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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Here it is analyzed the expression of a mini locus dual reporter construct composed by a micro-LCR and by the promoters for (A)gamma- and beta-globin gene, each one linked to a different Luciferase, in stably transfected GM979 cells for as long as 1-2 years from transfection. The transfected GM979 cells rapidly (within 1 month) evolved into a stable population which expresses constant levels of reporters for more than a year of continuous bulk culture. No silencing of the inserted construct was observed over time. In contrast, after 1 month, the reporter activity (both from (A)gamma- and beta-promoter) expressed per cell increased over time. The analysis of the Luciferase contained in single cell clones indicated that the higher reporter activity was due to increased gene expression per cell rather than to clonal selection of the most expressing clones. Since the activity driven by the beta-promoter increased 10-fold more than that driven by the (A)gamma one, the ratio between (A)gamma-driven/((A)gamma-driven + beta-driven) reporter activity in the cells decreased after 1 month and became similar to the gamma/(gamma + beta) globin mRNA ratio expressed by adult erythroid cells. Moreover, although both cells from early and late bulk culture responded to incubation with butyric acid, a known inducer of fetal globin gene expression, by increasing the reporter activity driven by the (A)gamma-promoter, only cells from late bulk culture decreased, as normal primary erythroblasts do, the activity of the reporter driven by the beta-promoter. These results suggest that the rapid changes in activity driven by the (A)gamma- and beta-globin promoters occurring during the first month after transfection may represent a novel in vitro model to study epigenetic regulation of the (A)gamma- and beta-promoter during the fetal to adult hemoglobin switch and confirm GM979 cells stably transfected with the dual reporter construct as a reliable assay for automated screening of chemical inducers of fetal globin gene activation.
Blood Cells Mol Dis
PMID:Spontaneous switch from Agamma- to beta-globin promoter activity in a stable transfected dual reporter vector. 1572 2

Chromatin insulators are regulatory elements that determine domains of genetic functions. We have previously described the characterization of a 265 bp insulator element, termed sns, localized at the 3' end of the early histone H2A gene of the sea urchin Paracentrotus lividus. This sequence contains three cis-acting elements (Box A, Box B, and Box C + T) all needed for the enhancer-blocking activity in both sea urchin and human cells. The goal of this study was to further characterize the sea urchin sns insulator in the erythroid environment. We employed colony assays in human (K562) and mouse (MEL) erythroid cell lines. We tested the capability of sns to interfere with the communication between the 5'HS2 enhancer of the human beta-globin LCR and the gamma-globin promoter. We found that the sns sequence displays directional enhancer-blocking activity. By the use of antibodies against known DNA binding proteins, in electrophoretic mobility shift assays, we demonstrated the binding of the erythroid-specific GATA-1 and the ubiquitous Oct-1 and Sp1 transcription factors. These factors bind to Box A, Box B, and Box C + T, respectively, in both K562 and MEL nuclear extracts. These results may have significant implications for the conservation of insulator function in evolutionary distant organisms and may prove to be of practical benefit in gene transfer applications for erythroid disorders such as hemoglobinopathies and thalassemias.
Blood Cells Mol Dis
PMID:Functional characterization of the sea urchin sns chromatin insulator in erythroid cells. 1618 1

A novel quantitative real-time PCR method using the duplex scorpion primer for detection of Chlamydia trachomatis DNA was developed and validated. The assay employs a duplex primer; its most important feature is the intramolecular probe-target interaction. The assay had many prominent characteristics. (i) The duplex probe is simpler to synthesize and significantly easier to purify than TaqMan probe because that the fluorescent dye pair and the quencher pair are in different oligonucleotides. (ii) The method has high sensitivity, specificity, intra- and interassay reproducibilities. (iii) The assay has a quantitative dynamic range of 25 to10(9) genome copies per reaction mixture. (iv) The scorpion system can identify 98.6% samples in the validation panel without retest. There were 81 positive samples and 67 negative samples, which were confirmed by two FDA-approved NAATs (the Roche Amplicor PCR assay, Abbott LCR kit) and our new method. Any two positive results out of the possible three-comparator results would define the infected-patient gold standard. Of the positive samples, 79 (97.5%) were found positive (ranging from 31 to 227,648 copies/microl, M=4219 copies/microl), whereas no negative samples were found positive by the assay. A quantitative, fast, and easy-to-handle diagnostic approach such as the MOMP-based real-time PCR described here might improve the detection of C. trachomatis infection.
Exp Mol Pathol 2007 Aug
PMID:Development of a novel quantitative real-time assay using duplex scorpion primer for detection of Chlamydia trachomatis. 1722 21

