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Intramural Papers of the Month

By Robin Arnette
March 2008

Atopic Asthma Risks to Women in the Agricultural Health Study

A team of researchers from the NIEHS, the National Institute for Occupational Safety and Health and the National Cancer Institute (NCI) determined that pesticides may contribute to atopic asthma, which is defined as asthma symptoms produced upon exposure to an environmental antigen. The results, generated from the 25,814 women who provided information on asthma status as part of the Agricultural Health Study, ( were published in the January 1 issue of the American Journal of Respiratory and Critical Care Medicine. The study was funded by the Intramural Research Program of the National Institutes of Health, the NIEHS and the NCI.

Farm women who spent their childhood on farms had a lower incidence of allergic asthma. There was additional protection for those women who grew up on farms and currently work with animals; however, women who did not grow up on farms had a higher risk if they currently worked with animals. Farm women who applied pesticides had a 50 percent increased risk of allergic asthma. Women who used parathion, phorate or coumaphos, among the most potent organophosphate pesticides, had a two-fold increased risk of atopic asthma. Pesticides were not associated with non-allergic asthma. 

The study is significant because it is the largest sample of farm women evaluated for asthma.

Citation: Hoppin JA, Umbach DM, London SJ, Henneberger PK, Kullman GJ, Alavanja MCR, Sandler DP. ( Exit NIEHS Website 2008. Pesticides and atopic and nonatopic asthma among farm women in the Agricultural Health Study. Am J Respir Crit Care Med 177(1):11-18.

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Nicotinamide Involvement in Hormone-Mediated Transcription

In a study funded by the Intramural Research Program of the NIH and NIEHS and published in the January issue of Molecular and Cellular Biology, investigators from the NIEHS Laboratory of Molecular Carcinogenesis reported that nicotinamide (NAM) uncouples promoter chromatin remodeling from the transcription preinitiation complex (PIC) assembly. This finding suggests that NAM blocks the regulation of a pathway that links the chromatin remodeling function with activation of gene expression.

The researchers wanted to know whether the sirtuin family of deacetylases, homologues of the yeast SIR2 family of deacetylases that require NAD as a co-substrate, was involved in progesterone receptor (PR)-mediated transcription. They monitored the effects of a sirtuin inhibitor, NAM, on PR-mediated transcription in T47D breast cancer cells using hormone treatments, siRNA knockdowns and ChIP assays.

Treating cells with NAM prior to hormone treatment suppressed hormone-dependent activation of PR- and glucocorticoid (GR)-regulated genes in a dose-dependent manner. Unexpectedly, an siRNA knockdown of SIRT1 and PARP1, two NAM sensitive proteins closely linked to transcription and gene regulation, determined that NAM inhibition occurred independently of these two factors. The ChIP data demonstrated that NAM inhibition of PR-regulated transcription occurred at the level of PIC formation which implied that the assembly of the basal transcription machinery was being disrupted.

The results of the research provide more information on the complexity of gene regulation.

Citation: Aoyagi S, Archer TK. Exit NIEHS Website( 2008. Nicotinamide uncouples hormone-dependent chromatin remodeling from transcription complex assembly. Mol Cell Biol 28(1):30-39.

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Crystallization of the Type II Dihydrofolate Reductase Complex

In the December 2007 issue of Biochemistry, scientists from the NIEHS and the University of Tennessee presented the first crystallographic data that explains how the Type II dihydrofolate reductase (DHFR) enzyme, with structurally similar binding sites, accommodates both the NADP+ cofactor and the dihydrofolate (DHF) substrate.

DHFR confers resistance to bacterial DHFR-targeted antifolate drugs and therefore plays an important role in the treatment of pathogenic and neoplastic diseases. DHFR, one of the smallest enzymes able to self-assemble, forms a homotetramer with its active site occurring within a central pore in the tetramer. The crystal structure provided several pieces of information about how the DHFR•DHF•NADP+ complex works, but a few of the important findings included the following conclusions: (1) two equivalent isoleucine residues bind structurally similar regions of the nicotinamide and pteridine rings and stabilize a catalytically-favorable interaction; (2) two pairs of residues, tyrosine 69 and glutamine 67, are hydrogen-bonded and provide a "clamp" which holds the rings in place during hydride transfer; and (3) the ternary complex explains how the enzyme retains activity even after massive mutations.

This study is the first to provide functional data on the DHFR•DHF•NADP+ enzyme which may lead to the development of inhibitors that can target Type II DHFR.

Citation: Krahn JM, Jackson MR, DeRose EF, Howell EE, London RE. ( NIEHS Website 2007. Crystal structure of a type II dihydrofolate reductase catalytic ternary complex. Biochemistry 46(51):14878-14888.

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The Roles of XRCC1 and Pol β in Repair of SSBs

Scientists from the NIEHS Laboratory of Structural Biology and the Laboratory of Molecular Carcinogenesis examined the scientific literature pertaining to the roles of X-ray cross-complementing group 1 (XRCC1) and DNA polymerase β (pol b) in protecting cells against DNA single-strand breaks (SSBs). The journal review article was published in the January issue of Cell Research and represented a thorough analysis of the research that had been performed to date on the topic.

The 70-kDa XRCC1 has no known enzymatic activity, but physically interacts with other proteins known to be involved in SSB and base excision repair (BER). The 39-kDa pol β contributes both polymerase and end-tailoring lyase activity to the predominant single-nucleotide BER pathway. Mouse cell lines that lacked XRCC1 and pol β (XRCC1-/- and pol β-/-, respectively) were especially hypersensitive to damage caused by the alkylating agent methyl methanesulfonate (MMS). Data from the comet assay, a technique that detects DNA damage in individual cells using electrophoresis, demonstrated elevated levels of SSBs, and diminished repair in XRCC1- and pol b-deficient cells following exposure to MMS.

The BRCT I domain of XRCC1 specifically interacts with activated poly(ADP-ribose) polymerase 1 (PARP-1) that is bound to damaged DNA. Since inhibition of PARP activity enhanced MMS-induced cell killing in XRCC1-/- cells, the authors concluded that PARP activation played a role in the modulation of cytotoxicity beyond recruitment of XRCC1 to sites of DNA damage.

Citation: Horton JK, Watson M, Stefanick DF, Shaughnessy DT, Taylor JA, Wilson SH. Exit NIEHS Website( 2008. XRCC1 and DNA polymerase beta in cellular protection against cytotoxic DNA single-strand breaks. Cell Res 18(1):48-63.

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