Search PubMed⌕ Search

Biomedical subjects

M R Johnson

Publications and source records attributed to M R Johnson.

At least 55 records · Page 3Linked to original sources
PubMed2001

Retroviral transduction of human dihydropyrimidine dehydrogenase cDNA confers resistance to 5-fluorouracil in murine hematopoietic progenitor cells and human CD34+-enriched peripheral blood progenitor cells.

Severe 5-fluorouracil (5-FU) toxicity has been reported among patients lacking dihydropyrimidine dehydrogenase (DPD) enzymatic activity. DPD is the principal enzyme involved in the degradation of 5-FU to 5'-6'-dihydrofluorouracil, which is further metabolized to fluoro-beta-alanine. We demonstrate here that overexpression of human DPD confers resistance to 5-FU in NIH3T3 cells, mouse bone marrow cells, and in human CD34+-enriched hematopoietic progenitor cells. An SFG-based dicistronic retroviral vector containing human DPD cDNA, an internal ribosomal entry site (IRES), and the neomycin phosphotransferase (Neo) gene was constructed (SFG-DPD-IRES-Neo). Transduced NIH3T3 cells demonstrated a 2-fold (ED50) increase in resistance to a 4-hour exposure of 5-FU in comparison to nontransduced cells. Expression of DPD was confirmed by Northern and Western blot analyses, and DPD enzyme activity was detectable only in transduced cells. Infection of mouse bone marrow cells with this retroviral construct resulted in an increased number of 5-FU-resistant CFU-GM colonies, compared to mock-transduced bone marrow in both 4-hour and 12- to 14-day exposures. Infection of human CD34+-enriched cells with this construct and incubation with 5-FU (10(-6) M) for 14 days also resulted in an increased number of 5-FU-resistant colonies. Retroviral transduction of human hematopoietic progenitor cells with a cDNA-expressing human DPD conferred resistance to 5-FU in NIH3T3 cells, mouse bone marrow cells, and human CD34+-enriched cells. These results encourage the use of this gene as a method to protect patients from 5-FU myelotoxicity.

3T3 Cells↗
PubMed2001

A characterization of solution gas flaring in Alberta.

Information reported here is the result of a detailed analysis of data on flared and vented solution gas in the Province of Alberta in 1999. A goal of characterizing these flares was to aid in the improved management of solution gas flaring. In total, 4499 oil and bitumen batteries reported flaring or venting with a combined gas volume of 1.42 billion m3. There was significant site-to-site variation in volumes of gas flared or vented, gas composition, and flare design. Approximately 5% of physical batteries generate 35.7% of the gas flared and vented from oil and bitumen batteries. Therefore, if one were to attempt to mitigate flaring, significant progress could be made by starting with only the largest sites. The monthly variability of gas volumes was considered because high variability could affect implementation of alternative technologies. It was found that slightly more than 40% of the sites were reasonably steady and had monthly deviations of 100% or less from the average flared volume. The variability in monthly volumes was less for the larger batteries. Data from individual well sites show significant variability in the relative concentrations of each of the major species contained in solution gas.

Air Pollution↗
PubMed2001

The role of [18F]fluoro-2-deoxyglucose-PET scanning in the diagnosis of paraneoplastic neurological disorders.

The detection of an occult tumour in a patient with a suspected paraneoplastic neurological disorder (PND) may be difficult because of the limitations of conventional imaging techniques. [(18)F]fluoro-2-deoxyglucose-PET (FDG-PET) can visualize a small tumour anywhere within the body. We retrospectively reviewed the case notes of 43 unselected patients with suspected PND referred for FDG-PET scanning to determine how useful this technique was when conventional imaging was negative. All patients had undergone standard radiological investigations and bronchoscopy (where appropriate) prior to PET scanning. There were discrete areas of hypermetabolism suggestive of malignancy (positive) in 16 patients (37%). A tissue diagnosis of cancer was subsequently made in seven patients (two at post-mortem), further radiological studies were suggestive of cancer in one patient, one patient subsequently presented with a metastatic deposit which was biopsied, and four patients died shortly afterwards without a post-mortem. In three patients, subsequent investigations were negative for cancer. Serum anti-neuronal antibodies were present in 43% and CSF oligoclonal bands were present in 46% of patients with positive PET scans compared with 16 and 26%, respectively, in PET-negative patients, but this was not significant. Only one patient with a negative scan has been diagnosed subsequently as having malignancy on prolonged follow-up. These findings confirm that FDG-PET scanning is a useful technique in the detection of small tumours in patients with suspected PND. False positives and false negatives do occur, but at a sufficiently low frequency to justify the clinical usefulness of this technique.

