    Loss-of-function mutations in the cystatin B (Cstb) gene cause a neurological disorder known as Unverricht Lundborg disease (EPM1) in human patients. Mice that lack Cstb provide a mammalian model for EPM1 by displaying progressive ataxia and myoclonic seizures. We analyzed RNAs from brains of Cstb-deficient mice by using modified differential display, oligonucleotide microarray hybridization and quantitative reverse transcriptase polymerase chain reaction to examine the molecular consequences of the lack of Cstb. We identified seven genes that have consistently increased transcript levels in neurological tissues from the knockout mice. These genes are cathepsin S, C1q B-chain of complement (C1qB), beta-2-microglobulin, glial fibrillary acidic protein (Gfap), apolipoprotein D, fibronectin 1 and metallothionein II, which are expected to be involved in increased proteolysis, apoptosis and glial activation. The molecular changes in Cstb-deficient mice are consistent with the pathology found in the mouse model and may provide clues towards the identification of therapeutic points of intervention for EPM1 patients.

  20. Gene therapy by allele selection in a mouse model of beta-thalassemia.

    PubMed

    Eckardt, Sigrid; Leu, N Adrian; Yanchik, Ashley; Hatada, Seigo; Kyba, Michael; McLaughlin, K John

    2011-02-01

    To be of therapeutic use, autologous stem cells derived from patients with inherited genetic disorders require genetic modification via gene repair or insertion. Here, we present proof of principle that, for diseases associated with dominant alleles (gain-of-function or haploinsufficient loss-of-function), disease allele–free ES cells can be derived from afflicted individuals without genome manipulation. This approach capitalizes on the derivation of uniparental cells, such as parthenogenetic (PG) ES cell lines from disease allele–free gametes. Diploid mammalian uniparental embryos with only maternally (oocyte-) or paternally (sperm-)derived genomes fail early in development due to the nonequivalence of parental genomes caused by genomic imprinting. However, these uniparental embryos develop to the blastocyst stage, allowing the derivation of ES cell lines. Using a mouse model for dominant beta-thalassemia, we developed disease allele–free PG ES cell lines from the oocytes of affected animals. Phenotype correction was obtained in donor-genotype recipients after transplantation of in vitro hematopoietic ES cell derivatives. This genetic correction strategy without gene targeting is potentially applicable to any dominant disease. It could also be the sole approach for larger or more complex mutations that cannot be corrected by homologous recombination.

  1. Chromosomal mapping, sequence and transcription analysis of the porcine fertilin beta gene (ADAM2).

    PubMed

    Day, A E; Quilter, C R; Sargent, C A; Mileham, A J

    2003-10-01

    Fertilin beta (ADAM2) forms a part of the heterodimeric surface protein fertilin, found on the plasma membrane of mammalian sperm, and has been implicated in the process of sperm-egg fusion. Analysis of cDNA products obtained from adult porcine testis mRNA has presented a sequence corresponding to 2620 bp of the ADAM2 gene. This sequence contained an open reading frame encoding a 735-amino acid protein and homologous to ADAM2 genes known in other mammalian species. Polymerase chain reaction (PCR) analysis of genomic DNA showed that the 2620 bp of cDNA sequence comprises at least 21 exons and spans approximately 76 kb of genomic DNA, with its size and structure being relatively conserved between mouse, human and pig. Fluorescence in situ hybridization was used to map ADAM2 to chromosome 15 of the pig, using a bacterial artificial chromosome clone from the PigE BAC library. This finding is consistent with comparative mapping experiments performed between pig and human chromosomes. Analysis of nine mRNA samples, by reverse transcriptase-PCR, from different porcine tissues has also suggested that expression of ADAM2 is limited to the testis, a finding that is consistent with other mammalian species.

  2. Extensive beta-glucuronidase activity in murine central nervous system after adenovirus-mediated gene transfer to brain.

    PubMed

    Ghodsi, A; Stein, C; Derksen, T; Yang, G; Anderson, R D; Davidson, B L

    1998-11-01

    Mucopolysaccharidosis type VII (MPS VII), caused by beta-glucuronidase deficiency, is a classic lysosomal storage disease. In the central nervous system (CNS), there is widespread pathology with distention of vacuoles in neurons and glia. An approach to therapy for MPS VII would require extensive delivery of enzyme to the CNS and subsequent uptake by the affected cells. In this study we show that intrastriatal injection of recombinant adenovirus encoding beta-glucuronidase (Ad betagluc) to MPS VII or wild-type mice results in focal, intense beta-glucuronidase mRNA expression near the injection site. Further, histochemical staining for enzyme activity showed that beta-glucuronidase activity extended well beyond transduced cells. Activity was detected throughout the ipsilateral striatum as well as in the corpus callosum, ventricles, and bilateral neocortex. Similarly, after injection into the right lateral ventricle or cisterna magna, enzyme activity was present in the ependymal cells of the ventricles, in the subarachnoid spaces, and also in the underlying cortex (150-500 microm from ependyma). The distribution of enzyme was most extensive 21 days after gene transfer to normal mouse brain, with more than 50% of the hemisphere positive for beta-glucuronidase activity. Eighty-four days after adenovirus injection a substantial level of enzyme expression remained (>40% of hemisphere positive for beta-glucuronidase activity). Histological sections from striatum of beta-glucuronidase-deficient mice injected with Ad betagluc showed a marked reduction in the number of distended vacuoles in both neurons and glia, as compared with uninjected striatum. Importantly, correction was noted in both hemispheres. Our finding that a relatively small number of transduced cells produce enzyme that reaches a large proportion of the CNS has favorable implications in developing direct gene transfer therapies for lysosomal storage disorders.

  3. Insertions of IS256-like element flanking the chromosomal beta-lactamase gene of Enterococcus faecalis CX19.

    PubMed Central

    Rice, L B; Marshall, S H

    1994-01-01

    We have previously identified an inverted repeat characteristic of staphylococcal beta-lactamase transposons adjacent to the chromosomal beta-lactamase genes of Enterococcus faecalis CH19 and its beta-lactamase-producing transconjugant CX19. Nucleotide sequence analysis of the CH19 beta-lactamase structural gene (blaZ) reveals it to be identical to the blaZ gene from E. faecalis HH22 and to the blaZ gene from the staphylococcal beta-lactamase transposon Tn552. We also report the presence of nucleotide sequence identical to a 317-bp region of the staphylococcal insertion sequence IS256 upstream of the blaZ gene in both CH19 and CX19. The identical segment of IS256 is present downstream of the blaZ gene of CX19, suggesting a second insertion of the element (in the inverted orientation) accompanying transfer to the recipient strain. Restriction analysis of the areas beyond the ClaI sites used to clone these regions suggests that full copies of the IS256-like element (designated IS256E) are present in all positions but that these elements were not directly involved in the transfer of the beta-lactamase gene to the recipient strain. We have also identified a region downstream of the second IS256E insertion site which exhibits substantial homology to ISSIW, an iso-ISSI insertion originally identified in Lactococcus lactis subsp. cremoris. These data suggest that the two enterococcal blaZ genes sequenced to date evolved from a common ancestor and may at one time have been incorporated into a transposon similar to Tn552. They also suggest that IS256-like elements are mobile in E. faecalis and capable of inserting in a manner consistent with the formation of novel composite transposons. Finally, they provide the first confirmation of the presence of an ISSI-like element in enterococci, raising the possibility that these elements play a role in the exchange of chromosomal antimicrobial resistance determinants. Images PMID:8031032

  4. Cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin

    PubMed Central

    1995-01-01

    Rabbit synovial fibroblasts (RSF) express basal levels of the metalloproteinases (MMP) collagenase, stromelysin-1 and 92-kD gelatinase when plated on intact fibronectin (FN), but elevated levels when plated on either the central RGD-containing cell-binding region of FN (120FN) or antibody against the alpha 5 beta 1 integrin, suggesting that domains outside 120FN may suppress the induction of MMP (Werb, Z., P. M. Tremble, O. Behrendtsen, E. Crowley, and C.H. Damsky. 1989. J. Cell Biol. 109:877-889). We therefore attempted to reconstitute the basal signaling of intact FN by plating RSF on 120FN together with domains of FN outside this region. Large COOH-terminal fragments containing both the heparin-binding and HICS domains suppressed MMP when combined with 120FN. To map the active sequences, peptides from this region and larger fragments that did, or did not, include the CS-1 portion of IIICS were tested. Only CS-1 peptide, or larger fragments containing CS-1, suppressed MMP expression induced by 120FN. In contrast, peptide V from the heparin-binding region, shown previously to stimulate focal contact formation, further enhanced MMP expression by RSF when present on the substrate with 120FN. RSF expressed alpha 4 beta 1 integrin, the receptor for CS-1, and the anti-alpha 4 mAb blocked the ability of CS-1 to suppress MMP induction by 120FN. These results show that signals modulating MMP expression and focal contact assembly are regulated independently, and that cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins plays a dominant role in regulating expression of these extracellular matrix-remodeling genes in response to FN. This work demonstrates directly the modular way in which information in the extracellular matrix is detected and processed by cell surface receptors. PMID:7537277

  5. Developmental and environmental regulation of a phenylalanine ammonia-lyase-beta-glucuronidase gene fusion in transgenic tobacco plants.

    PubMed Central

    Liang, X W; Dron, M; Schmid, J; Dixon, R A; Lamb, C J

    1989-01-01

    A 1.1-kilobase promoter fragment of the bean (Phaseolus vulgaris L.) phenylalanine ammonia-lyase (EC 4.3.1.5) gene PAL2 was translationally fused to the beta-glucuronidase reporter gene and transferred to tobacco by Agrobacterium tumefaciens-mediated leaf disk transformation. The distribution of beta-glucuronidase activity in these transgenic plants is very similar to that of endogenous PAL2 transcripts in bean, with very high levels in petals; marked accumulation in anthers, stigmas, roots, and shoots; and low levels in sepals, ovaries, and leaves. Histochemical analysis of the spatial pattern of beta-glucuronidase activity showed that the PAL2 promoter is highly active in the shoot apical meristem, the zone of cell proliferation immediately adjacent to the root apical meristem, and in the early stages of vascular development at the inception of xylem differentiation. Wounding and light evoke specific changes in the spatial pattern of beta-glucuronidase activity in stems, including induction in the epidermis. These data indicate that the PAL2 promoter transduces a complex set of developmental and environmental cues into an integrated spatial and temporal program of gene expression to regulate the synthesis of a diverse array of phenylpropanoid natural products. Images PMID:2594769

  6. beta-Glucosidase as a reporter for the gene expression studies in Thermus thermophilus and constitutive expression of DNA repair genes.

    PubMed

    Ohta, Toshihiro; Tokishita, Shin-Ichi; Imazuka, Reiko; Mori, Ichiro; Okamura, Jin; Yamagata, Hideo

    2006-07-01

    Thermus thermophilus is an extremely thermophilic eubacterium that grows optimally at 70-75 degrees C. Because the frequency of DNA damage, such as deamination, depurination and single-strand breaks, increases as the temperature rises, the regulation of expression as well as the specificities and activities of T.thermophilus DNA repair systems are of particular interest. To study those systems, we developed a gene expression vector using the T.thermophilus beta-glucosidase gene (bgl) with host strain JOS9 (Deltabgl) derived from the T.thermophilus wild-type strain HB27. Since HB27 has two putative beta-galactosidase genes, the use of a single bgl gene as a reporter in combination with a Deltabgl host strain permits the study of gene expression against a low background level. We assayed Bgl activity with 2-nitrophenyl-beta-d-glucopyranoside as the substrate at 80 degrees C. We measured the expression of seven genes involved in DNA repair--three nucleotide excision repair genes (uvrA, uvrB and uvrC) and four recombinational repair genes (recA, ruvA, ruvB and ruvC). Expression levels of uvrA and uvrB were about three times those of uvrC, while those of ruvA, ruvB and ruvC were almost equal. Both ruvA and ruvC formed an operon with their adjacent 5'-upstream gene paaG and ftsQAZ, respectively. recA was transcribed as an operon of four genes, amt-cinA-ligT-recA. All seven DNA repair genes were expressed constitutively, and the DNA damaging agent mitomycin C did not increase their expression.

  7. Pest protection conferred by a Beta vulgaris serine proteinase inhibitor gene.

    PubMed

    Smigocki, Ann C; Ivic-Haymes, Snezana; Li, Haiyan; Savić, Jelena

    2013-01-01

    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. Independently derived BvSTI transgenic tobacco T2 homozygous progeny were shown to have relatively high BvSTI gene transcript levels. BvSTI-specific polyclonal antibodies cross-reacted with the expected 30 kDA recombinant BvSTI protein on Western blots. In gel trypsin inhibitor activity assays revealed a major clear zone that corresponded to the BvSTI proteinase inhibitor that was not detected in the untransformed control plants. BvSTI-transgenic plants were bioassayed for resistance to five lepidopteran insect pests. Spodoptera frugiperda, S. exigua and Manduca sexta larvae fed BvSTI leaves had significant reductions in larval weights as compared to larvae fed on untransformed leaves. In contrast, larval weights increased relative to the controls when Heliothis virescens and Agrotis ipsilon larvae were fed on BvSTI leaves. As the larvae entered the pupal stage, pupal sizes reflected the overall larval weights. Some developmental abnormalities of the pupae and emerging moths were noted. These findings suggest that the sugar beet BvSTI gene may prove useful for effective control of several different lepidopteran insect pests in genetically modified tobacco and other plants. The sugar beet serine proteinase inhibitor may be more effective for insect control because sugar beet is cropped in restricted geographical areas thus limiting the exposure of the insects to sugar beet proteinase inhibitors and build up of non-sensitive midgut proteases.

  8. A second glutamine synthetase gene with expression in the gills of the gulf toadfish (opsanus beta)

    SciTech Connect

    Walsh, Patrick J.; Mayer, Gregory D.; Medina, Monica; Bernstein, Matthew L.; Barimo, John F.; Mommsen, Thomas P.

    2003-05-08

    Enzyme and molecular biology approaches were used to more completely characterize the expression of the nitrogen metabolism enzyme glutamine synthetase [GSase; L-glutamate: ammonia ligase (ADP-forming), E.C. 6.3.1.2] in a variety of tissues of the gulf toadfish (Opsanus beta) subjected to unconfined (ammonotelic) and confined (ureotelic) conditions. Enzymological results demonstrate that while weight-specific GSase activities rank in the order of brain > liver > stomach {approx} kidney > intestine > gill> heart/spleen > muscle, when tissue mass is used to calculate a glutamine synthetic potential, the liver has the greatest, followed by muscle > stomach and intestine with minor contributions from the remaining tissues. Additionally, during confinement stress, GSase activity only increases significantly in liver (5-fold) and muscle (2-fold), tissues which previously showed significant expression of the other enzymes of urea synthesis. RT PCR and RACE PCR revealed the presence of a second GSas e cDNA from gill tissue that appears to share relatively low nucleotide and amino acid sequence similarity ({approx}73 percent) with the original GSase cloned from liver, and furthermore lacks a mitochondrial leader targeting sequence. RT PCR and restriction digestion experiments demonstrated that mRNA from the original ''liver'' GSase is expressed in all tissues examined (liver, gill, stomach, intestine, kidney, brain and muscle), whereas the new ''gill'' form shows expression primarily in the gill. Enzyme activities of gill GSase also exhibit a different subcellular compartmentation with apparent exclusive expression in the soluble compartment, whereas other tissues expressing the ''liver'' form show both cytoplasmic and mitochondrial activities. Finally, phylogenetic analysis of a number of GSases demonstrates that the toadfish gill GSase has a greater affinity for a clade that includes the Xenopus GSase genes and one of two Fugu GSase genes, than it has for a clade

  9. Baseline Gene Expression Signatures in Monocytes from Multiple Sclerosis Patients Treated with Interferon-beta

    PubMed Central

    Bustamante, Marta F.; Nurtdinov, Ramil N.; Río, Jordi; Montalban, Xavier; Comabella, Manuel

    2013-01-01

    Background A relatively large proportion of relapsing-remitting multiple sclerosis (RRMS) patients do not respond to interferon-beta (IFNb) treatment. In previous studies with peripheral blood mononuclear cells (PBMC), we identified a subgroup of IFNb non-responders that was characterized by a baseline over-expression of type I IFN inducible genes. Additional mechanistic experiments carried out in IFNb non-responders suggested a selective alteration of the type I IFN signaling pathway in the population of blood monocytes. Here, we aimed (i) to investigate whether the type I IFN signaling pathway is up-regulated in isolated monocytes from IFNb non-responders at baseline; and (ii) to search for additional biological pathways in this cell population that may be implicated in the response to IFNb treatment. Methods Twenty RRMS patients classified according to their clinical response to IFNb treatment and 10 healthy controls were included in the study. Monocytes were purified from PBMC obtained before treatment by cell sorting and the gene expression profiling was determined with oligonucleotide microarrays. Results and discussion Purified monocytes from IFNb non-responders were characterized by an over-expression of type I IFN responsive genes, which confirms the type I IFN signature in monocytes suggested from previous studies. Other relevant signaling pathways that were up-regulated in IFNb non-responders were related with the mitochondrial function and processes such as protein synthesis and antigen presentation, and together with the type I IFN signaling pathway, may also be playing roles in the response to IFNb. PMID:23637780

  10. Is the staple diet eaten in Medawachchiya, Sri Lanka, a predisposing factor in the development of chronic kidney disease of unknown etiology? - A comparison based on urinary β2-microglobulin measurements

    PubMed Central

    2014-01-01

    Background Exact mechanism of causation of chronic kidney disease of unknown etiology (CKDu) in Sri Lanka is not described to date, despite the identification of possible multiple risk factors. Questions have been raised as to why only some are affected while others remain intact, though they are inhabitants of the same locality. Methods Comparative studies were carried out, assessing urinary β2 microglobulin (β2m) and the dietary patterns of CKDu patients and age sex matched non-CKDu subjects. Urinary β2m levels of spot urine samples were analyzed using the Enzyme-linked Immunosorbent assay (ELISA) and dietary patterns were studied using twenty four hour dietary recalls and frequency consumption of foods of animal origin performed on three occasions at six months intervals within a period of one and half years. Results The mean urinary β2m level of CKDu patients from Medawachchiya was significantly (p < 0.05) higher when compared with that of the non-CKDu subjects. The mean urinary β2m level of the non-CKDu subjects was within the reference limits for spot urine samples (0 – 0.3 μg/mL). White raw rice was the staple diet of both CKDu patients and non-CKDu subjects and the level of consumption was almost the same. The consumption of fresh water fish products of CKDu patients under high (14, 14%), moderate (36, 36%), low (26, 26%) and less (20, 20%) categories did not show significant variations (p > 0.05) compared to non-CKDu subjects. Conclusions Staple food in diet and the consumption pattern of CKDu patients from Medawachchiya were similar to that of non-CKDu subjects from the same area despite their urinary β2m concentration being significantly higher. PMID:24988874

  11. The β2-microglobulin-free heterodimerization of rhesus monkey MHC class I A with its normally spliced variant reduces the ubiquitin-dependent degradation of MHC class I A.

    PubMed

    Dai, Zheng-Xi; Zhang, Gao-Hong; Zhang, Xi-He; Xia, Hou-Jun; Li, Shao-You; Zheng, Yong-Tang

    2012-03-01

    The MHC class I (MHC I) molecules play a pivotal role in the regulation of immune responses by presenting antigenic peptides to CTLs and by regulating cytolytic activities of NK cells. In this article, we show that MHC I A in rhesus macaques can be alternatively spliced, generating a novel MHC I A isoform (termed "MHC I A-sv1") devoid of α(3) domain. Despite the absence of β2-microglobulin (β2m), the MHC I A-sv1 proteins reached the cell surface of K562-transfected cells as endoglycosidase H-sensitive glycoproteins that could form disulfide-bonded homodimers. Cycloheximide-based protein chase experiments showed that the MHC I A-sv1 proteins were more stable than the full-length MHC I A in transiently or stably transfected cell lines. Of particular interest, our studies demonstrated that MHC I A-sv1 could form β2m-free heterodimers with its full-length protein in mammalian cells. The formation of heterodimers was accompanied by a reduction in full-length MHC I A ubiquitination and consequent stabilization of the protein. Taken together, these results demonstrated that MHC I A-sv1 and MHC I A can form a novel heterodimeric complex as a result of the displacement of β2m and illustrated the relevance of regulated MHC I A protein degradation in the β2m-free heterodimerization-dependent control, which may have some implications for the MHC I A splice variant in the fine tuning of classical MHC I A/TCR and MHC I A/killer cell Ig-like receptor interactions.

  12. Laser-induced propagation and destruction of amyloid beta fibrils.

    PubMed

    Yagi, Hisashi; Ozawa, Daisaku; Sakurai, Kazumasa; Kawakami, Toru; Kuyama, Hiroki; Nishimura, Osamu; Shimanouchi, Toshinori; Kuboi, Ryoichi; Naiki, Hironobu; Goto, Yuji

    2010-06-18

    The amyloid deposition of amyloid beta (Abeta) peptides is a critical pathological event in Alzheimer disease (AD). Preventing the formation of amyloid deposits and removing preformed fibrils in tissues are important therapeutic strategies against AD. Previously, we reported the destruction of amyloid fibrils of beta(2)-microglobulin K3 fragments by laser irradiation coupled with the binding of amyloid-specific thioflavin T. Here, we studied the effects of a laser beam on Abeta fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed Abeta fibrils. However, irradiation during spontaneous fibril formation resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils. The explosive propagation was caused by an increase in the number of active ends due to breakage. The results not only reveal a case of fragmentation-induced propagation of fibrils but also provide insights into therapeutic strategies for AD.

  13. Assignment of the gene for the. beta. subunit of thyroid-stimulating hormone to the short arm of human chromosome 1

    SciTech Connect

    Dracopoli, N.C.; Rettig, W.J.; Whitfield, G.K.; Darlington, G.J.; Spengler, B.A.; Biedler, J.L.; Old, L.J.; Kourides, I.A.

    1986-03-01

    The chromosomal locations of the genes for the ..beta.. subunit of human thyroid-stimulating hormone (TSH) and the glycoprotein hormone ..cap alpha.. subunit have been determined by restriction enzyme analysis of DNA extracted from rodent-human somatic cell hybrids. Human chorionic gonadotropin (CG) ..cap alpha..-subunit cDNA and a cloned 0.9-kilobase (kb) fragment of the human TSH ..beta..-subunit gene were used as hybridization probes in the analysis of Southern blots of DNA extracted from rodent-human hybrid clones. Analysis of the segregation of 5- and 10-kb EcoRI fragments hybridizing to CG ..cap alpha..-subunit cDNA confirmed the previous assignment of this gene to chromosome 6. Analysis of the patterns of segregation of a 2.3-kb EcoRI fragment containing human TSH ..beta..-subunit sequences permitted the assignment of the TSH ..beta..-subunit gene to human chromosome 1. The subregional assignment of TSH ..beta.. subunit to chromosome 1p22 was made possible by the additional analysis of a set of hybrids containing partially overlapping segments of this chromosome. Human TSH ..beta.. subunit is not syntenic with genes encoding the ..beta.. subunits of CG, luteinizing hormone, or follicle-stimulating hormone and is assigned to a conserved linkage group that also contains the structural genes for the ..beta.. subunit of nerve growth factor (NGFB) and the proto-oncogene N-ras (NRAS).

  14. Expression of human beta-hexosaminidase alpha-subunit gene (the gene defect of Tay-Sachs disease) in mouse brains upon engraftment of transduced progenitor cells.

    PubMed

    Lacorazza, H D; Flax, J D; Snyder, E Y; Jendoubi, M

    1996-04-01

    In humans, beta-hexosaminidase alpha-subunit deficiency prevents the formation of a functional beta-hexosaminidase A heterodimer resulting in the severe neurodegenerative disorder, Tay-Sachs disease. To explore the feasibility of using ex vivo gene transfer in this lysosomal storage disease, we produced ecotropic retroviruses encoding the human beta-hexosaminidase alpha-subunit cDNA and transduced multipotent neural cell lines. Transduced progenitors stably expressed and secreted high levels of biologically active beta-hexosaminidase A in vitro and cross-corrected the metabolic defect in a human Tay-Sachs fibroblasts cell line in vitro. These genetically engineered CNS progenitors were transplanted into the brains of both normal fetal and newborn mice. Engrafted brains, analyzed at various ages after transplant, produced substantial amounts of human beta-hexosaminidase alpha-subunit transcript and protein, which was enzymatically active throughout the brain at a level reported to be therapeutic in Tay-Sachs disease. These results have implications for treating neurologic diseases characterized by inherited single gene mutations.

  15. Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4-8- alpha/beta T cells demonstrates preferential use of several V beta genes and an invariant TCR alpha chain

    PubMed Central

    1993-01-01

    CD4-CD8- (double negative [DN]) alpha/beta T cells are a largely uncharacterized subpopulation of unknown function. To investigate whether these cells are selected to recognize particular antigens or antigen-presenting molecules, DN alpha/beta T cells were purified from the peripheral blood of five normal donors and their T cell receptor (TCR) alpha and beta chains were examined. Random cloning of TCR alpha chains by single-sided polymerase chain reaction (PCR) amplification identified an invariant rearrangement between V alpha 24 and J alpha Q, with no N region diversity, which was expressed preferentially by DN alpha/beta T cells from all donors. Random cloning also identified a precise V alpha 7.2-J alpha (IGRJa14) rearrangement, with two variable amino acids encoded in the V-J junction, which was enriched in the DN alpha/beta T cell preparations from some, but not all, donors. Analysis of TCR beta chains by quantitative PCR amplification demonstrated that the expression of four V beta gene families, V beta 2, 8, 11, and 13, was markedly increased in these DN alpha/beta T cell preparations. The expression of particular TCRs by DN alpha/beta T cells from multiple donors indicates that these cells, or at least a subpopulation of cells with this phenotype, recognize a limited spectrum of antigens and suggests that they may use nonpolymorphic antigen-presenting molecules. PMID:8391057

  16. Age-related changes in relative expression of real-time PCR housekeeping genes in human skeletal muscle.

    PubMed

    Touchberry, Chad D; Wacker, Michael J; Richmond, Scott R; Whitman, Samantha A; Godard, Michael P

    2006-04-01

    The purpose of this investigation was to examine the expression of three commonly used housekeeping genes -- glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta(2)-microglobulin (beta(2)M), and RNA polymerase 2a (polR2a) -- in elderly (E) compared to young (Y) subjects. Nine young subjects (22.7 +/- 3.4 yrs) and 11 elderly subjects (73.0 +/- 9.5 yrs) underwent a percutaneous skeletal muscle biopsy of the vastus lateralis. Equal concentrations of isolated mRNA from these samples were used to perform real-time polymerase chain reaction with primer/probe combinations specific to each gene of interest. The expression of GAPDH, beta(2)M, and polR2a was obtained as the value of cycle threshold (C(T)). An independent t-test with a level of significance at p < or = 0.05 was used to determine differences between groups. There was no difference in average C(T) of GAPDH between groups (p=0.869) (Y = 16.92 +/- 2.25 vs. E = 17.08 +/- 2.09) and polR2a (p = 0.089) (Y = 28.00 +/- 0.89 vs. E = 26.73 +/- 1.91). However, there was a significant difference (p < or = 0.05) in the average C(T) of beta(2)M (Y =21.79 +/- 0.44 vs. E = 21.05 +/- 0.51). The results indicate that special consideration needs to be made when selecting housekeeping genes for comparisons in real-time reverse-transcriptase polymerase chain reaction, depending upon the age of the populations of interest.

  17. Novel mutations within the RFX-B gene and partial rescue of MHC and related genes through exogenous class II transactivator in RFX-B-deficient cells.

    PubMed

    Nagarajan, U M; Peijnenburg, A; Gobin, S J; Boss, J M; van den elsen, P J

    2000-04-01

    MHC class II deficiency or bare lymphocyte syndrome is a severe combined immunodeficiency caused by defects in MHC-specific regulatory factors. Fibroblasts derived from two recently identified bare lymphocyte syndrome patients, EBA and FZA, were found to contain novel mutations in the RFX-B gene. RFX-B encodes a component of the RFX transcription factor that functions in the assembly of multiple transcription factors on MHC class II promoters. Unlike RFX5- and RFXAP-deficient cells, transfection of exogenous class II transactivator (CIITA) into these RFX-B-deficient fibroblasts resulted in the induction of HLA-DR and HLA-DP and, to a lesser extent, HLA-DQ. Similarly, CIITA-mediated induction of MHC class I, beta2-microglobulin, and invariant chain genes was also found in these RFX-B-deficient fibroblasts. Expression of wild-type RFX-B completely reverted the noted deficiencies in these cells. Transfection of CIITA into Ramia cells, a B cell line that does not produce a stable RFX-B mRNA, resulted in induction of an MHC class II reporter, suggesting that CIITA overexpression may partially override the RFX-B defect.

