Depressive symptoms in mid-pregnancy, lifetime stressors and the 5-HTTLPR genotype.

Few studies of gene-environment interactions for the serotonin transporter promoter polymorphism (5-HTTLPR), life stressors and depression have considered women separately or examined specific types of stressful life events. None have looked at depression during pregnancy. In the Pregnancy Outcomes and Community Health (POUCH) Study, women were queried about history of stressful life events and depressive symptoms at the time of enrollment (15-27 weeks gestation). Stressful life events were grouped a priori into "subconstructs" (e.g. economic, legal, abuse, loss) and evaluated by subconstruct, total subconstruct score and total stressful life event score. The effect of genotype on the association between stressful life events and elevated depressive symptoms was assessed in 568 white non-Hispanic participants. The relationship between exposure to abuse and elevated depressive symptoms was more pronounced in the s/s group (OR = 24.5) than in the s/l group (OR = 3.0) and the l/l group (OR = 7.7), but this significant interaction was detected only after excluding 73 (13%) women with recent use of psychotropic medications. There was no evidence of gene-environment interaction in analytic models with other stressful life events subconstructs, total subconstruct score or total stressful life events score. These data offer modest support to other reports of gene-environment interaction and highlight the importance of considering specific stressful life events.

Use of PCR amplification of cDNA to study mechanisms of human cell mutagenesis and malignant transformation.

PCR is widely employed to amplify short segments of genomic DNA to determine if a specific change has occurred. But some investigators need to sequence the entire coding region of mammalian genes to determine what specific changes have occurred. In 1989, we [Yang et al: Gene 83:347-354] described a method to copy mRNA of the hypoxanthine (guanine) phosphoribosyl transferase (HPRT) gene directly from the lysate of a clone of 6-thioguanine-resistant mutant diploid human fibroblasts without the need for RNA extraction or DNA template purification. To avoid detecting random changes introduced by polymerases, 100 to 500 cells from an individual clone, each containing the identical mutation, are lysed and the cDNA is amplified 10(10)-to 10(11)-fold to obtain 5 to 10 micrograms of DNA. The consensus sequence of the cDNA is determined by direct nucleotide sequencing. Using this method, we have investigated the kinds of mutations induced by carcinogens in the coding region of the HPRT gene and their location in the gene and examined the role of DNA repair in this process. Normal repair-proficient human cells and cells deficient in DNA repair were exposed to mutagens in exponential growth or synchronized and exposed at the beginning of S phase or in G1 phase several hr prior to DNA replication. The kinds and location of mutations in the HPRT gene were determined and knowledge of the nature of the DNA lesions formed by the various mutagens allowed assignment of the DNA strand in which the premutagenic lesion that gave rise to the mutation had been located. Related assays involving PCR have been used to determine the nature of mutations in the coding region of the H-, N-, or K-ras genes of tumor-derived malignant human cells and to determine whether or not such cells express specific growth factor genes.

Induction of mitochondrial metabolism and pH-modulated ammoniagenesis by rocking LLC-PK1 cells.

In an effort to find a model system in which the regulation of renal ammoniagenesis could be delineated, the LLC-PK1 line of cultured pig kidney epithelial cells was examined. Ammonia production by normally cultured LLC-PK1 cells (monolayers on 75-cm2 flasks, under 6 ml still serum-containing medium) was found to be neither modulated by direct (3 h) nor adapted by chronic (3 day) manipulation of medium pH (production at pH 7.05, 7.40, or 7.60 not significantly different). Considering that the mitochondrial glutamine to alpha-ketoglutarate pathway is the major regulatory site of ammoniagenesis, and that mitochondrial metabolism might be restricted under the normal lactate-generating or hypoxic culture conditions, we examined ammonia production by rocked flasks of LLC-PK1 cells. Flask were rocked continually at a rate of 2.5 oscillations/min, thereby exposing the cells to the atmosphere 40-50% of the time and also maximizing medium O2 tension and nutrient-waste exchange. Mitochondrial enzyme activities in rocked LLC-PK1 cells were shown to be consistently 1.5-fold greater than those in normally cultured cells. Ammonia production by rocked cells was not only directly modulated by short incubations with low and high pH media (production at pH 7.05 greater than 7.40 greater than 7.60) but was adapted by chronic (3 day) manipulations of medium pH (16 h after return to pH 7.40 medium production by pH 7.05- greater than 7.40- greater than 7.60-adapted cells). Thus rocker culture of the LLC-PK1 cell line both induces mitochondrial metabolism and converts cellular ammoniagenesis to a pH-sensitive phenomenon.(ABSTRACT TRUNCATED AT 250 WORDS)