A 213 kb human beta-globin locus yeast artificial chromosome (beta-YAC) was modified by homologous recombination to delete 2.9 kb of cross-species conserved sequence similarity encompassing the LCR 5' hypersensitive site (HS) 4 (Delta5'HS4 beta-YAC). In three transgenic mouse lines, completion of the gamma- to beta-globin switch during definitive erythropoiesis was delayed relative to wild-type beta-YAC mice. In addition, quantitative per-copy human beta-like globin mRNA levels were similar to wild-type beta-YAC transgenic lines, although beta-globin gene expression was slightly decreased in the day 12 fetal liver of Delta5'HS4 beta-YAC mice. A 0.8 kb 5'HS1 fragment was similarly deleted in the YAC. Three Delta5'HS1 beta-YAC transgenic lines were established. epsilon-globin gene expression was markedly reduced, approximately 16 fold, during primitive erythropoiesis compared to wild-type beta-YAC mice, but gamma-globin expression levels were unaffected. However, during the fetal stage of definitive erythropoiesis, gamma-globin gene expression was decreased approximately 4 fold at day 12 and approximately 5 fold at day 14. Temporal developmental expression profiles of the beta-like globin genes were unaffected by deletion of 5'HS1. Decreased expression of the epsilon- and gamma-globin genes is the first phenotype ascribed to a 5'HS1 mutation in the human beta-globin locus, suggesting that this HS does indeed have a role in LCR function beyond simply a combined synergism with the other LCR HSs.
Blood Cells Mol Dis
PMID:Deletion of the human beta-globin LCR 5'HS4 or 5'HS1 differentially affects beta-like globin gene expression in beta-YAC transgenic mice. 1743 33

Understanding the risk of offspring inheriting rare mutations, and the frequencies at which these mutations are present in germ cells can be explored with direct analysis of human semen samples. The present work utilized the ultrasensitive PCR/RE/LCR mutation assay to detect, identify and determine the prevalence single base substitution mutations in the TP53 and KRAS genes in human sperm. Four disease-associated base sites in the TP53 and KRAS genes, three of which are known to be heritable to live, term offspring, were studied in sperm from eleven human semen specimens. Eight of the specimens (73%) displayed single base substitution mutations, and 30% of all base sites tested were found to harbor mutations ranging in prevalence from 1 x 10(-6) to 1 x 10(-5) wild type sperm. These germ cell single base substitution mutation frequencies are very similar to somatic tissue TP53 and KRAS mutation frequencies. Equivalent single base mutation frequencies in both germ and somatic cells suggest that there is no unusual selection or mutation protective process operating premeiotically in the germline, and that a selection bias at the level of sperm viability, conception, early cleavage, implantation, and/or embryogenesis operates to exclude the majority of these TP53 mutations and all of the activating KRAS mutations.
Environ Mol Mutagen 2008 Jul
PMID:Human germline and somatic cells have similar TP53 and Kirsten-RAS gene single base mutation frequencies. 1841 64