  18. Structural and phylogenetic analysis of the MHC class I-like Fc receptor gene

    SciTech Connect

    Kandil, Eman; Ishibashi, Teruo; Kasahara, Masanori

    1995-06-01

    The intestinal epithelium of neonatal mice and rats expresses an Fc receptor that mediates selective uptake of IgG in mothers`milk. This receptor (FcRn), which helps newborn animals to acquire passive immunity, is an MHC class I-like heterodimer made up of a heavy chain and {beta}{sub 2}-microglobulin. In the present study, we determined the genomic structure of a mouse gene (FcRn) encoding the heavy of FcRn. The overall exon-intron organization of the Fcrn gene was similar to that of the Fcrn gene, thus providing structural evidence that Fcrn os a bona fide class I gene. The 5{prime}-flanking region of the Fcrn gene contained the binding motifs for two cytokine-inducible transcription factors, NF-IL6 and NF1. However, regulatory elements found in MHC class I genes (enhancer A, enhancer B, and the IFN response element) were absent. Phylogenetic tree analysis suggested that, like the MICA, AZGP1, and CD1 genes, the Fcrn gene diverged form MHC class I genes after the emergence of amphibians but before the split of placental and marsupial mammals. Consistent with this result, Southern blot analysis with a mouse Fcrn cDNA probe detected cross-hybridizing bands in various mammalian species and chickens. Sequence analysis of the Fcrn gene isolated from eight mouse strains showed that the membrane-distal domain of FcRn has at least three amino acid variants. The fact that Fcrn is a single copy gene indicates that it is expressed in both the neonatal intestine and the fetal yolk sac. 74 refs., 7 figs., 2 tabs.

  19. The Beta-Binomial Distribution for Estimating the Number of False Rejections in Microarray Gene Expression Studies.

    PubMed

    Hunt, Daniel L; Cheng, Cheng; Pounds, Stanley

    2009-03-15

    In differential expression analysis of microarray data, it is common to assume independence among null hypotheses (and thus gene expression levels). The independence assumption implies that the number of false rejections V follows a binomial distribution and leads to an estimator of the empirical false discovery rate (eFDR). The number of false rejections V is modeled with the beta-binomial distribution. An estimator of the beta-binomial false discovery rate (bbFDR) is then derived. This approach accounts for how the correlation among non-differentially expressed genes influences the distribution of V. Permutations are used to generate the observed values for V under the null hypotheses and a beta-binomial distribution is fit to the values of V. The bbFDR estimator is compared to the eFDR estimator in simulation studies of correlated non-differentially expressed genes and is found to outperform the eFDR for certain scenarios. As an example, this method is also used to perform an analysis that compares the gene expression of soft tissue sarcoma samples to normal tissue samples.

  20. Effects of Ghrelin on Sexual Behavior and Luteinizing Hormone Beta-subunit Gene Expression in Male Rats

    PubMed Central

    Babaei-Balderlou, Farrin; Khazali, Homayoun

    2016-01-01

    Background: The hormones of hypothalamo-pituitary-gonadal (HPG) axis have facilitative effects on reproductive behavior in mammals. Ghrelin as a starvation hormone has an inhibitory effect on HPG axis’ function. Hence, it is postulated that ghrelin may reduce the sexual behavior through inhibiting of HPG axis. The aim of this study was to examine the effects of ghrelin and its antagonist, [D-Lys3 ]-GHRP-6, on sexual behavior and LH beta-subunit gene expression in male rats. Methods: In this experimental study, 128 male Wistar rats were divided into two groups. Each group was further subdivided into eight subgroups (n=8 rats/subgroup) including the animals that received saline, ghrelin (2, 4 or 8 nmol), [D-Lys3 ]-GHRP-6 (5 or 10 nmol) or co-administration of ghrelin (4 nmol) and [D-Lys3 ]-GHRP-6 (5 or 10 nmol) through the stereotaxically implanted cannula into the third cerebral ventricle. The sexual behavior of male rats encountering with females and the hypo-physeal LH beta-subunit gene expression were evaluated at two different groups. Data were analyzed by ANOVA and p<0.05 was considered statistically significant. Results: Ghrelin injection (4 and 8 nmol) significantly (p<0.01) increased the latencies to the first mount, intromission and ejaculation as well as the post-ejaculatory interval. Also, 4 and 8 nmol ghrelin significantly (p<0.05) increased the number of mount and decreased the number of ejaculation. In co-administrated groups, [D-Lys3 ]-GHRP-6 antagonized the effects of ghrelin. Ghrelin injection (4 and 8 nmol) reduced the LH beta-subunit gene expression while pretreatment with [D-Lys3 ]-GHRP-6 improved the gene expression. Conclusion: Ghrelin decreased the sexual behavior and LH beta-subunit gene expression in male rats, whereas [D-Lys3 ]-GHRP-6 antagonizes these effects. PMID:27141463

  1. Exonic Sp1 sites are required for neural-specific expression of the glycine receptor beta subunit gene.

    PubMed Central

    Tintrup, H; Fischer, M; Betz, H; Kuhse, J

    2001-01-01

    The gene encoding the beta subunit of the inhibitory glycine receptor (GlyR) is widely expressed throughout the mammalian central nervous system. To unravel the elements regulating its transcription, we isolated its 5' non-coding and upstream flanking regions from mouse. Sequence analysis revealed significant differences between the 5' region of the beta subunit gene and the corresponding regions of the homologous GlyR alpha subunit genes; it also identified a novel exon (exon 0) that encodes most of the 5'-untranslated portion of the GlyR beta mRNA. Primer extension experiments disclosed multiple transcriptional start sites. Transfection experiments with luciferase reporter gene constructs showed that sequences encompassing 1.58 kb of upstream flanking region and 180 bp of exon 0 displayed high promoter activity in two neuroblastoma cell lines but not in non-neural cells. Analysis of various deletion constructs showed that the 5' flanking region preceding the transcriptional start sites silences expression in non-neural cells but is not essential for general promoter activity. In contrast, the deletion of sequences within exon 0 drastically decreased or abolished transcription; the removal of sequences harbouring Sp1 consensus sequences within exon 0 decreased expression specifically in a neuroblastoma cell line. Band-shift assays confirmed the binding of Sp1 to sites within the deleted sequence. Our results indicate that neural-specific expression of the GlyR beta subunit gene might depend on a direct interaction of Sp1 transcription factors with cis elements located downstream from transcription initiation sites. PMID:11256962

  2. Mutations in the dopamine beta-hydroxylase gene are associated with human norepinephrine deficiency

    NASA Technical Reports Server (NTRS)

    Kim, Chun-Hyung; Zabetian, Cyrus P.; Cubells, Joseph F.; Cho, Sonhae; Biaggioni, Italo; Cohen, Bruce M.; Robertson, David; Kim, Kwang-Soo

    2002-01-01

    Norepinephrine (NE), a key neurotransmitter of the central and peripheral nervous systems, is synthesized by dopamine beta-hydroxylase (DBH) that catalyzes oxidation of dopamine (DA) to NE. NE deficiency is a congenital disorder of unknown etiology, in which affected patients suffer profound autonomic failure. Biochemical features of the syndrome include undetectable tissue and circulating levels of NE and epinephrine, elevated levels of DA, and undetectable levels of DBH. Here, we report identification of seven novel variants including four potentially pathogenic mutations in the human DBH gene (OMIM 223360) from analysis of two unrelated patients and their families. Both patients are compound heterozygotes for variants affecting expression of DBH protein. Each carries one copy of a T-->C transversion in the splice donor site of DBH intron 1, creating a premature stop codon. In patient 1, there is a missense mutation in DBH exon 2. Patient 2 carries missense mutations in exons 1 and 6 residing in cis. We propose that NE deficiency is an autosomal recessive disorder resulting from heterogeneous molecular lesions at DBH. Copyright 2002 Wiley-Liss, Inc.

  3. Pit-1/GHF-1 binds to TRH-sensitive regions of the rat thyrotropin beta gene.

    PubMed

    Mason, M E; Friend, K E; Copper, J; Shupnik, M A

    1993-08-31

    Three regions within the 5'-flanking region of the TSH beta gene have A-T-rich sequences which have sequence similarity to binding sites for the pituitary-specific POU domain transcription factor Pit-1/GHF-1. These three regions have been termed TSH A (-274 to -258 bp), TSH B (-336 to -326 bp), and TSH C (-402 to -384 bp). TSH A and TSH C are able to confer 2-6-fold TRH stimulation to the heterologous viral thymidine kinase (tk) promoter in transient expression assays in GH3 pituitary cells; TSH C can confer a 3-10-fold increase in basal enhancer activity as well. TSH A, B, and C DNAs all bound Pit-1 from GH3 cell nuclear extracts, based on gel mobility shift analysis in which antibody against Pit-1 prevented the formation of specific DNA-GH3 nuclear protein complexes. TSH A and TSH C also each formed several additional DNA-nuclear protein complexes which were not observed with TSH B. Some of these complexes may contain Pit-1 as their formation was inhibited by the addition of Pit-1 antibody; other complexes, however, were not altered by antibody treatment. All three A-T-rich elements bound in vitro translated Pit-1, with calculated affinities of 360 (A), 125 (B), and 38 (C) nM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Beta-globin gene cluster haplotypes in sickle cell patients from southwest Iran.

    PubMed

    Rahimi, Z; Karimi, M; Haghshenass, M; Merat, A

    2003-11-01

    Sickle cell anemia in Iran is accompanied by a high level of HbF and mild clinical presentation. Here we report haplotypes of the beta gene cluster found in 81 randomly selected sickle cell patients, including 47 sickle cell anemia (SS), 17 sickle cell trait (AS), and 17 sickle/thalassemia (S/thal) from southwest Iran. We found all five common typical haplotypes as well as five atypical haplotypes in our patients. Except for four patients with homozygous Benin haplotype, none of the other African typical haplotypes were found in a homozygous state. Arab-Indian was found to be the most prevalent haplotype in the study population. This haplotype accounted for 51.1% as the homozygous form in SS patients, where 69.1% of chromosomes in these patients had the Arab-Indian haplotype. Bantu A2 was the second most prevalent haplotype among all patients. The mean %HbF in SS patients was 27.83 and in the homozygous Arab-Indian haplotype it was still higher (30.40%), while in AS patients the %HbF was only 1.20. The high %Ggamma chain (71.81) in the Arab-Indian homozygous haplotype was concomitant with the presence of an Xmn I site in both chromosomes. The presence of the Arab-Indian haplotype as the predominant haplotype might be suggestive of a gene flow to/from Saudi Arabia or India. More haplotype investigations of a normal population can clarify the high incidence of Bantu A2 haplotype in our population.

  5. Human-Specific SNP in Obesity Genes, Adrenergic Receptor Beta2 (ADRB2), Beta3 (ADRB3), and PPAR γ2 (PPARG), during Primate Evolution

    PubMed Central

    Takenaka, Akiko; Nakamura, Shin; Mitsunaga, Fusako; Inoue-Murayama, Miho; Udono, Toshifumi; Suryobroto, Bambang

    2012-01-01

    Adrenergic-receptor beta2 (ADRB2) and beta3 (ADRB3) are obesity genes that play a key role in the regulation of energy balance by increasing lipolysis and thermogenesis. The Glu27 allele in ADRB2 and the Arg64 allele in ADRB3 are associated with abdominal obesity and early onset of non-insulin-dependent diabetes mellitus (NIDDM) in many ethnic groups. Peroxisome proliferator-activated receptor γ (PPARG) is required for adipocyte differentiation. Pro12Ala mutation decreases PPARG activity and resistance to NIDDM. In humans, energy-expense alleles, Gln27 in ADRB2 and Trp64 in ADRB3, are at higher frequencies than Glu27 and Arg64, respectively, but Ala12 in PPARG is at lower frequency than Pro12. Adaptation of humans for lipolysis, thermogenesis, and reduction of fat accumulation could be considered by examining which alleles in these genes are dominant in non-human primates (NHP). All NHP (P. troglodytes, G. gorilla, P. pygmaeus, H. agilis and macaques) had energy-thrifty alleles, Gly16 and Glu27 in ADRB2, and Arg64 in ADRB3, but did not have energy-expense alleles, Arg16, Gln27 and Trp64 alleles. In PPARG gene, all NHP had large adipocyte accumulating type, the Pro12 allele. Conclusions These results indicate that a tendency to produce much more heat through the energy-expense alleles developed only in humans, who left tropical rainforests for savanna and developed new features in their heat-regulation systems, such as reduction of body hair and increased evaporation of water, and might have helped the protection of entrails from cold at night, especially in glacial periods. PMID:22937051

  6. Interleukin (IL)1beta, IL-1alpha, and IL-1 receptor antagonist gene polymorphisms in patients with temporal lobe epilepsy.

    PubMed

    Kanemoto, K; Kawasaki, J; Miyamoto, T; Obayashi, H; Nishimura, M

    2000-05-01

    Proinflammatory cytokines, including interleukin (IL)-1beta, are known to modulate effects of neurotoxic neurotransmitters discharged during excitation or inflammation in the central nervous system (CNS). They also regulate development of glial scars at sites of CNS injury. To elucidate a genetic predisposition of temporal lobe epilepsy with hippocampal sclerosis (TLE-HS+), we studied polymorphisms in the IL-1beta, IL-1alpha, and IL-1 receptor antagonist (IL-1RA) genes in 50 patients with TLE-HS+ and in 112 controls. Fifty-three patients who had TLE without HS were also examined (TLE-HS-) as disease controls. The distribution of the biallelic polymorphism in the promoter region at position -511 of the IL-1beta gene (IL-1B-511) was significantly different both between TLE-HS+ patients and controls and between TLE-HS+ and TLE-HS- patients. The differences were due to overrepresentation of the homozygotes for IL-1B-511*2, which is suggested to be a high producer of IL-1beta, in TLE-HS+ patients compared with both controls and TLE-HS- patients. In contrast, there was no difference between TLE-HS- patients and controls. Our data suggest that, in the homozygotes for IL-IB-511*2, minor events in development such as febrile convulsions could set up a cascade leading to HS.

  7. Occurrence of bacteria producing broad-spectrum beta-lactamases and qnr genes in hospital and urban wastewater samples.

    PubMed

    Röderová, Magdaléna; Sedláková, Miroslava Htoutou; Pudová, Vendula; Hricová, Kristýna; Silová, Romana; Imwensi, Peter Eghonghon Odion; Bardoň, Jan; Kolář, Milan

    2016-04-01

    The aims were to investigate the level of antibiotic-resistant bacteria in hospital and urban wastewater and to determine the similarity of isolates obtained from wastewater and hospitalized patients. Wastewater samples were collected in September 2013 and 2014. After identification using MALDI-TOF MS, beta-lactamase production was determined by relevant phenotypic tests. Genes responsible for the production of single beta-lactamase groups and Qnr proteins were established. The epidemiological relationship of the isolates from wastewater and hospitalized patients was determined by PFGE. A total of 51 isolates of enterobacteria were obtained. Overall, 45.1% of them produced broad-spectrum beta-lactamases. Genes encoding TEM, SHV, CTX-M, CIT, DHA and EBC types of enzymes and Qnr proteins were detected. No broad-spectrum beta-lactamase production was confirmed in the urban wastewater treatment plant. The most important finding was the detection of two identical isolates of K. pneumoniae in 2013, one from a patient's urinary catheter and the other from a wastewater sample.

  8. Functional analysis of a proline to serine mutation in codon 453 of the thyroid hormone receptor {beta}1 gene

    SciTech Connect

    Ozata, M.; Suzuki, Satoru; Takeda, Teiji

    1995-10-01

    Mutations in the gene encoding human thyroid hormone receptor {beta}(hTR{beta}) have been associated with generalized resistance to thyroid hormone (GRTH). This disorder is associated with significant behavoral abnormalities. We examined the hTR{beta} gene in a family with members who manifest inappropriately normal TSH, elevated free T{sub 4}, and free and total T{sub 3}. Sequence analysis showed a cytosine to thymine transition at nucleotide 1642 in one allele of the index patient`s genomic DNA. This altered proline to serine at codon 453. The resulting mutant receptor when expressed in vitro bound DNA with high affinity, but the T{sub 3} affinity of the receptor was impaired. The mutant TR demonstrated a dominant negative effect when cotransfected with two isoforms of wild-type receptor and also in the presence of TR variant {alpha}2 in COS-1 cells. Mutations of codon 453 occur more frequently than at other sites, and four different amino acid substitutions have been reported. Significant differences in phenotype occur among affected individuals, varying from normality to moderately severe GRTH. There is no clear correlation between K{sub a} or in vitro function of the mutant receptor, and phenotype. This study extends the association between GRTH and illness, and indicates that early diagnosis and counseling are needed in families with TR{beta}1 abnormalities. 34 refs., 5 figs., 2 tabs.

  9. Sequential mutations in the interleukin-3 (IL3)/granulocyte-macrophage colony-stimulating factor/IL5 receptor beta-subunit genes are necessary for the complete conversion to growth autonomy mediated by a truncated beta C subunit.

    PubMed

    Hannemann, J; Hara, T; Kawai, M; Miyajima, A; Ostertag, W; Stocking, C

    1995-05-01

    An amino-terminally truncated beta C receptor (beta C-R) subunit of the interleukin-3 (IL3)/granulocyte-macrophage colony-stimulating factor/IL5 receptor complex mediates factor-independent and tumorigenic growth in two spontaneous mutants of a promyelocytic cell line. The constitutive activation of the JAK2 protein kinase in these mutants confirms that signaling occurs through the truncated receptor protein. Noteworthily, in addition to a 10-kb deletion in the beta C-R subunit gene encoding the truncated receptor, several secondary and independent mutations that result in the deletion or functional inactivation of the allelic beta C-R subunit and the closely related beta IL3-R subunit genes were observed in both mutants, suggesting that such mutations are necessary for the full oncogenic penetrance of the truncated beta C-R subunit. Reversion of these mutations by the expression of the wild-type beta C-R in the two mutants resulted in a fivefold decrease in cloning efficiency of the mutants in the absence of IL3, confirming a functional interaction between the wild-type and truncated proteins. Furthermore, expression of the truncated beta C-R subunit in factor-dependent myeloid cells did not immediately render the cells autonomous but increased the spontaneous frequency to factor-independent growth by 4 orders of magnitude. Implications for both leukemogenic progression and receptor-subunit interaction and signaling are discussed.

  10. [Effects of intergenic interaction of the high pigmentation gene hp-2(dg) (high pigment-2 dark green) with the gene B (beta-carotene) in tomato].

    PubMed

    Kuzemenskiĭ, A V

    2008-01-01

    It was shown that during intergenic interaction of genes hp-2(dg) and B in dihomozygote an additive factor is formed activating biogenesis of beta-carotene in tomato fruits. In the genotype B/B//hp-2(dg)/hp-2(dg) there is preserved the positive effects of the gene hp-2(dg) on the content of ascorbic acid and the negative one on the content of titrated acids. With this stabilization of the gene hp-2(dg) genetic depression is observed, which is manifested in the increased productivity of B/B//hp-2(dg)/hp-2(dg)-genotypes.

  11. Mapping of the gene encoding the. beta. -amyloid precursor protein and its relationship to the Down syndrome region of chromosome 21

    SciTech Connect

    Patterson, D.; Gardiner, K.; Kao, F.T.; Tanzi, R.; Watkins, P.; Gusella, J.F. )

    1988-11-01

    The gene encoding the {beta}-amyloid precursor protein has been assigned to human chromosome 21, as has a gene responsible for at least some cases of familial Alzheimer disease. Linkage studies strongly suggest that the {beta}-amyloid precursor protein and the product corresponding to familial Alzheimer disease are from two genes, or at least that several million base pairs of DNA separate the markers. The precise location of the {beta}-amyloid precursor protein gene on chromosome 21 has not yet been determined. Here the authors show, by using a somatic-cell/hybrid-cell mapping panel, in situ hybridization, and transverse-alternating-field electrophoresis, that the {beta}-amyloid precursor protein gene is located on chromosome 21 very near the 21q21/21q/22 border and probably within the region of chromosome 21 that, when trisomic, results in Down syndrome.

  12. Cloning and characterization of two genes from Bacillus polymyxa expressing beta-glucosidase activity in Escherichia coli.

    PubMed Central

    González-Candelas, L; Aristoy, M C; Polaina, J; Flors, A

    1989-01-01

    DNA fragments from Bacillus polymyxa which encode beta-glucosidase activity were cloned in Escherichia coli by selection of yellow transformants able to hydrolyze the artificial chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside. Restriction endonuclease maps and Southern analysis of the cloned fragments showed the existence of two different genes. Expression of either one of these genes allowed growth of E. coli in minimal medium with cellobiose as the only carbon source. One of the two enzymes was found in the periplasm of E. coli, hydrolyzed arylglucosides more actively than cellobiose, and rendered glucose as the only product upon cellobiose hydrolysis. The other enzyme was located in the cytoplasm, was more active toward cellobiose, and hydrolyzed this disaccharide, yielding glucose and another, unidentified compound, probably a phosphorylated sugar. Images PMID:2515802

  13. Sardinian delta beta zero-thalassemia: a further example of a C to T substitution at position -196 of the A gamma globin gene promoter.

    PubMed

    Ottolenghi, S; Giglioni, B; Pulazzini, A; Comi, P; Camaschella, C; Serra, A; Guerrasio, A; Saglio, G

    1987-04-01

    Selective overexpression (50- to 100-fold) in adult erythroid cells of either G gamma or A gamma fetal globin gene is observed in hereditary conditions known as delta beta zero-thalassemia and hereditary persistence of fetal hemoglobin (HPFH). Recently, a C----T change at position -196 of an overexpressed A gamma globin gene from an Italian HPFH was hypothesized, on the basis of indirect evidence, to represent the cause of the functional defect. We now show that the same mutation is present in a different overexpressed A gamma-globin gene from a Sardinian patient with a different syndrome (delta beta zero-thalassemia). The Sardinian A gamma globin gene differs from both the HPFH and the normal A gamma globin gene at nucleotide 1,560 in the noncoding portion of the third exon, where an A is deleted. In addition, the mutant -196 A gamma-globin gene is linked to a normal beta globin gene in HPFH, and to a beta-thalassemic gene (beta 39CAG----TAG) in delta beta zero-thalassemia. These data strengthen the suggestion that -196 mutation is causally linked to the abnormal phenotype and raise the question of whether the same or multiple mutational events are responsible for the appearance of the -196 mutation in different syndromes.

  14. Induction of cytochrome P450IA1 gene expression in rat epidermis and human keratinocytes by. beta. -napthoflavone and benzanthracene

    SciTech Connect

    Khan, I.U.; Mukhtar, H.; Bickers, D.R.; Haqqi, T.M. )

    1991-03-15

    Cytochrome P450IA1 (P450IA1) plays a major role in the bioactivation of procarcinogens in various tissues including skin. However, factors controlling the expression of P450IA1 gene message in mammalian skin are unknown. In this study, the polymerase chain reaction (PCR) using specific primers was employed to study the expression of P450IA1 mRNA transcripts in rat epidermis and human keratinocytes (HK) treated with {beta}-napthoflavone ({beta}NF) and benzanthracene (BA). Total RNA was extracted from the epidermis of control and inducer-treated 4-day-old and adult Sprague Dawley rats, and from control and inducer-treated HL. cDNAs were synthesized using random primers and reverse transcriptase. PCR products were analyzed on agarose gel and quantitated by densitometry. Inducer treatment of rats and HK resulted in several-fold increases in aryl hydrocarbon hydroxylase (AHH) activity. The level of P450IA1 gene message increased 2-5-fold in treated animals as compared to controls; higher basal level and inducibility in adult than in 4-day-old rats. This induction occurred as early as 4 h after {beta}NF application, reached a maximum at 16 h and returned to basal levels by 36 h. Exposure to {beta}NF and BA resulted in 2-3-fold increase in gene message in HK. Northern blot analysis complemented PCR data. These results indicate that in mammalian skin P450IA1 gene expression is increased by the inducers of epidermal AHH activity.

  15. Cloning of the RHO1 gene from Candida albicans and its regulation of beta-1,3-glucan synthesis.

    PubMed Central

    Kondoh, O; Tachibana, Y; Ohya, Y; Arisawa, M; Watanabe, T

    1997-01-01

    The Saccharomyces cerevisiae RHO1 gene encodes a low-molecular-weight GTPase. One of its recently identified functions is the regulation of beta-1,3-glucan synthase, which synthesizes the main component of the fungal cell wall (J. Drgonova et al., Science 272:277-279, 1996; T. Mazur and W. Baginsky, J. Biol. Chem. 271:14604-14609, 1996; and H. Qadota et al., Science 272:279-281, 1996). From the opportunistic pathogenic fungus Candida albicans, we cloned the RHO1 gene by the PCR and cross-hybridization methods. Sequence analysis revealed that the Candida RHO1 gene has a 597-nucleotide region which encodes a putative 22.0-kDa peptide. The deduced amino acid sequence predicts that Candida albicans Rho1p is 82.9% identical to Saccharomyces Rho1p and contains all the domains conserved among Rho-type GTPases from other organisms. The Candida albicans RHO1 gene could rescue a S. cerevisiae strain containing a rho1 deletion. Furthermore, recombinant Candida albicans Rho1p could reactivate the beta-1,3-glucan synthesis activities of both C. albicans and S. cerevisiae membranes in which endogenous Rho1p had been depleted by Tergitol NP-40-NaCl treatment. Candida albicans Rho1p was copurified with the beta-1,3-glucan synthase putative catalytic subunit, Candida albicans Gsc1p, by product entrapment. Candida albicans Rho1p was shown to interact directly with Candida albicans Gsc1p in a ligand overlay assay and a cross-linking study. These results indicate that Candida albicans Rho1p acts in the same manner as Saccharomyces cerevisiae Rho1p to regulate beta-1,3-glucan synthesis. PMID:9401032

  16. Two translational initiation sites in the infB gene are used to express initiation factor IF2 alpha and IF2 beta in Escherichia coli.

    PubMed Central

    Plumbridge, J A; Deville, F; Sacerdot, C; Petersen, H U; Cenatiempo, Y; Cozzone, A; Grunberg-Manago, M; Hershey, J W

    1985-01-01

    The gene infB codes for the two forms of translational initiation factor IF2: IF2 alpha (97 300 daltons) and IF2 beta (79 700 daltons). To determine whether the two forms differ at their N terminus, purified IF2 alpha and IF2 beta were subjected to 11 or more steps of Edman degradation. The N-terminal amino acid sequences are completely different, but match perfectly the DNA sequences at the beginning of the infB open reading frame and an in-phase region 471 bp downstream. A fusion was constructed between the proximal half of the infB gene and the lacZ gene lacking the region coding for the first eight amino acids. The fused gene expresses two products of 170 000 and 150 000 daltons, corresponding to the fused proteins IF2 alpha-beta-galactosidase and IF2 beta-beta-galactosidase, which confirms in vivo that the IF2 forms differ at their N terminus. A deletion of the 5'-non-translated region of the fused gene, including the Shine/Dalgarno ribosomal binding site, results in the expression of IF2 beta-beta-galactosidase but not IF2 alpha-beta-galactosidase. This strongly suggests that IF2 beta results from independent translation rather than from a precise proteolytic cleavage of IF2 alpha. Further evidence for initiation of protein synthesis at the putative IF2 alpha and IF2 beta start sites was sought by using an in vitro dipeptide synthesis assay. A DNA fragment containing the entire infB gene was cloned into three plasmid vectors and the resulting recombinant DNAs were used as templates in assays containing fMet-tRNA and various labelled aminoacyl-tRNAs.(ABSTRACT TRUNCATED AT 250 WORDS) Images Fig. 2. PMID:3894004

  17. Analysis of CD2 and TCR-{beta} gene expression in jurkat cell mutants suggests a cis regulation of gene transcription

    SciTech Connect

    Kamoun, M.; Woods, J.S.; Sano, N.