Adult↗
PubMed2001

Role of matrix metalloproteinases in colorectal carcinogenesis.

OBJECTIVE: To measure coexpression of matrix metalloproteinase (MMP)-2, MMP-7, and MMP-9 genes by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in benign and malignant phases of colorectal carcinogenesis. SUMMARY BACKGROUND DATA: Matrix metalloproteinases degrade and remodel the extracellular matrix and have been implicated in facilitating carcinoma cells to invade and metastasize. MMP-2, MMP-7, and MMP-9 have been shown to be overexpressed in various carcinomas; however, simultaneous examination of these enzymes in human normal mucosa, adenoma, and carcinoma has not been performed to date. METHODS: Between January 1, 1998, and June 15, 2000, 40 patients underwent colectomy and harvest and snap-freezing of normal mucosa, adenoma, and carcinoma. Five patients had adenoma and carcinoma in the same specimen; 35 had either adenoma (n = 6) or carcinoma (n = 29). Taqman qRT-PCR methodology was used to measure MMP gene copy number and normalized to beta-actin RNA expression. RESULTS: The mean age was 62 +/- 4 years, with 22 men and 18 women. One fifth of the adenomas exhibited severe dysplasia. MMP-7 gene expression was significantly increased in adenomas (43 times normal mucosa) but did not increase further in carcinomas (50 times normal mucosa). MMP-2 and MMP-9 were not different in adenomas (1.8 and 1.4 times normal mucosa, respectively) but were elevated in carcinomas (2.2 and 1.8 times normal mucosa, respectively). There was no correlation between size or dysplasia in adenomas or AJCC stage in carcinomas and MMP gene expression. CONCLUSIONS: Overexpression of MMP-7 is an early event in the adenoma-to-carcinoma pathway, and expression does not appear to increase further in carcinomas. MMP-2 and MMP-9 appear to be primarily overexpressed in carcinomas. This may be one mechanism by which adenoma cells gain the ability to invade and carcinoma cells to metastasize.

Adenoma↗
PubMed2001

Biochemical markers of maternal bone turnover are elevated in pre-eclampsia.

OBJECTIVES: To investigate the hypothesis that bone turnover is reduced in pre-eclampsia compared with normal pregnancy. DESIGN: A prospective cross-sectional study. SETTING: Obstetric unit at the Chelsea and Westminster Hospital, London. METHODS: Third trimester maternal plasma levels of the cross-linked carboxyl-terminal telo-peptide of type I collagen (ICTP), (a marker of bone resorption) and the carboxyl terminal pro-peptide of type I pro-collagen (PICP), (a marker of bone formation) were compared in 25 women with pre-eclampsia and in 24 normal pregnant controls. The subjects were matched for maternal age, booking body mass index and gestational age. PICP and ICTP levels were measured by radio-immunoassay. RESULTS: ICTP and PICP levels were significantly increased in women with pre-eclampsia compared with the normal pregnant controls (P = 0.0001 and P = 0.004, for ICTP and PICP respectively, Wilcoxon signed ranked test). There was no significant correlation between either of the markers and booking body mass index, blood pressure, serum uric acid levels or platelet count. CONCLUSIONS: These data suggest that bone turnover is increased in established pre-eclampsia compared with normal pregnancy. Further studies are required to investigate whether this precedes the onset of the disease.

Adult↗
PubMed2001

Fetal bone metabolism in normal and rhesus isoimmunised pregnancies.

OBJECTIVE: To construct gestation-specific reference intervals for fetal concentrations of biochemical markers of bone metabolism and assess the effect of rhesus isoimmunisation on these. METHODS: Fetal blood samples were obtained by cordocentesis from 175 pregnancies (43 complicated by rhesus isoimmunisation) and assayed for carboxy terminal pro-peptide of type I pro-collagen (PICP) and cross-linked carboxyterminal telopeptide of type I collagen (ICTP) which directly monitor the rate of bone formation and resorption respectively. RESULTS: Both plasma PICP and ICTP were negatively correlated with gestational age (r = -0.351 and -0.472 for PICP and ICTP, respectively, and P < 0.001 for both). In fetuses affected by rhesus isoimmunisation PICP levels were lower (P=0.030) and more variable (P <0.001) than expected, compared with normal unaffected fetuses. However, no such differences were found in the ICTP levels. In the fetuses affected by rhesus isoimmunisation there was a significant correlation between haemoglobin concentration and both PICP (r = 0.504, P = 0.001) and ICTP (r = 0.343, P = 0.030). CONCLUSIONS: Fetal bone turnover declines from early second trimester to term, and may be deranged in fetuses affected by rhesus isoimmunisation.

