Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.61 (porphobilinogen deaminase)
637 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protoporphyrin IX (PpIX) synthesis by malignant cells is clinically exploited for photodiagnosis and photodynamic therapy following administration of 5-aminolevulinic acid (ALA). The expression and activity of the housekeeping porphobilinogen deaminase (PBGD) was correlated to PpIX synthesis in differentiating B16 melanoma cells. Differentiation was stimulated by two inducers, butyrate and hexamethylene bisacetamide (HMBA), both of which promote the formation of typical melanosomes and melanin, as well as morphological changeover. A marked decrease in total PBGD activity and PpIX synthesis was observed following stimulation by butyrate, while HMBA induced an opposite effect. In contrast, ferrochelatase levels remained unchanged. Photodynamic inactivation of the cells undergoing differentiation was largely dependent on the PpIX accumulation, which was modulated by the two inducers butyrate and HMBA. Fluorescence immunostaining with anti-PBGD antibodies revealed a major PBGD fraction in the nucleus and a minor fraction in the cytosol. This nuclear localisation pattern was confirmed by expression of PBGD fused to green fluorescence protein. We suggest that efficient photodynamic therapy of cancer facilitated by ALA administration can be enhanced using combined therapeutic modalities.
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PMID:Differentiation-dependent photodynamic therapy regulated by porphobilinogen deaminase in B16 melanoma. 1515 May 93

For interpretation of quantitative gene expression measurements in clinical tumor samples, a normalizer is necessary to correct expression data for differences in cellular input, RNA quality, and RT efficiency between samples. In many studies, a single housekeeping gene is used for normalization. However, no unequivocal single reference gene (with proven invariable expression between cells) has been identified yet. As the best alternative, the mean expression of multiple housekeeping genes can be used for normalization. In this study, no attempt was made to determine the gold-standard gene for normalization, but to identify the best single housekeeping gene that could accurately replace the measurement of multiple genes. Expression patterns of 13 frequently used housekeeping genes were determined in 80 normal and tumor samples from colorectal, breast, prostate, skin, and bladder tissues with real-time quantitative RT-PCR. These genes included, large ribosomal protein, beta-actin, cyclophilin A, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerokinase 1, beta-2-microglobin, beta-glucuronidase, hypoxanthine ribosyltransferase (HPRT), TATA-box-binding protein, transferrin receptor, porphobilinogen deaminase, ATP synthase 6, and 18S ribosomal RNA. Principal component analysis was used to analyze these expression patterns, independent of the level of expression. Our approach identified HPRT as the single best reference gene that could be used as an accurate and economic alternative for the measurement of multiple housekeeping genes. We recommend this gene for future studies to standardize gene expression measurements in cancer research and tumor diagnostics until a definite gold standard has been determined.
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PMID:Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. 1554 3

The polycomb group protein enhancer of zeste 2 (EZH2) is a transcriptional repressor involved in the control of cellular proliferation and oncogenesis. The aim of the present study was to quantify EZH2 expression in bladder carcinomas and to correlate the data with clinicopathological findings. EZH2 mRNA expression was measured by real-time reverse transcription-polymerase chain reaction in tumor tissue specimens obtained from 37 patients with urothelial carcinomas of the bladder and in four bladder cancer cell lines. EZH2 levels were normalized to expression of the housekeeping porphobilinogen deaminase gene. EZH2 transcripts were commonly detected in tumor tissue. Transcript levels correlated significantly with the invasiveness of bladder tumors (p = 0.029) with elevated EZH2 mRNA expression measured in invasive bladder carcinomas (median value, 38.92) compared with non-invasive tumors (15.51). In addition, levels of expression were significantly higher in high-grade (G3) than in low-grade (G1/2) lesions (p < 0.001). EZH2 mRNA levels in bladder carcinoma cell lines were within the range of high-grade invasive bladder cancers. In conclusion, expression levels of EZH2 are elevated in aggressive and invasive urothelial carcinomas, suggesting that deregulated EZH2 expression may be involved in the progression of bladder tumors.
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PMID:Expression levels of the EZH2 polycomb transcriptional repressor correlate with aggressiveness and invasive potential of bladder carcinomas. 1601 74