    1995-10-15

    Thirty CD2{sup -} J32 stable clones, derived by mutagenesis and subsequent immunoselection with anti-CD2 Ab, were used to study the regulation of CD2 and TCR gene expression. Analysis of RNA expression revealed that the loss of surface expression of CD2 was due to a lack of expression of CD2 mRNA and was associated with a lack of expression of VDJ TCR-{beta} transcripts in 12 of these mutants, sparing the expression of DJ TCR-{beta}, TCR-{alpha}, CD3{gamma}, {delta}, {epsilon}, and {zeta} RNA. The expression of other differentiation molecules was unaffected, except for CD1, CD4, and CD5, which were either decreased or absent in most of these mutants. A gain in the expression of TCR-{gamma} transcripts was observed in each of these mutants, while, as expected, no TCR-{gamma} transcripts were detected in wild-type J32 cells. Several mutants were able to use the human CD2 enhancer and the murine TCR-{beta} enhancer and promoter to activate transcription from reporter genes in the context of heterologous promoters, indicating that the mutation(s) does not affect transcription pathways. Consistent with this finding is the adequate expression in these mutants of several lineage-specific transcription factors. The expression of CD2 in several of these mutants was rescued by gene transfer using a genomic 28.5-kb CD2 fragment, suggesting that the enchancer function of this gene may be dependent on the enhancer site. These observations suggest that the coordinate expressions of CD2 and TCR-{beta} genes share common regulatory mechanisms involving factors regulating chromatin structure and accessibility. 51 refs., 6 figs., 3 tabs.

  18. Genomics and genetics of gonadotropin beta-subunit genes: Unique FSHB and duplicated LHB/CGB loci

    PubMed Central

    Nagirnaja, Liina; Rull, Kristiina; Uusküla, Liis; Hallast, Pille; Grigorova, Marina; Laan, Maris

    2010-01-01

    The follicle stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (HCG) play a critical role in human reproduction. Despite the common evolutionary ancestry and functional relatedness of the gonadotropin hormone beta (GtHB) genes, the single-copy FSHB (at 11p13) and the multi-copy LHB/CGB genes (at 19q13.32) exhibit locus-specific differences regarding their genomic context, evolution, genetic variation and expressional profile. FSHB represents a conservative vertebrate gene with a unique function and it is located in a structurally stable gene-poor region. In contrast, the primate-specific LHB/CGB gene cluster is located in a gene-rich genomic context and demonstrates an example of evolutionary young and unstable genomic region. The gene cluster is shaped by a constant balance between selection that acts on specific functions of the loci and frequent gene conversion events among duplicons. As the transcription of the GtHB genes is rate-limiting in the assembly of respective hormones, the genomic and genetic context of the FSHB and the LHB/CGB genes largely affects the profile of the hormone production. PMID:20488225

  19. The functional importance of a cap site-proximal region of the human prointerleukin 1 beta gene is defined by viral protein trans-activation.

    PubMed Central

    Hunninghake, G W; Monks, B G; Geist, L J; Monick, M M; Monroy, M A; Stinski, M F; Webb, A C; Dayer, J M; Auron, P E; Fenton, M J

    1992-01-01

    Prointerleukin 1 beta (IL-1 beta) is a cytokine that mediates a broad range of biological activities. Genomic sequences that regulate IL-1 beta transcription include both inducible regulatory elements located more than 2,700 bp upstream of the transcriptional start site (cap site) and proximal elements located near the TATA box of this gene. In this study, we focused on the identification and characterization of trans-acting nuclear regulatory proteins that bind to the cap site-proximal region of the human IL-1 beta gene. We identified a protein, termed NFIL-1 beta A (NF beta A), that binds to a highly conserved 12-bp DNA sequence (-49 to -38) located upstream of the TATA box motif in both the human and murine IL-1 beta genes. The IL-1 alpha gene, which lacks a TATA motif, does not possess an NF beta A-binding sequence within the promoter region, suggesting that NF beta A may selectively regulate IL-1 beta expression. Using electrophoretic mobility shift assays, we identified several distinct DNA-protein complexes that are expressed in a cell-type-specific manner. In monocytic cell lines, the relative abundance of these complexes varies rapidly following stimulation of the cells with phorbol esters or lipopolysaccharide. UV cross-linking analysis identified two distinct DNA-binding polypeptides that comprise distinct complexes. The functional role of NF beta A was assessed in transient transfection assays. These data indicate that NF beta A is required for both basal and inducible promoter activity in monocytic cells. Furthermore, the human cytomegalovirus immediate-early 1 gene product requires the presence of NF beta A in order to trans-activate the proximal IL-1 beta promoter in a monocytic cell line. We propose that NF beta A is a factor that mediates either direct or indirect activation by the immediate-early 1 gene product. The proximity of this essential factor to the TATA motif suggests a possible role in transcriptional initiation. Images PMID:1630455

  20. PARP-1 and YY1 are important novel regulators of CXCL12 gene transcription in rat pancreatic beta cells.

    PubMed

    Marković, Jelena; Grdović, Nevena; Dinić, Svetlana; Karan-Djurašević, Teodora; Uskoković, Aleksandra; Arambašić, Jelena; Mihailović, Mirjana; Pavlović, Sonja; Poznanović, Goran; Vidaković, Melita

    2013-01-01

    Despite significant progress, the molecular mechanisms responsible for pancreatic beta cell depletion and development of diabetes remain poorly defined. At present, there is no preventive measure against diabetes. The positive impact of CXCL12 expression on the pancreatic beta cell prosurvival phenotype initiated this study. Our aim was to provide novel insight into the regulation of rat CXCL12 gene (Cxcl12) transcription. The roles of poly(ADP-ribose) polymerase-1 (PARP-1) and transcription factor Yin Yang 1 (YY1) in Cxcl12 transcription were studied by examining their in vitro and in vivo binding affinities for the Cxcl12 promoter in a pancreatic beta cell line by the electrophoretic mobility shift assay and chromatin immunoprecipitation. The regulatory activities of PARP-1 and YY1 were assessed in transfection experiments using a reporter vector with a Cxcl12 promoter sequence driving luciferase gene expression. Experimental evidence for PARP-1 and YY1 revealed their trans-acting potential, wherein PARP-1 displayed an inhibitory, and YY1 a strong activating effect on Cxcl12 transcription. Streptozotocin (STZ)-induced general toxicity in pancreatic beta cells was followed by changes in Cxcl12 promoter regulation. PARP-1 binding to the Cxcl12 promoter during basal and in STZ-compromised conditions led us to conclude that PARP-1 regulates constitutive Cxcl12 expression. During the early stage of oxidative stress, YY1 exhibited less affinity toward the Cxcl12 promoter while PARP-1 displayed strong binding. These interactions were accompanied by Cxcl12 downregulation. In the later stages of oxidative stress and intensive pancreatic beta cell injury, YY1 was highly expressed and firmly bound to Cxcl12 promoter in contrast to PARP-1. These interactions resulted in higher Cxcl12 expression. The observed ability of PARP-1 to downregulate, and of YY1 to upregulate Cxcl12 promoter activity anticipates corresponding effects in the natural context where the functional

  1. Identification of Novel Tumor Suppressor Genes in Human Breast Cancer Using Nonsense-Mediated mRNA Decay Inhibition (NMDI)-Microarray Analysis

    DTIC Science & Technology

    2007-08-01

    of apoptosis and regulating heat shock protein Beta- 2 -microglobulin B2M Del CT in (CT)4 nt 37–44 LoVo MHC class I receptor activity Abbreviations...inappropriate location by interaction with components of the spliceosome that remained associated with the 2 downstream exon - exon junction following splicing...valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE 01-08-2007 2 . REPORT TYPE Final 3. DATES COVERED

  2. Active macrophage-associated TGF-beta co-localizes with type I procollagen gene expression in atherosclerotic human pulmonary arteries.

    PubMed Central

    Bahadori, L.; Milder, J.; Gold, L.; Botney, M.

    1995-01-01

    Vascular remodeling in adult atherosclerotic pulmonary arteries is characterized by discrete areas of neointimal smooth muscle cell extracellular matrix gene expression in close proximity to non-foamy macrophages, suggesting regulation by local macrophage-associated factors. The purpose of these studies was to begin addressing the role of putative macrophage-associated factors such as transforming growth factor-beta (TGF-beta), by determining the spatial relationship between TGF-beta and neointimal matrix gene expression in human atherosclerotic pulmonary arteries. For example, the participation of TGF-beta in vascular remodeling could be inferred by its colocalization with non-foamy macrophages in areas of active matrix synthesis. In situ hybridization and immunohistochemistry demonstrated focal neointimal procollagen gene expression in close association with non-foamy but not foamy macrophages. Immunohistochemistry with isoform-specific anti-TGF-beta antibodies demonstrated all three isoforms of TGF-beta associated with non-foamy macrophages, but foamy macrophages were not immunoreactive. Neointimal and medial smooth muscle cells stained lightly. In contrast, intense TGF-beta immunoreactivity was also associated with medial smooth muscle cells in normal nonremodeling vessels. Immunohistochemistry with antibodies specific for latent TGF-beta was similar to immunohistochemistry for mature TGF-beta in both remodeling and nonremodeling vessels. Finally, using an antibody specific for active TGF-beta 1, immunoreactivity was only seen in non-foamy neointimal macrophages but not in foamy macrophages or medial smooth muscle cells from hypertensive or normal vessels. These observations suggest non-foamy macrophages may participate in modulating matrix gene expression in atherosclerotic remodeling via a TGF-beta-dependent mechanism. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7747808

  3. Lysyl oxidase like 4, a novel target gene of TGF-{beta}1 signaling, can negatively regulate TGF-{beta}1-induced cell motility in PLC/PRF/5 hepatoma cells

    SciTech Connect

    Kim, Dong Joon; Lee, Dong Chul; Yang, Suk-Jin; Lee, Jung Ju; Bae, Eun Mi; Kim, Dong Min; Min, Sang Hyun; Kim, Soo Jung; Kang, Dong Chul; Sang, Byung Chan; Myung, Pyung Keun; Park, Kyung Chan Yeom, Young Il

    2008-09-05

    Transforming growth factor-{beta}1 (TGF-{beta}1) is a multi-functional cytokine involved in the regulation of cell proliferation, differentiation and extracellular matrix formation. In search for novel genes mediating the TGF-{beta}1 function at downstream signaling, we performed a cDNA microarray analysis and identified 60 genes whose expression is regulated by TGF-{beta}1 in the liver cancer cell line PLC/PRF/5. Among them, we report here lysyl oxidase like 4 (LOXL4) as a novel target of TGF-{beta}1 signaling, and provide experimental evidence for its expression regulation and function. LOXL4 was found to be the only member of LOX family whose expression is induced by TGF-{beta}1 in hepatoma cells. Deletion mapping of the LOXL4 promoter indicated that the TGF-{beta}1 regulation of LOXL4 expression is mediated through the binding of AP1 transcription factor to a conserved region of the promoter. This was confirmed by the chromatin immunoprecipitation assay that captured c-Fos-bound chromatin from TGF-{beta}1-treated cells. Forced expression of LOXL4 in PLC/PRF/5 cells resulted in inhibition of cell motility through Matrigel in the presence of TGF-{beta}1 treatment. In parallel, LOXL4 suppressed the expression of laminins and {alpha}3 integrin and the activity of MMP2. These results suggest that LOXL4 may function as a negative feedback regulator of TGF-{beta}1 in cell invasion by inhibiting the metabolism of extracellular matrix (ECM) components.

  4. The bla gene of the cephamycin cluster of Streptomyces clavuligerus encodes a class A beta-lactamase of low enzymatic activity.

    PubMed Central

    Pérez-Llarena, F; Martín, J F; Galleni, M; Coque, J J; Fuente, J L; Frère, J M; Liras, P

    1997-01-01

    A gene (bla) encoding a beta-lactamase is present in the cephamycin gene cluster of Streptomyces clavuligerus, the strain producing clavulanic acid and a beta-lactamase inhibitory protein. The bla gene is located 5.1 kb downstream from and in the opposite orientation to cefE, encoding the deacetoxycephalosporin C synthase. The bla gene encodes a 332-residue protein (Mr, 35,218), similar to other class A beta-lactamases produced by actinomycetes. Modification (to SDG) of the SDN conserved motif of class A beta-lactamases as well as of amino acids in otherwise conserved regions in the molecule may explain the low penicillinase and cephalosporinase activities of the protein. The beta-lactamase has been purified to homogeneity and found to bind [3H]benzylpenicillin, a result reflecting a rate-limiting deacylation step. Nucleotide sequences homologous to bla were found in all tested cephamycin producers, but several other Streptomyces species which produce a beta-lactamase do not contain genes for beta-lactam antibiotic biosynthesis. PMID:9324249

  5. Mutant HNF-1{alpha} and mutant HNF-1{beta} identified in MODY3 and MODY5 downregulate DPP-IV gene expression in Caco-2 cells

    SciTech Connect

    Gu Ning; Adachi, Tetsuya; Matsunaga, Tetsuro; Takeda, Jun; Tsujimoto, Gozoh; Ishihara, Akihiko; Yasuda, Koichiro; Tsuda, Kinsuke . E-mail: jinkan@tom.life.h.kyoto-u.ac.jp

    2006-08-04

    Dipeptidylpeptidase IV (DPP-IV) is a well-documented drug target for the treatment of type 2 diabetes. Hepatocyte nuclear factors (HNF)-1{alpha} and HNF-1{beta}, known as the causal genes of MODY3 and MODY5, respectively, have been reported to be involved in regulation of DPP-IV gene expression. But, it is not completely clear (i) that they play roles in regulation of DPP-IV gene expression, and (ii) whether DPP-IV gene activity is changed by mutant HNF-1{alpha} and mutant HNF-1{beta} in MODY3 and MODY5. To explore these questions, we investigated transactivation effects of wild HNF-1{alpha} and 13 mutant HNF-1{alpha}, as well as wild HNF-1{beta} and 2 mutant HNF-1{beta}, on DPP-IV promoter luciferase gene in Caco-2 cells by means of a transient experiment. Both wild HNF-1{alpha} and wild HNF-1{beta} significantly transactivated DPP-IV promoter, but mutant HNF-1{alpha} and mutant HNF-1{beta} exhibited low transactivation activity. Moreover, to study whether mutant HNF-1{alpha} and mutant HNF-1{beta} change endogenous DPP-IV enzyme activity, we produced four stable cell lines from Caco-2 cells, in which wild HNF-1{alpha} or wild HNF-1{beta}, or else respective dominant-negative mutant HNF-1{alpha}T539fsdelC or dominant-negative mutant HNF-1{beta}R177X, was stably expressed. We found that DPP-IV gene expression and enzyme activity were significantly increased in wild HNF-1{alpha} cells and wild HNF-1{beta} cells, whereas they decreased in HNF-1{alpha}T539fsdelC cells and HNF-1{beta}R177X cells, compared with DPP-IV gene expression and enzyme activity in Caco-2 cells. These results suggest that both wild HNF-1{alpha} and wild HNF-1{beta} have a stimulatory effect on DPP-IV gene expression, but that mutant HNF-1{alpha} and mutant HNF-1{beta} attenuate the stimulatory effect.

  6. The alpha- and beta-expansin and xyloglucan endotransglucosylase/hydrolase gene families of wheat: molecular cloning, gene expression, and EST data mining.

    PubMed

    Liu, Yong; Liu, Dongcheng; Zhang, Haiying; Gao, Hongbo; Guo, Xiaoli; Wang, Daowen; Zhang, Xiangqi; Zhang, Aimin

    2007-10-01

    Expansins and xyloglucan endotransglucosylase/hydrolases (XTHs) are families of extracellular proteins with members that have been shown to play an important role in cell wall growth. In this study, three, six, and five members of the wheat alpha-expansin (TaEXPA1 to TaEXPA3), beta-expansin (TaEXPB1 to TaEXPB6), and XTH (TaXTH1 to TaXTH5) gene families, respectively, were isolated from a dwarf wheat line. The mRNA expression analysis by real-time RT-PCR indicates that these genes display different transcription levels in different stages/organs/treatments, possibly suggesting their functional roles in the cell wall expansion process. Moreover, the comparison of the expression levels reveals that most of the expansins show lower expression than the XTHs. Finally, we present the analysis of wheat alpha- and beta-expansins and XTH families by expressed sequence tag data mining.

  7. Mutations in the lysosomal [beta]-galactosidase gene that cause the adult form of GMI gangliosidosis

    SciTech Connect

    Chakraborty, S.; Rafi, M.A.; Wenger, D.A. )

    1994-06-01

    Three adult patients with acid-galactosidase deficiency/GM1 gangliosidosis who were from two unrelated families of Scandinavian descent were found to share a common point mutation in the coding region of the corresponding gene. The patients share common clinical features, including early dysarthria, mild ataxia, and bone abnormalities. When cDNA from the two patients in family 1 was PCR amplified and sequenced, most (39/41) of the clones showed a C-to-T transition (C[yields]T) at nucleotide 245 (counting from the initiation codon). This mutation changes the codon for the Thr(ACG) to Met(ATG). Mutant and normal sequences were also found in that position in genomic DNA, indicating the presence of another mutant allele. Genomic DNA from the patient in family 2 revealed the same point mutation in one allele. It was determined that in each family only the father carried the C[yields]T mutation. Expression studies showed that this mutation produced 3%-4% of [beta]-galactosidase activity, confirming its deleterious effects. The cDNA clones from the patients in family 1 that did not contain the C[yields]T revealed a 20-bp insertion of intronic sequence between nucleotides 75 and 76, the location of the first intron. Further analysis showed the insertion of a T near the 5[prime] splice donor site which led to the use of a cryptic splice site. It appears that the C[yields]T mutation results in enough functional enzyme to produce a mild adult form of the disease, even in the presence of a second mutation that likely produces nonfunctional enzyme. 31 refs., 7 figs., 1 tab.

  8. Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker.

    PubMed Central

    Orbach, M J; Porro, E B; Yanofsky, C

    1986-01-01

    We cloned the beta-tubulin gene of Neurospora crassa from a benomyl-resistant strain and determined its nucleotide sequence. The gene encodes a 447-residue protein which shows strong homology to other beta-tubulins. The coding region is interrupted by six introns, five of which are within the region coding for the first 54 amino acids of the protein. Intron position comparisons between the N. crassa gene and other fungal beta-tubulin genes reveal considerable positional conservation. The mutation responsible for benomyl resistance was determined; it caused a phenylalanine-to-tyrosine change at position 167. Codon usage in the beta-tubulin gene is biased, as has been observed for other abundantly expressed N. crassa genes such as am and the H3 and H4 histone genes. This bias results in pyrimidines in the third positions of 96% of the codons in codon families in which there is a choice between purines and pyrimidines in this position. Bias is also evident by the absence of 19 of the 61 sense codons. We demonstrated that benomyl resistance is due to the cloned beta-tubulin gene of strain Bml511(r)a and that this gene can be used as a dominant selectable marker in N. crassa transformation. PMID:2946938

  9. Gene polymorphism in transforming growth factor-beta codon 10 is associated with susceptibility to Giardiasis.

    PubMed

    Taherkhani, H; Hajilooi, M; Fallah, M; Khyabanchi, O; Haidari, M

    2009-12-01

    Secretory immunoglobulin A (S-IgA) antibodies have a central role in anti-Giardial defence. It has been demonstrated that transforming growth factor-beta1 (TGF-beta1) stimulates B lymphocytes to produce and secrete S-IgA. We sought to determine the association between TGF-beta1 polymorphism (T+869C) with susceptibility to Giardiasis. The TGF-beta1 genotypes and levels of salivary (S-IgA) were analysed in individuals with Giardiasis (97 symptomatic and 57 asymptomatic) and controls (n = 92). Individuals with symptomatic Giardiasis had the lowest levels of S-IgA compared to individuals in asymptomatic Giardiasis and control groups (97%, 73% and 43%, <1 g L(-1), respectively, P = 0.002). The frequency of allele C and CC genotypes of TGF-beta1 polymorphism was significantly higher among symptomatic patients than asymptomatic and control groups. Logistic regression analysis demonstrated that the individuals homozygous for allele C of TGF-beta1 had a significantly higher risk for symptomatic Giardiasis with odds ratio of 2.76 (95% CI: 3.88, 1.71, P = 0.007). Among the participants with TT genotype per cent of individuals with S-IgA level of more than 1 g L(-1) was almost twice the percentage in CC genotype individuals (14% versus 7% respectively P = 0.01). Our data suggest that CC genotype of TGF-beta1 polymorphism at codon 10 is associated with occurrence of Giardiasis.

  10. Provocative pattern of rearrangements of the genes for the. gamma. and. beta. chains of the T-cell receptor in human leukemias

    SciTech Connect

    Goorha, R.; Bunin, N.; Mirro, J. Jr.; Murphy, S.B.; Cross, A.H.; Behm, F.G.; Quertermous, T.; Seidman, J.; Kitchingman, G.R.

    1987-07-01

    To examine the distribution of rearrangements of the ..gamma..- and ..beta..-chain T-cell receptor (TCR) genes in T- and non-T acute lymphoblastic leukemias (ALLs), and potentially to determine which genes rearrange first in ontogeny, the authors analyzed high molecular weight DNA from 102 patients with acute leukemia. Rearranged ..gamma..- and ..beta..-chain genes were found in all T-cell ALLs (22/22) examined. Overall, 27% (18/66) of B-lineage ALLs had ..beta..-chain gene rearrangements, and 41% (24/58) had ..gamma..-chain gene rearrangements, but the distribution of rearranged genes varied according to the stage of B-cell differentiation. The ..gamma..-chain genes were rearranged in 11% (1/9) of the B-lineage patients negative for the common acute lymphoblastic leukemia antigen (cALLA) and 50% (23/46) of cALLA/sup -/= ALL patients, while the ..beta..-chain genes were not rearranged in any of the 7 cALLA/sup -/ ALL patients examined but were rearranged in 32% (18/56) of the cALLA/sup +/ patients. Of the 44 cALLA/sup +/ patients in which a direct comparison of ..gamma..- and ..beta..-chain gene rearrangements could be made, 34% had both genes rearranged, 16% had only ..gamma..-chain gene rearrangements, and the remaining 50% had both genes in the germ-line configuration. ..beta..-Chain rearrangements have not been found in the absence of ..gamma..-chain rearrangements, thus supporting a proposed hierarchy of TCR gene rearrangements.

  11. A positive regulatory element is involved in the induction of the beta-galactosidase gene from Kluyveromyces lactis.

    PubMed Central

    Das, S; Breunig, K D; Hollenberg, C P

    1985-01-01

    The regulation of the LAC4 gene encoding beta-galactosidase in the yeast Kluyveromyces lactis has been studied. The expression of cloned LAC4 gene present on autonomously replicating plasmids was normally regulated by lactose or galactose as inducers. The LAC4 transcription initiation sites were mapped on two plasmids, PTY75-LAC4 and pKL2. The sites were found to be dependent on the level of gene expression and on the plasmid used. Under induced conditions, the normal cluster of initiation sites was used on both plasmids, whereas under non-induced conditions LAC4 on pKL2 showed additional sites. Deletion mapping of the 5' regulatory region of the LAC4 gene revealed a DNA element required for induction, presumably for the binding of a positive regulator. Images Fig. 2. Fig. 3. Fig. 6. PMID:3924596

  12. Cloning and characterization of genes encoding alpha and beta subunits of glutamate-gated chloride channel protein in Cylicocyclus nassatus.

    PubMed

    Tandon, Ritesh; LePage, Keith T; Kaplan, Ray M

    2006-11-01

    The invertebrate glutamate-gated chloride channels (GluCls) are receptor molecules and targets for the avermectin-milbemycin (AM) group of anthelmintics. Mutations in GluCls are associated with ivermectin resistance in the soil dwelling nematode Caenorhabditis elegans and the parasitic nematode Cooperia oncophora. In this study, full-length cDNAs encoding alpha and beta subunits of GluCl were cloned and sequenced in Cylicocyclus nassatus, a common and important cyathostomin nematode parasite of horses. Both genes possess the sequence characteristics typical of GluCls, and phylogenetic analysis confirms that these genes are evolutionarily closely related to GluCls of other nematodes and flies. Complete coding sequences of C. nassatus GluCl-alpha and GluCl-beta were subcloned into pTL1 mammalian expression vector, and proteins were expressed in COS-7 cells. Ivermectin-binding characteristics were determined by incubating COS-7 cell membranes expressing C. nassatus GluCl-alpha and GluCl-beta proteins with [(3)H]ivermectin. In competitive binding experiments, fitting the data to a one site competition model, C. nassatus GluCl-alpha was found to bind [(3)H]ivermectin with a high amount of displaceable binding (IC(50)=208 pM). Compared to the mock-transfected COS-7 cells, the means of [(3)H]ivermectin binding were significantly different for C. nassatus GluCl-alpha and the Haemonchus contortus GluCl (HcGluCla) (p=0.018 and 0.023, respectively) but not for C. nassatus GluCl-beta (p=0.370). This is the first report of orthologs of GluCl genes and in vitro expression of an ivermectin-binding protein in a cyathostomin species. These data suggest the likelihood of a similar mechanism of action of AM drugs in these parasites, and suggest that mechanisms of resistance may also be similar.

  13. Spectrum of Beta Globin Gene Mutations in Egyptian Children with β-Thalassemia

    PubMed Central

    El-Shanshory, MR; Hagag, AA; Shebl, SS; Badria, IM; Abd Elhameed, AH; Abd El-Bar, ES; Al-Tonbary, Y; Mansour, A; Hassab, H; Hamdy, M; Alfy, M; Sherief, L; Sharaf, E

    2014-01-01

    Background The molecular defects resulting in a β-thalassemia phenotype, in the Egyptian population, show a clear heterogenic mutations pattern. PCR-based techniques, including direct DNA sequencing are effective on the molecular detection and characterization of these mutations. The molecular characterization of β-thalassemia is necessary for carrier screening, genetic counseling, and to offer prenatal diagnosis. The aim of the work was to evaluate the different β-globin gene mutations in two hundred β-thalassemic Egyptian children. Subjects and Methods This study was carried out on two hundred β-thalassemic Egyptian children covering most Egyptian Governorates including 158 (79%) children with thalassemia major (TM) and 42 (21%) children with thalassemia intermedia(TI). All patients were subjected to meticulous history taking, clinical examination, complete blood count, hemoglobin electrophoresis, serum ferritin and direct fluorescent DNA sequencing of the β-globin gene to detect the frequency of different mutations. Results The most common mutations among patients were IVS I-110(G>A) 48%, IVS I-6(T>C) 40%, IVS I-1(G>A) 24%, IVS I-5(G>C)10%, IVS II-848 (C>A) 9%, IVS II-745(C>G) 8%, IVS II-1(G>A) 7%, codon “Cd”39(C> T) 4%, −87(C>G) 3% and the rare mutations were: Cd37 (G>A), Cd8 (−AA), Cd29(−G), Cd5 (−CT), Cd6(−A), Cd8/9(+G), Cd 106/107(+G), Cd27(C>T), IVS II-16(G> C), Cd 28 (−C), Cap+1(A>C), −88(C>A), all of these rare mutations were present in 1%. There was a considerable variation in phenotypic severity among patients resulting from the interaction of different β∘ and β+mutations. Furthermore, no genotype-phenotype association was found both among the cases with thalassemia major and the cases with thalassemia intermedia. Conclusion Direct DNA sequencing provides insights for the frequency of different mutations in patients with β-thalassemia including rare and/or unknown ones. The most common mutations in Egyptian children with

  14. Cystathionine beta synthase gene dose dependent vascular remodeling in murine model of hyperhomocysteinemia.

    PubMed

    Tyagi, Neetu; Qipshidze, Natia; Sen, Utpal; Rodriguez, Walter; Ovechkin, Alexander; Tyagi, Suresh C

    2011-09-30

    Although children born with severe homocystinurea (i.e. cystathionine beta synthase homozygote knockout, CBS-/-) develop deleterious vascular complications with structural malformation and do not live past teenage, the heterozygote (CBS-/+) lives with apparently normal phenotype. Interestingly, this differential role of CBS expression in vascular remodeling is unclear. Peroxisome proliferator activated receptor gamma (PPARγ) is nuclear transcription factor that mitigates vascular complications. The hypothesis was that homocysteine (Hcy) decreased thioredoxin (Trx), peroxiredoxin (Prx), increased NADPH oxidase (NOX1), mitochondrial nitric oxide synthase (mtNOS) activity and reactive oxygen species (ROS) in mitochondria in a CBS gene dose-dependent manner. ROS transduced matrix metalloproteinase (MMP) activation causing thickening (fibrosis) of the basement membrane, rendering ineffective endothelial nitric oxide synthase (eNOS) and promoted endothelial-smooth muscle disconnection/uncoupling by antagonizing PPARγ. Wild type (WT-CBS+/+), CBS-/+ and CBS -/- mice were treated with or without ciglitazone (CZ, a PPARγ agonist) in food at birth. Aortic nuclear PPARγ expression was measured by EMSA. Aortic mtNOS activity and ROS production was measured using NO- and H(2)O(2)-electrodes, respectively. Aorta was analyzed for Trx, Prx, by Western blot, and PCR. MMP activity was by in situ zymography. Aortic function was measured in tissue myobath. The results suggested 90% morbidity in CBS-/- allele at 12 wks. However, treatment with the PPARγ agonist, CZ significantly reduced the morbidity to 20%. In addition, CZ restored the PPARγ activity in CBS-/+ and -/- mice to normal levels. The oxidative stress was alleviated by CZ treatment. In situ labeling with mito-tracker suggests co-localization of ROS with mitochondrial mitophagy. The mtNOS activity was increased in HHcy compared to WT. The data support the notion that Hcy decreases redoxins, increases mtNOS activity and

  15. SNP analyses of growth factor genes EGF, TGF{beta}-1, and HGF reveal haplotypic association of EGF with autism

    SciTech Connect

    Toyoda, Takao; Thanseem, Ismail; Kawai, Masayoshi; Sekine, Yoshimoto; Nakamura, Kazuhiko; Anitha, Ayyappan; Suda, Shiro . E-mail: nakamura@hama-med.ac.jp; Yamada, Kazuo; Tsujii, Masatsugu |; Iwayama, Yoshimi; Hattori, Eiji; Toyota, Tomoko; Yoshikawa, Takeo; Miyachi, Taishi; Tsuchiya, Kenji; Sugihara, Gen-ichi; Matsuzaki, Hideo; Iwata, Yasuhide; Suzuki, Katsuaki; Mori, Norio |; Ouchi, Yasuomi |; Sugiyama, Toshiro; Takei, Nori

    2007-09-07

    Autism is a pervasive neurodevelopmental disorder diagnosed in early childhood. Growth factors have been found to play a key role in the cellular differentiation and proliferation of the central and peripheral nervous systems. Epidermal growth factor (EGF) is detected in several regions of the developing and adult brain, where, it enhances the differentiation, maturation, and survival of a variety of neurons. Transforming growth factor-{beta} (TGF{beta}) isoforms play an important role in neuronal survival, and the hepatocyte growth factor (HGF) has been shown to exhibit neurotrophic activity. We examined the association of EGF, TGF{beta}1, and HGF genes with autism, in a trio association study, using DNA samples from families recruited to the Autism Genetic Resource Exchange; 252 trios with a male offspring scored for autism were selected for the study. Transmission disequilibrium test revealed significant haplotypic association of EGF with autism. No significant SNP or haplotypic associations were observed for TGF{beta}1 or HGF. Given the role of EGF in brain and neuronal development, we suggest a possible role of EGF in the pathogenesis of autism.