A new series of 4 beta-anilino-podophyllotoxin analogs have been designed, synthesized and evaluated their bioactivities as novel DNA-topoisomerase II poisons as well as P-glycoprotein (P-gp)-dependent multidrug resistance (MDR) inhibitors. The new compounds show improved potency and efficacy with respect to the parent molecule etoposide (VP-16), one of the semisynthetic derivatives of podophyllotoxin. The treatment of 5k-n in KB/VCR cells caused G(2)/M phase arrest and finally induced apoptosis. Furthermore, molecular docking is applied to testify that 5k-n could not be the substrates of P-gp, which is consistent with the result of MDR1 and P-glycoprotein express tests. The most potent compound 5n is chosen for in vivo studies, the administration of 5n was effective in treatment of cancer with a lower dose than VP-16 in drug-sensitive xenograft model and drug-resistant xenograft model. Compound 5n is a potential drug candidate for anticancer chemotherapy.
Mol Biosyst 2010 Feb
PMID:Novel 4 beta-anilino-podophyllotoxin derivatives: design synthesis and biological evaluation as potent DNA-topoisomerase II poisons and anti-MDR agents. 2009 61

Invertase (INV) hydrolyzes sucrose into glucose and fructose, thereby playing key roles in primary metabolism and plant development. Based on their pH optima and sub-cellular locations, INVs are categorized into cell wall, cytoplasmic, and vacuolar subgroups, abbreviated as CWIN, CIN, and VIN, respectively. The broad importance and implications of INVs in plant development and crop productivity have attracted enormous interest to examine INV function and regulation from multiple perspectives. Here, we review some exciting advances in this area over the last two decades, focusing on (1) new or emerging roles of INV in plant development and regulation at the post-translational level through interaction with inhibitors, (2) cross-talk between INV-mediated sugar signaling and hormonal control of development, and (3) sugar- and INV-mediated responses to drought and heat stresses and their impact on seed and fruit set. Finally, we discuss major questions arising from this new progress and outline future directions for unraveling mechanisms underlying INV-mediated plant development and their potential applications in plant biotechnology and agriculture.
Mol Plant 2010 Nov
PMID:Sugar input, metabolism, and signaling mediated by invertase: roles in development, yield potential, and response to drought and heat. 2072 75

The demonstration of human papillomavirus (HPV) in 99.7% of cervical carcinoma surgical specimens from around the world required investigations by multiple alternative polymerase chain reaction (PCR) assays. A similar approach may therefore be necessary to best characterize HPV prevalence and genotype distribution among cervical cytology samples. In an earlier study, 752 of 799 (94.1%) abnormal and 82 of 300 (27.3%) normal cytology specimens tested HPV positive after PCR using GP5+/6+primers. This study has reinvestigated the "HPV negative" abnormal samples (20 atypical squamous cells of undetermined significance, 5 low-grade squamous intraepithelial lesion, 14 atypical squamous cells, cannot exclude HSIL, 6 high-grade squamous intraepithelial lesion) and an age-matched cohort of "HPV negative" normal (negative for an intraepithelial lesion or malignancy) samples by PCR using PGMY09/11, FAP59/64, and LCR-E7 primers. PGMY09/11-GP5+/6+ nested PCR was performed on samples that were HPV negative by PGMY09/11 PCR. After the first 3 assays, HPV was detected in 41 of 45 (91.1%) abnormal and in 10 of 47 (21.3%) normal samples (P<0.0001). Eighteen HPV genotypes were detected and in some samples the genotype that was identified differed between the tests. The nondetection of common HPV genotypes (eg, HPVs 6, 11, 16, and 18) was notable. High-grade histopathology was found for 2 patients with HPV52-positive cytopathology. Combined with our earlier study, HPV (40 different genotypes) is shown in 99.5% of abnormal samples (99.8% inclusive of the nested PCR data). These findings show that HPV genotype and prevalence estimates are dependent on the method(s) of detection and indicate that suboptimal analytical sensitivity for one or more of the less common high-risk HPV genotypes could lead to impaired clinical sensitivity. HPV may be causal in almost every instance of abnormal cervical cytology; however, passenger HPV that is incidental to an abnormality may also have been detected.
Diagn Mol Pathol 2010 Sep
PMID:HPV is detectable in virtually all abnormal cervical cytology samples after reinvestigation of HPV negatives with multiple alternative PCR tests. 2073 43