Adolescent↗
PubMed2001

The clinical impact of epilepsy genetics.

An overemphasis on molecular genetic advances in epilepsy is in danger of missing the major contribution that clinical genetic studies make in predicting the likely benefit of molecular research efforts. Genetic epidemiology, twin, and family studies suggest that some individual epilepsy genes raise the risk for developing many different types of epilepsy but that specific combinations of these genes determine specific epilepsy phenotypes. Experimental data show how differences in drug response can result from inherited differences in drug sensitivity and there is emerging data on the genetic basis of harmful adverse drug reactions and teratogenesis. These developments predict a future in which epilepsy is ultimately classified on the basis of its oligogenic architecture, effective treatments are tailored to the appropriate patient and harmful adverse drug reactions avoided in those who are sensitive.

Epilepsy↗
PubMed2000

Molecular cloning and characterization of the human dihydropyrimidine dehydrogenase promoter.

Several studies have demonstrated that dihydropyrimidine dehydrogenase (EC 1.3.1.2) has a critical role in the pharmacokinetics of the anticancer agent 5-fluorouracil. We previously reported the structural organization of the human DPYD gene. In this article, we describe the molecular cloning and functional characterization of 1.2 kb of the 5' flanking region of the DPYD gene. Sequence analysis demonstrated that this region of the DPYD gene lacks the typical TATA or CCAAT boxes with several GC-rich regions containing potential cis-regulatory elements. Progressive 5' deletions of the 5' flanking region were fused to the luciferase reporter gene and transient expression measured following transfection into HeLa and 293 cells. Comparative analysis of luciferase activity revealed that a 208 bp region of the DPYD gene (-121/+86) contained equivalent transcriptional activity to the complete 1.2 kb 5' flanking region of the DPYD gene. Site-directed mutagenesis of the luciferase reporter constructs demonstrated that the -72/-23 sequence contained two regulatory regions (designated elements I and II) essential for promoter activity. Gel shift experiments demonstrated that both regulatory elements specifically bind with protein(s) from nuclear extracts of 293 cells. Competitive binding experiments with 293 nuclear extracts and radiolabeled oligonucleotides (corresponding to elements I and II) suggest that the same protein(s) bind to both regulatory elements. We conclude that constitutive expression of the DPYD gene involves a limited GC-rich region of the 5' flanking sequence of the DPYD gene which contains two regulatory elements.

Amino Acid Sequence↗
PubMed2000

Activation of protein kinase C induces nuclear translocation of RFX1 and down-regulates c-myc via an intron 1 X box in undifferentiated leukemia HL-60 cells.

Treatment of human promyelocytic leukemia cells (HL-60) with phorbol 12-myristate 13-acetate (PMA) is known to decrease c-myc mRNA by blocking transcription elongation at sites near the first exon/intron border. Treatment of HL-60 cells with either PMA or bryostatin 1, which acutely activates protein kinase C (PKC), decreased the levels of myc mRNA and Myc protein. The inhibition of Myc synthesis accounted for the drop in Myc protein, because PMA treatment had no effect on Myc turnover. Treatment with PMA or bryostatin 1 increased nuclear protein binding to MIE1, a c-myc intron 1 element that defines an RFX1-binding X box. RFX1 antiserum supershifted MIE1-protein complexes. Increased MIE1 binding was independent of protein synthesis and abolished by a selective PKC inhibitor, which also prevented the effect of PMA on myc mRNA and protein levels and Myc synthesis. PMA treatment increased RFX1 in the nuclear fraction and decreased it in the cytosol without affecting total RFX1. Transfection of HL-60 cells with myc reporter gene constructs showed that the RFX1-binding X box was required for the down-regulation of reporter gene expression by PMA. These findings suggest that nuclear translocation and binding of RFX1 to the X box cause the down-regulation of myc expression, which follows acute PKC activation in undifferentiated HL-60 cells.