Defects in the enzyme porphobilinogen deaminase (PBG-D) are associated with acute intermittent porphyria (AIP). Human PBG-D is transcribed into a housekeeping or an erythroid form as a result of differential promoter usage and splicing. In addition, three pairs of isoallelic forms have been described. However, whether the enzymatic properties of housekeeping and erythroid forms differ is unknown. In this study the two isoallelic forms, K210 and E210, were cloned and expressed in Escherichia coli together with three mutations associated with a clinical AIP phenotype. The mutations were introduced in the K210 isoallelic background and expressed as both the housekeeping and the erythroid form. The proteins were expressed as GST fusions and purified to homogeneity. Initial experiments revealed that the GST-PBG-D fusions and the purified PBG-D obtained by proteolytic removal of the GST moiety had enzymatic properties that were indistinguishable. Consequently, all analyses with mutant PBG-D were performed on the GST-fusion proteins. Comparison of the wild-type proteins revealed a significant difference in Km between isoalleles with a Km of 9 microM for K210 and 7 microM for E210, whereas no significant difference in activity or kinetics between the housekeeping and the erythroid isoforms was observed. The mutant proteins showed 0.3-1.0% wild-type activity, depending on mutation. There was a clear correlation between yield of recombinant protein and CRIM status of patients. Furthermore, co-expression of the mutant proteins with the bacterial chaperone GroESL did not affect protein yield or function to any significant extent, supporting the view that the investigated mutations primarily influence structure and function and not folding of the proteins.
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PMID:Characterization of two isoalleles and three mutations in both isoforms of purified recombinant human porphobilinogen deaminase. 1602 32

The purpose of this study was to investigate whether levels of hTERT mRNA, as determined by real-time RT-PCR, are associated with prognosis and clinical course in AML patients. Fifty-four bone marrow specimens from 21 patients diagnosed with de-novo AML were included. The level of hTERT mRNA was measured with the Telo TAGGG hTERT Quantification Kit (Roche Diagnostics, Mannheim, Germany), using a LightCycler Instrument (Roche Diagnostics). The level of hTERT mRNA was determined as the relative ratio (RR), which was calculated by dividing the level of hTERT mRNA by the level of the porphobilinogen deaminase (PBGD) housekeeping gene in the same samples [1,000x(hTERT/PBGD)]. The expression rates of hTERT mRNA were significantly higher at diagnosis (73%) and during relapse (80%) than during remission (27%) (P<0.05). The median RR for diagnosis or relapse was significantly higher than that for patients in remission (P<0.05). hTERT mRNA expression was not correlated with CD34 expression, blast counts, white blood cell counts, or chromosomal abnormality (P>0.05). Two patients who showed hTERT mRNA expression during remission (RR 3.14 and 7.15, respectively) relapsed after 1 month. Among seven patients with high hTERT mRNA levels (RR>9.51), 4 failed to achieve complete remission (CR), whereas 4 of 5 patients without hTERT mRNA expression at diagnosis or during relapse achieved CR (P>0.05). Patients showing a trend of increasing hTERT mRNA levels failed to reach a second CR after relapse, while those with a trend toward decreasing hTERT mRNA did achieve CR. Among eight samples showing hTERT mRNA expression in remission (RR>0), 5 were obtained from patients who had received GCSF within 14 days. The expression rate and level of hTERT mRNA during remission were significantly higher in patients who had previously received GSCF (56%, RR=0.15) than in other patients (15%, RR=0) (P<0.05). Serial and quantitative analysis of hTERT mRNA may be a useful marker for prediction of prognosis and monitoring in AML patients.
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PMID:hTERT mRNA levels by real-time RT-PCR in acute myelogenous leukemia. 1604 49