There is an intense interest in differentiating embryonic stem cells to engineer biological pacemakers as an alternative to electronic pacemakers for patients with cardiac pacemaker function deficiency. Embryonic stem cell-derived cardiocytes (ESCs), however, often exhibit dysrhythmic excitations. Using Ca(2+) imaging and patch-clamp techniques, we studied requirements for generation of spontaneous rhythmic action potentials (APs) in late-stage mouse ESCs. Sarcoplasmic reticulum (SR) of ESCs generates spontaneous, rhythmic, wavelet-like Local Ca(2+)Releases (LCRs) (inhibited by ryanodine, tetracaine, or thapsigargin). L-type Ca(2+)current (I(CaL)) induces a global Ca(2+) release (CICR), depleting the Ca(2+) content SR which resets the phases of LCR oscillators. Following a delay, SR then generates a highly synchronized spontaneous Ca(2+)release of multiple LCRs throughout the cell. The LCRs generate an inward Na(+)/Ca(2+)exchanger (NCX) current (absent in Na(+)-free solution) that ignites the next AP. Interfering with SR Ca(2+) cycling (ryanodine, caffeine, thapsigargin, cyclopiazonic acid, BAPTA-AM), NCX (Na(+)-free solution), or I(CaL) (nifedipine) results in dysrhythmic excitations or cessation of automaticity. Inhibition of cAMP/PKA signaling by a specific PKA inhibitor, PKI, decreases SR Ca(2+) loading, substantially reducing both spontaneous LCRs (number, size, and amplitude) and rhythmic AP firing. In contrast, enhancing PKA signaling by cAMP increases the LCRs (number, size, duration) and converts irregularly beating ESCs to rhythmic "pacemaker-like" cells. SR Ca(2+) loading and LCR activity could be also increased with a selective activation of SR Ca(2+) pumping by a phospholamban antibody. We conclude that SR Ca(2+) loading and spontaneous rhythmic LCRs are driven by inherent cAMP/PKA activity. I(CaL) synchronizes multiple LCR oscillators resulting in strong, partially synchronized diastolic Ca(2+) release and NCX current. Rhythmic ESC automaticity can be achieved by boosting "coupling" factors, such as cAMP/PKA signaling, that enhance interactions between SR and sarcolemma.
J Mol Cell Cardiol 2011 Jan
PMID:Rhythmic beating of stem cell-derived cardiac cells requires dynamic coupling of electrophysiology and Ca cycling. 2092 May 9

Our data on 114 Iranian individuals with thalassemia intermedia phenotype revealed homozygous or compound heterozygous beta-globin mutations to be the predominant disease factor in 86.2% of cases. However, 8.2% of these individuals were found to be heterozygous or wild type for beta-globin mutations. In search for determinants outside of the beta-globin gene, which could be responsible for the unexpected thalassemia intermedia phenotype in these subjects, we screened the alpha-globin genes, the 5'HS3 and 5'HS4 regions of the beta-globin LCR, and the NF-E2 transcription factor for sequence variations in selected individuals. The -3.7 deletion was the only alpha-globin mutation detected, and no alterations were found in 5'HS3 and NF-E2. Sequence analysis of the 5'HS4 LCR core region identified three known SNPs in a single patient, who required irregular blood transfusions. The A/G polymorphism in the 5'HS4 palindromic region was also observed to be variable. Family studies were carried out on a female G/G homozygous patient, who received irregular blood transfusions. Her father, who had the same heterozygous IVSII-1 beta-globin mutation but the A/G genotype at the 5'HS4 palindromic site, presented with mild anemia and no requirement for blood transfusions. This suggests an impact of SNPs in the 5'HS4 LCR core region on the thalassemia phenotype and offers an interesting subject for further investigations in the Iranian population.
Blood Cells Mol Dis 2011 Mar 15
PMID:Analyzing 5'HS3 and 5'HS4 LCR core regions and NF-E2 in Iranian thalassemia intermedia patients with normal or carrier status for beta-globin mutations. 2123 98


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