  16. The beta-glucoside genes of Klebsiella aerogenes: conservation and divergence in relation to the cryptic bgl genes of Escherichia coli.

    PubMed

    Raghunand, Tirumalai R; Mahadevan, S

    2003-06-27

    The ability to metabolize aromatic beta-glucosides such as salicin and arbutin varies among members of the Enterobacteriaceae. The ability of Escherichia coli to degrade salicin and arbutin appears to be cryptic, subject to activation of the bgl genes, whereas many members of the Klebsiella genus can metabolize these sugars. We have examined the genetic basis for beta-glucoside utilization in Klebsiella aerogenes. The Klebsiella equivalents of bglG, bglB and bglR have been cloned using the genome sequence database of Klebsiella pneumoniae. Nucleotide sequencing shows that the K. aerogenes bgl genes show substantial similarities to the E. coli counterparts. The K. aerogenes bgl genes in multiple copies can also complement E. coli mutants deficient in bglG encoding the antiterminator and bglB encoding the phospho-beta-glucosidase, suggesting that they are functional homologues. The regulatory region bglR of K. aerogenes shows a high degree of similarity of the sequences involved in BglG-mediated regulation. Interestingly, the regions corresponding to the negative elements present in the E. coli regulatory region show substantial divergence in K. aerogenes. The possible evolutionary implications of the results are discussed.

  17. Frequent deletions and mutations of the beta-catenin gene are associated with overexpression of cyclin D1 and fibronectin and poorly differentiated histology in childhood hepatoblastoma.

    PubMed

    Takayasu, H; Horie, H; Hiyama, E; Matsunaga, T; Hayashi, Y; Watanabe, Y; Suita, S; Kaneko, M; Sasaki, F; Hashizume, K; Ozaki, T; Furuuchi, K; Tada, M; Ohnuma, N; Nakagawara, A

    2001-04-01

    Hepatoblastoma (HBL) is the most common malignant liver tumor in young children. Recent reports have shown that the beta-catenin gene was frequently mutated or deleted in HBLS: To elucidate the role of beta-catenin abnormalities in HBLs, we searched for mutations of beta-catenin and APC as well as expression of the target genes, cyclin D1, c-myc, and fibronectin, in 68 primary HBLS: The mutation analysis revealed that 44 (65%) tumors carried missense mutations or deletions of beta-catenin, all of which were somatic and targeted to the exon 3 encoding the amino acid residues involved in its degradation. However, no loss of function mutation of the APC gene was detected by the yeast functional assay. Of interest, beta-catenin mutation was significantly correlated with overexpression of the target genes, cyclin D1 and fibronectin, but not with that of c-myc in HBLs as measured by quantitative real-time reverse transcription-PCR. The immunohistochemical studies in 15 HBLs demonstrated that the nuclear/cytoplasmic accumulation of beta-catenin was positive in 13 tumors, 9 of which had the deletion or mutation of the gene. The significant correlation between the beta-catenin gene abnormality and the positive staining of cyclin D1 was also confirmed. Furthermore, the nuclear accumulation of beta-catenin was strongly associated with the poorly differentiated tumor cell components as well as with the positive staining of cyclin D1 within the tumor. Thus, our present results suggested that the gain of function mutation of beta-catenin played a crucial role in the malignant progression of HBL in vivo.

  18. T cell receptor repertoire differences between African Americans and Caucasians associated with polymorphism of the TCRBV3S1 (V{beta}3.1) gene

    SciTech Connect

    De Inocencio, J.; Glass, D.N.; Hirsch, R.

    1995-05-01

    The generation of TCR diversity occurs primarily through rearrangement of germline DNA. Genetic polymorphism of the TCR chains appears to be a rarer mechanism for generating repertoire differences between races. Flow cytometric analysis of the TCR V{beta} repertoire in a population of healthy African Americans (n = 30) and Caucasians (n = 30) revealed a significant difference in the frequency of cells bearing V{beta}3.1, but not V{beta}2, V{beta}5.1, V{beta}5.2-5.3, V{beta}6.7, V{beta}8.1-8.2, V{beta}12.1, V{beta}13.3, or V{beta}19. African Americans had a significantly lower frequency of V{beta}3.1{sup +} cells, in both the CD4{sup +} (2.55 {+-} 0.36% vs 4.85 {+-} 0.43%, p = 0.0001) and the CD8{sup +} (3.03 {+-} 0.54% vs 5.32 {+-} 0.57%, p = 0.004) population than did Caucasians, and this difference was independent of the age of the individuals. Analysis of genomic DNA revealed that the observed differences in frequency of V{beta}3.1{sup +} cells correlated with a recently described polymorphism of the recombination signal sequence of the TCRBV3S1 gene. Allele 1, associated with a lower frequency of V{beta}3.1{sup +} cells, was more commonly present in African Americans (0.68 vs 0.43), whereas allele 2, associated with a higher frequency of V{beta}3.1{sup +} cells, was more commonly present in Caucasians (0.31 vs 0.56). This study demonstrates the potential for TCR repertoire differences, based on genetic polymorphism, between African Americans and Caucasians. 31 refs., 2 figs., 5 tabs.

  19. Mechanism of fiber carcinogenesis: from reactive radical species to silencing of the beta igH3 gene.

    PubMed

    Hei, Tom K; Xu, An; Huang, Sarah X; Zhao, Yongliang

    2006-11-01

    Although the U.S. Environmental Protection Agency has restricted the industrial use of regulated forms of asbestos in the United States since the early 1970s, environmental exposure to asbestos remains a health concern in the United States and is a significant health issue among developing countries. Exposure to asbestos is associated with chronic pulmonary diseases and cancer of the lung, pleura, and peritoneum. The mechanism of fiber carcinogenesis is far from clear and is likely to be complex, depending on fiber dimensions, surface properties, and physical durability. The induction of reactive oxygen and nitrogen species upon phagocytosis of fibers plays an important role in fiber genotoxicity. The beta igH3, a secreted protein induced by the transforming growth factor-beta and essential for cell adhesion, is downregulated in asbestos-induced tumorigenic human bronchial epithelial cells. Ectopic expression of the beta igH3 gene abrogates the tumorigenic phenotype and suggests that the gene plays a causal role in fiber carcinogenesis. A better understanding of the carcinogenic mechanism of asbestos and other mineral fibers will provide useful information on interventional and preventive measures for asbestos-mediated diseases such as human pleural and peritoneal mesotheliomas.

  20. Identification of a mutation in the human raloxifene response element of the transforming growth factor-beta 3 gene.

    PubMed Central

    Han, K. O.; Kang, Y. S.; Hwang, C. S.; Moon, I. G.; Yim, C. H.; Chung, H. Y.; Jang, H. C.; Yoon, H. K.; Han, I. K.; Choi, Y. K.

    2001-01-01

    The human transforming growth factor-beta 3 (TGF-beta 3) is an important cytokine to maintain bone mass by inhibiting osteoclast differentiation. Recently raloxifene response element (RRE), a new enhancer with a polypurine sequence for estrogen receptor (ER)-mediated gene activation, was identified on the TGF-beta 3 gene. Functional analysis of the RRE-mediated pathway has shown that this would be an important pathway for bone preserving effect. We found a novel mutation in the RRE sequence by single-strand conformational polymorphism analysis in one of 200 Korean women. Cloning and sequencing revealed a heterozygote in which one allele had an insertion of 20 nucleotides (AGAGAGGGAGAGGGAGA GGG) between nucleotide +71 and +72 and a point mutation at nucleotide +75 (G-A transition), and the other allele had normal sequence. The insertion was a nearly perfect tandem duplication of the wild type DNA sequence. The bone mineral density of the affected woman was not much lower than that of age-matched controls. Transient transfection of the mutant allele showed no significantly different activity compared with that of the wild type allele. These observations suggest that the heterozygote variation of the RRE sequence seems not to be operative in determination of bone mass. PMID:11641521

  1. Dimorphisms of the proteasome subunit beta type 8 gene (PSMB8) of ectothermic tetrapods originated in multiple independent evolutionary events.

    PubMed

    Huang, Ching-Huei; Tanaka, Yuta; Fujito, Naoko T; Nonaka, Masaru

    2013-11-01

    The proteasome subunit beta type 8 gene (PSMB8) encodes one of the beta subunits of the immunoproteasome responsible for the generation of peptides presented by major histocompatibility complex class I molecules. Dimorphic alleles of the PSMB8 gene, termed A and F types, based on the deduced 31st amino acid residue of the mature protein have been reported from various vertebrates. Phylogenetic analysis revealed the presence of dichotomous ancient lineages, one comprising the F-type PSMB8 of basal ray-finned fishes, and the other comprising the A-type PSMB8 of these animals and both the F- and A-type PSMB8 of Xenopus and acanthopterygians, indicating that evolutionary history of the PSMB8 dimorphism was not straightforward. We analyzed the PSMB8 gene of five reptile and one amphibian species and found both the A and F types from all six. Phylogenetic analysis indicated that the PSMB8 F type was apparently regenerated from the PSMB8 A type at least five times independently during tetrapod evolution. Genomic typing of wild individuals of geckos and newts indicated that the frequencies of the A- and F-type alleles are not highly biased in these species. Phylogenetic analysis of each exon of the reptile PSMB8 gene suggested interallelic sequence homogenization as a possible evolutionary mechanism for the apparent recurrent regeneration of PSMB8 dimorphism in tetrapods. An extremely strong balancing selection acting on PSMB8 dimorphism was implicated in an unprecedented pattern of allele evolution.

  2. EpCAM Intracellular Domain Promotes Porcine Cell Reprogramming by Upregulation of Pluripotent Gene Expression via Beta-catenin Signaling

    PubMed Central

    Yu, Tong; Ma, Yangyang; Wang, Huayan

    2017-01-01

    Previous study showed that expression of epithelial cell adhesion molecule (EpCAM) was significantly upregulated in porcine induced pluripotent stem cells (piPSCs). However, the regulatory mechanism and the downstream target genes of EpCAM were not well investigated. In this study, we found that EpCAM was undetectable in fibroblasts, but highly expressed in piPSCs. Promoter of EpCAM was upregulated by zygotic activated factors LIN28, and ESRRB, but repressed by maternal factors OCT4 and SOX2. Knocking down EpCAM by shRNA significantly reduced the pluripotent gene expression. Conversely, overexpression of EpCAM significantly increased the number of alkaline phosphatase positive colonies and elevated the expression of endogenous pluripotent genes. As a key surface-to-nucleus factor, EpCAM releases its intercellular domain (EpICD) by a two-step proteolytic processing sequentially. Blocking the proteolytic processing by inhibitors TAPI-1 and DAPT could reduce the intracellular level of EpICD and lower expressions of OCT4, SOX2, LIN28, and ESRRB. We noticed that increasing intracellular EpICD only was unable to improve activity of EpCAM targeted genes, but by blocking GSK-3 signaling and stabilizing beta-catenin signaling, EpICD could then significantly stimulate the promoter activity. These results showed that EpCAM intracellular domain required beta-catenin signaling to enhance porcine cell reprogramming. PMID:28393933

  3. Localization of eight additional genes in the human major histocompatibility complex, including the gene encoding the casein kinase II {beta} subunit (CSNK2B)

    SciTech Connect

    Albertella, M.R.; Jones, H.; Thomson, W.

    1996-09-01

    A wide range of autoimmune and other diseases are known to be associated with the major histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility antigens in the class I and class II regions, but some appear to be more strongly associated with genes in the central 1100-kb class III region, making it important to characterize this region fully for the presence of novel genes. An {approximately}220-kb segment of DNA in the class III region separating the Hsp70 (HSPA1L) and BAT1 (D6S8IE) genes, which was previously known to contain 14 genes. Genomic DNA fragments spanning the gaps between the known genes were used as probes to isolate cDNAs corresponding to five new genes within this region. Evidence from Northern blot analysis and exon trapping experiments that suggested the presence of at least two more new genes was also obtained. Partial cDNA and complete exonic genomic sequencing of one of the new genes has identified it as the casein kinase II{beta} subunit (CSNK2B). Two of the other novel genes lie within a region syntenic to that implicated in susceptibility to experimental allergic orchitis in the mouse, an autoimmune disease of the testis, and represent additional candidates for the Orch-1 locus associated with this disease. In addition, characterization of the 13-kb intergenic gap separating the RD (D6545) and G11 (D6S60E) genes has revealed the presence of a gene encoding a 1246-amino-acid polypeptide that shows significant sequence similarity to the yeast anti-viral Ski2p gene product. 49 refs., 8 figs.

  4. Pancreatic beta-cell-specific targeted disruption of glucokinase gene. Diabetes mellitus due to defective insulin secretion to glucose.

    PubMed

    Terauchi, Y; Sakura, H; Yasuda, K; Iwamoto, K; Takahashi, N; Ito, K; Kasai, H; Suzuki, H; Ueda, O; Kamada, N

    1995-12-22

    Mice carrying a null mutation in the glucokinase (GK) gene in pancreatic beta-cells, but not in the liver, were generated by disrupting the beta-cell-specific exon. Heterozygous mutant mice showed early-onset mild diabetes due to impaired insulin-secretory response to glucose. Homozygotes showed severe diabetes shortly after birth and died within a week. GK-deficient islets isolated from homozygotes showed defective insulin secretion in response to glucose, while they responded to other secretagogues: almost normally to arginine and to some extent to sulfonylureas. These data provide the first direct proof that GK serves as a glucose sensor molecule for insulin secretion and plays a pivotal role in glucose homeostasis. GK-deficient mice serve as an animal model of the insulin-secretory defect in human non-insulin-dependent diabetes mellitus.

  5. Evidence that a human soluble beta-galactoside-binding lectin is encoded by a family of genes.

    PubMed Central

    Gitt, M A; Barondes, S H

    1986-01-01

    Two cDNA clones were isolated by immunoscreening a human hepatoma cDNA library with an antiserum that bound specifically to a human soluble beta-galactoside-binding lectin with Mr of approximately 14,000. The deduced amino acid sequences of the inserts of these two clones show considerable homology with each other, the sequence of chicken skin beta-galactoside-binding lectin, and eight peptides derived from purified human lung lectin of Mr approximately 14,000. However, the sequence differences between the two hepatoma clones as well as among each clone and the lung peptides suggest that at least three variants of the gene encoding this lectin are expressed in human tissue. Images PMID:3020551

  6. Augmentation of lung liquid clearance via adenovirus-mediated transfer of a Na,K-ATPase beta1 subunit gene.

    PubMed Central

    Factor, P; Saldias, F; Ridge, K; Dumasius, V; Zabner, J; Jaffe, H A; Blanco, G; Barnard, M; Mercer, R; Perrin, R; Sznajder, J I

    1998-01-01

    Previous studies have suggested that alveolar Na,K-ATPases play an important role in active Na+ transport and lung edema clearance. We reasoned that overexpression of Na,K-ATPase subunit genes could increase Na,K-ATPase function in lung epithelial cells and edema clearance in rat lungs. To test this hypothesis we produced replication deficient human type 5 adenoviruses containing cDNAs for the rat alpha1 and beta1 Na,K-ATPase subunits (adMRCMValpha1 and adMRCMVbeta1, respectively). As compared to controls, adMRCMVbeta1 increased beta1 subunit expression and Na,K-ATPase function by 2. 5-fold in alveolar type 2 epithelial cells and rat airway epithelial cell monolayers. No change in Na,K-ATPase function was noted after infection with adMRCMValpha1. Rat lungs infected with adMRCMVbeta1, but not adMRCMValpha1, had increased beta1 protein levels and lung liquid clearance 7 d after tracheal instillation. Alveolar epithelial permeability to Na+ and mannitol was mildly increased in animals infected with adMRCMVbeta1 and a similar Escherichia coli lacZ-expressing virus. Our data shows, for the first time, that transfer of the beta1 Na,K-ATPase subunit gene augments Na,K-ATPase function in epithelial cells and liquid clearance in rat lungs. Conceivably, overexpression of Na,K-ATPases could be used as a strategy to augment lung liquid clearance in patients with pulmonary edema. PMID:9769335

  7. Priming affects the activity of a specific region of the promoter of the human beta interferon gene.

    PubMed Central

    Dron, M; Lacasa, M; Tovey, M G

    1990-01-01

    Treatment of Daudi or HeLa cells with human interferon (IFN) alpha 8 before induction with either poly(I)-poly(C) or Sendai virus resulted in an 8- to 100-fold increase in IFN production. The extent of priming in Daudi cells paralleled the increase in the intracellular content of IFN-beta mRNA. IFN-alpha mRNA remained undetectable in poly(I)-poly(C)-treated Daudi cells either before or after priming. An IFN-resistant clone of Daudi cells was found to produce 4- to 20-fold more IFN after priming, indicating that priming was unrelated to the phenotype of IFN sensitivity. IFN treatment of either Daudi or HeLa cells transfected with the human IFN-beta promoter (-282 to -37) linked to the chloramphenicol acetyltransferase (CAT) gene resulted in an increase in CAT activity after induction with poly(I)-poly(C) or Sendai virus. A synthetic double-stranded oligonucleotide corresponding to an authentic 30-base-pair (bp) region of the human IFN-beta promoter between positions -91 and -62 was found to confer virus inducibility upon the reporter CAT gene in HeLa cells. IFN treatment of HeLa cells transfected with this 30-bp region of the IFN-beta promoter in either the correct or reversed orientation also increased CAT activity upon subsequent induction. IFN treatment alone had no detectable effect on the activity of either the 30-bp region or the complete human IFN promoter. Images PMID:2153928

  8. Interactions between beta-2 adrenoceptor gene variation, cardiovascular control and dietary sodium in healthy young adults.

    PubMed

    Eisenach, John H; Schroeder, Darrell R; Pavey, Emily S; Penheiter, Alan R; Knutson, Jean N; Turner, Stephen T; Joyner, Michael J

    2014-12-01

    Dietary sodium affects function of the beta-2 adrenoceptor (ADRB2). We tested the hypothesis that haplotype variation in the ADRB2 gene would influence the cardiovascular and regional vasodilator responses to sympathoexcitatory manoeuvres following low, normal and high sodium diets, and ADRB2-mediated forearm vasodilation in the high sodium condition. Seventy-one healthy young adults were grouped by double homozygous haplotypes: Arg16+Gln27 (n = 31), the rare Gly16+Gln27 (n = 10) and Gly16+Glu27 (n = 30). Using a randomized cross-over design, subjects were studied following 5 days of controlled low, normal and high sodium with 1 month or longer between diets (and low hormone phase of the menstrual cycle). All three visits utilized ECG and finger plethysmography for haemodynamic measures, and the high sodium visit included a brachial arterial catheter for forearm vasodilator responses to isoprenaline with plethysmography. Lymphocytes were sampled for ex vivo analysis of ADRB2 density and binding conformation. We found a main effect of haplotype on ADRB2 density (P = 0.03) with the Gly16+Glu27 haplotype having the greatest density (low, normal, high sodium: 12.9 ± 0.9, 13.5 ± 0.9 and 13.6 ± 0.8 fmol mg(-1) protein, respectively) and Arg16+Gln27 having the least (9.3 ± 0.6, 10.1 ± 0.5 and 10.3 ± 0.6  fmol mg(-1) protein, respectively), but there were no sodium or haplotype effects on receptor binding conformation. In the mental stress trial, there was a main effect of haplotype on cardiac output (P = 0.04), as Arg16+Gln27 had the lowest responses. Handgrip and forearm vasodilation yielded no haplotype differences, and no correlations were present for ADRB2 density and haemodynamics. Our findings support cell-based evidence that ADRB2 haplotype influences ADRB2 protein expression independent of dietary sodium, yet the haemodynamic consequences appear modest in healthy humans.

  9. Mutations in the latent TGF-beta binding protein 3 (LTBP3) gene cause brachyolmia with amelogenesis imperfecta

    PubMed Central

    Huckert, Mathilde; Stoetzel, Corinne; Morkmued, Supawich; Laugel-Haushalter, Virginie; Geoffroy, Véronique; Muller, Jean; Clauss, François; Prasad, Megana K.; Obry, Frédéric; Raymond, Jean Louis; Switala, Marzena; Alembik, Yves; Soskin, Sylvie; Mathieu, Eric; Hemmerlé, Joseph; Weickert, Jean-Luc; Dabovic, Branka Brukner; Rifkin, Daniel B.; Dheedene, Annelies; Boudin, Eveline; Caluseriu, Oana; Cholette, Marie-Claude; Mcleod, Ross; Antequera, Reynaldo; Gellé, Marie-Paule; Coeuriot, Jean-Louis; Jacquelin, Louis-Frédéric; Bailleul-Forestier, Isabelle; Manière, Marie-Cécile; Van Hul, Wim; Bertola, Debora; Dollé, Pascal; Verloes, Alain; Mortier, Geert; Dollfus, Hélène; Bloch-Zupan, Agnès

    2015-01-01

    Inherited dental malformations constitute a clinically and genetically heterogeneous group of disorders. Here, we report on four families, three of them consanguineous, with an identical phenotype, characterized by significant short stature with brachyolmia and hypoplastic amelogenesis imperfecta (AI) with almost absent enamel. This phenotype was first described in 1996 by Verloes et al. as an autosomal recessive form of brachyolmia associated with AI. Whole-exome sequencing resulted in the identification of recessive hypomorphic mutations including deletion, nonsense and splice mutations, in the LTBP3 gene, which is involved in the TGF-beta signaling pathway. We further investigated gene expression during mouse development and tooth formation. Differentiated ameloblasts synthesizing enamel matrix proteins and odontoblasts expressed the gene. Study of an available knockout mouse model showed that the mutant mice displayed very thin to absent enamel in both incisors and molars, hereby recapitulating the AI phenotype in the human disorder. PMID:25669657

  10. The mec-7 beta-tubulin gene of Caenorhabditis elegans is expressed primarily in the touch receptor neurons.

    PubMed Central

    Hamelin, M; Scott, I M; Way, J C; Culotti, J G

    1992-01-01

    Mutants of the mec-7 beta-tubulin gene of Caenorhabditis elegans lack the large diameter 15-protofilament microtubules normally found only in the set of six touch receptor neurons. Both a mec-7-lacZ reporter gene and affinity-purified anti-mec-7 antibodies were used to show that mec-7 is expressed primarily in the touch neurons. These data are consistent with a possible instructive role for the mec-7 tubulin in determining microtubule protofilament number. The antibodies and the mec-7-lacZ transgene were also used to examine mec-7 expression in mutants affecting the generation, differentiation or maintenance of the touch neurons. Decreased expression was observed in mutants of unc-86 and mec-3, genes that encode transcription factors essential for touch receptor neuron generation and differentiation, respectively. Images PMID:1639062

  11. Mutations in the latent TGF-beta binding protein 3 (LTBP3) gene cause brachyolmia with amelogenesis imperfecta.

    PubMed

    Huckert, Mathilde; Stoetzel, Corinne; Morkmued, Supawich; Laugel-Haushalter, Virginie; Geoffroy, Véronique; Muller, Jean; Clauss, François; Prasad, Megana K; Obry, Frédéric; Raymond, Jean Louis; Switala, Marzena; Alembik, Yves; Soskin, Sylvie; Mathieu, Eric; Hemmerlé, Joseph; Weickert, Jean-Luc; Dabovic, Branka Brukner; Rifkin, Daniel B; Dheedene, Annelies; Boudin, Eveline; Caluseriu, Oana; Cholette, Marie-Claude; Mcleod, Ross; Antequera, Reynaldo; Gellé, Marie-Paule; Coeuriot, Jean-Louis; Jacquelin, Louis-Frédéric; Bailleul-Forestier, Isabelle; Manière, Marie-Cécile; Van Hul, Wim; Bertola, Debora; Dollé, Pascal; Verloes, Alain; Mortier, Geert; Dollfus, Hélène; Bloch-Zupan, Agnès

    2015-06-01

    Inherited dental malformations constitute a clinically and genetically heterogeneous group of disorders. Here, we report on four families, three of them consanguineous, with an identical phenotype, characterized by significant short stature with brachyolmia and hypoplastic amelogenesis imperfecta (AI) with almost absent enamel. This phenotype was first described in 1996 by Verloes et al. as an autosomal recessive form of brachyolmia associated with AI. Whole-exome sequencing resulted in the identification of recessive hypomorphic mutations including deletion, nonsense and splice mutations, in the LTBP3 gene, which is involved in the TGF-beta signaling pathway. We further investigated gene expression during mouse development and tooth formation. Differentiated ameloblasts synthesizing enamel matrix proteins and odontoblasts expressed the gene. Study of an available knockout mouse model showed that the mutant mice displayed very thin to absent enamel in both incisors and molars, hereby recapitulating the AI phenotype in the human disorder.

  12. BLG-e1 - a novel regulatory element in the distal region of the beta-lactoglobulin gene promoter.

    PubMed

    Reichenstein, Moshe; German, Tania; Barash, Itamar

    2005-04-11

    beta-Lactoglobulin (BLG) is a major ruminant milk protein. A regulatory element, termed BLG-e1, was defined in the distal region of the ovine BLG gene promoter. This 299-bp element lacks the established cis-regulatory sequences that affect milk-protein gene expression. Nevertheless, it alters the binding of downstream BLG sequences to histone H4 and the sensitivity of the histone-DNA complexes to trichostatin A treatment. In mammary cells cultured under favorable lactogenic conditions, BLG-e1 acts as a potent, position-independent silencer of BLG/luciferase expression, and similarly affects the promoter activity of the mouse whey acidic protein gene. Intragenic sequences upstream of BLG exon 2 reverse the silencing effect of BLG-e1 in vitro and in transgenic mice.