DNA topoisomerase I (Topo I) is involved in DNA replication, transcription, recombination and repair. Clinical interest has focused on Topo I as it is the molecular target of camptothecin (CPT), used in first and second lines of treatment for different cancer types. Furthermore, it is well demonstrated that the patients who best responded to CPT-based chemotherapy were generally those with the greatest tumoral Topo I expression and/or activity. We developed a sensitive, simple and reproducible method to measure Topo I mRNA expression in human cancer samples. Experiments were performed in two steps. First, we checked the accuracy of the reverse transcription-polymerase chain reaction (RT-PCR) method by testing intra- and interassay reproducibility of Topo I and G6PDH gene amplification in different cell types. We observed that crossing-points (Cps) were different, depending on the cell type, dilution or cDNA concentration, but that the intra- and interassay Cp standard deviation (SD) never exceeded 0.77% and 1.39% for Topo I amplification, or 1.63% and 2.9% for G6PDH amplification, respectively. Secondly, we used our method to measure Topo I mRNA levels in primary tumor samples obtained from 27 patients with advanced colorectal cancer and 10 patients with pharyngeal/laryngeal cancer. The accuracy of G6PDH as a housekeeping gene was tested by analyzing its correlation with the mRNA level of a second housekeeping gene, porphobilinogen deaminase (PBG-D) in the tumoral samples. We found that the normalized Topo I/G6PDH mRNA ratios were significantly correlated with that of Topo I/PBGD in colorectal tumors (r(2)=0.47, p=0.02) but not in pharyngeal/laryngeal tumors (r(2)=0.35, p=0.3). Neither ratio showed any significant association with clinicopathological parameters, such as gender, age, tumor size, or grade and lymph node status. We believe that RT-PCR is a reliable and highly reproducible technique. However, the choice of the reference gene is an important point and must be defined based on the samples studied.
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PMID:A quantitative RT-PCR method to determine topoisomerase I mRNA levels in human tissue samples. 1620 29

Heat stress perturbs prolactin (PRL) release and affects dairy cow lactational performance and immune cell function. We hypothesized that greater PRL concentration in plasma of heat-stressed cows relative to cooled cows would decrease expression of prolactin receptor (PRL-R) mRNA and increase mRNA expression of suppressors of cytokine signaling (SOCS) in lymphocytes, altering their cytokine production. To test this hypothesis, multiparous Holstein cows were dried off 46 d before their expected calving date and assigned randomly to heat stress (HT; n=9) or cooling (CL; n=7) during the entire dry period. A second study was conducted the following year with an additional 21 cows (12 HT; 9 CL). Lymphocytes were isolated from cows at -46, -20, +2, and +20 d relative to expected calving date and mRNA expression of PRL-R, SOCS-1, SOCS-2, SOCS-3, cytokine-inducible SH2-containing protein (CIS), and heat shock protein 70 KDa A5 (HSPA5), and housekeeping genes hydroxymethylbilane synthase (HMBS), ATP synthase, H+ transporting mitochondrial F1 complex, beta subunit (ATP5B), and ribosomal protein S9 (RPS9) was analyzed by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Cows exposed to HT had greater PRL concentration in plasma compared with CL cows. Measurement of lymphocyte proliferation indicated that lymphocytes of CL cows proliferated more than those from HT cows and exressed more PRL-R mRNA and less SOCS-1 and SOCS-3 mRNA relative to HT cows. Further, lymphocytes from CL cows produced more tumor necrosis factor-alpha (TNF-alpha) than those from HT cows. These results suggest that changes in PRL-signaling pathway genes during heat stress are associated with differential cytokine secretion by lymphocytes and may regulate lymphocyte proliferation in dairy cows.
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PMID:Heat stress abatement during the dry period influences prolactin signaling in lymphocytes. 1973 97

