Renal cell carcinoma after therapy for neuroblastoma.L F Donnelly, I O Rencken, K Shardell, K K Matthay, C R Miller, R K Vartanian and C A GoodingAudio Available | Share
Several genes involved in the metabolism of carcinogens have been found to be polymorphic in human populations and are associated with increased risk of cancer at some sites.This study focuses on the polymorphic enzyme glutathione transferase u (GT pu).Smokers with low lymphocyte GT p. activity are at an approximately 2-fold higher risk for lung cancer and an approximately 3-fold higher risk for stomach and colon adenocarcinomas.Recent cloning and sequencing of the GSTI gene has allowed the development of convenient genotyping methods based on restriction fragment length polymorphisms (RFLP) or the polymerase chain reaction (PCR).The GSTI polymorphism has been shown to be a deletion of the gene locus.To detect the presence or absence of the gene we amplified exons 4-5 and/or exons 6-7 of the GSTI gene by PCR.PCR amplification produced bands of 215-bp or 273-bp from individuals with one or two copies of the GSTI allele and no band ifthe individual was homozygously deleted (0/0).In the exon 6-7 PCR, we co-amplified a 268- bp portion of the P-globin gene as an internal reference standard for quantitative analysis of product yield.This allowed homozygote individuals ( + / + ) to be distinguished from heterozygotes ( + /0).We have compared the GSTI genotype to lymphocyte GT p. activity measured on trans-stilbene oxide (TSO) in the lymphocytes of 45 individuals.Low GT p. activity (< 67 pmole/min/107 cells) was strongly associated (24/24) with the GSTI 0/0 genotype.With the exception of one individual, activities greater than 67 pmole/ min/107 were associated with the presence of the GSTI allele (20/21).Individuals with the highest GT-TSO activity were found to be homozygous for GSTI ( + / + ), while heterozygotes ( + /0) generally had lower activity, suggesting a gene dosage effect in lymphocytes.The allele distribution among four sampled populations varied considerably.In a North Carolina population, 51% (65/127) were GSTI 0/0, and this finding is consistent with those of other studies based on phenotypic analysis.In three smaller cohorts, the GSTI 0/0 genotype was observed to occur in: 30% (14/47) of Finnish foundry workers, 33% (18/54) of Georgia dye workers, and 62% (74/120) of Thiwanese placental samples.In the future, we shall investigate the mechanistic link between polymorphisms in carcinogen metabolism genes and interindividual variation in measures of DNA damage, such as DNA adducts and hprt mutation frequency.
It is well established that gut and pancreas development depend on epithelial-mesenchymal interactions. We show here that several Wnt, Frizzled, and secreted frizzled-related protein (sFRP) encoding mRNAs are present during mouse pancreatic morphogenesis. Wnt5a and 7b mRNA is broadly expressed in foregut mesenchyme starting around embryonic day 10 in mice. Other members expressed are Wnt2b, Wnt5b, and Wnt11. In addition, genes for the Wnt receptors, Frizzled2, 3, 4, 5, 6, 7, 8, and 9 are expressed. To understand potential Wnt functions in pancreas and foregut development in vivo, we analyzed transgenic F0 mouse fetuses expressing Wnt1 and 5a cDNAs under control of the PDX-1 gene promoter. In PDX-Wnt1 fetuses, the foregut region normally comprising the proximal duodenum instead resembles a posterior extension of the stomach, often associated with complete pancreatic and splenic agenesis. Furthermore, the boundary between expression domains of gastric and duodenal markers is shifted in a posterior direction. In PDX-Wnt5a fetuses, several structures derived from the proximal foregut are reduced in size, including the pancreas, spleen, and stomach, without any apparent shift in the stomach to duodenum transition. In these fetuses, overall pancreatic morphology is changed and the pancreatic epithelium is dense and compact, consistent with Wnt5A effects on cell movements and/or attachment. Taken together, these results suggest that Wnt genes participate in epithelial-mesenchymal signaling and may specify region identity in the anterior foregut.