  13. Homozygous deletion of the. alpha. - and. beta. sub 1 -interferon genes in human leukemia and derived cell lines

    SciTech Connect

    Diaz, M.O.; Ziemin, S.; Le Beau, M.M.; Pitha, P.; Smith, S.D.; Chilcote, R.R.; Rowley, J.D. )

    1988-07-01

    The loss of bands p21-22 from one chromosome 9 homologue as a consequence of a deletion of the short arm (del(9p)), unbalanced translocation, or monosomy 9 is frequently observed in the malignant cells of patients with lymphoid neoplasias, including acute lymphoblastic leukemia and non-Hodgkin lymphoma. The {alpha}- and {beta}{sub 1}-interferon genes have been assigned to this chromosome region (9p21-22). The authors now present evidence of the homozygous deletion of the interferon genes in neoplastic hematopoietic cell lines and primary leukemia cells in the presence or absence of chromosomal deletions that are detectable at the level of the light microscope. In these cell lines, the deletion of the interferon genes is accompanied by a deficiency of 5{prime}-methylthioadenosine phosphorylase, an enzyme of purine metabolism. These homozygous deletions may be associated with the loss of a tumor-suppressor gene that is involved in the development of these neoplasias. The relevant genes may be either the interferon genes themselves or a gene that has a tumor-suppressor function and is closely linked to them.

  14. Gene correction of integrin beta4-dependent pyloric atresia-junctional epidermolysis bullosa keratinocytes establishes a role for beta4 tyrosines 1422 and 1440 in hemidesmosome assembly.

    PubMed

    Dellambra, E; Prislei, S; Salvati, A L; Madeddu, M L; Golisano, O; Siviero, E; Bondanza, S; Cicuzza, S; Orecchia, A; Giancotti, F G; Zambruno, G; De Luca, M

    2001-11-02

    The cytoplasmic domain of beta4 integrin contains two pairs of fibronectin-like repeats separated by a connecting segment. The connecting segment harbors a putative tyrosine activation motif in which tyrosines 1422 and 1440 are phosphorylated in response to alpha6beta4 binding to laminin-5. Primary beta4-null keratinocytes, obtained from a newborn suffering from lethal junctional epidermolysis bullosa, were stably transduced with retroviruses carrying a full-length beta4 cDNA or a beta4 cDNA with phenylalanine substitutions at Tyr-1422 and Tyr-1440. Hemidesmosome assembly was evaluated on organotypic skin cultures. beta4-corrected keratinocytes were indistinguishable from normal cells in terms of alpha6beta4 expression, the localization of hemidesmosome components, and hemidesmosome structure and density, suggesting full genetic and functional correction of beta4-null keratinocytes. In cultures generated from beta4(Y1422F/Y1440F) keratinocytes, beta4 mutants as well as alpha6 integrin, HD1/plectin, and BP180 were not concentrated at the dermal-epidermal junction. Furthermore, the number of hemidesmosomes was strikingly reduced as compared with beta4-corrected keratinocytes. The rare hemidesmosomes detected in beta4(Y1422F/Y1440F) cells were devoid of sub-basal dense plates and of inner cytoplasmic plaques with keratin filament insertion. Collectively, our data demonstrate that the beta4 tyrosine activation motif is not required for the localization of alpha6beta4 at the keratinocyte plasma membrane but is essential for optimal assembly of bona fide hemidesmosomes.

  15. CCAAT-binding factor regulates expression of the beta1 subunit of soluble guanylyl cyclase gene in the BE2 human neuroblastoma cell line

    NASA Technical Reports Server (NTRS)

    Sharina, Iraida G.; Martin, Emil; Thomas, Anthony; Uray, Karen L.; Murad, Ferid

    2003-01-01

    Soluble guanylyl cyclase (sGC) is a cytosolic enzyme producing the intracellular messenger cyclic guanosine monophosphate (cGMP) on activation with nitric oxide (NO). sGC is an obligatory heterodimer composed of alpha and beta subunits. We investigated human beta1 sGC transcriptional regulation in BE2 human neuroblastoma cells. The 5' upstream region of the beta1 sGC gene was isolated and analyzed for promoter activity by using luciferase reporter constructs. The transcriptional start site of the beta1 sGC gene in BE2 cells was identified. The functional significance of consensus transcriptional factor binding sites proximal to the transcriptional start site was investigated by site deletions in the 800-bp promoter fragment. The elimination of CCAAT-binding factor (CBF) and growth factor independence 1 (GFI1) binding cores significantly diminished whereas deletion of the NF1 core elevated the transcription. Electrophoretic mobility-shift assay (EMSA) and Western analysis of proteins bound to biotinated EMSA probes confirmed the interaction of GFI1, CBF, and NF1 factors with the beta1 sGC promoter. Treatment of BE2 cells with genistein, known to inhibit the CBF binding to DNA, significantly reduced protein levels of beta1 sGC by inhibiting transcription. In summary, our study represents an analysis of the human beta1 sGC promoter regulation in human neuroblastoma BE2 cells and identifies CBF as a critically important factor in beta1 sGC expression.

  16. Analysis of Claviceps africana and C. sorghi from India using AFLPs, EF-1alpha gene intron 4, and beta-tubulin gene intron 3.

    PubMed

    Tooley, Paul W; Bandyopadhyay, Ranajit; Carras, Marie M; Pazoutová, Sylvie

    2006-04-01

    Isolates of Claviceps causing ergot on sorghum in India were analysed by AFLP analysis, and by analysis of DNA sequences of the EF-1alpha gene intron 4 and beta-tubulin gene intron 3 region. Of 89 isolates assayed from six states in India, four were determined to be C. sorghi, and the rest C. africana. A relatively low level of genetic diversity was observed within the Indian C. africana population. No evidence of genetic exchange between C. africana and C. sorghi was observed in either AFLP or DNA sequence analysis. Phylogenetic analysis was conducted using DNA sequences from 14 different Claviceps species. A multigene phylogeny based on the EF-1alpha gene intron 4, the beta-tubulin gene intron 3 region, and rDNA showed that C. sorghi grouped most closely with C. gigantea and C. africana. Although the Claviceps species we analysed were closely related, they colonize hosts that are taxonomically very distinct suggesting that there is no direct coevolution of Claviceps with its hosts.

  17. Transforming growth factors beta 1 and 2 transcriptionally regulate human papillomavirus (HPV) type 16 early gene expression in HPV-immortalized human genital epithelial cells.

    PubMed Central

    Woodworth, C D; Notario, V; DiPaolo, J A

    1990-01-01

    Human papillomavirus type 16 (HPV16) early proteins E6 and E7 have been implicated in maintenance of the malignant phenotype in cervical cancer. Transforming growth factors beta one and two (TGF betas 1 and 2), polypeptides that regulate cellular growth and differentiation, reversibly inhibited expression of the HPV16 E6 and E7 genes in several immortal genital epithelial cell lines. Loss of E6 and E7 protein expression followed a dramatic time- and dose-dependent decrease in E6 and E7 RNA levels and was accompanied by cessation of cell proliferation. TGF betas 1 and 2 inhibited HPV16 RNA expression at the transcriptional level; inhibition was dependent upon ongoing protein synthesis. TGF betas 1 and 2 also induced a six- to sevenfold increase in TGF beta 1 RNA. Cells became partially resistant to the inhibitory effects of TGF beta 1 on cell growth and HPV early gene expression after prolonged cultivation in vitro or after malignant transformation. Thus, TGF beta 1 may function as an autocrine regulator of HPV gene expression in infected genital epithelial cells. Images PMID:2168964

  18. T-cell tolerance toward a transgenic beta-cell antigen and transcription of endogenous pancreatic genes in thymus.

    PubMed Central

    Jolicoeur, C; Hanahan, D; Smith, K M

    1994-01-01

    Transgenic mice expressing T antigen (Tag) in pancreatic beta cells establish systemic tolerance toward this self-protein. The self-tolerance in two families of rat insulin promoter (RIP)-Tag mice, expressing different levels of Tag protein, has been characterized. These mice have impaired antibody responses to Tag, show diminished Tag-specific T-cell proliferation, and evidence an inability to generate Tag-specific cytotoxic T cells. The existence of systemic tolerance toward a beta-cell-specific protein motivated examination of transgene expression in the thymus. Indeed, low levels of Tag mRNA were detected intrathymically. Remarkably, this expression is a valid property of the insulin gene regulatory region, since insulin RNA was also expressed in the thymus of nontransgenic mice. RNA for other pancreatic genes was also detected in the thymus, thus raising the possibility that many tissue-specific genes could be expressed intrathymically during immunological development and induction of self-tolerance. These results raise important questions for future research into the role of the thymus in tolerance induction toward so-called tissue-specific antigens. Images PMID:8022837

  19. [C825T polymorphism of the GNB3 gene codifying the G-protein beta3-subunit and cardiovascular risk].

    PubMed

    Sartori, Michelangelo; Parotto, Emanuela; Ceolotto, Giulio; Papparella, Italia; Lenzini, Livia; Calò, Lorenzo A; Semplicini, Andrea

    2004-01-01

    Hypertension is a common disorder of multifactorial origin that constitutes a major risk factor for cardiovascular events such as stroke and myocardial infarction. The subunits of the heterotrimeric G proteins are attractive candidate gene products for both susceptibility to essential hypertension and interindividual variation in blood pressure. A polymorphism (825C/T) in exon 10 of the GNB3 gene, that encodes for the beta3 subunit, has recently been described. The 825T allele is associated with alternative splicing of the gene and formation of a truncated but functionally active beta3 subunit. Carriers of the 825T allele appear to have an increased risk for hypertension, obesity, insulin-resistance and left ventricular hypertrophy. Moreover, 825T allele carriers respond with a stronger decrease in blood pressure to therapy with a thiazide diuretic and with clonidine. GNB3 825T allele may be regarded as a potential genetic marker for a better definition of the risk profile of hypertensive subjects, but further studies are needed to precisely define the impact of T allele on the prognosis of such patients.

  20. Cloning and characterization of the major promoter of the human protein kinase C beta gene. Regulation by phorbol esters.

    PubMed

    Obeid, L M; Blobe, G C; Karolak, L A; Hannun, Y A

    1992-10-15

    The expression of the beta isoenzyme for protein kinase C is regulated developmentally and in response to inducers of cell differentiation (such as phorbol esters and 1 alpha,25-dihydroxyvitamin D3). The 5' segment of the gene for protein kinase C beta was cloned from a human leukocyte genomic library in EMBL3 bacteriophage. This segment of the gene (greater than 54 kilobases in length) encompassed the coding sequence for the amino-terminal regulatory domain of the enzyme, the 5'-untranslated region, and the 5'-flanking region. Initiation of transcription was identified by S1 nuclease analysis and confirmed by RNase protection analysis at 197 base pairs 5' of the initiator ATG. Sequence analysis of the 5'-flanking region revealed it to be extremely G+C-rich (> 80%) with many features of a CpG island. Comparison of sequence with known cis-regulatory motifs disclosed a number of potential regulatory elements including an octamer binding motif at -76, Sp1-binding sites at -94 and -63, E boxes at -110, -26, and +18, an AP-1 site at -442, and an AP-2 site at -330. To demonstrate promoter activity, a 630-base pair fragment extending from -587 to +43 was subcloned in front of a promoterless luciferase gene. This fragment was able to drive the expression of luciferase in transient transfections of human hematopoietic cells. Deletion analysis demonstrated that a fragment -111 to +43 was necessary and sufficient for promoter activity; this fragment did not contain TATA or CAAT motifs. The promoter was stimulated 8-20-fold by phorbol esters accounting for the previously observed transcriptional activation of protein kinase C beta. This phorbol ester responsiveness was conferred by the basal promoter (-111 to +43) and was independent of the AP-1 site. These results define a novel mechanism of protein kinase C autoregulation at a transcriptional level.

  1. Efficient inhibition of beta-secretase gene expression in HEK293 cells by tRNAVal-driven and CTE-helicase associated hammerhead ribozymes.

    PubMed

    Nawrot, Barbara; Antoszczyk, Slawomir; Maszewska, Maria; Kuwabara, Tomoko; Warashina, Masaki; Taira, Kazunari; Stec, Wojciech J

    2003-10-01

    The beta-amyloid peptide (Abeta) is a major component of toxic amyloid plaques found in the brains of patients with Alzheimer's disease. Abeta is liberated by sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. The level of Abeta depends directly on the hydrolytic activity of beta-secretase. Therefore, beta-secretase is an excellent target for drug design. An approach based on RNA-cleaving ribozymes was developed to control expression of beta-secretase. Two sites of mRNA coding beta-site APP cleaving enzyme were chosen as target sequences for endogenously delivered ribozymes. The ribozyme cassette was designed to constitute a catalytic hammerhead core and substrate recognition arms, flanked at the 5'-terminus by tRNAVal and at the 3'-terminus by constitutive transport element sequences. Ribozyme cassettes were cloned into a pUC19 plasmid and used for transient transfection of HEK293 cells. We demonstrate that such ribozymes efficiently inhibit beta-secretase gene expression at both the mRNA (up to 95%) and the protein (up to 90%) levels. Inhibition of beta-site APP cleaving enzyme activity directly influences the intra- and extracellular population of Abeta peptide. Therefore, such ribozymes may be considered as molecular tools for silencing the beta-secretase activity, and further, as therapeutic agents for anti-amyloid treatment.

  2. Identification of the human {beta}A2 crystallin gene (CRYBA2): Localization of the gene on human chromosome 2 and of the homologous gene on mouse chromosome 1

    SciTech Connect

    Hulsebos, T.J.M.; Cerosaletti, K.M.; Fournier, R.E.K.

    1995-08-10

    By using primers synthesized on the basis of the bovine {beta}A2 crystalline gene sequence, we amplified exons 5 and 6 of the human gene (CRYBA2). CRYBA2 was assigned to human chromosome 2 by concordance analysis in human x rodent somatic cell hybrids using the amplified PCR products as probe. Regional localization to 2q34-q36 was established by hybridizing the CRYBA2 probe to microcell and radiation hybrids containing defined fragments of chromosome 2 as the only human contribution. The CRYBA2 probe was also used to localize, by interspecific backcross mapping, the mouse gene (Cryba2) to the central portion of chromosome 1 in a region of known human chromosome 2 homology. Finally, we demonstrate that in both species the {beta}A2 crystallin gene is linked but separable from the {gamma}A crystallin gene. The {beta}A2 crystallin gene is a candidate gene for human and mouse hereditary cataract. 32 refs., 4 figs.

  3. Phylogeny of the Enterobacteriaceae based on genes encoding elongation factor Tu and F-ATPase beta-subunit.

    PubMed

    Paradis, Sonia; Boissinot, Maurice; Paquette, Nancy; Bélanger, Simon D; Martel, Eric A; Boudreau, Dominique K; Picard, François J; Ouellette, Marc; Roy, Paul H; Bergeron, Michel G

    2005-09-01

    The phylogeny of enterobacterial species commonly found in clinical samples was analysed by comparing partial sequences of their elongation factor Tu gene (tuf) and of their F-ATPase beta-subunit gene (atpD). An 884 bp fragment for tuf and an 884 or 871 bp fragment for atpD were sequenced for 96 strains representing 78 species from 31 enterobacterial genera. The atpD sequence analysis exhibited an indel specific to Pantoea and Tatumella species, showing, for the first time, a tight phylogenetic affiliation between these two genera. Comprehensive tuf and atpD phylogenetic trees were constructed and are in agreement with each other. Monophyletic genera are Cedecea, Edwardsiella, Proteus, Providencia, Salmonella, Serratia, Raoultella and Yersinia. Analogous trees based on 16S rRNA gene sequences available from databases were also reconstructed. The tuf and atpD phylogenies are in agreement with the 16S rRNA gene sequence analysis, and distance comparisons revealed that the tuf and atpD genes provide better discrimination for pairs of species belonging to the family Enterobacteriaceae. In conclusion, phylogeny based on tuf and atpD conserved genes allows discrimination between species of the Enterobacteriaceae.

  4. Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation.

    PubMed

    Zhang, Juan; Tang, Hongju; Zhang, Yuqing; Deng, Ruyuan; Shao, Li; Liu, Yun; Li, Fengying; Wang, Xiao; Zhou, Libin

    2014-05-01

    Quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important in the effort to gain insight into the molecular mechanisms underlying adipogenesis. However, the expression profile of a target gene may be misinterpreted due to the unstable expression of the reference genes under different experimental conditions. Therefore, in this study, we investigated the expression stability of 10 commonly used reference genes during 3T3-L1 adipocyte differentiation. The mRNA expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transferrin receptor (TFRC) significantly increased during the course of 3T3-L1 adipocyte differentiation, which was decreased by berberine, an inhibitor of adipogenesis. Three popular algorithms, GeNorm, NormFinder and BestKeeper, identified 18 ribosomal RNA and hydroxymethylbilane synthase (HMBS) as the most stable reference genes, while GAPDH and TFRC were the least stable ones. Peptidylprolyl isomerase A [PIPA (cyclophilin A)], ribosomal protein, large, P0 (36-B4), beta-2-microglobulin (B2M), α1-tubulin, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and β-actin showed relatively stable expression levels. The choice of reference genes with various expression stabilities exerted a profound influence on the expression profiles of 2 target genes, peroxisome proliferator-activated receptor (PPAR)γ2 and C/EBPα. In addition, western blot analysis revealed that the increased protein expression of GAPDH was markedly inhibited by berberine during adipocyte differentiation. This study highlights the importance of selecting suitable reference genes for qRT-PCR studies of gene expression during the process of adipogenesis.

  5. Cloning and Functional Analysis of a beta-amyrin synthase gene associated with oleanolic acid biosynthesis in Gentiana straminea MAXIM.

    PubMed

    Liu, Yanling; Cai, Yunfei; Zhao, Zhongjuan; Wang, Junfeng; Li, Jing; Xin, Wei; Xia, Guangmin; Xiang, Fengning

    2009-05-01

    Phytosterols and triterpenes are synthesized by oxidosqualene cyclases (OSCs) via the isoprenoid pathway. Here, GsAS1--a full-length beta-amyrin synthase cDNA isolated from Gentiana straminea MAXIM.--was characterized. Its open reading frame consists of 2268 bp, predicted to encode a 756 residue protein containing four QW and one Asp-Cys-Thr-Ala-Glu (DCTAE) motifs, which are both well conserved among known triterpene synthases. The deduced GsAS1 peptide sequence shares 76.2% homology with that of Panax ginseng beta-amyrin synthase. A phylogenetic analysis showed that GsAS1 is closely related to other plant OSCs, and particularly to the beta-amyrin synthases. When the GsAS1 sequence was heterologously expressed in Escherichia coli, an 88 kDa gene product was produced, and this reacted with the appropriate antibody. The sequence was also heterologously expressed in the Pichia pastoris yeast. GsAS1 is expressed in a tissue-specific manner, with its expression in the leaf being ca. 4.5-fold than that in the root, and nearly three-fold than that in the stem. GsAS1 expression was up-regulated by treatment with methyl jasmonate (MeJA) over a period from 6 h to 10 d post treatment. The accumulation oleanolic acid increased after induction by MeJA.

  6. Wound induced Beta vulgaris polygalacturonase-inhibiting protein genes encode a longer leucine-rich repeat domain and inhibit fungal polygalacturonases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins involved in plant defense. Sugar beet (Beta vulgaris L.) PGIP genes, BvPGIP1, BvPGIP2 and BvPGIP3, were isolated from two breeding lines, F1016 and F1010. Full-length cDNA sequences of the three BvPGIP genes encod...

  7. Contribution of enzymatic properties, cell permeability, and enzyme expression to microbiological activities of beta-lactams in three Bacteroides fragilis isolates that harbor a metallo-beta-lactamase gene.

    PubMed Central

    Rasmussen, B A; Yang, Y; Jacobus, N; Bush, K

    1994-01-01

    The metallo-beta-lactamase gene, ccrA, has been cloned from three clinical isolates of Bacteroides fragilis, TAL3636, QMCN3, and QMCN4. Although all three isolates harbored a gene encoding a potent beta-lactamase, the MICs of benzylpenicillin, piperacillin, cefotaxime, ceftazidime, imipenem, and biapenem for the three isolates varied from 4- to > 128-fold. QMCN4 was the most susceptible of the three isolates, followed by QMCN3. TAL3636 was resistant to all of the beta-lactams. Previous DNA sequence analysis of the three ccrA genes revealed that the enzymes differed at 5 amino acid residues (B. A. Rasmussen, Y. Gluzman, and F. P. Tally, Mol. Microbiol. 5:1211-1219, 1991). Biochemical characterization of the three enzymes revealed only small differences in kcat and Km values for the majority of beta-lactams tested. Thus, the 5 amino acid substitutions affected the hydrolyzing activity of the enzymes only modestly. Crypticity differences between the three isolates showed that QMCN4 was the least permeable of the isolates to cephaloridine, followed by TAL3636, and that QMCN3 was highly permeable to cephaloridine. Therefore, neither catalytic activity nor permeability was a major contributor to the dramatic differences in the MICs. Instead, microbiological susceptibility was closely related to the level of metallo-beta-lactamase present in each isolate. Both biochemical and physical studies indicated that TAL3636 produced 5- to 10-fold and 50- to 100-fold more metallo-beta-lactamase than QMCN3 and QMCN4, respectively. Therefore, the level of CcrA enzyme production is the dominant contributing factor to high-level resistance among strains harboring a ccrA gene. Images PMID:7811029

  8. Contribution of enzymatic properties, cell permeability, and enzyme expression to microbiological activities of beta-lactams in three Bacteroides fragilis isolates that harbor a metallo-beta-lactamase gene.

    PubMed

    Rasmussen, B A; Yang, Y; Jacobus, N; Bush, K

    1994-09-01

    The metallo-beta-lactamase gene, ccrA, has been cloned from three clinical isolates of Bacteroides fragilis, TAL3636, QMCN3, and QMCN4. Although all three isolates harbored a gene encoding a potent beta-lactamase, the MICs of benzylpenicillin, piperacillin, cefotaxime, ceftazidime, imipenem, and biapenem for the three isolates varied from 4- to > 128-fold. QMCN4 was the most susceptible of the three isolates, followed by QMCN3. TAL3636 was resistant to all of the beta-lactams. Previous DNA sequence analysis of the three ccrA genes revealed that the enzymes differed at 5 amino acid residues (B. A. Rasmussen, Y. Gluzman, and F. P. Tally, Mol. Microbiol. 5:1211-1219, 1991). Biochemical characterization of the three enzymes revealed only small differences in kcat and Km values for the majority of beta-lactams tested. Thus, the 5 amino acid substitutions affected the hydrolyzing activity of the enzymes only modestly. Crypticity differences between the three isolates showed that QMCN4 was the least permeable of the isolates to cephaloridine, followed by TAL3636, and that QMCN3 was highly permeable to cephaloridine. Therefore, neither catalytic activity nor permeability was a major contributor to the dramatic differences in the MICs. Instead, microbiological susceptibility was closely related to the level of metallo-beta-lactamase present in each isolate. Both biochemical and physical studies indicated that TAL3636 produced 5- to 10-fold and 50- to 100-fold more metallo-beta-lactamase than QMCN3 and QMCN4, respectively. Therefore, the level of CcrA enzyme production is the dominant contributing factor to high-level resistance among strains harboring a ccrA gene.

  9. Provocative pattern of rearrangements of the genes for the gamma and beta chains of the T-cell receptor in human leukemias.

    PubMed Central

    Goorha, R; Bunin, N; Mirro, J; Murphy, S B; Cross, A H; Behm, F G; Quertermous, T; Seidman, J; Kitchingman, G R

    1987-01-01

    To examine the distribution of rearrangements of the gamma- and beta-chain T-cell receptor (TCR) genes in T- and non-T acute lymphoblastic leukemias (ALLs), and potentially to determine which genes rearrange first in ontogeny, we analyzed high molecular weight DNA from 102 patients with acute leukemia. Rearranged gamma- and beta-chain genes were found in all T-cell ALLs (22/22) examined. Overall, 27% (18/66) of B-lineage ALLs had beta-chain gene rearrangements, and 41% (24/58) had gamma-chain gene rearrangements, but the distribution of rearranged genes varied according to the stage of B-cell differentiation. The gamma-chain genes were rearranged in 11% (1/9) of the B-lineage patients negative for the common acute lymphoblastic leukemia antigen (cALLA) and 50% (23/46) of cALLA+ ALL patients, while the beta-chain genes were not rearranged in any of the 7 cALLA- ALL patients examined but were rearranged in 32% (18/56) of the cALLA+ patients. Neither TCR gene was found to be rearranged in acute nonlymphoid leukemia patients (0/12) or in patients with B-cell (surface immunoglobulin-positive) leukemia (0/3). Of the 44 cALLA+ patients in which a direct comparison of gamma- and beta-chain gene rearrangements could be made, 34% had both genes rearranged, 16% had only gamma-chain gene rearrangements, and the remaining 50% had both genes in the germ-line configuration. beta-Chain rearrangements have not been found in the absence of gamma-chain rearrangements, thus supporting a proposed hierarchy of TCR gene rearrangements. A provocative finding was that only a small percentage (11%) of the patients with cALLA- B precursor cell ALLs had rearranged TCR genes, while 50% of the cALLA+ leukemia patients had at least gamma-chain rearrangement, raising a question as to whether indeed cALLA- cells are precursors to cALLA+ cells. Interestingly, 18% (2/11) of the cytoplasmic immunoglobulin (cIg)-positive cALLA+ (pre-B) ALLs involved TCR gene rearrangements, compared to 60% (21/35) of

  10. The Effect of Estradiol-17(beta), Goitrogen (T3), and Flutamide on Gene Expression in Medaka, Oryzias latipes

    SciTech Connect

    E.Haut, J

    2005-09-06

    Concern has been generated over the discovery of endocrine disrupting chemicals in rivers near sewage outflows. The presence of endocrine disrupting chemicals such as estradiol-17{beta} has been associated with a reduction of reproductive success in fish and an increase in the female phenotype and gonadal intersex in fish downstream of sewage treatment facilities. Such effects are believed to result from a disruption in the normal estrogenic pathways since estrogen plays a vital role in reproduction, sexual differentiation, the developments of secondary sex characteristics, and ovulation. Most studies have focused on the effect of a single endocrine disruptor on a single gene which does not provide for the interaction between genes. Microarray technology has made it possible to put an entire genome on a single chip so that researchers can get a clearer picture of the interaction of genes expressed in a cell and changes of said interactions when those cells are exposed to various conditions. Medaka males were exposed to known endocrine disruptors, estradial-17{beta} and goitrogen, and medaka females were exposed to flutamide. All treatments were then compared to controls. Total RNA was extracted from the livers of both treated and untreated males and hybridized to a microarray chip designed to have EST sequences specific to medaka. ESTs were identified through two-channel microarray analysis and compared to GenBank using blastn searches to identify up regulated genes. Choriogenins H and L, zona radiata, and vitellogenin, previously shown to be estrogen-induced in male fish were identified. Heat shock proteins (hsp70, hsp90, and hsp8) were also induced by estradiol-17{beta}, as was choriogenin Hminor. Exposure to goitrogen (T3) resulted in the induced expression of glutathione S-transferase and a GABA receptor protein in male medaka. Treatment with flutamide, an antiandrogen, caused the up regulation of choriogenin L, choriogenin Hminor, and zona radiata-2 in female

  11. An Atropa belladonna hyoscyamine 6beta-hydroxylase gene is differentially expressed in the root pericycle and anthers.

    PubMed

    Suzuki, K; Yun, D J; Chen, X Y; Yamada, Y; Hashimoto, T

    1999-05-01

    The AbH6H gene for hyoscyamine 6beta-hydroxylase (H6H), which converts hyoscyamine to scopolamine, was isolated from Atropa belladonna. This plant also possesses a related sequence, Ab psiH6H, which appears to be a non-functional pseudo-gene. AbH6H RNA was detected in cultured root, native root and anther, but not in stem, leaf, pistil, petal, and sepal tissues. In situ hybridization, immunohistochemistry and promoter::GUS transgene analysis showed that AbH6H is expressed specifically in root pericycle cells, and in tapetum and pollen mother cells. A 671 bp 5'-upstream region from AbH6H was sufficient for pericycle-specific expression in hairy roots of A. belladonna and Hyoscyamus niger, which both produce scopolamine, but cell-specific regulation was severely compromised in tobacco hairy roots, which do not produce scopolamine.

  12. Cloning, Characterization, and Molecular Application of a Beta-Agarase Gene from Vibrio sp. Strain V134▿

    PubMed Central

    Zhang, Wei-wei; Sun, Li

    2007-01-01

    V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40°C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria. PMID:17337564

  13. Cyclic stretch induces cyclooxygenase-2 gene expression in vascular endothelial cells via activation of nuclear factor kappa-{beta}

    SciTech Connect

    Zhao, Haige; Hiroi, Toyoko; Hansen, Baranda S.; Rade, Jeffrey J.

