Evaluation of an automated extraction system in combination with Affigene CMV Trender for CMV DNA quantitative determination: comparison with nested PCR and pp65 antigen test.

CMV viral load quantitation is a powerful tool to assist clinicians in making accurate diagnoses, managing post-transplant CMV disease and monitoring antiviral therapy. The aim of this study was to evaluate the performance of Affigene CMV Trender for CMV viral load determination used in combination with a non-dedicated nucleic acid extraction system (BioRobot MDx) for high-throughput routine. Linearity, reproducibility and sensitivity were examined. Clinical samples were used to compare results obtained with the Affigene CMV Trender, with an "in house" nested PCR used for routine diagnosis and with pp65 antigenemia. The results indicated that the test is linear in the range of 1.81-5.18 Logcopies/ml and that sensitivity is 77 copies/ml. The concordance of the Affigene CMV Trender with nested PCR was high, (k=0.91, IC 95%=0.82-1.00), whereas a substantial concordance with pp65 antigenemia was observed (k=0.64, IC 95%=0.54-0.73). In conclusion, combined use of a non-dedicated automated nucleic acid extraction method with the Affigene CMV Trender results in an accurate high throughput system, suitable for routine laboratory monitoring of CMV infection.

Insulin-like growth factor 1 (IGF-I) effects on sex-specific cytochrome P450 enzymes in normal and hypophysectomised male rats.

The role of growth hormone (GH) in the regulation of the sex-differentiated rat cytochrome P450 (CYP) enzymes has been extensively studied. However, little is known about the involvement of insulin-like growth factor I (IGF-I) as a mediator in this regulation. We wanted to study if IGF-I had effects on sex-differentiated CYP enzymes and to compare the effects of IGF-I to the effects of GH. IGF-I, GH or saline was administered continuously via osmotic minipumps to normal and hypophysectomised rats for seven days. After treatment, the expression of several sex-differentiated liver enzymes (CYP2C11, CYP2C12), the female-dominant steroid 5alpha-reductase, and the male-dominant CYP3A2 enzyme was studied at mRNA, protein and/or functional levels. Our results demonstrate that IGF-I has marked effects on the sex-specific expression of CYP2C11 and CYP2C12. The effects of IGF-I were similar to those of GH. In contrast, in hypophysectomised rats IGF-I gave effects opposite to those observed after GH treatment to normal rats on the CYP3A-associated cortisol 6beta-hydroxylation. No effects of IGF-I on the steroid 5alpha-reductase activity were observed.

Gene expression of a novel cytochrome P450 of the CYP4F subfamily in human seminal vesicles.

19R-Hydroxyprostaglandins are major components of human seminal fluid. They are apparently formed in the seminal vesicles by NADPH-dependent omega2-hydroxylation. The hydroxylase is likely a cytochrome P450 (CYP), which has not been identified. To address this issue we studied gene expression of CYPs in human seminal vesicles (n = 4) with reverse-transcription polymerase chain reaction (RT-PCR). CYP1B1, CYP2E1, CYP2J2, CYP3A5, CYP4B1, and CYP4B1 with insertion of three nucleotides (Ser207) were detected in all subjects. RT-PCR with degenerate primers for the CYP4 family yielded a novel cDNA sequence, which was derived from a previously reported genomic sequence on chromosome 19p13.1 and present in all subjects. cDNA cloning showed that the deduced amino acid sequence consisted of 520 amino acids. Northern blot analysis demonstrated mRNA transcripts of approximately 2.1 and approximately 2.3 kb. The deduced protein showed 81.2 and 76.7% amino acid identity with the human enzymes CYP4F2 and CYP4F3. The novel CYP was designated CYP4F8.

Independent patterns of cytochrome P450 gene expression in liver and blood in patients with suspected liver disease.

OBJECTIVE: Assessment of liver metabolism using blood samples was tested by comparison of cytochrome P450 (CYP) gene expression in paired liver and blood samples from 13 individuals. METHODS: Total RNA was isolated from percutaneous needle biopsies and blood collected simultaneously. Gene expression for CYP1A2, CYP1B1, CYP2E1 and CYP3A4 was studied using a real-time reverse-transcription polymerase chain reaction (RT-PCR) method. RESULTS: All CYP mRNA species were expressed in all liver biopsies but at varying levels. The highest and lowest levels of expression were observed for CYP2E1 and CYP1B1, respectively. The expression patterns differed between blood and liver. CYP1B1 was expressed in all blood samples at a 20% higher level than in the liver. CYP1A2, CYP2E1 and CYP3A4 were expressed in blood at 35- to 5,000-fold lower levels than in liver. None of the transcripts in blood showed any correlation with the expression in liver. CONCLUSION: We conclude that blood cannot serve as a surrogate organ for assessment of the expression of the studied CYP genes in liver.