Abstract Influenza and other respiratory viruses represent a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as the current COVID-19 crisis. One of the greatest barriers to the development of effective therapeutic agents to treat influenza, coronaviruses, and many other infections of the respiratory tract is the absence of a robust preclinical model. Preclinical studies currently rely on high-throughput, low-fidelity in vitro screening with cell lines and/or low-throughput animal models that often provide a poor correlation to human clinical responses. Here, we introduce a human primary airway epithelial cell-based model integrated into a high-throughput platform where tissues are cultured at an air-liquid interface (PREDICT96-ALI). We present results on the application of this platform to influenza and coronavirus infections, providing multiple readouts capable of evaluating viral infection kinetics and potentially the efficacy of therapeutic agents in an in vitro system. Several strains of influenza A virus are shown to successfully infect the human primary cell-based airway tissue cultured at an air-liquid interface (ALI), and as a proof-of-concept, the effect of the antiviral oseltamivir on one strain of Influenza A is evaluated. Human coronaviruses NL63 (HCoV-NL63) and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for protein priming, and we confirm expression of both in our ALI model. We also demonstrate coronavirus infection in this system with HCoV-NL63, observing sufficient viral propagation over 96 hours post-infection to indicate successful infection of the primary cell-based model. This new capability has the potential to address a gap in the rapid assessment of therapeutic efficacy of various small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.
Directed sort-and-combine strategies offer the efficiency of combinatorial split-and-mix synthesis, with the discrimination to produce only the desired compounds. The technique involve the placement of the microspheres into the reaction vessels, namely random (split-and-mix) versus directed (sort-and-combine). Therefore, it is a requirement of directed sort-and-combine strategies for the microspheres to be pre-encoded to determine the appropriate reaction vessel for each microsphere. An instrumental technique was also developed to use the fluorescently labeled microspheres to produce a large oligonucleotide library via a directed sort-and-combine process.
Effect of Different Levels and Periods of Lead Exposure on Tissue Levels and Excretion of Lead, Zinc, and Calcium in the Rat. Victery, W., MILLER, C. R., ZHU, S.-Y., AND GOYER, R. A. (1987). Fundam. Appl. Toxicol. 8, 506–516. Influence of lead on tissue content and urinary excretion of lead, zinc, and calcium in rats was studied following various exposure periods. Weanling male rats were fed a trace mineral-sufficient diet with either 0, 200, 500, or 1000 ppm lead (as acetate) in drinking water for 4, 8, or 12 weeks. Blood lead ranged from 40 to over 100 μg/dl; kidney lead was highest at 4 weeks. Urinary lead excretion was highest at 4 weeks and declined with longer exposure. Urinary zinc excretion correlated positively with lead excretion at the lower excretion rates but plateaued at higher lead excretion rates. After 12 weeks exposure at each lead dose employed, decreased zinc concentration was observed in testes, bone, and brain. Plasma, erythrocyte, and kidney zinc were not affected, while pancreas and liver zinc were slightly elevated. Urine calcium was increased significantly only in rats exposed to 1000 ppm, possibly reflecting renal cell damage as determined by elevated renal calcium levels. These results indicate that lead dose is more important than exposure period for determining kidney lead levels, while urinary lead excretion rate is both dose and time dependent. Blood lead clearance values are relatively independent of dose and fall as exposure continues. Essential trace metal balance for zinc, especially, and to a lesser extent for calcium, is affected by the dose and length of chronic lead exposure.
Since real time PCR was first developed, several approaches to estimating the initial quantity of template in an RT-PCR reaction have been tried. While initially only the early thermal cycles corresponding to exponential duplication were used, lately there has been an effort to use all of the cycles in a PCR. The efforts have included both fitting empirical sigmoid curves and more elaborate mechanistic models that explore the chemical reactions taking place during each cycle. The more elaborate mechanistic models require many more parameters than can be fit from a single amplification, while the empirical models provide little insight and are difficult to tailor to specific reactants.We directly estimate the initial amount of amplicon using a simplified mechanistic model based on chemical reactions in the annealing step of the PCR. The basic model includes the duplication of DNA with the digestion of Taqman probe and the re-annealing between previously synthesized DNA strands of opposite orientation. By modelling the amount of Taqman probe digested and matching that with the observed fluorescence, the conversion factor between the number of fluorescing dye molecules and observed fluorescent emission can be estimated, along with the absolute initial amount of amplicon and the rate parameter for re-annealing. The model is applied to several PCR reactions with known amounts of amplicon and is shown to work reasonably well. An expanded version of the model allows duplication of amplicon without release of fluorescent dye, by adding 1 more parameter to the model. The additional process is helpful in most cases where the initial primer concentration exceeds the initial probe concentration. Software for applying the algorithm to data may be downloaded at http://www.niehs.nih.gov/research/resources/software/pcranalyzer/We present proof of the principle that a mechanistically based model can be fit to observations from a single PCR amplification. Initial amounts of amplicon are well estimated without using a standard solution. Using the ratio of the predicted initial amounts of amplicon from 2 PCRs is shown to work well even when the absolute amounts of amplicon are underestimated in the individual PCRs.
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