    2009-11-27

    Vascular endothelial cells respond to biomechanical forces, such as cyclic stretch and shear stress, by altering gene expression. Since endothelial-derived prostanoids, such as prostacyclin and thromboxane A{sub 2}, are key mediators of endothelial function, we investigated the effects of cyclic stretch on the expression of genes in human umbilical vein endothelial cells controlling prostanoid synthesis: cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostacyclin synthase (PGIS) and thromboxane A{sub 2} synthase (TXAS). COX-2 and TXAS mRNAs were upregulated by cyclic stretch for 24 h. In contrast, PGIS mRNA was decreased and stretch had no effect on COX-1 mRNA expression. We further show that stretch-induced upregulation of COX-2 is mediated by activation of the NF-{kappa}{beta} signaling pathway.

  14. Polymorphism of follicle stimulating hormone beta (FSHβ) subunit gene and its association with litter traits in giant panda.

    PubMed

    Huang, Xiaoyu; Li, Desheng; Wang, Jiwen; Huang, Yan; Han, Chunchun; Zhang, Guiquan; Huang, Zhi; Wu, Honglin; Wei, Ming; Wang, Guosong; Hu, Haiping; Deng, Tao; He, Tao; Zhou, Yingming; Song, Shixian; Luo, Bo; Zhang, Heming

    2013-11-01

    The different SSCP patterns of the follicle stimulating hormone beta (FSHβ) gene amplified by three pairs of primers were sequenced. Comparisons among the three nucleotide sequences of three genotypes indicated that three base substitutions (A213T, A91G, and A89C) were detected in FSHβ gene, which A213T substitution led to one amino acids mutation (Lys > Met), and the other two substitutions were synonymous mutations. The AA, AB and BB genotypes patterns obtained by FSHβ primer1 had evident relation with the litter traits, but the SSCP genotypes patterns obtained by FSHβ primer2 and primer3 had no evident relation with the litter traits in giant panda. The giant panda with AA and AB genotype had the largest litter size and multiparity rate compared with the BB genotypes (P < 0.05). We speculated that the giant pandas with the A allele have better litter traits than those with the B allele.

  15. IL-1beta stimulates argininosuccinate synthetase gene expression through NF-kappaB in Caco-2 cells.

    PubMed

    Brasse-Lagnel, Carole; Lavoinne, Alain; Fairand, Alain; Vavasseur, Karine; Husson, Annie

    2005-05-01

    Argininosuccinate synthetase (ASS) is limiting the arginine synthesis and can be stimulated by immunostimulants. We previously identified a putative NF-kappaB element in the human ASS gene promoter but its functionality was unknown (Husson et al., Eur. J. Biochem. 270 (2003) 1887). In the present study, using Caco-2 cells, a human enterocyte line, we demonstrate that IL-1beta rapidly induces the expression of the ASS gene at a transcriptional level through NF-kappaB activation. Using gel shift assay and double-strand oligonucleotide sequence of the identified putative NF-kappaB binding site of the ASS promoter, we provide evidence that NF-kappaB may functionally interact with this element.

  16. Linker scanning mutagenesis of the 5'-flanking region of the mouse beta-major-globin gene: sequence requirements for transcription in erythroid and nonerythroid cells.

    PubMed

    Charnay, P; Mellon, P; Maniatis, T

    1985-06-01

    We analyzed the sequences required for transcription of the mouse beta-major-globin gene by introducing deletion and linker scanning mutations into the 5'-flanking region and then studying the effects of these mutations on beta-globin gene transcription in a HeLa cell transient expression assay or after stable introduction into mouse erythroleukemia cells. Consistent with earlier studies, we found that three distinct regions upstream from the RNA capping site are required for efficient beta-globin gene transcription in HeLa cells: the ATA box located 30 base pairs upstream from the mRNA capping site (-30), the CCAAT box located at -75, and the distal sequence element CCACACCC located at -90. In the ATA and CAAT box regions, the sequences necessary for efficient transcription extend beyond the limits of the canonical sequences. Mutations in the sequences located between the three transcriptional control elements do not significantly affect transcription in HeLa cells. Although the promoter defined in HeLa cell transfection experiments is also required for efficient transcription in mouse erythroleukemia cells, none of the mutations tested affects the regulation of beta-globin gene transcription during mouse erythroleukemia cell differentiation. Thus, DNA sequences downstream from the mRNA cap site appear to be sufficient for the regulation of beta-globin gene expression during the differentiation of mouse erythroleukemia cells in culture.

  17. RNA-silencing in Penicillium chrysogenum and Acremonium chrysogenum: validation studies using beta-lactam genes expression.

    PubMed

    Ullán, Ricardo V; Godio, Ramiro P; Teijeira, Fernando; Vaca, Inmaculada; García-Estrada, Carlos; Feltrer, Raúl; Kosalkova, Katarina; Martín, Juan F

    2008-10-01

    In this work we report the development and validation of a new RNA interference vector (pJL43-RNAi) containing a double-stranded RNA expression cassette for gene silencing in the filamentous fungi Penicillium chrysogenum and Acremonium chrysogenum. Classical targeted gene disruption in these fungi is very laborious and inefficient due to the low frequency of homologous recombination. The RNAi vector has been validated by testing the attenuation of two different genes of the beta-lactam pathway; pcbC in P. chrysogenum and cefEF in A. chrysogenum. Quantification of mRNA transcript levels and antibiotic production showed knockdown of pcbC and cefEF genes in randomly isolated transformants of P. chrysogenum and A. chrysogenum, respectively. The process is efficient; 15 to 20% of the selected transformants were found to be knockdown mutants showing reduced penicillin or cephalosporin production. This new RNAi vector opens the way for exploring gene function in the genomes of P. chrysogenum and A. chrysogenum.

  18. Extended spectrum beta-lactamase and fluoroquinolone resistance genes and plasmids among Escherichia coli isolates from zoo animals, Czech Republic.

    PubMed

    Dobiasova, Hana; Dolejska, Monika; Jamborova, Ivana; Brhelova, Eva; Blazkova, Lucie; Papousek, Ivo; Kozlova, Marketa; Klimes, Jiri; Cizek, Alois; Literak, Ivan

    2013-09-01

    Commensal Escherichia coli isolates from healthy zoo animals kept in Ostrava Zoological Garden, Czech Republic, were investigated to evaluate the dissemination of extended-spectrum beta-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes. A total of 160 faecal samples of various animal species were inoculated onto MacConkey agar with cefotaxime (2 mg L(-1)) or ciprofloxacin (0.05 mg L(-1)) to obtain ESBL- or PMQR-positive E. coli isolates. Clonality of E. coli isolates was investigated by multilocus sequence typing and pulsed-field gel electrophoresis. Plasmids carrying ESBL or PMQR genes were typed by PCR-based replicon typing, plasmid multilocus sequence typing and restriction fragment length polymorphism. Forty-nine (71%, n = 69) cefotaxime-resistant and 15 (16%, n = 94) ciprofloxacin-resistant E. coli isolates harboured ESBL or PMQR genes. Isolates were assigned to 18 sequence types (ST) and 20 clusters according to their macrorestriction patterns by pulsed-field gel electrophoresis. The genes blaCTX -M-1 and qnrS1 were detected on highly related IncI1 plasmids assigned to clonal complex 3 (ST3, ST38) and on non-related IncN plasmids of ST1 and ST3, respectively. The gene qnrS1 was located on related IncX1 plasmids. Dissemination of antibiotic resistance is associated with spreading of particular E. coli clones and plasmids of specific incompatibility groups among various animal species.

  19. Gene expression and molecular phylogenetic analyses of beta-glucosidase in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae).

    PubMed

    Shimada, Keisuke; Maekawa, Kiyoto

    2014-06-01

    Beta-glucosidase (BG) is known as a multifunctional enzyme for social maintenance in terms of both cellulose digestion and social communication in termites. However, the expression profiles of each BG gene and their evolutionary history are not well understood. First, we cloned two types of BG homologs (RsBGI and RsBGII) from the termite Reticulitermes speratus (Kolbe). Gene expression analyses showed that RsBGI expression levels of primary queens and kings from 30 to 100 days after colony foundation were high, but those of reproductives dropped after day 400. Extremely low gene expression levels of RsBGI were observed in eggs, whereas workers had significantly higher expression levels than those of soldiers and other colony members. Consequently, RsBGI gene expression levels changed among each developmental stage, and RsBGI was shown to be involved in cellulose digestion. On the other hand, the RsBGII gene was consistently expressed in all castes and developmental stages examined, and notable expression changes were not observed among them, including in eggs. It was indicated that RsBGII is a main component involved in social communication, for example, the egg-recognition pheromone shown in this species previously. Finally, we obtained partial gene homologs from other termite and cockroach species, including the woodroach (genus Cryptocercus), which is the sister group to termites, and performed molecular phylogenetic analyses. The results showed that the origin of the BG gene homologs preceded the divergence of termites and cockroaches, suggesting that the acquisition of multifunctionality of the BG gene also occurred in cockroach lineages.

  20. The cotton-top tamarin (Saguinus oedipus) has five beta-microseminoprotein genes, two of which are pseudogenes.

    PubMed

    Valtonen-André, Camilla; Lundwall, Ake

    2008-01-01

    beta-Microseminoprotein (MSP) is one of the most abundant proteins in human seminal plasma and is secreted from the prostate gland. Its evolution can be traced from primates down to nonvertebrate species such as amphioxus, despite substantial differences in the primary structure. Most mammals are known to have one single MSP gene, but we have previously shown that the cotton-top tamarin and the common marmoset-two New World monkeys-carry several MSP genes. In this study we continue our characterization of MSP genes in the cotton-top tamarin by presenting the full nucleotide sequence of the three previously identified genes, mspA, mspE, and mspJ. A promoter analysis using the luciferase reporter showed that mspE is as transcriptionally active as the single human MSP gene, whereas mspA and mspJ display no activity with this assay. Two novel MSP genes were also identified, mspB and mspH, both of which are pseudogenes. MspB has a frameshift mutation in the third exon resulting in a new C-terminus and premature stop of translation. MspH has the features of a processed pseudogene, originating from a transcript of mspE. It is integrated into the genome together with another processed pseudogene originating from a transcript of the nucleoporin gene NUP88. The MSP genes described in this study probably arose by phylogenetically rather late duplication or retrotransposition, suggesting that they are confined to a limited number of New World monkeys.

  1. A Polymorphism Within the Promoter of the TGF{beta}1 Gene Is Associated With Radiation Sensitivity Using an Objective Radiologic Endpoint

    SciTech Connect

    Kelsey, Chris R.; Jackson, Lauren; Langdon, Scott; Owzar, Kouros; Hubbs, Jessica; Vujaskovic, Zeljko; Das, Shiva; Marks, Lawrence B.

    2012-02-01

    Purpose: To evaluate whether single nucleotide polymorphisms (SNPs) in the transforming growth factor-{beta}1 (TGF{beta}1) gene are associated with radiation sensitivity using an objective radiologic endpoint. Methods and Materials: Preradiation therapy and serial postradiation therapy single photon emission computed tomography (SPECT) lung perfusion scans were obtained in patients undergoing treatment for lung cancer. Serial blood samples were obtained to measure circulating levels of TGF{beta}1. Changes in regional perfusion were related to regional radiation dose yielding a patient-specific dose-response curve, reflecting the patient's inherent sensitivity to radiation therapy. Six TGF{beta}1 SNPs (-988, -800, -509, 869, 941, and 1655) were assessed using high-resolution melting assays and DNA sequencing. The association between genotype and slope of the dose-response curve, and genotype and TGF{beta}1 ratio (4-week/preradiation therapy), was analyzed using the Kruskal-Wallis test. Results: 39 white patients with preradiation therapy and {>=}6-month postradiation therapy SPECT scans and blood samples were identified. Increasing slope of the dose-response curve was associated with the C(-509)T SNP (p = 0.035), but not the other analyzed SNPs. This SNP was also associated with higher TGF{beta}1 ratios. Conclusions: This study suggests that a polymorphism within the promoter of the TGF{beta}1 gene is associated with increased radiation sensitivity (defined objectively by dose-dependent changes in SPECT lung perfusion).

  2. Derepression of Polycomb targets during pancreatic organogenesis allows insulin-producing beta-cells to adopt a neural gene activity program

    PubMed Central

    van Arensbergen, Joris; García-Hurtado, Javier; Moran, Ignasi; Maestro, Miguel Angel; Xu, Xiaobo; Van de Casteele, Mark; Skoudy, Anouchka L.; Palassini, Matteo; Heimberg, Harry; Ferrer, Jorge

    2010-01-01

    The epigenome changes that underlie cellular differentiation in developing organisms are poorly understood. To gain insights into how pancreatic beta-cells are programmed, we profiled key histone methylations and transcripts in embryonic stem cells, multipotent progenitors of the nascent embryonic pancreas, purified beta-cells, and 10 differentiated tissues. We report that despite their endodermal origin, beta-cells show a transcriptional and active chromatin signature that is most similar to ectoderm-derived neural tissues. In contrast, the beta-cell signature of trimethylated H3K27, a mark of Polycomb-mediated repression, clusters with pancreatic progenitors, acinar cells and liver, consistent with the epigenetic transmission of this mark from endoderm progenitors to their differentiated cellular progeny. We also identified two H3K27 methylation events that arise in the beta-cell lineage after the pancreatic progenitor stage. One is a wave of cell-selective de novo H3K27 trimethylation in non-CpG island genes. Another is the loss of bivalent and H3K27me3-repressed chromatin in a core program of neural developmental regulators that enables a convergence of the gene activity state of beta-cells with that of neural cells. These findings reveal a dynamic regulation of Polycomb repression programs that shape the identity of differentiated beta-cells. PMID:20395405

  3. Cloning, characterization, and nucleotide sequence of a gene encoding Microbispora bispora BglB, a thermostable beta-glucosidase expressed in Escherichia coli.

    PubMed Central

    Wright, R M; Yablonsky, M D; Shalita, Z P; Goyal, A K; Eveleigh, D E

    1992-01-01

    Genomic DNA fragments encoding beta-glucosidase activities of the thermophilic actinomycete Microbispora bispora were cloned into Escherichia coli. Transformants expressing beta-glucosidase activity were selected by their ability to hydrolyze the fluorogenic substrate 4-methylumbelliferyl-beta-D-glucoside. Two genes encoding beta-glucosidase activity were isolated and distinguished by restriction analysis, Southern hybridization, and the substrate specificities of the encoded enzymes. One gene, bglB, encoded a beta-glucosidase that was expressed intracellularly in E. coli. It exhibited a molecular mass of approximately 52,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and 51,280 Da by nondenaturing gradient PAGE, a pI of 4.6, and temperature and pH optima of 60 degrees C and 6.2, respectively. Cloned BglB showed greater activity against cellobiose than against aryl-beta-D-glucosides and was thermostable, retaining about 70% of its activity after 48 h at 60 degrees C. BglB activity is activated two- to threefold in the presence of 2 to 5% (0.1 to 0.3 M) glucose. The DNA sequence of the 2.2-kb insert carrying bglB has been determined. An open reading frame which codes for a protein of 473 amino acids with a predicted molecular mass of 52,227 Da showed significant homology (40 to 47% identity) with beta-glucosidases from glycosal hydrolase family 1. Images PMID:1482172

  4. Induction of stable prenatal tolerance to beta-galactosidase by in utero gene transfer into preimmune sheep fetuses.

    PubMed

    Tran, N D; Porada, C D; Almeida-Porada, G; Glimp, H A; Anderson, W F; Zanjani, E D

    2001-06-01

    The successful transduction of hematopoietic stem cells and long-term (28 months) transgene expression within the hematopoietic system following the direct injection of high-titer retroviral vectors into preimmune fetal sheep was previously demonstrated. The present studies extended these analyses for 40 months postinjection and evaluated whether the longevity of transgene expression in this model system was the result of induction of prenatal tolerance to the transgene product. The intraperitoneal injection of retroviral vectors into preimmune sheep fetuses transduces thymic epithelial cells thought to present antigen and thus define self during immune system development. To directly demonstrate induction of tolerance, postnatal sheep were boosted with purified beta-galactosidase and showed that the peripheral blood lymphocytes from in utero-transduced sheep exhibited significantly lower stimulation indices to transduced autologous cells than did control animals and that the in utero-transduced sheep had a reduced ability to mount an antibody response to the vector-encoded beta-galactosidase protein compared with control sheep. Collectively, our results provide evidence that the direct injection of retroviral vectors into preimmune sheep fetuses induces cellular and humoral tolerance to the vector/transgene products and provide an explanation for the duration and stability of transgene expression seen in this model. These results also suggest that even relatively low levels of gene transfer in utero may render the recipient tolerant to the exogenous gene and thus potentially permit the successful postnatal treatment of the recipient. (Blood. 2001;97:3417-3423)

  5. apl-1, a Caenorhabditis elegans gene encoding a protein related to the human beta-amyloid protein precursor.

    PubMed Central

    Daigle, I; Li, C

    1993-01-01

    The major component of senile plaques found in the brains of Alzheimer disease patients is the beta-amyloid peptide, which is derived from a larger amyloid precursor protein (APP). Recently, a number of APP and APP-related proteins have been identified in different organisms and constitute the family of APP proteins. We have isolated several cDNAs encoding an APP-related protein in the nematode Caenorhabditis elegans and have designated the corresponding gene as apl-1. The apl-1 transcripts undergo two forms of posttranscriptional modification: trans-splicing and alternative polyadenylylation. In vitro translation of an apl-1 cDNA results in a protein of approximately the expected size. Similar to the Drosophila, human, and mouse APP-related proteins, APL-1 does not appear to contain the beta-amyloid peptide. Because APP-related proteins seem to be conserved through evolution, the apl-1 gene from C. elegans should be important for determining the normal function of human APP. Images Fig. 2 Fig. 3 PMID:8265668

  6. Frequency of Hereditary Hemochromatosis (HFE) Gene Mutations in Egyptian Beta Thalassemia Patients and its Relation to Iron Overload

    PubMed Central

    Enein, Azza Aboul; El Dessouky, Nermine A.; Mohamed, Khalda S.; Botros, Shahira K.A.; Abd El Gawad, Mona F.; Hamdy, Mona; Dyaa, Nehal

    2016-01-01

    AIM: This study aimed to detect the most common HFE gene mutations (C282Y, H63D, and S56C) in Egyptian beta thalassemia major patients and its relation to their iron status. SUBJECTS AND METHODS: The study included 50 beta thalassemia major patients and 30 age and sex matched healthy persons as a control group. Serum ferritin, serum iron and TIBC level were measured. Detection of the three HFE gene mutations (C282Y, H63D and S65C) was done by PCR-RFLP analysis. Confirmation of positive cases for the mutations was done by sequencing. RESULTS: Neither homozygote nor carrier status for the C282Y or S65C alleles was found. The H63D heterozygous state was detected in 5/50 (10%) thalassemic patients and in 1/30 (3.3%) controls with no statistically significant difference between patients and control groups (p = 0.22). Significantly higher levels of the serum ferritin and serum iron in patients with this mutation (p = 001). CONCLUSION: Our results suggest that there is an association between H63D mutation and the severity of iron overload in thalassemic patients. PMID:27335591

  7. Development a diagnostic pan-dermatophyte TaqMan probe real-time PCR assay based on beta tubulin gene.

    PubMed

    Mirhendi, Hossein; Motamedi, Marjan; Makimura, Koichi; Satoh, Kazuo

    2016-08-01

    Early differentiation of dermatophytosis from other cutaneous mycoses is essential to avoid inaccurate therapy. DNA-based techniques including real-time PCR have increasingly been considered for detection of fungal elements in clinical specimens. In this study, after partial sequence analysis of beta tubulin (BT2) gene in 13 common and rare pathogenic dermatophyte species, a pan-dermatophyte primer and probe set was designed in a TaqMan probe-based PCR format. The sensitivity and specificity of the system was tested with 22 reference strains of dermatophytes, 234 positive clinical specimens, 32 DNA samples extracted from normal nails, several fungi other than dermatophytes and human DNAs. Analytical detection limit of the designed PCR on serially diluted DNAs of prepared recombinant plasmid indicated that only five molecules per sample are the minimum number for reliable detection by the assay. A total of 226 out of 234 (96.5%) DNAs extracted from clinical samples, but none of the 32 nail samples, from healthy volunteers were positive in PCR. The real-time PCR targeted beta tubulin gene established in this study could be a sensitive diagnostic tool which is significantly faster than the conventional culture method and should be useful in the clinical settings, in large-scale epidemiological studies and in clinical trials of antifungal therapy.

  8. Overexpression of transforming growth factor-. beta. in transgenic mice carrying the human T-cell lymphotropic virus type I tax gene

    SciTech Connect

    Kim, Seongjin; Winokur, T.S.; Lee, Hyde; Danielpour, D.; Kim, Kyung Young; Geiser, A.G.; Sporn, M.B.; Roberts, A.B. ); Chen, Liansheng; Jay, G. )

    1991-10-01

    Human T-cell lymphotropic virus type I (HTLV-I) has been associated with an adult form of T-cell leukemia as well as tropical spastic paraparesis, a neurodegenerative disease. Adult T-cell leukemia patients express high levels of the type 1 isoform of transforming growth factor-{beta} (TGF-{beta}1), which is mediated by the effects of the HTLV-I Tax transactivator protein on the TGF-{beta}1 promoter. To understand further the regulation of TGF-{beta}1 expression by Tax, the authors examined its expression in transgenic mice carrying the HTLV-I tax gene. They show that tumors from these mice and other tissues, such as submaxillary glands and skeletal muscle, which express high levels of tax mRNA selectively express high levels of TGF-{beta}1 mRNA and protein. Moreover, TGF-{beta}1 significantly stimulated the incorporation of tritiated thymidine into one of three cells lines derived from neurofibromas of tax-transgenic mice, which suggest that the excessive production of TGF-{beta}1 may play a role in tumorigenesis and that these mice may serve as a useful model for studying the biological effects of TGF-{beta} in vivo.

  9. Two beta-glycanase genes are clustered in Bacillus polymyxa: molecular cloning, expression, and sequence analysis of genes encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase.

    PubMed

    Gosalbes, M J; Pérez-González, J A; González, R; Navarro, A

    1991-12-01

    Two genes, xynD and gluB, encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase (lichenase) from Bacillus polymyxa have been cloned and expressed in Escherichia coli and Bacillus subtilis. A sequenced DNA fragment of 4,466 bp contains both genes, which are separated by 155 bp. The xynD and gluB genes encode proteins of 67.8 kDa (XYND) and 27 kDa (GLUB). Two peptides with molecular masses of 62 and 53 kDa appear in cell extracts of E. coli and culture supernatants of B. subtilis clones containing the xynD gene. Both peptides show xylanase activity in zymogram analysis. The XYND enzyme also shows alpha-L-arabinofuranosidase activity. The XYND peptide and the xylanase XYNZ from Clostridium thermocellum (O. Grépinet, M. C. Chebrou, and P. Béguin, J. Bacteriol. 170:4582-4588, 1988) show 64% homology in a stretch of about 280 amino acids.

  10. Two beta-glycanase genes are clustered in Bacillus polymyxa: molecular cloning, expression, and sequence analysis of genes encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase.

    PubMed Central

    Gosalbes, M J; Pérez-González, J A; González, R; Navarro, A

    1991-01-01

    Two genes, xynD and gluB, encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase (lichenase) from Bacillus polymyxa have been cloned and expressed in Escherichia coli and Bacillus subtilis. A sequenced DNA fragment of 4,466 bp contains both genes, which are separated by 155 bp. The xynD and gluB genes encode proteins of 67.8 kDa (XYND) and 27 kDa (GLUB). Two peptides with molecular masses of 62 and 53 kDa appear in cell extracts of E. coli and culture supernatants of B. subtilis clones containing the xynD gene. Both peptides show xylanase activity in zymogram analysis. The XYND enzyme also shows alpha-L-arabinofuranosidase activity. The XYND peptide and the xylanase XYNZ from Clostridium thermocellum (O. Grépinet, M. C. Chebrou, and P. Béguin, J. Bacteriol. 170:4582-4588, 1988) show 64% homology in a stretch of about 280 amino acids. Images FIG. 3 PMID:1938968

  11. Regulatory elements in the first intron contribute to transcriptional regulation of the beta 3 tubulin gene by 20-hydroxyecdysone in Drosophila Kc cells.

    PubMed Central

    Bruhat, A; Tourmente, S; Chapel, S; Sobrier, M L; Couderc, J L; Dastugue, B

    1990-01-01

    We have studied the transcriptional regulation of the beta 3 tubulin gene by the steroid hormone 20-hydroxyecdysone (20-OH-E) in Drosophila Kc cells. A series of hybrid genes with varying tubulin gene lengths driving the bacterial chloramphenicol acetyl transferase (CAT) gene were constructed. The promoter activity was assayed after transient expression in Kc cells, in the presence or absence of 20-OH-E. We find that 0.91Kb upstream from the transcription start site contain one or several hormone independent positive cis-acting elements, responsible for the constitutive expression of the beta 3 tubulin gene. In the large (4.5 Kb) first intron of this gene, we identified additional hormone dependent negative and positive regulatory elements, which can act in both directions and in a position-independence manner. Then, the negative intron element(s), which repress the transcription in the absence of 20-OH-E has characteristics of silencer. Images PMID:2349088

  12. Disruption of the gene encoding the latent transforming growth factor-beta binding protein 4 (LTBP-4) causes abnormal lung development, cardiomyopathy, and colorectal cancer.

    PubMed

    Sterner-Kock, Anja; Thorey, Irmgard S; Koli, Katri; Wempe, Frank; Otte, Jürgen; Bangsow, Thorsten; Kuhlmeier, Katharina; Kirchner, Thomas; Jin, Shenchu; Keski-Oja, Jorma; von Melchner, Harald

    2002-09-01

    Transforming growth factor-betas (TGF-betas) are multifunctional growth factors that are secreted as inactive (latent) precursors in large protein complexes. These complexes include the latency-associated propeptide (LAP) and a latent transforming growth factor-beta binding protein (LTBP). Four isoforms of LTBPs (LTBP-1-LTBP-4) have been cloned and are believed to be structural components of connective tissue microfibrils and local regulators of TGF-beta tissue deposition and signaling. By using a gene trap strategy that selects for integrations into genes induced transiently during early mouse development, we have disrupted the mouse homolog of the human LTBP-4 gene. Mice homozygous for the disrupted allele develop severe pulmonary emphysema, cardiomyopathy, and colorectal cancer. These highly tissue-specific abnormalities are associated with profound defects in the elastic fiber structure and with a reduced deposition of TGF-beta in the extracellular space. As a consequence, epithelial cells have reduced levels of phosphorylated Smad2 proteins, overexpress c-myc, and undergo uncontrolled proliferation. This phenotype supports the predicted dual role of LTBP-4 as a structural component of the extracellular matrix and as a local regulator of TGF-beta tissue deposition and signaling.

  13. Microarray analysis of liver gene expression in iron overloaded patients with sickle cell anemia and beta-thalassemia.

    PubMed

    Flanagan, Jonathan M; Steward, Shirley; Hankins, Jane S; Howard, Thad M; Neale, Geoffrey; Ware, Russell E

    2009-06-01

    Chronic transfusion therapy is used clinically to supply healthy erythrocytes for patients with sickle cell anemia (SCA) or beta-thalassemia major (TM). Despite the benefits of red blood cell transfusions, chronic transfusions lead to iron accumulation in key tissues such as the heart, liver, and endocrine glands. Transfusion-acquired iron overload is recognized as a cause of morbidity and mortality among patients receiving chronic transfusions. At present, there is little understanding of molecular events that occur during transfusional iron loading and the reasons for the large inter-individual variation observed clinically in transfusion-acquired iron accumulation. To address these issues, we examined whether any liver-expressed genes in SCA or TM patients with transfusional iron overload were associated with the degree of iron accumulation. Specifically, we performed microarray analysis on liver biopsy specimens comparing SCA patients with mild or severe iron overload and also compared SCA with TM patients. Fifteen candidate genes were identified with significantly differential expression between the high and low liver iron concentrations. SCA patients and 20 candidate genes were detected between the SCA and TM patient comparison. Subsequent quantitative PCR experiments validated 12 candidate genes; with GSTM1, eIF5a, SULF2, NTS, and HO-1 being particularly good prospects as genes that might affect the degree of iron accumulation. Future work will determine the baseline expression of these genes prior to transfusional iron overload and elucidate the full impact of these genes on the inter-individual variation observed clinically in transfusion-acquired iron accumulation.

  14. Gene cloning, homology comparison and analysis of the main functional structure domains of beta estrogen receptor in Jining Gray goat.