Acute intermittent porphyria (AIP) is an autosomal dominant disorder caused by molecular abnormalities in the HMBS gene. This gene is transcribed from two promoters to produce ubiquitous and erythroid specific isoforms of porphobilinogen deaminase (PBGD). In the classical form of AIP, both isoforms are deficient, but about 5% of families have the non-erythroid variant in which only the ubiquitous isoform is affected. Only one mutation sited in the housekeeping promoter has been previously reported as causative for this form of AIP. In this study, we identified one small deletion and six nucleotide substitutions within the 5'UTR and the housekeeping promoter of HMBS gene: c.1-440_-427del14bp; c.1-421G>A; c.1-331C>T; c.1-270G>A; c.1-122T>A; c.1-103C>T; c.1-28A>C. Using luciferase reporter assays and quantitative PCR experiments, we characterized the functional role of these seven novel genetic variants demonstrating that all mutations cause a significant loss of transcriptional activity. Our investigations suggest that these nucleotide substitutions may alter critical binding sites for transcriptional factors, which confirms that these regions represent an important molecular target for pathogenesis of non-erythroid form of acute intermittent porphyria.
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PMID:Seven novel genetic mutations within the 5'UTR and the housekeeping promoter of HMBS gene responsible for the non-erythroid form of acute intermittent porphyria. 2769 11

Comprehensive analyses of gene expression have been carried out by the development of microarrays and deep sequencers. However, it is difficult to obtain comprehensive information on gene expression from a small amount of ribonucleic acid (RNA). Therefore, we investigated the reproducibility and application of T7 RNA polymerase-mediated transcription, adaptor ligation and polymerase chain reaction (PCR) amplification, followed by T7 transcription (TALPAT), an efficient method for amplifying poly (A)-positive RNA, such as messenger RNA (mRNA). When amplified complementary RNA (cRNA) was electrophoresed, a large number of amplified cRNA was detected in the size of 0.2-0.5 kb. This indicates that the region up to 0.2-0.5 kb from the 3' end of the original mRNA was amplified by the TALPAT method. Seven housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT1), ribosomal protein L13a (RPL13A), succinate dehydrogenase complex (SDHA), TATA box-binding protein (TBP) and ubiquitin C (UBC), showed high reproducibility (square of the correlation coefficient, R²=0.9954), according to scatter plots of Ct values obtained in the real-time PCR analysis of amplified cRNA. In addition, relative expression ratios of amplified cRNA of the seven housekeeping genes were approximately equal to the ratio of the original RNA solution. Furthermore, cRNA was amplified from 20 pg total RNA. In the present study, we confirmed the characteristics of mRNA amplification using the TALPAT method. This method may be applicable to mRNA and poly (A)-positive non-coding RNA amplification, using a small amount of RNA from single, laser-captured and sorted cells, as well as exosomes from serum, urine and body fluids.
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PMID:An efficient method for high-fidelity messenger RNA amplification from a small amount of total RNA. 2464 3

The identification of human body fluids or tissues through mRNA-based profiling is very useful for forensic investigations. Previous studies have shown mRNA biomarkers are effective to identify the origin of biological samples. In this study, we selected 16 tissue specific biomarkers to evaluate their specificities and sensitivities for human body fluids and tissues identification, including porphobilinogen deaminase (PBGD), hemoglobin beta (HBB) and Glycophorin A (GLY) for circulatory blood, protamine 2 (PRM2) and transglutaminase 4 (TGM4) for semen, mucin 4 (MUC4) and human beta defensin 1(HBD1) for vaginal secretion, matrix metalloproteinases 7 and 11 (MMP7 and MMP11) for menstrual blood, keratin 4(KRT4) for oral mucosa, loricrin (LOR) and cystatin 6 (CST6) for skin, histatin 3(HTN3) for saliva, statherin (STATH) for nasal secretion, dermcidin (DCD) for sweat and uromodulin (UMOD) for urine. The above mentioned ten common forensic body fluids or tissues were used in the evaluation. Based on the evaluation, a reverse transcription (RT) PCR multiplex assay, XCYR1, which includes 12 biomarkers (i.e., HBB, GLY, HTN3, PRM2, KRT4, MMP11, MUC4, DCD, UMOD, MMP7, TGM4, and STATH) and 2 housekeeping genes [i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18SrRNA], was developed. This assay was further validated with real casework samples and mock samples (with both single source and mixture) and it was approved that XCYR1 is effective to identify common body fluids or tissues (i.e., circulatory blood, saliva, semen, vaginal secretion, menstrual blood, oral mucosa, nasal secretion, sweat and urine) in forensic casework samples.
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PMID:Development of highly sensitive and specific mRNA multiplex system (XCYR1) for forensic human body fluids and tissues identification. 2499 6


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