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Intramural Papers of the Month

By Robin Arnette
October 2007

Susceptibility to Human Acute Lung Injury

The transcription factor, NRF2, confers protection against oxidant injury, but mutations in the gene may help identify patients who are susceptible to oxidant-induced acute lung injury (ALI). The results were published in The FASEB Journal by a team of scientists from NIEHS, the University of Pennsylvania School of Medicine, Johns Hopkins Bloomberg School of Public Health, Children's Hospital of Philadelphia and the University of Tsukuba.

The researchers used a positional cloning approach to identify Nrf2 as a susceptibility gene in inbred mice, and then sequenced NRF2 in four ethnically diverse human populations. They identified six novel single nucleotide polymorphisms (SNPs) in NRF2, and one of these, -617 (C/A), significantly affected basal NRF2 expression and function. They then asked whether functional NRF2 SNPs associated with ALI in a prospective cohort of patients with major trauma. Compared to trauma patients with normal NRF2, those trauma patients with a -617 A SNP had a 6-fold higher risk of developing ALI.

This translational investigation provides novel insight into the molecular mechanisms of susceptibility to ALI, and may help to identify patients who are predisposed to develop ALI under at-risk conditions, and other oxidative stress-related illnesses. 

Citation: Marzec JM, Christie JD, Reddy SP, Jedlicka AE, Vuong H, Lanken PN, Aplenc R, Yamamoto T, Yamamoto M, Cho H-Y, Kleeberger, SR. ( Exit NIEHS 2007. Functional polymorphisms in the transcription factor NRF2 in humans increase the risk of acute lung injury. FASEB J 21(9):2237-2246.

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Activation-induced Deaminase Deficiency Prevents Lupus Symptoms in Mice

In an NIEHS-funded study published in The Journal of Immunology, scientists from NIEHS and Pathology Associates reported new findings related to systemic lupus erythematosus. Their investigation showed that a strain of mice that usually develop many characteristics of human form of the disease don't develop symptoms if a key gene is removed. The results are an important step in the fight against lupus, which affects an estimated 1.5 million Americans.

MRL/lpr mice generally develop kidney disease, secrete hypermutated autoantibodies and have enlarged spleens and lymph nodes as do humans with lupus, but MRL/lpr mice that lack the activation-induced deaminase (AID) gene experienced a significant decrease in the aforementioned symptoms and exhibited increased survival rates. Most importantly, however, these mice contain high serum levels of autoreactive, unmutated IgM antibodies and fully functioning B cells.

The researchers theorized that since the AID-deficient MRL/lpr mice were unable to undergo somatic hypermutation (SHM) and isotype class switch recombination (CSR)- two programmed processes that occur during an immune response-IgM-bearing B cells accumulated and conferred some protection against lupus-like symptoms.

Citation: Jiang C, Foley J, Clayton N, Kissling G, Jokinen M, Herbert R, Diaz M. ( Exit NIEHS 2007. Abrogation of lupus nephritis in activation-induced deaminase-deficient MRL/lpr mice. J Immunol 178(11):7422-7431.

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The Involvement of p38 MAPK and Hsp27 in Bronchiolitis

In an NIEHS-funded study published in the American Journal of Physiology-Lung Cellular and Molecular Physiology, researchers from the Laboratory of Respiratory Biology reported that they were able to pinpoint how changes in endothelial and epithelial membrane integrity led to fluid build-up in the lungs of children, the elderly and immuno-compromised patients with bronchiolitis.

The respiratory syncytial virus (RSV) is a negatively-stranded non-segmented RNA virus and is the major cause of bronchiolitis. The researchers infected primary human bronchial epithelial and A549 human alveolar epithelial cells with RSV. They measured the cells' permeability, also known as trans-epithelial resistance (TEpR), using an electrical cell-substrate impedance sensing system. They detected a decrease in TEpR five to ten hours after the infection and a continued 30 percent decrease over time.

Since earlier data from these researchers and others have shown that the MAPK pathway was involved in endothelial permeability, they treated cells with a variety of MAPK protein inhibitors and performed Western blots looking at the phosphorylation of Hsp27, a 27kDa heat shock protein. Their research indicated that the decrease in TEpR could be weakened by using p38 MAPK inhibitors, but a decrease in TEpR also corresponded to an increase in Hsp27 phosphorylation and actin microfilament rearrangement. This is the first report to detail the mechanism of fluid build-up in bronchiolitis.

Citation: Singh D, McCann KL, Imani F. ( Exit NIEHS 2007. MAPK and heat shock protein 27 activation are associated with respiratory syncytial virus induction of human bronchial epithelial monolayer disruption. Am J Physiol Lung Cell Mol Physiol 293(2):L436-L445.

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DNA Replication Fidelity-Active Site is Conserved in Yeast

Researchers from NIEHS in collaboration with investigators from UmeÅ University in Sweden have determined that the methionine at position 644 (M644) within the yeast DNA polymerase ? (pol ?) active site plays an important role in replication fidelity. The findings were published in Nucleic Acids Research.

Earlier sequence identity studies revealed that M644 in pol ? corresponded to similar active site positions in other Saccharomyces cerevisiae polymerases such as T4 pol, pol ? and pol ?. They also knew that the side chain of M644 specifically interacted with a tyrosine that contacts the sugar of the incoming dNTP. Therefore, the research team used site-directed mutagenesis to replace the M644 with leucine (M644L), tryptophan (M644W) and phenylalanine (M644F).

They discovered that M644L and M644W correctly synthesized DNA (high fidelity), but that M644F had significantly increased replication errors (low fidelity). The team theorized that the aromatic ring present in the amino acid phenylalanine possibly altered the conformation of the binding pocket, leading the M644F mutant pol ? to insert the wrong nucleotides. The data from this project and others indicated that the position occupied by M644 in pol ? is an important determinant of replication fidelity in all three B family polymerases.

Enzymes with amino acid replacements at this position are being used to probe the roles of the B family polymerases in leading and lagging strand replication of normal DNA, as well as in replication of DNA that has been damaged as a result of environmental stress.

Citation: Pursell ZF, Isoz I, Lundström E-B, Johansson, Kunkel TA. ( NIEHS 2007. Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase e. Nucleic Acids Res 35(9):3076-3086.

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