    PubMed

    Liu, Hai-gang; Li, Hong-mei; Wang, Shu-ying; Huang, Li-bo; Guo, Hui-jun

    2014-08-01

    To clarify the molecular evolution and characteristic of beta estrogen receptor (ERβ) gene in Jining Gray goat in China, the entire ERβ gene from Jining Gray goat ovary was amplified, identified and sequenced, and the gene sequences were compared with those of other animals. Functional structural domains and variations in DNA binding domains (DBD) and ligand binding domains (LBD) between Jining Gray goat and Boer goat were analyzed. The results indicate that the ERβ gene in Jining Gray goat includes a 1584bp sequence with a complete open-reading-frame (ORF), encoding a 527 amino acid (aa) receptor protein. Compared to other species, the nucleotide homology is 73.9-98.9% and the amino acid homology is 79.5-98.5%. The main antigenic structural domains lie from the 97th aa to the 286th aa and from the 403rd aa to the 527th aa. The hydrophilicity and the surface probability of the structural domains are distributed throughout a range of amino acids. There are two different amino acids in the DBD and three different amino acids in the LBD between Jining Gray and Boer goats, resulting in dramatically different spatial structures for ERβ protein. These differences may explain the different biological activities of ERβ between the two goat species. This study firstly acquired the whole ERβ gene sequence of Jining Gray goat with a complete open reading frame, and analyzed its gene evolutionary relationship and predicted its mainly functional structural domains, which may very help for further understanding the genome evolution and gene diversity of goat ERβ.

  15. Efficient and persistent expression of beta-glucuronidase gene in CD34+ cells from human umbilical cord blood by retroviral vector.

    PubMed

    Ohashi, T; Iizuka, S; Sly, W S; Machiki, K; Eto, Y

    1998-10-01

    We succeeded in efficiently transferring the beta-glucuronidase gene in a retroviral vector to human hematopoietic progenitor cells using a centrifugation enhancement protocol. The transduction efficiency in CFU-GM was highly variable (23-100%) with an average of 66.8%. In the case of BFU-E, efficiency was 83% and 76% in 2 separate experiments. In LTCIC (long-term culture-initiating cell), transduction efficiency were 20% and 50% in 2 experiments. The enzymatic activity of beta-glucuronidase in transduced cells were increased above the control level up to 5 wk. Considering that correction of the enzyme deficiency in a small number of hematopoietic cells can be therapeutic for the Sly disease mouse, our data provide encouragement that human trials of gene therapy based on transferring beta-glucuronidase gene to hematopoietic cells may be efficacious.

  16. Rat mandibular distraction osteogenesis: II. Molecular analysis of transforming growth factor beta-1 and osteocalcin gene expression.

    PubMed

    Mehrara, B J; Rowe, N M; Steinbrech, D S; Dudziak, M E; Saadeh, P B; McCarthy, J G; Gittes, G K; Longaker, M T

    1999-02-01

    Distraction osteogenesis is a powerful technique capable of generating viable osseous tissue by the gradual separation of osteotomized bone edges. Although the histologic and ultrastructural changes associated with this process have been extensively delineated, the molecular events governing these changes remain essentially unknown. We have devised a rat model of mandibular distraction osteogenesis that facilitates molecular analysis of this process. Such information has significant clinical implications because it may enable targeted therapeutic manipulations designed to accelerate osseous regeneration. In this study, we have evaluated the expression of transforming growth factor beta-1, a major regulator of osteogenesis during endochondral bone formation and development, and osteocalcin, an abundant noncollagenous extracellular matrix protein implicated in the regulation of mineralization and bone turnover. The right hemimandible of 36 adult male rats was osteotomized, and a customized distraction device was applied. Animals were allowed to recover and, after a 3-day latency period, were distracted at a rate of 0.25 mm twice daily for 6 days followed by a 2- or 4-week consolidation period. Distraction regenerate was harvested after the latency period, days 2, 4, or 6 of distraction, and after 2 or 4 weeks of consolidation and processed for Northern analysis (n = 4 at each time point) and immunohistochemical localization of TGF-beta1 (n = 2 at each time point). Six sham-operated animals (i.e., skin incision without osteotomy) were also killed (immediately postoperatively), and the mandibles were harvested and prepared in a similar fashion. Equal loading and transfer of RNA for Northern analysis was ensured by stripping and probing membranes with a probe against GAPDH (a housekeeping gene). Our results demonstrate that the spatial and temporal patterns of TGF-beta1 mRNA expression and protein production coincide with osteoblast migration, differentiation, and

  17. Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor beta pathway components.

    PubMed Central

    Savage, C; Das, P; Finelli, A L; Townsend, S R; Sun, C Y; Baird, S E; Padgett, R W

    1996-01-01

    Although transforming growth factor beta (TGF-beta) superfamily ligands play critical roles in diverse developmental processes, how cells transduce signals from these ligands is still poorly understood. Cell surface receptors for these ligands have been identified, but their cytoplasmic targets are unknown. We have identified three Caenorhabditis elegans genes, sma-2, sma-3, and sma-4, that have mutant phenotypes similar to those of the TGF-beta-like receptor gene daf-4, indicating that they are required for daf-4-mediated developmental processes. We show that sma-2 functions in the same cells as daf-4, consistent with a role in transducing signals from the receptor. These three genes define a protein family, the dwarfins, that includes the Mad gene product, which participates in the decapentaplegic TGF-beta-like pathway in Drosophila [Sekelsky, J. J., Newfeld, S. J., Raftery, L. A., Chartoff, E. H. & Gelbart, W. M. (1995) Genetics 139, 1347-1358]. The identification of homologous components of these pathways in distantly related organisms suggests that dwarfins may be universally required for TGF-beta-like signal transduction. In fact, we have isolated highly conserved dwarfins from vertebrates, indicating that these components are not idiosyncratic to invertebrates. These analyses suggest that dwarfins are conserved cytoplasmic signal transducers. Images Fig. 1 PMID:8570636

  18. Integration and gene replacement in the Lactococcus lactis lac operon: induction of a cryptic phospho-beta-glucosidase in LacG-deficient strains.

    PubMed Central

    Simons, G; Nijhuis, M; de Vos, W M

    1993-01-01

    Insertions, replacement mutations, and deletions were introduced via single or double crossover recombination into the lacE (enzyme IIlac) and lacG (phospho-beta-galactosidase) genes of the Lactococcus lactis chromosomal lacABCDFEGX operon. LacG production was abolished in strains missing the lacG gene or carrying multicopy insertions in the lacE gene that affected expression of the lacG gene. However, these LacG-deficient strains could still ferment lactose slowly and were found to contain an enzymatic activity that hydrolyzed the chromogenic substrate o-nitrophenyl-beta-D-galactopyranoside phosphate. Induction of this phospho-beta-glycohydrolase activity coincided with the appearance of a new 55-kDa protein cross-reacting with anti-LacG antibodies that had a size similar to that of LacG but a higher isoelectric point (pI 5.2) and was not found in wild-type cells during growth on lactose. Since the phospho-beta-glycohydrolase activity and this protein with a pI of 5.2 were highly induced in both mutant and wild-type cells during growth on cellobiose that is likely to be transported via a phosphoenolpyruvate-dependent phosphotransferase system, we propose that this induced activity is a phospho-beta-glucosidase that also hydrolyzes lactose-6-phosphate. Images PMID:8349556

  19. Transforming growth factor-beta/Smads signaling induces transcription of the cell type-restricted ankyrin repeat protein CARP gene through CAGA motif in vascular smooth muscle cells.

    PubMed

    Kanai, H; Tanaka, T; Aihara, Y; Takeda, S; Kawabata, M; Miyazono, K; Nagai, R; Kurabayashi, M

    2001-01-19

    Transforming growth factor (TGF)-beta plays a major role in the development of vascular diseases. Despite the pleiotropic effects of TGF-ss on vascular smooth muscle cells (VSMCs), only a few genes have been characterized as direct targets of TGF-beta in VSMCs. Cardiac ankyrin repeat protein (CARP) has been thought to be expressed exclusively in the heart. In the present study, we showed that CARP is expressed in the vasculature after balloon injury and in cultured VSMCs in response to TGF-beta. Analysis of a half-life of the cytoplasmic CARP mRNA levels and the transient transfection of the CARP promoter/luciferase gene indicates that the regulation of CARP expression is increased by TGF-beta at the transcriptional level. Transfection of expression vectors encoding Smads significantly activated the CARP promoter/luciferase activity. Deletion analysis and site-specific mutagenesis of the CARP promoter indicate that TGF-beta response element is localized to CAGA motif at -108 bp relative to the transcription start site. Electrophoretic mobility shift assays showed that the binding activity to the CAGA motif was increased in nuclear extracts of cultured VSMCs by TGF-beta. Cells transfected with adenovirus vector expressing CARP showed a significant decrease in DNA synthesis. Overexpression of CARP enhanced the TGF-beta-mediated inhibition of the DNA synthesis. These data indicate that CARP is a downstream target of TGF-beta/Smad signaling in VSMCs and suggest a role of CARP in mediation of the inhibitory effects of TGF-beta on the proliferation of VSMCs.

  20. DNA methylation profiles in the human genes for tumor necrosis factors. alpha. and. beta. in subpopulations of leukocytes and in leukemias

    SciTech Connect

    Kochanek, S.; Radbruch, A.; Tesch, H.; Renz, D.; Doerfler, W. )

    1991-07-01

    The genomic sequencing technique has been applied to assess the state of methylation in the DNA from human leukocyte subpopulations from healthy individuals and in the DNA from several individuals with myeloid or lymphatic leukemias or non-Hodgkin lymphomas. Leukocyte populations were purified by the high-gradient magnetic cell sorting technique. In the human tumor necrosis factor {alpha} (TNF-{alpha}) gene segment between nucleotides 300 and 1150, the specific methylation profile in the DNA from human granulocytes and monocytes is maintained in three cases of myeloid leukemia. In the TNF-{beta} gene, DNA methylation is decreased in several examples of acute or chronic myeloid leukemias in comparison to normal human granulocytes or monocytes, whose DNA is almost completely methylated between nucleotides 700 and 900. In human T and B lymphocytes, the main producers of TNF-{beta}, in three instances of chronic lymphatic leukemias and two cases of non-Hodgkin lymphomas, all 5{prime}-CG-3{prime} sequences are unmethylated in this region. The DNA from the human HeLa cell line is highly methylated at all 5{prime}-CG-3{prime} sequences in the TNF-{alpha} and -{beta} genes. The TNF-{alpha} gene is transcribed in the cells of one case of acute myeloid leukemia in which the analyzed region of the TNF-{alpha} gene is completely unmethylated. The TNF-{beta} gene is not transcribed in any of the malignant cells tested.

  1. Functional screening yields a new beta-1,4-endoglucanase gene from Heterodera glycines that may be the product of recent gene duplication.

    PubMed

    Yan, Y; Smant, G; Davis, E

    2001-01-01

    Clones with secreted cellulolytic activity were identified when a cDNA library constructed from poly A(+) RNA of preparasitic second-stage juveniles of Heterodera glycines, the soybean cyst nematode, was expressed in the Escherichia coli SOLR strain and overlaid with a carboxymethylcellulose (CMC) substrate. Twenty CMC-degrading clones were analyzed, and all were either identical or strongly similar to a beta-1,4-endoglucanase gene (HG-eng-2), previously isolated from H. glycines. A subgroup of identical "HG-eng-2-like" clones had considerable differences in the 5' untranslated region compared with HG-eng-2 and were designated HG-eng-3. One H. glycines genomic clone contained HG-eng-2 and HG-eng-3 full-length genes, separated by a distance of approximately 8 kb, and a second genomic clone contained two copies of HG-eng-2, separated by approximately 6.5 kb, suggesting the presence of endoglucanase gene clusters in H. glycines. The HG-eng-2 and HG-eng-3 genes were in opposite transcriptional orientation, with considerable nucleotide differences in their 5' flanking regions. The highly conserved nucleotide sequence in the introns and exons and their close proximity within the genome suggest that HG-eng-2 and HG-eng-3 are the products of recent gene duplication and inversion.

  2. Calcium/calmodulin-dependent protein kinase type IV is a target gene of the Wnt/beta-catenin signaling pathway.

    PubMed

    Arrázola, Macarena S; Varela-Nallar, Lorena; Colombres, Marcela; Toledo, Enrique M; Cruzat, Fernando; Pavez, Leonardo; Assar, Rodrigo; Aravena, Andrés; González, Mauricio; Montecino, Martín; Maass, Alejandro; Martínez, Servet; Inestrosa, Nibaldo C

    2009-12-01

    Calcium/calmodulin-dependent protein kinase IV (CaMKIV) plays a key role in the regulation of calcium-dependent gene expression. The expression of CaMKIV and the activation of CREB regulated genes are involved in memory and neuronal survival. We report here that: (a) a bioinformatic analysis of 15,476 promoters of the human genome predicted several Wnt target genes, being CaMKIV a very interesting candidate; (b) CaMKIV promoter contains TCF/LEF transcription motifs similar to those present in Wnt target genes; (c) biochemical studies indicate that lithium and the canonical ligand Wnt-3a induce CaMKIV mRNA and protein expression levels in rat hippocampal neurons as well as CaMKIV promoter activity; (d) treatment of hippocampal neurons with Wnt-3a increases the binding of beta-catenin to the CaMKIV promoter: (e) In vivo activation of the Wnt signaling improve spatial memory impairment and restores the expression of CaMKIV in a mice double transgenic model for Alzheimer's disease which shows decreased levels of the kinase. We conclude that CaMKIV is regulated by the Wnt signaling pathway and that its expression could play a role in the neuroprotective function of the Wnt signaling against the Alzheimer's amyloid peptide.

  3. Short- and long-term changes in sugarbeet (Beta vulgaris L.) gene expression due to postharvest jasmonic acid treatment - Data.

    PubMed

    de Oliveira, Lucilene Silva; Fugate, Karen Klotz; Ferrareze, Jocleita Perruzo; Bolton, Melvin D; Deckard, Edward L; Finger, Fernando L

    2017-04-01

    Jasmonic acid is a natural plant hormone that induces native defense responses in plants. Sugarbeet (Beta vulgaris L.) root unigenes that were differentially expressed 2 and 60 days after a postharvest jasmonic acid treatment are presented. Data include changes in unigene expression relative to water-treated controls, unigene annotations against nonredundant (Nr), Swiss-Prot, Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) protein databases, and unigene annotations with Gene Ontology (GO) terms. Putative defense unigenes are compiled and annotated against the sugarbeet genome. Differential gene expression data were generated by RNA sequencing. Interpretation of the data is available in the research article, "Jasmonic acid causes short- and long-term alterations to the transcriptome and the expression of defense genes in sugarbeet roots" (K.K. Fugate, L.S. Oliveira, J.P. Ferrareze, M.D. Bolton, E.L. Deckard, F.L. Finger, 2017) [1]. Public dissemination of this dataset will allow further analyses of the data.

  4. Computational identification and characterization of conserved miRNAs and their target genes in beet (Beta vulgaris).

    PubMed

    Li, J L; Cui, J; Cheng, D Y

    2015-08-07

    Highly conserved endogenous non-coding microRNAs (miRNAs) play important roles in plants and animals by silencing genes via destruction or blocking of translation of homologous mRNA. Sugar beet, Beta vulgaris, is one of the most important sugar crops in China, with properties that include wide adaptability and strong tolerance to salinity and impoverished soils. Seedlings of B. vulgaris can grow in soils containing up to 0.6% salt; it is important to understand the molecular mechanisms of salt tolerance to enrich genetic resources for breeding salt-tolerant sugar beets. Here, we report 13 mature miRNAs from 12 families, predicted using an in silico approach from 29,857 expressed sequence tags and 279,223 genome survey sequences. The psRNATarget server predicted 25 target genes for the 13 miRNAs. Most of the target genes appeared to encode transcription factors or were involved in metabolism, signal transduction, stress response, growth, and development. These results improve our understanding of the molecular mechanisms of miRNA in beet and may aid in the development of novel and precise techniques for understanding post-transcriptional gene-silencing mechanisms in response to stress tolerance.

  5. Polymorphism analysis in estrogen receptors alpha and beta genes and their association with infertile population in Pakistan

    PubMed Central

    Liaqat, Sinha; Hasnain, Shahida; Muzammil, Saima; Hayat, Sumreen

    2015-01-01

    Studies on polymorphism of estrogen receptor (ESR) alpha and beta genes have been mostly implicated in infertility, but the results have been controversial due to lack of comprehensive data. The present study focused on association of ESR genes with both male and female infertility. In ESRα, PvuII (rs2234693) and XbaI (rs9340799) were studied while in ESRβ gene, risk of infertility was determined for silent G/A RsaI (rs1256049) polymorphism. Total 124 subjects (74 cases and 50 controls) were part of this study having primary infertility. Restriction fragment length polymorphism (RFLP) was performed with PvuII, XbaI and RsaI to determine polymorphism. Correlation between age and follicle stimulating hormone (FSH) of cases and controls was determined and no association was found between infertility and FSH hormone. Heterozygous AG genotype of XbaI polymorphism (P= 2.505e-06) and heterozygous TC genotype (P= 0.00003) in PvuII polymorphism were strongly associated with risk of infertility. In ESRβ gene, there was lack of polymorphism for RsaI in our population as all subjects were homozygous (GG). Haplotype frequencies showed that XbaI and PvuII polymorphisms are in strong linkage disequilibrium. This study shows that in our population XbaI and PvuII polymorphisms of ESRα are associated with risk of infertility. PMID:27065769

  6. The Polymorphisms of Ser49Gly and Gly389Arg in Beta-1-Adrenergic Receptor Gene in Major Depression

    PubMed Central

    KOKUT, Süleyman; ATAY, İnci Meltem; UZ, Efkan; AKPINAR, Abdullah; DEMİRDAŞ, Arif

    2015-01-01

    Introduction It was reported that the genetic susceptibility of major depressive disorder (MDD) is related with genetic polymorphisms. The aim of this study was to investigate the possible association of the genotype and allele frequencies of Ser49Gly and Arg389Gly polymorphisms in MDD by comparing them with healthy subjects. Methods A total of 144 patients with MDD diagnosed according to Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV) criteria and 105 healthy controls were included in the study. Polymerase chain reaction (PCR) with restriction fragment length polymorphism (RFLP) was used for genotyping. Results Of the 144 participants in the MDD group, 77 (53.5%) had homozygous wild type (AA), 57 (39.6%) had heterozygous type (AG), and 10 (6.9%) had mutant (GG) genotype for Ser49Gly, whereas 75 (52.1%) had homozygous wild type (GG), 59 (41.0%) had heterozygous (GC) type, and 10 (6.9%) had mutant homozygous (CC) genotype for Gly386Arg. There were no significant difference in the allele and genotype frequencies of the beta-1-adrenergic receptor (ADRB1) gene for Ser49Gly and Arg389Gly polymorphisms after comparing with healthy controls (p=0.626; p=0.863 and p=0.625; p=0.914). Conclusion The results of our study did not reveal a major effect of the polymorphism of Ser49Gly and Gly389Arg in the ADRB1 gene in MDD. Further studies with larger sample size are required to elucidate the role of other beta-1 adrenergic gene polymorphisms in MDD. PMID:28360691

  7. Expression of human. alpha. -globin and mouse/human hybrid. beta. -globin genes in murine hemopoietic stem cells transduced by recombinant retroviruses

    SciTech Connect

    Li, C.L.; Dwarki, V.J.; Verma, I.M. )

    1990-06-01

    Murine cell lines releasing helper-free recombinant retroviruses containing human {alpha}-globin and mouse/human hybrid {beta}-globin genes were generated. The expression of the hybrid {beta}-globin gene but not the human {alpha}-globin gene was regulated appropriately in infected mouse erythroid leukemia (MEL) cells. Murine bone marrow cells were infected by coculture with virus-producing cells and transplanted into lethally irradiated syngeneic recipients. Greater than 90% of the spleen colonies (12-15 days), which are derived from hemopoietic multipotential stem cells, showed proviral integration. Various levels of expression of the transduced globin genes were detected in all of the provirus-positive spleen colonies. Proviral sequences and transcripts from the transduced globin genes could also be detected in a few long-term reconstituted recipients in an observation period of 10 months after transplantation.

  8. MDA5 and PTPN2, two candidate genes for type 1 diabetes, modify pancreatic beta-cell responses to the viral by-product double-stranded RNA.

    PubMed

    Colli, Maikel L; Moore, Fabrice; Gurzov, Esteban N; Ortis, Fernanda; Eizirik, Decio L

    2010-01-01

    beta-Cell destruction in type 1 diabetes (T1D) is at least in part consequence of a 'dialog' between beta-cells and immune system. This dialog may be affected by the individual's genetic background. We presently evaluated whether modulation of MDA5 and PTPN2, two candidate genes for T1D, affects beta-cell responses to double-stranded RNA (dsRNA), a by-product of viral replication. These genes were selected following comparison between known candidate genes for T1D and genes expressed in pancreatic beta-cells, as identified in previous array analysis. INS-1E cells and primary fluorescence-activated cell sorting-purified rat beta-cells were transfected with small interference RNAs (siRNAs) targeting MDA5 or PTPN2 and subsequently exposed to intracellular synthetic dsRNA (polyinosinic-polycitidilic acid-PIC). Real-time RT-PCR, western blot and viability assays were performed to characterize gene/protein expression and viability. PIC increased MDA5 and PTPN2 mRNA expression, which was inhibited by the specific siRNAs. PIC triggered apoptosis in INS-1E and primary beta-cells and this was augmented by PTPN2 knockdown (KD), although inhibition of MDA5 did not modify PIC-induced apoptosis. In contrast, MDA5 silencing decreased PIC-induced cytokine and chemokine expression, although inhibition of PTPN2 induced minor or no changes in these inflammatory mediators. These findings indicate that changes in MDA5 and PTPN2 expression modify beta-cell responses to dsRNA. MDA5 regulates inflammatory signals, whereas PTPN2 may function as a defence mechanism against pro-apoptotic signals generated by dsRNA. These two candidate genes for T1D may thus modulate beta-cell apoptosis and/or local release of inflammatory mediators in the course of a viral infection by acting, at least in part, at the pancreatic beta-cell level.

  9. Inhibin beta E is upregulated by drug-induced endoplasmic reticulum stress as a transcriptional target gene of ATF4

    SciTech Connect

    Brüning, Ansgar Matsingou, Christina; Brem, German Johannes; Rahmeh, Martina; Mylonas, Ioannis

    2012-10-15

    Inhibins and activins are gonadal peptide hormones of the transforming growth factor-β super family with important functions in the reproductive system. By contrast, the recently identified inhibin βE subunit, primarily expressed in liver cells, appears to exert functions unrelated to the reproductive system. Previously shown downregulation of inhibin βE in hepatoma cells and anti-proliferative effects of ectopic inhibin βE overexpression indicated growth-regulatory effects of inhibin βE. We observed a selective re-expression of the inhibin βE subunit in HepG2 hepatoblastoma cells, MCF7 breast cancer cells, and HeLa cervical cancer cells under endoplasmic reticulum stress conditions induced by tunicamycin, thapsigargin, and nelfinavir. Analysis of XPB1 splicing and ATF4 activation revealed that inhibin βE re-expression was associated with induction of the endoplasmic reticulum stress reaction by these drugs. Transfection of an ATF4 expression plasmid specifically induced inhibin βE expression in HeLa cells and indicates inhibin βE as a hitherto unidentified target gene of ATF4, a key transcription factor of the endoplasmic reticulum stress response. Therefore, the inhibin βE subunit defines not only a new player but also a possible new marker for drug-induced endoplasmic reticulum stress. -- Highlights: ► Endoplasmic reticulum stress induces inhibin beta E expression. ► Inhibin beta E is regulated by the transcription factor ATF4. ► Inhibin beta E expression can be used as a marker for drug-induced ER stress.

  10. Site-specific deletions involving the tal-1 and sil genes are restricted to cells of the T cell receptor alpha/beta lineage: T cell receptor delta gene deletion mechanism affects multiple genes

    PubMed Central

    1993-01-01

    Site-specific deletions in the tal-1 gene are reported to occur in 12- 26% of T cell acute lymphoblastic leukemias (T-ALL). So far two main types of tal-1 deletions have been described. Upon analysis of 134 T- ALL we have found two new types of tal-1 deletions. These four types of deletions juxtapose the 5' part of the tal-1 gene to the sil gene promoter, thereby deleting all coding sil exons but leaving the coding tal-1 exons undamaged. The recombination signal sequences (RSS) and fusion regions of the tal-1 deletion breakpoints strongly resemble the RSS and junctional regions of immunoglobulin/T cell receptor (TCR) gene rearrangements, which implies that they are probably caused by the same V(D)J recombinase complex. Analysis of the 134 T-ALL suggested that the occurrence of tal-1 deletions is associated with the CD3 phenotype, because no tal-1 deletions were found in 25 TCR-gamma/delta + T-ALL, whereas 8 of the 69 CD3- T-ALL and 11 of the 40 TCR-alpha/beta + T-ALL contained such a deletion. Careful examination of all TCR genes revealed that tal-1 deletions exclusively occurred in CD3- or CD3+ T- ALL of the alpha/beta lineage with a frequency of 18% in T-ALL with one deleted TCR-delta allele, and a frequency of 34% in T-ALL with TCR- delta gene deletions on both alleles. Therefore, we conclude that alpha/beta lineage commitment of the T-ALL and especially the extent of TCR-delta gene deletions determines the chance of a tal-1 deletion. This suggests that tal-1 deletions are mediated via the same deletion mechanism as TCR-delta gene deletions. PMID:8459224

  11. Molecular Cloning and Sequencing of Hemoglobin-Beta Gene of Channel Catfish, Ictalurus Punctatus Rafinesque

    Technology Transfer Automated Retrieval System (TEKTRAN)

    : Hemoglobin-y gene of channel catfish , lctalurus punctatus, was cloned and sequenced . Total RNA from head kidneys was isolated, reverse transcribed and amplified . The sequence of the channel catfish hemoglobin-y gene consists of 600 nucleotides . Analysis of the nucleotide sequence reveals one o...

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  13. Fusion of platelet-derived growth receptor {beta} to a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation

    SciTech Connect

    Golub, T.; Barker, G.; Gilliland, D.G.

    1994-09-01

    Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome characterized by abnormal clonal myeloid proliferation, and by progression to acute myelogenous leukemia (AML). A recently recognized subgroup of CMML has a t(5;12) (q33;p13) balanced translocation. Fluorescence in situ hybridization (FISH) localized the translocation breakpoint near the CSF1 receptor (CSF1R) locus on chromosome 5q. Pulsed-field gel electrophoresis confirmed rearrangements near CSF1R, but involvement of CSF1R itself was excluded. Southern blotting showed a rearrangement within the closely linked PDGF receptor {beta} (PDGFR{beta}) gene. Ribonuclease protection assays localized the translocation breakpoint to nucleotide 1766 in PDGFR{beta} RNA. Anchored PCR was used to identify the chromosome 12 fusion partner, a novel ets-like protein, tel. Tel contains a highly conserved carboxy terminal ets-like DNA-binding domain, and an amino terminal domain with a predicted helix-loop-helix (HLH) secondary structure. The consequence of the t(5;12) translocation is fusion of the tel HLH domain to the PDGFR{beta} transmembrane and tyrosine kinase domains. The tel HLH domain may contribute a dimerization motif which serves to constitutively activate PDGFR{beta} tyrosine kinase activity. The tel-PDGFR{beta} fusion demonstrates the oncogenic potential of PDGFR{beta}, and may provide a paradigm for early events in the pathogenesis of AML.

  14. The beta-globin gene cluster haplotypes in sickle cell anemia patients from Northeast Brazil: a clinical and molecular view.

    PubMed

    Adorno, Elisângela Vitória; Zanette, Angela; Lyra, Isa; Souza, Cyntia Cajado; Santos, Leandro Ferraz; Menezes, Joelma Figueiredo; Dupuit, Marie France; Almeida, Mari Ney Tavares; Reis, Mitermayer Galvão; Gonçalves, Marilda Souza

    2004-08-01

    The beta(S)-globin haplotypes were studied in 78 sickle cell Brazilian patients from Bahia, Northeast Brazil, that has a large population of African origin. Hemoglobin (Hb) profiles were developed by high-performance liquid chromatography (HPLC), and beta(S)-globin gene haplotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. We identified 44 (55.0%) patients with the CAR/Ben (Central African Republic/Benin) genotype, 16 (20.0%) Ben/Ben, 13 (16.2%) CAR/CAR and seven (8.8%) with other genotypes. Analyses of the phenotypes showed clinical differences related only to Hb F levels and blood transfusion therapy; the presence of -alpha(-3.7)-thalassemia (thal) demonstrated statistical significance when associated with hematocrit (p=0.044), MCV (p=0.0007), MCH (p=0.012) and spleen sequestration events. The haplotype diversity found in the present study can be justified by information about the origin of the slave traffic period in Bahia during the 19th century. The specific characteristics described among the Bahian sickle cell patients could be confirmed by increasing the number of patients with specific genotypes and further studies of genetic markers.

