Quantification of mRNA expression levels using real-time reverse transcription PCR (RT-PCR) is increasingly used to validate results of DNA microarrays or GeneChips. It requires an improved method that is more robust and more suitable for high-throughput measurements. In this report, we compare a user non-influent, second derivative method with that of a user influent, fit point method that is widely used in the literature. We also describe the advantage of using a double correction: one correction using the expression levels of a housekeeping gene of an experiment as an internal standard and a second using reference expression levels of the same housekeeping gene in the tissue or cells. The first correction permits one to decrease errors due to sample preparation and handling, while the second correction permits one to avoid the variation of the results with the variability of housekeeping in each tissue, especially in experiments using various treatments. The data indicate that the real-time PCR method is highly efficient with an efficiency coefficient close to the theoretical value of two. The results also show that the second derivative method is more accurate than the fit point method in quantifying low gene expression levels. Using triplicate experiments, we show that measurement variations using our method are low with a mean of variation coefficients of <1%.

Blocking morning light exposure with dark goggles can contribute to the adjustment to night work but these glasses are incompatible with driving. Recently, it was discovered that the biological clock is most sensitive to short wavelengths (blue light). Therefore, we tested the hypothesis that cutting the blue portion of the light spectrum with orange lens glasses (blue blockers) would prevent the light-induced melatonin suppression, a test broadly used as an indirect assessment of the circadian clock sensitivity. Fourteen normal subjects were exposed at night to a 60 min bright light pulse (1300 lx behind filters) between 01:00 and 02:00 hr while wearing orange lens glasses (experimental condition) or grey lens glasses (control condition). The amount of salivary melatonin change observed during the light pulse was compared with a melatonin baseline obtained the night before. Although both glasses transmitted the same illuminance (1300 lx) but at an irradiance 25% higher for the orange lens (408 microW/cm2) compared with the grey lens (327 microW/cm2), a non-significant increase of 6% (95% CI, -20% to 9%) was observed with the orange lens whereas a significant (P < 0.05) reduction of 46% (95% CI, 35-57%) was observed with the grey lens. Blue blockers represent an elegant means to prevent the light-induced melatonin suppression. Further studies are needed to show that these glasses, which are suitable for driving, could facilitate adaptation to night work.

The best known activity of steroid 5 alpha-reductase is the transformation of testosterone into dihydrotestosterone, the most potent androgen. Two types of human steroid 5 alpha-reductase cDNAs and the type I gene have previously been isolated and characterized. This report describes the isolation and characterization of the human type II 5 alpha-reductase gene, the gene most likely responsible for male pseudohermaphroditism due to 5 alpha-reductase deficiency as well as the one presumed to be involved in a major androgen-related diseases such as prostate cancer and benign prostatic hyperplasia. The type II 5 alpha-reductase gene contains five exons of 352, 164, 102, 151 and 1695 bp, respectively, which share 43.8% to 64.1% homology with exons of the corresponding type I gene. These exons are separated by four introns of greater than 29, and approximately 2.3, 2.0 and 3.0 kb. Analysis of primer extension products by polyacrylamide gel electrophoresis as well as by subcloning and sequencing reveals a start site located 71 nucleotides upstream the ATG initiating codon.

Metal toxicity toward microorganisms is usually evaluated by determining growth inhibition. To achieve a mechanistic interpretation of such toxic effects, the intricate coupling between cell growth kinetics and metal partitioning dynamics at the cell-solution interface over time must be considered on a quantitative level. A formalism is elaborated to evaluate cell-surface-bound, internalized, and extracellular metal fractions in the limit where metal uptake kinetics is controlled by internalization under noncomplexing medium conditions. Cell growth kinetics is tackled using the continuous logistic equation modified to include growth inhibition by metal accumulation to intracellular or cell surface sites. The theory further includes metal-proton competition for adsorption at cell-surface binding sites, as well as possible variation of cell size during exposure to metal ions. The formalism elucidates the dramatic impacts of initial cell concentration on metal bioavailability and toxicity over time, in agreement with reported algae bioassays. It further highlights that appropriate definition of toxicity endpoints requires careful inspection of the ratio between exposure time scale and time scale of metal depletion from bulk solution. The latter depends on metal internalization-excretion rate constants, microorganism growth, and the extent of metal adsorption on nonspecific, transporter, and growth inhibitory sites. As an application of the theory, Cd toxicity in the algae Pseudokirchneriella subcapitata is interpreted from constrained modeling of cell growth kinetics and of interfacial Cd-partitioning dynamics measured under various exposure conditions.

Dehydroepiandrosterone sulfotransferase catalyzes the transformation of dehydroepiandrosterone to dehydroepiandrosterone sulfate, the most abundant steroid in circulation in the human and primate. Dehydroepiandrosterone sulfate serves as precursor for the formation of active androgens and estrogens in peripheral target tissues. In addition, blockade at the dehydroepiandrosterone level could give raise to high level of DHEA and thus disorders due to mild excess of androgen. Recently, the cDNA encoding dehydroepiandrosterone sulfotransferase has been isolated from a human liver cDNA library. To study the regulation and expression, as well as the possible defect linked to DHEA sulfotransferase gene, we have isolated and characterized its structure by screening a lambda EMBL3 library of human leukocyte genomic DNA using human dehydroepiandrosterone sulfotransferase cDNA as a probe. Sequencing of the gene shows that it is included in approximately 17 kb and contains six exons separated by five introns. Northern blot analysis shows a strong signal in the adrenals and liver, whereas no signal was detected in the spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocytes, heart, brain, placenta, lung, skeletal muscle, kidney, or pancreas. Using primer extension analysis, the transcription start site is located at nucleotide 98 upstream from the ATG initiating codon. Putative TATA and CAAT boxes are situated at positions 72 and 96 upstream from the transcription start site, respectively. Using DNA from a panel of human/rodent somatic cell hybrids, and amplification of the gene by the polymerase chain reaction, the human dehydroepiandrosterone sulfotransferase gene has been assigned to chromosome 19.