Active Transport, Cell Nucleus↗
PubMed2000

Identification of a serine hydrolase as a key determinant in the microbial degradation of polychlorinated biphenyls.

The ability of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD) of Burkholderia cepacia LB400 to hydrolyze polychlorinated biphenyl (PCB) metabolites was assessed by determining its specificity for monochlorinated HOPDAs. The relative specificities of BphD for HOPDAs bearing chlorine substituents on the phenyl moiety were 0.28, 0.38, and 1.1 for 8-Cl, 9-Cl, and 10-Cl HOPDA, respectively, versus HOPDA (100 mm phosphate, pH 7.5, 25 degrees C). In contrast, HOPDAs bearing chlorine substituents on the dienoate moiety were poor substrates for BphD, which hydrolyzed 3-Cl, 4-Cl, and 5-Cl HOPDA at relative maximal rates of 2.1 x 10(-3), 1.4 x 10(-4), and 0.36, respectively, versus HOPDA. The enzymatic transformation of 3-, 5-, 8-, 9-, and 10-Cl HOPDAs yielded stoichiometric quantities of the corresponding benzoate, indicating that BphD catalyzes the hydrolysis of these HOPDAs in the same manner as unchlorinated HOPDA. HOPDAs also underwent a nonenzymatic transformation to products that included acetophenone. In the case of 4-Cl HOPDA, this transformation proceeded via the formation of 4-OH HOPDA (t(12) = 2.8 h; 100 mm phosphate, pH 7.5, 25 degrees C). 3-Cl HOPDA (t(12) = 504 h) was almost 3 times more stable than 4-OH HOPDA. Finally, 3-Cl, 4-Cl and 4-OH HOPDAs competitively inhibited the BphD-catalyzed hydrolysis of HOPDA (K(ic) values of 0.57 +/- 0. 04, 3.6 +/- 0.2, and 0.95 +/- 0.04 microm, respectively). These results explain the accumulation of HOPDAs and chloroacetophenones in the microbial degradation of certain PCB congeners. More significantly, they indicate that in the degradation of PCB mixtures, BphD would be inhibited, thereby slowing the mineralization of all congeners. BphD is thus a key determinant in the aerobic microbial degradation of PCBs.

Bacterial Proteins↗
PubMed2000

Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction.

Several recent studies have reported a correlation between intratumor dihydropyrimidine dehydrogenase (DPD) messenger RNA (mRNA) levels and sensitivity to 5-fluorouracil (5-FU). However, significant tissue requirements and labor-intensive methodology have limited the large-scale studies necessary for statistical validation. In addition, the semiquantitative results obtained by these methods further limit their application. We have developed a real-time reverse transcription-PCR (RT-PCR) assay, based on TaqMan fluorescence methodology, capable of rapid and accurate quantitation of DPD mRNA levels in biopsy-sized tissue samples. Results obtained with this approach indicate a linear dynamic range of 10(8)-10(3) DPD mRNA copies, with an intra-assay variation of <5%. We evaluated the data using three different methods (absolute standard curve, relative standard curve, and comparative C(T)) and show them to be equivalent. This RT-PCR assay was validated by quantitative comparison to Northern blot analysis in five tissues. In addition, analysis of 18 colorectal tumor and liver tissue specimens demonstrated a significant correlation (r(2) = 0.90) between DPD enzyme activity and mRNA levels. This method provides the first high-throughput, reproducible, and sensitive technique capable of determining DPD mRNA expression levels in nanogram amounts of total RNA.

Dihydrouracil Dehydrogenase (NADP)↗
PubMed2000

Bupropion-SR in treatment of social phobia.

A 12-week, open label flexible dosing study was conducted to evaluate the efficacy of bupropion-SR in the treatment of generalized social phobia. The primary outcome measures include the Clinical Global Impression of Improvement (CGI-I) and the Brief Social Phobia Rating Scale (BSPS). A total of 18 subjects were enrolled. Five of the ten subjects who completed all 12 weeks were considered as responders. Response to treatment was defined as a CGI-I score of 1 or 2, ("much improved" or "very much improved," respectively) and a > 50% decrease in BSPS score. The final doses for the completers ranged between 200 and 400 mg/day (mean 366 +/- 68 mg/day). The medication was generally well tolerated. Findings from this open-label trial suggest that bupropion-SR may be useful in treating generalized social phobia.

