Melissa Standley

Comprehensive maternal serum proteomic profiles of preclinical and clinical preeclampsia

We systematically characterized maternal serum proteome in women with clinical preeclampsia (PE) and asymptomatic women in early pregnancy that subsequently developed PE. Clinical PE cohort comprised 30 patients with mild PE, 30 with severe PE, and 58 normotensive women. Preclinical PE cohort included 149 women whose serum samples were collected at 8-14 gestational weeks and in whom 30 women later developed mild and 40 severe PE. Serum proteome was analyzed and enzyme-linked immunosorbent assays were used for protein quantification. In Clinical PE, fibronectin, pappalysin-2, choriogonadotropin-beta, apolipoprotein C-III, cystatin-C, vascular endothelial growth factor receptor-1, and endoglin were more abundant compared to normotensive women. In preclinical PE, differently expressed proteins included placental, vascular, transport, matrix, and acute phase proteins. Angiogenic and antiangiogenic proteins were not significant. We conclude that placental and antiangiogenic proteins are abundant in clinical PE. In preclinical PE, proteomic profile is distinct and different from that in clinical PE.

Proteomic Profiling of Mature CD10+ B-Cell Lymphomas

Proteomic profiling with protein-chip technology has been used successfully to discover biomarkers with potential clinical usefulness in several cancer types. Little proteomic study has been done in B-cell lymphomas. We determined whether the expression of a set of proteins by protein-chip technology coupled with new informatics tools could be used to build a model to molecularly classify B-cell lymphoma subgroups. We used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to analyze 18 CD10+ B-cell lymphomas, including 6 grade 1 (G1) follicular lymphomas (FLs), 7 grade 3 (G3) FLs, and 5 Burkitt lymphomas. We used 7 reactive follicular hyperplasia cases as a control group. By using SAX2 ProteinChip arrays (Ciphergen Biosystems, Fremont, CA), we found a unique protein expression profile for each type of lesion. Two-way hierarchical clustering analysis of these protein expression profiles differentiated reactive follicular hyperplasia, FL, and Burkitt lymphoma, with 5 major clusters of differentially expressed protein peaks. In addition, we identified histone H4 as a potential differentially expressed protein marker that seems to distinguish G1 from G3 FL. To our knowledge, this is the first proteomic study using protein-chip technology for molecular classification of B-cell lymphoma subtypes with clinical samples.

Genotoxicants Target Distinct Molecular Networks in Neonatal Neurons

Background Exposure of the brain to environmental agents during critical periods of neuronal development is considered a key factor underlying many neurologic disorders. Objectives In this study we examined the influence of genotoxicants on cerebellar function during early development by measuring global gene expression changes. Methods We measured global gene expression in immature cerebellar neurons (i.e., granule cells) after treatment with two distinct alkylating agents, methylazoxymethanol (MAM) and nitrogen mustard (HN2). Granule cell cultures were treated for 24 hr with MAM (10–1,000 μM) or HN2 (0.1–20 μM) and examined for cell viability, DNA damage, and markers of apoptosis. Results Neuronal viability was significantly reduced (p < 0.01) at concentrations > 500 μM for MAM and > 1.0 μM for HN2; this correlated with an increase in both DNA damage and markers of apoptosis. Neuronal cultures treated with sublethal concentrations of MAM (100 μM) or HN2 (1.0 μM) were then examined for gene expression using large-scale mouse cDNA microarrays (27,648). Gene expression results revealed that a) global gene expression was predominantly up-regulated by both genotoxicants; b) the number of down-regulated genes was approximately 3-fold greater for HN2 than for MAM; and c) distinct classes of molecules were influenced by MAM (i.e, neuronal differentiation, the stress and immune response, and signal transduction) and HN2 (i.e, protein synthesis and apoptosis). Conclusions These studies demonstrate that individual genotoxicants induce distinct gene expression signatures. Further study of these molecular networks may explain the variable response of the developing brain to different types of environmental genotoxicants.

Molecular networks perturbed in a developmental animal model of brain injury

Methylazoxymethanol (MAM) is widely used as a developmental neurotoxin and exposure to its glucoside (i.e., cycasin) is associated with the prototypical neurological disorder western Pacific ALS/PDC. However, the specific molecular targets that play a key role in MAM-induced brain injury remain unclear. To reveal potential molecular networks targeted by MAM in the developing nervous system, we examined characteristic phenotypic changes (DNA damage, cytoarchitecture) induced by MAM and their correlation with gene expression differences using microarray assays (27,648 genes). Three day-old postnatal C57BL/6 mice (PND3) received a single injection of MAM and the cerebellum and cerebral cortex of PND4, 8, 15, and 22 mice were analyzed. DNA damage was detected in both the cerebellum (N7-mGua, TUNEL labeling) and cerebral cortex (N7-mGua) of PND4 mice, but progressive disruption of the cytoarchitecture was restricted to the cerebellum. A majority (>75%) of the genes affected (cerebellum 636 genes, cortex 1080 genes) by MAM were developmentally regulated, with a predominant response early (PND4) in the cerebellum and delayed (PND8 and 15) in the cerebral cortex. The genes and pathways (e.g., proteasome) affected by MAM in the cerebellum are distinct from cortex. The genes perturbed in the cerebellum reflect critical cellular processes such as development (17%), cell cycle (7%), protein metabolism (12%), and transcriptional regulation (9%) that could contribute to the observed cytoarchitectural disruption of the cerebellum. This study demonstrates for the first time that specific genes and molecular networks are affected by MAM during CNS development. Further investigation of these targets will help to understand how disruption of these developmental programs could contribute to chronic brain injury or neurodegenerative disease.

Mycobacterium smegmatis RoxY Is a Repressor of oxyS and Contributes to Resistance to Oxidative Stress and Bactericidal Ubiquitin-Derived Peptides

The mycobactericidal properties of macrophages include the generation of reactive oxygen intermediates and the delivery of bacteria to a hydrolytic lysosome enriched in bactericidal ubiquitin-derived peptides (Ub-peptides). To better understand the interactions of ubiquitin-derived peptides with mycobacteria and identify putative mycobacterial intrinsic resistance mechanisms, we screened for transposon mutants with increased susceptibility to the bactericidal Ub-peptide Ub2. We isolated 27 Mycobacterium smegmatis mutants that were hypersusceptible to Ub2. Two mutants were isolated that possessed mutations in the msmeg_0166 gene, which encodes a transcriptional regulator. The msmeg_0166 mutants were also hypersusceptible to other host antimicrobial peptides and oxidative stress. In characterizing msmeg_0166, we found that it encodes a repressor of oxyS, and therefore we have renamed the gene roxY. We demonstrate that RoxY and OxyS contribute to M. smegmatis resistance to oxidative stress. An ahpD transposon mutant was also isolated in our screen for Ub-peptide hypersusceptibility. Overexpression of oxyS in M. smegmatis reduced transcription of the ahpCD genes, which encode a peroxide detoxification system. Our data indicate that RoxY, OxyS, and AhpD play a role in the mycobacterial oxidative stress response and are important for resistance to host antimicrobial peptides.