  15. Exploring divergent antibiotic resistance genes in ancient metagenomes and discovery of a novel beta-lactamase family.

    PubMed

    Rascovan, Nicolás; Telke, Amar; Raoult, Didier; Rolain, Jean Marc; Desnues, Christelle

    2016-08-12

    Antibiotic resistance in pathogenic bacteria is a major problem for human health. We analyzed metagenomic datasets from ancient and remote samples from diverse environmental sources and observed the presence of all the eleven antibiotic resistance genes (ARG) groups evaluated. Since ancient samples are not subjected to modern effects of antibiotic misuse, they represent a clean model to explore the natural diversity of ARG in the environment. Most sequences showed high divergence compared with known ARG, representing a much larger universe than the currently known and characterized ARGs. We explored whether proteins within the "divergent resistome" may correspond to functional ARG by characterizing a beta-lactamase hit with very low similarity to any known sequence (<45% to best BLAST hit in NCBI). By starting from purely in-silico data, we revived a new family of class B beta-lactamases from ancient medieval samples, which exhibited a very high penicillinase activity. In this work, we explored ancient resistomes and added novel support to previous works showing that the universe of ARG is naturally vast and diverse in microbial communities. Our results bring a new perspective to the exploration of environmental ARG and indicate that this gigantic reservoir represents a natural endless source of emerging resistances.

  16. SH2B1beta (SH2-Bbeta) enhances expression of a subset of nerve growth factor-regulated genes important for neuronal differentiation including genes encoding urokinase plasminogen activator receptor and matrix metalloproteinase 3/10.

    PubMed

    Chen, Linyi; Maures, Travis J; Jin, Hui; Huo, Jeffrey S; Rabbani, Shafaat A; Schwartz, Jessica; Carter-Su, Christin

    2008-02-01

    Previous work showed that the adapter protein SH2B adapter protein 1beta (SH2B1) (SH2-B) binds to the activated form of the nerve growth factor (NGF) receptor TrkA and is critical for both NGF-dependent neurite outgrowth and maintenance. To identify SH2B1beta-regulated genes critical for neurite outgrowth, we performed microarray analysis of control PC12 cells and PC12 cells stably overexpressing SH2B1beta (PC12-SH2B1beta) or the dominant-negative SH2B1beta(R555E) [PC12-SH2B1beta(R555E)]. NGF-induced microarray expression of Plaur and Mmp10 genes was greatly enhanced in PC12-SH2B1beta cells, whereas NGF-induced Plaur and Mmp3 expression was substantially depressed in PC12-SH2B1beta(R555E) cells. Plaur, Mmp3, and Mmp10 are among the 12 genes most highly up-regulated after 6 h of NGF. Their protein products [urokinase plasminogen activator receptor (uPAR), matrix metalloproteinase 3 (MMP3), and MMP10] lie in the same pathway of extracellular matrix degradation; uPAR has been shown previously to be critical for NGF-induced neurite outgrowth. Quantitative real-time PCR analysis revealed SH2B1beta enhancement of NGF induction of all three genes and the suppression of NGF induction of all three when endogenous SH2B1 was reduced using short hairpin RNA against SH2B1 and in PC12-SH2B1beta(R555E) cells. NGF-induced levels of uPAR and MMP3/10 and neurite outgrowth through Matrigel (MMP3-dependent) were also increased in PC12-SH2B1beta cells. These results suggest that SH2B1beta stimulates NGF-induced neuronal differentiation at least in part by enhancing expression of a specific subset of NGF-sensitive genes, including Plaur, Mmp3, and/or Mmp10, required for neurite outgrowth.

  17. Familial hypertrophic cardiomyopathy. Microsatellite haplotyping and identification of a hot spot for mutations in the beta-myosin heavy chain gene.

    PubMed

    Dausse, E; Komajda, M; Fetler, L; Dubourg, O; Dufour, C; Carrier, L; Wisnewsky, C; Bercovici, J; Hengstenberg, C; al-Mahdawi, S

    1993-12-01

    Familial hypertrophic cardiomyopathy (FHC) is a clinically and genetically heterogeneous disease. The first identified disease gene, located on chromosome 14q11-q12, encodes the beta-myosin heavy chain. We have performed linkage analysis of two French FHC pedigrees, 720 and 730, with two microsatellite markers located in the beta-myosin heavy chain gene (MYO I and MYO II) and with four highly informative markers, recently mapped to chromosome 14q11-q12. Significant linkage was found with MYO I and MYO II in pedigree 720, but results were not conclusive for pedigree 730. Haplotype analysis of the six markers allowed identification of affected individuals and of some unaffected subjects carrying the disease gene. Two novel missense mutations were identified in exon 13 by direct sequencing, 403Arg-->Leu and 403Arg-->Trp in families 720 and 730, respectively. The 403Arg-->Leu mutation was associated with incomplete penetrance, a high incidence of sudden deaths and severe cardiac events, whereas the consequences of the 403Arg-->Trp mutation appeared less severe. Haplotyping of polymorphic markers in close linkage to the beta-myosin heavy chain gene can, thus, provide rapid analysis of non informative pedigrees and rapid detection of carrier status. Our results also indicate that codon 403 of the beta-myosin heavy chain gene is a hot spot for mutations causing FHC.

  18. The Saccharomyces cerevisiae SPR1 gene encodes a sporulation-specific exo-1,3-beta-glucanase which contributes to ascospore thermoresistance.

    PubMed Central

    Muthukumar, G; Suhng, S H; Magee, P T; Jewell, R D; Primerano, D A

    1993-01-01

    A number of genes have been shown to be transcribed specifically during sporulation in Saccharomyces cerevisiae, yet their developmental function is unknown. The SPR1 gene is transcribed during only the late stages of sporulation. We have sequenced the SPR1 gene and found that it has extensive DNA and protein sequence homology to the S. cerevisiae EXG1 gene which encodes an exo-1,3-beta-glucanase expressed during vegetative growth (C. R. Vasquez de Aldana, J. Correa, P. San Segundo, A. Bueno, A. R. Nebrada, E. Mendez, and F. del Ray, Gene 97:173-182, 1991). We show that spr1 mutant cells do not hydrolyze p-nitrophenyl-beta-D-glucoside or laminarin in a whole-cell assay for exo-1,3-beta-glucanases. In addition to the absence of this enzymatic activity, spr1 mutant spores exhibit reduced thermoresistance relative to isogenic wild-type spores. These observations are consistent with the notion that SPR1 encodes a sporulation-specific exo-1,3-beta-glucanase. Images PMID:8419289

  19. Comparative analysis of the locus control region of the rabbit beta-like gene cluster: HS3 increases transient expression of an embryonic epsilon-globin gene.

    PubMed Central

    Hardison, R; Xu, J; Jackson, J; Mansberger, J; Selifonova, O; Grotch, B; Biesecker, J; Petrykowska, H; Miller, W

    1993-01-01

    The rabbit homolog to the locus control region (LCR) of the human beta-like globin gene cluster was isolated, and long segments containing the DNase I hypersensitive sites (HS) were sequenced. The order and spacing of HS4, HS3, HS2 and HS1 are conserved between rabbit and human. Alignment of these sequences with their homologs from human, goat, and mouse shows that very long segments of DNA match between species, for over a thousand base pairs on either side of the previously identified functional cores, indicating that some important functions are found outside the cores. The activity of rabbit HS2 and HS3 was tested by attaching each to a novel reporter gene constructed by inserting the luciferase coding region into the rabbit epsilon-globin gene. In contrast to previous reports showing no effect of human or mouse HS3 on transient expression, both the rabbit HS2 and HS3 DNA fragments separately increased transient expression from the epsilon-luciferase hybrid gene and expression from stably integrated constructs in K562 erythroleukemia cells. PMID:8464710

  20. Definition of the minimal requirements within the human beta-globin gene and the dominant control region for high level expression.

    PubMed Central

    Collis, P; Antoniou, M; Grosveld, F

    1990-01-01

    The human beta-globin dominant control region (DCR) was previously identified as a region from the 5' end of the human beta-globin locus which directs high level, site of integration-independent, copy number-dependent expression on a linked human beta-globin gene in transgenic mice and stably transfected mouse erythroleukaemia (MEL) cells. We have now analysed the elements comprising the DCR by systematic deletion mutagenesis in stable MEL transfectants. We have identified two independent elements within the DNase I hypersensitive sites 2 and 3, containing fragments which direct strong transcriptional inducibility of a beta-globin gene. Whilst the remaining two hypersensitive sites do not direct significant transcriptional induction, our data suggest that all four sites may be necessary for the fully regulated expression conferred by the DCR. We have also tested a number of beta-globin minigene constructs under the control of the DCR to assess if any of the local sequences from the gene may be removed without loss of expression. We find that the 3' enhancer may be removed without affecting expression, but there is an absolute requirement for the presence of the second intron, not related to the enhancer present in that intron. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2295312

  1. Wide dissemination of Pseudomonas aeruginosa producing beta-lactamase blaKPC-2 gene in Colombia.

    PubMed

    Cuzon, Gaelle; Naas, Thierry; Villegas, Maria-Virginia; Correa, Adriana; Quinn, John P; Nordmann, Patrice

    2011-11-01

    Ten bla(KPC-2)-harboring Pseudomonas aeruginosa isolates from hospitals located in five different Colombian cities have been characterized. Isolates were multidrug resistant, belonged to five different pulsotypes, and possessed naturally chromosome-encoded bla(AmpC) and bla(OXA-50) genes and the acquired bla(KPC-2) gene. In most cases, the bla(KPC-2) genes were carried by plasmids of different sizes and were associated with Tn4401b or a new structure containing only part of the Tn4401 sequence. This study revealed that several clones of P. aeruginosa producing bla(KPC-2) are disseminating in Colombia.

  2. Mutations in cystathionine beta-synthase or methylenetetrahydrofolate reductase gene increase N-homocysteinylated protein levels in humans.

    PubMed

    Jakubowski, Hieronim; Boers, Godfried H J; Strauss, Kevin A

    2008-12-01

    Severely elevated plasma homocysteine (Hcy) levels observed in genetic disorders of Hcy metabolism are associated with pathologies in multiple organs and lead to premature death due to vascular complications. In addition to elevating plasma Hcy, mutations in cystathionine beta-synthase (CBS) or methylenetetrahydrofolate reductase (MTHFR) gene lead to markedly elevated levels of circulating Hcy-thiolactone. The thiooester chemistry of Hcy-thiolactone underlies its ability to form isopeptide bonds with protein lysine residues (N-Hcy-protein), which may impair or alter the protein's function. However, it was not known whether genetic deficiencies in Hcy metabolism affect N-Hcy-protein levels in humans. Here we show that plasma N-Hcy-protein levels are significantly elevated in CBS- and MTHFR-deficient patients. We also show that CBS-deficient patients have significantly elevated plasma levels of prothrombotic N-Hcy-fibrinogen. These results provide a possible explanation for increased atherothrombosis observed in CBS-deficient patients.

  3. Cloning and sequencing of beta-1,4-endoglucanase gene (celA) from Pseudomonas sp. YD-15.

    PubMed

    Her, S; Lee, H S; Choi, S J; Choi, S W; Choi, H J; Yoon, S S; Oh, D H

    1999-12-01

    A beta-1,4-endoglucanase gene (celA) from Pseudomonas sp. YD-15 was cloned in Escherichia coli DH5 alpha and its nucleotide sequence determined. The open reading frame of celA was 1830 base pairs and the enzyme was composed of 609 amino acids with a molecular weight of 63,617 Da. The deduced amino acid sequence and putative active site of CelA had high amino acid homology with family E cellulases. By dot blot analysis, the induction of celA according to carbon sources was determined. The transcripts hybridizing to the internal fragment of celA were detected in total RNA isolated from Pseudomonas sp. YD-15 cells grown on avicel and glycerol, but not from cells grown on glucose and cellobiose.

  4. Expression of gibberellin 3 beta-hydroxylase gene in a gravi-response mutant, weeping Japanese flowering cherry

    NASA Technical Reports Server (NTRS)

    Sugano, Mami; Nakagawa, Yuriko; Nyunoya, Hiroshi; Nakamura, Teruko

    2004-01-01

    Expressions of the gibberellin biosynthesis gene were investigated in a normal upright type and a gravi-response mutant, a weeping type of Japanese flowering cherry (Prunus spachiana), that is unable to support its own weight and elongates downward. A segment of the gibberellin 3 beta-hydroxylase cDNA of Prunus spachiana (Ps3ox), which is responsible for active gibberellin synthesis, was amplified by using real-time RT-PCR. The content of Ps3ox mRNA in the weeping type was much greater than that in the upright type, while the endogenous gibberellin level was much higher in the elongating zone of the weeping type. These results suggest that the amount and distribution of synthesized gibberellin regulate secondary xylem formation, and the unbalanced distribution of gibberellin affects the gravi-response of the Prunus tree.

  5. Two mutations in the transforming growth factor beta-induced gene associated with familial Lattice corneal dystrophy

    PubMed Central

    Cao, Wen-Ping; Yuan, Hai-Gang; Liu, Ping; Li, Xue; Hu, Qi

    2017-01-01

    AIM To report a phenotypic variant pedigree of lattice corneal dystrophy (LCD) associated with two mutations, R124C and A546D, in the transforming growth factor beta-induced gene (TGFBI). METHODS A detailed ocular examination was taken for all participants of a LCD family. Peripheral blood leukocytes from each participant were extracted to obtain the DNA. Polymerase chain reaction (PCR) of all seventeen exons of TGFBI gene was performed. The products were sequenced and analyzed. Histological examination was carried out after a penetrating keratoplasty from the right eye of proband. RESULTS Genetic analysis showed that the proband and all 6 affected individuals harbored both a heterozygous CGC to TGC mutation at codon 124 and a heterozygous GCC to GAC mutation at codon 546 of TGFBI. None of the 100 control subjects and unaffected family members was positive for these two mutations. Ocular examination displayed multiple refractile lattice-like opacities in anterior stroma of the central cornea and small granular deposits in the peripheral cornea. The deposits were stained positively with Congo red indicating be amyloid in nature and situated mainly in the anterior and middle stroma. CONCLUSION We observed a novel LCD family which carried two pathogenic mutations (R124C and A546D) in the TGFBI gene. The phenotypic features were apparently different from those associated with corresponding single mutations. The result reveals that although the definite mutation is the most important genetic cause of the disease, some different modifier alleles may influence the phenotype.

  6. Expression, purification and characterization of zinc-finger nuclease to knockout the goat beta-lactoglobulin gene.

    PubMed

    Song, Yujie; Cui, Chenchen; Zhu, Hongmei; Li, Qian; Zhao, Fan; Jin, Yaping

    2015-08-01

    Engineered zinc-finger nucleases (ZFNs) have been widely used for precise genome editing. ZFNs can induce DNA double-strand breaks at specific genomic locations and drive the introduction of an insertion or deletion of base pairs at the targeted region, consequently resulting in a loss-of-function mutation. In this study, we investigated the cloning, expression and purification of ZFN fusion proteins targeting the goat beta-lactoglobulin (BLG) gene and detected the cleavage activities of these ZFN proteins in vitro and in cells, respectively. The results showed that the pET-BLG-LFN and pET-BLG-RFN prokaryotic expression plasmids can be constructed correctly and expressed efficiently in Escherichia coli BL21 (DE3) cells to produce the 6× His-tagged ZFN proteins that can be purified by Ni-IDA-Sefinose Column. The predetermined sequence of BLG can be recognized and excised both in vitro and in goat fibroblasts by the purified ZFN fusion proteins, which demonstrated that the purified ZFN fusion proteins can be used as gene modification tools to knock out the BLG gene. Furthermore, these results lay the foundation for eliminating allergen BLG from goat milk and improving the quality of goat milk products.

  7. Morbidity, beta S haplotype and alpha-globin gene patterns among sickle cell anemia patients in Kuwait.

    PubMed

    Adekile, A D; Haider, M Z

    1996-01-01

    Admission records of children with sickle cell anemia (SS), in the two main teaching hospitals in Kuwait, were reviewed for a 1-year period. The haplotypes of 92 beta s chromosomes (from 39 SS, 11 AS, 2 S beta-thalassemia [S beta-thal] and 1 SD individuals) were determined using an allele-specific oligonucleotide (ASO) hybridization technique, while the alpha-globin gene status of 27 SS and 33 AS individuals, i.e. 120 chromosomes, was determined with a combination of polymerase chain reaction and AS techniques. A vasooclusive crisis was the most common (60.0%) cause of hospitalization, followed by infections (20%). Hospital admissions were most common during the hottest month of the year (July). Few complications of the disease were seen among patients on follow-up; however, splenomegaly was present in 24.0%, hepatomegaly in 15.2%, gallstones in 15.2% and aseptic necrosis of the femoral head in 6.1%. Haplotype 31 (Saudi Arabia/India) is the most frequent in this community, being present in 80.4% of the chromosomes tested; Benin haplotype 19 was found in 12.0% and Bantu haplotype 20 in 6.5%. Hb F in the haplotype 31 homozygotes and heterozygotes ranged from 11.4 to 35.1% (mean 22.5 +/- 5.2%). The frequency of alpha-thal determinants in the study was 40.0%, the commonest being the -alpha 3.7-kb deletion (27.5%), the alpha 2 polyadenylation signal (AATAAA-> AATAAG) mutation (10.2%) and the IVS-I 5' end GAGGT-GAGG->GAGG pentanucleotide (5 nt) deletion (3.3%). SS patients with coexistent alpha-thal trait did not have severe recurrent infections and none had gallstones. The high frequencies of the Saudi Arabia/India beta s haplotype and alpha-thalassemia trait contribute to the mild nature of SS disease among Kuwaiti Arabs comparable to that in eastern Saudi Arabia.

  8. Molecular analysis of the beta-glucuronidase gene: novel mutations in mucopolysaccharidosis type VII and heterogeneity of the polyadenylation region.

    PubMed

    Vervoort, R; Buist, N R; Kleijer, W J; Wevers, R; Fryns, J P; Liebaers, I; Lissens, W

    1997-04-01

    We used polymerase chain reaction (PCR)/single-strand conformation polymorphism analysis and direct sequencing of the coding region of the beta-glucuronidase cDNA and gene to detect mutations causing beta-glucuronidase enzyme deficiency in five MPS VII patients. Four patients presented with hydrops fetalis, one with an early infantile form of the disease. Genetic heterogeneity of MPS VII alleles was further confirmed in this study. Recurrent mutations were observed in patients of related origin. Previously unknown alleles detected were RII0X, F361delta9, 1270 + 1G-->A, S52F and 1480delta4. Reverse transcription/PCR analysis of the 1270 + 1G-->A messenger showed aberrant splicing: inclusion of intron 7 or skipping of exons 6-7 and 9. Messenger RNA transcribed from the R110X and 1480delta4 alleles was unstable. We detected a 2154A/G change in the 3' non-coding region of the gene, in the neighbourhood of the two consensus polyadenylation sites. 3'-Rapid amplification of cDNA ends/PCR of fibroblast cDNA revealed equal usage of two alternative polyadenylation sites. The 2154A/G substitution did not influence adenylation-site choice, nor the amount of stable messenger produced. The finding that 2 out of 30 normal controls carried the 2154G allele indicated that the 2154A/G substitution is a harmless polymorphism. The S52F and F361delta9 cDNAs were constructed in vitro and used to transfect COS cells transiently. Both mutations completely abolished enzyme activity.

  9. Correlation of phenotypic tests with the presence of the blaZ gene for detection of beta-lactamase.

    PubMed

    Ferreira, Adriano Martison; Martins, Katheryne Benini; Silva, Vanessa Rocha da; Mondelli, Alessandro Lia; Cunha, Maria de Lourdes Ribeiro de Souza da

    Staphylococcus aureus and Staphylococcus saprophyticus are the most common and most important staphylococcal species associated with urinary tract infections. The objective of the present study was to compare and to evaluate the accuracy of four phenotypic methods for the detection of beta-lactamase production in Staphylococcus spp. Seventy-three strains produced a halo with a diameter ≤28mm (penicillin resistant) and all of them were positive for the blaZ gene. Among the 28 susceptible strain (halo ≥29mm), 23 carried the blaZ gene and five did not. The zone edge test was the most sensitive (90.3%), followed by MIC determination (85.5%), but the specificity of the former was low (40.0%). The nitrocefin test was the least sensitive (28.9%). However, the nitrocefin test together with the disk diffusion method showed the highest specificity (100%). The present results demonstrated that the zone edge test was the most sensitive phenotypic test for detection of beta-lactamase, although it is still not an ideal test to detect this type of resistance since its specificity was low. However, the inhibition halo diameter of the penicillin disk can be used together with the zone edge test since the same disk is employed in the two tests. Combined analysis of the two tests shows a sensitivity of 90.3% and specificity of 100%, proving better sensitivity, especially for S. saprophyticus. This is a low-cost test of easy application and interpretation that can be used in small and medium-sized laboratories where susceptibility testing is usually performed by the disk diffusion method.

  10. The utility of DNA sequences of an intron from the beta-fibrinogen gene in phylogenetic analysis of woodpeckers (Aves: Picidae).

    PubMed

    Prychitko, T M; Moore, W S

    1997-10-01

    Estimating phylogenies from DNA sequence data has become the major methodology of molecular phylogenetics. To date, molecular phylogenetics of the vertebrates has been very dependent on mtDNA, but studies involving mtDNA are limited because the several genes comprising the mt-genome are inherited as a single linkage group. The only apparent solution to this problem is to sequence additional genes, each representing a distinct linkage group, so that the resultant gene trees provide independent estimates of the species tree. There exists the need to find novel gene sequences which contain enough phylogenetic information to resolve relationships between closely related species. A possible source is the nuclear-encoded introns, because they evolve more rapidly than exons. We designed primers to amplify and sequence the 7 intron from the beta-fibrinogen gene for a recently evolved group, the woodpeckers. We sequenced the entire intron for 10 specimens representing five species. Nucleotide substitutions are randomly distributed along the length of the intron, suggesting selective neutrality. A preliminary analysis indicates that the phylogenetic signal in the intron is as strong as that in the mitochondrial encoded cytochrome b (cyt b) gene. The topology of the beta-fibrinogen tree is identical to that of the cyt b tree. This analysis demonstrates the ability of the 7 intron of beta-fibrinogen to provide well resolved, independent gene trees for recently evolved groups and establishes it as a source of sequences to be used in other phylogenetic studies.

  11. Type 1 Diabetes Candidate Genes Linked to Pancreatic Islet Cell Inflammation and Beta-Cell Apoptosis

    PubMed Central

    Størling, Joachim; Pociot, Flemming

    2017-01-01

    Type 1 diabetes (T1D) is a chronic immune-mediated disease resulting from the selective destruction of the insulin-producing pancreatic islet β-cells. Susceptibility to the disease is the result of complex interactions between environmental and genetic risk factors. Genome-wide association studies (GWAS) have identified more than 50 genetic regions that affect the risk of developing T1D. Most of these susceptibility loci, however, harbor several genes, and the causal variant(s) and gene(s) for most of the loci remain to be established. A significant part of the genes located in the T1D susceptibility loci are expressed in human islets and β cells and mounting evidence suggests that some of these genes modulate the β-cell response to the immune system and viral infection and regulate apoptotic β-cell death. Here, we discuss the current status of T1D susceptibility loci and candidate genes with focus on pancreatic islet cell inflammation and β-cell apoptosis. PMID:28212332

  12. Type 1 Diabetes Candidate Genes Linked to Pancreatic Islet Cell Inflammation and Beta-Cell Apoptosis.

    PubMed

    Størling, Joachim; Pociot, Flemming

    2017-02-16

    Type 1 diabetes (T1D) is a chronic immune-mediated disease resulting from the selective destruction of the insulin-producing pancreatic islet β-cells. Susceptibility to the disease is the result of complex interactions between environmental and genetic risk factors. Genome-wide association studies (GWAS) have identified more than 50 genetic regions that affect the risk of developing T1D. Most of these susceptibility loci, however, harbor several genes, and the causal variant(s) and gene(s) for most of the loci remain to be established. A significant part of the genes located in the T1D susceptibility loci are expressed in human islets and β cells and mounting evidence suggests that some of these genes modulate the β-cell response to the immune system and viral infection and regulate apoptotic β-cell death. Here, we discuss the current status of T1D susceptibility loci and candidate genes with focus on pancreatic islet cell inflammation and β-cell apoptosis.

  13. Genes, Gender, Environment, and Novel Functions of Estrogen Receptor Beta in the Susceptibility to Neurodevelopmental Disorders

    PubMed Central

    Varshney, Mukesh; Nalvarte, Ivan

    2017-01-01

    Many neurological disorders affect men and women differently regarding prevalence, progression, and severity. It is clear that many of these disorders may originate from defective signaling during fetal or perinatal brain development, which may affect males and females differently. Such sex-specific differences may originate from chromosomal or sex-hormone specific effects. This short review will focus on the estrogen receptor beta (ERβ) signaling during perinatal brain development and put it in the context of sex-specific differences in neurodevelopmental disorders. We will discuss ERβ’s recent discovery in directing DNA de-methylation to specific sites, of which one such site may bear consequences for the susceptibility to the neurological reading disorder dyslexia. We will also discuss how dysregulations in sex-hormone signaling, like those evoked by endocrine disruptive chemicals, may affect this and other neurodevelopmental disorders in a sex-specific manner through ERβ. PMID:28241485

  14. Genes, Gender, Environment, and Novel Functions of Estrogen Receptor Beta in the Susceptibility to Neurodevelopmental Disorders.

    PubMed

    Varshney, Mukesh; Nalvarte, Ivan

    2017-02-23

    Many neurological disorders affect men and women differently regarding prevalence, progression, and severity. It is clear that many of these disorders may originate from defective signaling during fetal or perinatal brain development, which may affect males and females differently. Such sex-specific differences may originate from chromosomal or sex-hormone specific effects. This short review will focus on the estrogen receptor beta (ERβ) signaling during perinatal brain development and put it in the context of sex-specific differences in neurodevelopmental disorders. We will discuss ERβ's recent discovery in directing DNA de-methylation to specific sites, of which one such site may bear consequences for the susceptibility to the neurological reading disorder dyslexia. We will also discuss how dysregulations in sex-hormone signaling, like those evoked by endocrine disruptive chemicals, may affect this and other neurodevelopmental disorders in a sex-specific manner through ERβ.

  15. Detection of beta2 and major toxin genes by PCR in Clostridium perfringens field isolates of domestic animals suffering from enteritis or enterotoxaemia.

    PubMed

    Sting, Reinhard

    2009-01-01

    The production of Clostridium (C.) perfringens toxins in the intestine is an important cause of enteritis and enterotoxaemia in livestock. In the present study, the alpha toxin and the genes encoding beta2 and epsilon toxin could be frequently detected by means of phenotypical and PCR examinations in these bacteria. The C. perfringens isolates originated from 1213 field samples taken from diseased or perished livestock located in the north-eastern administrative districts of Baden-Württemberg (Germany) from 2005 to 2008. The beta2 toxin gene of C perfringens was detected in all animal species examined, comprising pigs, the small ruminants sheep and goats, cattle, horses, rabbits, alpacas and lamas, and fallow deer. Among all the animal species included in this study, pigs attracted attention by a high quota of 74.2% (610 of 822) cpb2-positive C. perfringens isolates in comparison to the other animal species tested, revealing a quota of 20.8% (72 of 346). Beta2 toxigenic isolates could be predominantly cultivated from the faeces of young piglets. The beta toxin gene was detected in isolates from piglets and small ruminants only, amounting to 82.5% (33 of 40) in piglets in combination with the cpb2 gene. In this context, cpb2/cpb-positive C. perfringens isolates of piglets could be clearly detected more often in the intestine of perished animals (18 of 158) than in faeces (15 of 629). Furthermore, cpb2-bearing C. perfringens isolates were detected in cattle, horses, rabbits, alpacas and lamas, and fallow deer to a notable degree. The detection of C. perfringens isolates carrying the epsilon toxin gene was restricted to sheep and goats. Of a total of 242 small ruminants that succumbed to sudden death, 71 (29.3%) harboured epsilon toxin-positive C. perfringens isolates in their intestines. These cases clustered seasonally in the second quarter (April, May, and June) of the year. Neither the isolates bearing the beta2 nor beta toxin gene nor those carrying the epsilon

  16. Intron-exon organization of the active human protein S gene PS. alpha. and its pseudogene PS. beta. : Duplication and silencin