Adult↗
PubMed2000

A longitudinal study of maternal plasma insulin-like growth factor binding protein-1 concentrations during normal pregnancy and pregnancies complicated by pre-eclampsia.

Insulin-like growth factor binding protein-1 (IGFBP-1) is synthesized by the decidual stroma, and is thought to act locally to inhibit IGF activity and so limit trophoblast invasion. Cross-sectional studies have reported conflicting data on maternal circulating concentrations of IGFBP-1 in early pregnancy before the development of pre-eclampsia. A longitudinal study was performed in 10 women who went on to develop pre-eclampsia and a group of 12 normal pregnant controls, chosen to be similar for maternal age, booking body mass index (BMI) and gestational age. Maternal IGFBP-1 concentrations were measured in plasma obtained at 16, 20, 24, 28, 32 and 36 weeks. Plasma IGFBP-1 concentrations were unchanged over this period in normal pregnancy. In contrast, the concentrations in women who developed pre-eclampsia increased progressively. At 16, 20, and 24 weeks the concentrations were significantly lower compared to normal pregnancy, at 28 and 32 weeks, similar, but by 36 weeks the concentrations were significantly greater than the normal controls. The data show that circulating IGFBP-1 concentrations are lower in early pregnancy before the development of pre-eclampsia. Thus, it is suggested that IGFBP-1-induced inhibition of IGF activity is unlikely to be responsible for the impaired trophoblast invasion observed in pre-eclampsia.

Adult↗
PubMed2000

Antenatal dexamethasone and the growth hormone-insulin-like growth factor axis.

Dexamethasone administration has marked effects on the growth hormone-insulin-like growth factor axis (GH-IGF) in animal and human studies. During pregnancy in the rat, it is associated with fetal growth restriction due to inhibition of IGF bioactivity. In the human only repeated dosages have been associated with fetal growth restriction. The aim of this study is to test the hypothesis that antenatal dexamethasone administration to pregnant women is associated with reduced activity of the GH-IGF axis. To achieve this blood samples were taken from 12 pregnant women pre- and at 24 h and 48 h after dexamethasone administration. In these samples GH, IGF-I, IGF bioactivity and IGF binding protein (IGFBP)-3 protease activity were measured. In view of the interaction between insulin and the GH-IGF axis, glucose and insulin concentrations were also measured. There were no significant differences between the concentrations of GH, IGF-I, IGF bioactivity and IGFBP-3 protease activity before and after dexamethasone. The concentrations of glucose and insulin were significantly higher at 24 h, but not 48 h post-dexamethasone. It is concluded that a single antenatal course of dexamethasone does not alter the GH-IGF-I axis in pregnant women at the time points studied.

Adult↗
PubMed2000

The feto-placental unit stimulates the pregnancy-associated increase in maternal bone metabolism.

The aim of the study was to investigate role of the feto-placental unit in the pregnancy-induced increase in maternal bone metabolism. To achieve this, circulating concentrations of carboxy terminal pro-peptide of type I pro-collagen (PICP, a marker of bone formation) and cross-linked carboxy terminal telopeptide of type I collagen (ICTP, a marker of bone resorption) were measured in three groups of pregnant women. Group 1 comprised 12 women with singleton pregnancies; group 2, nine women with twin pregnancies; and group 3, 19 women with multifetal pregnancies (> or =3 fetuses) before and after selective fetal reduction to twin pregnancies. Blood samples were obtained at 10-12 weeks gestation (groups 1-3, pre-fetal reduction in group 3) and 4 weeks and 8 weeks later (groups 2 and 3). Before fetal reduction there was a significant correlation between the number of fetuses and the concentrations of both PICP and ICTP (r = 0.503 and P = 0.001 and r = 0.573 and P < 0.001 respectively). The circulating concentrations of PICP and ICTP were significantly higher in the pre-reduction multifetal pregnancies than in the twin pregnancies (P < 0.001 and P = 0.0013 respectively). The circulating concentrations of ICTP in multifetal pregnancies fell by 4 weeks after fetal reduction to those observed in control twins. Concentrations of PICP were unaltered after fetal reduction. Higher order multiple pregnancies had the greatest decline in ICTP concentrations. These data suggest that the increased bone turnover observed in the multifetal pregnancies is due to a factor derived from the feto-placental unit and that this factor acts primarily to stimulate bone resorption.

Biomarkers↗