Quantitation of cytochrome P450 mRNAs in patients with suspected liver diseases as assessed by reverse transcriptase-polymerase chain reaction.

The cytochrome P450 system (CYP) is vital for the oxidation and detoxification of numerous drugs and other xenobiotics in the liver. Many of the CYP enzymes are polymorphically expressed and may be induced or inhibited by xenobiotics including drugs and alcohol. The measurement of gene expression is thus important in studies of the mechanisms of interaction with and function of the CYP system. We have developed a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method for the study of the mRNA expression of three CYP enzymes--2E1, 1A2, and 3A4--in snap-frozen percutaneous liver biopsy samples. The method was made quantitative by the introduction of a recombinant RNA internal standard that contains a transcript of the beta-globin gene and sequences specific for the studied CYP enzymes. The method allows the analysis of mRNA expression of several enzymes in as little as 5 mg of liver tissue. Liver tissue specimens from 19 patients with suspected liver disease were analyzed for CYP-specific mRNA expression. The mean mRNA concentrations for CYP1A2, 2E1, and 3A4 were 0.16, 0.74, and 0.32 amol of specific mRNA per nanogram of poly (A+) mRNA, respectively, but a large interindividual variation was observed. CYP3A7 primers were included in the internal standard. However, because of low expression it was not possible to quantitate the enzyme. This quantitative RT-PCR method is of value for studies of the mechanisms of variation and interactions with the members of the CYP enzyme family in healthy and diseased liver and other organs.

Amino acid differences at positions 10, 11, and 104 explain the profound catalytic differences between two murine pi-class glutathione S-transferases.

The glutathione S-transferases play a pivotal role in the detoxification of toxic and carcinogenic electrophiles. We have previously reported the isolation of two actively transcribed murine pi-class glutathione S-transferase genes. In this study the two proteins encoded by these genes, Gst p-1 and Gst p-2, were expressed in Escherichia coli and found to exhibit profoundly different catalytic activities, the activity of Gst p-2 toward a panel of electrophilic substrates being 1-3 orders of magnitude lower than that of Gst p-1. In order to establish the basis for the difference between these highly homologous proteins, mutants were generated where specific amino acids had been exchanged. Kinetic analysis of the wild-type and mutant enzymes revealed that the amino acid differences occurring at positions 10 (Val/Ser), 11 (Arg/Pro), and 104 (Val/Gly) are responsible for the reduced enzymatic activity of Gst p-2. This analysis together with computer graphics modeling for Gst p-2 indicated that these changes affected both substrate and glutathione binding to the enzyme.

Intra-individual variation and sex differences in gene expression of cytochromes P450 in circulating leukocytes.

To clarify partly inconsistent results in gene expression of cytochromes P450 (CYP) in the circulation, we undertook a systematic study over a long time period in 19 healthy men and women. CYP specific mRNA for 1A2, 1B1, 2E1 and 3A4 was studied in the leukocytes collected repeatedly on 20 occasions over a 10-week period. Our study revealed a varying pattern of CYP expression over time. CYP3A4 specific mRNA exhibited the largest intra-individual variation with an average coefficient of variation between 40 and 250%. CYP1B1 and CYP2E1 did not vary as much (39-110%). CYP1A2 was sporadically detected in only ten individuals, but varied considerably when measurable (61-256%). The expression in CYP1B1 was significantly higher in women than in men (P = 0.02). We conclude that CYP gene expression in blood varies considerably over time. It is conceivable that the variation reflects a hitherto unknown influence of exogenous or endogenous factors such as hormones, cytokines, and other circulating factors on the hematogeneous cytochromes.

Detection of cytochrome P450 mRNA transcripts in prostate samples by RT-PCR.

BACKGROUND: The expression of cytochrome P450 (CYP)-dependent mono-oxygenases in the prostate is important, as it will determine the rate of activation of potential carcinogens as well as the metabolism of hormones with implications in diseases of the prostate. In addition, the levels of cytochromes P450 in prostatic tumours may well be determinants of the outcome of therapy involving P450 substrates such as anti-androgens. METHODS: The gene expression of 12 different CYP genes was measured by reverse transcription-polymerase chain reaction (RT-PCR) in a total of 28 human prostatic tumour and nontumour samples. RESULTS: Intriguingly, a large number of CYP mRNAs were detected in the prostate samples, including CYP1A2, -1B1, -2C19, -2D6, -3A4, -3A5, -3A7 and -4B1. CYP1B1 was consistently expressed and CYP3A5 and CYP4B1 were expressed in a majority of the samples tested. CONCLUSIONS: These data demonstrate a wide range of CYP genes being expressed in the prostate. The relative importance of these enzymes in the pathogenesis and treatment of prostatic disease remains an important theme for further study.