Construction of heparan sulfate microarray for investigating the binding of specific saccharide sequences to proteins.

Heparan sulfate (HS) is a heterogeneous, extracellular glycan that interacts with proteins and other molecules affecting many biological processes. The specific binding motifs of HS interactions are of interest, but have not been extensively characterized. Glycan microarrays are valuable tools that can be used to probe the interactions between glycans and their ligands while relying on relatively small amounts of samples. Recently, chemoenzymatic synthesis of HS has been employed to produce specific HS structures that can otherwise be difficult to produce. In this study, a microarray of diverse chemoenzymatically synthesized HS structures was developed and HS interactions were characterized. Fluorescently labeled antithrombin III (AT) and fibroblast growth factor-2 (FGF2) were screened against 95 different HS structures under three different printing concentrations to confirm the utility of this microarray. Specific sulfation patterns were found to be important for binding to these proteins and results are consistent with previous specificity studies. Furthermore, the binding affinities (KD,surf) of AT and FGF2 to multiple HS structures were determined using a microarray technique and is consistent with previous reports. Lastly, the 95-compound HS microarray was used to determine the distinct binding profiles for interleukin 12 and platelet factor 4. This technique is ideal for rapid expansion and will be pivotal to the high-throughput characterization of biologically important structure/function relationships.

Reprogramming CBX8-PRC1 function with a positive allosteric modulator.

Canonical targeting of Polycomb repressive complex 1 (PRC1) to repress developmental genes is mediated by cell-type-specific, paralogous chromobox (CBX) proteins (CBX2, 4, 6, 7, and 8). Based on their central role in silencing and their dysregulation associated with human disease including cancer, CBX proteins are attractive targets for small-molecule chemical probe development. Here, we have used a quantitative and target-specific cellular assay to discover a potent positive allosteric modulator (PAM) of CBX8. The PAM activity of UNC7040 antagonizes H3K27me3 binding by CBX8 while increasing interactions with nucleic acids. We show that treatment with UNC7040 leads to efficient and selective eviction of CBX8-containing PRC1 from chromatin, loss of silencing, and reduced proliferation across different cancer cell lines. Our discovery and characterization of UNC7040 not only reveals the most cellularly potent CBX8-specific chemical probe to date, but also corroborates a mechanism of Polycomb regulation by non-specific CBX nucleotide binding activity.

Introduction--the methyl parathion story: a chronicle of misuse and preventable human exposure.

In the fall of 1994, Lorain County, Ohio, became the site of the first investigation of several large-scale incidences in which the organophosphate pesticide methyl parathion was illegally applied to private residences. The extent of potential human exposure to this pesticide led the Ohio Department of Health to formally request technical assistance from the Centers for Disease Control and Prevention (CDC). This article describes the initial investigation of 64 homes in Ohio and introduces the method of using both biological markers of exposure (p-nitrophenol levels in human urine samples) and environmental markers of contamination in dust and air samples when making public health decisions about the cleanup of homes sprayed with methyl parathion. The results of the CDC rapid investigation led the U.S. Environmental Protection Agency to declare the contaminated homes in Lorain County a Superfund cleanup site. Seven years after the Lorain incident, and after subsequent Superfund actions had been implemented in Illinois and Mississippi, researchers participated in an expanded session devoted to methyl parathion at the 11th Annual Meeting of the International Society of Exposure Analysis held in Charleston, South Carolina, in the fall of 2001. The articles included in this monograph are based on presentations at that meeting. They report previously unpublished data that tell the methyl parathion story from different perspectives, each providing in-depth information about separate aspects of this multistate, multiagency, and multimillion dollar chemical exposure. This monograph is the methyl parathion story.

Assessment of human exposure and human health effects after indoor application of methyl parathion in Lorain County, Ohio, 1995-1996.

In January 1995 the U.S. Environmental Protection Agency declared methyl parathion-contaminated homes in Lorain County, Ohio, as a Superfund cleanup site. During the 2-year cleanup, the Centers for Disease Control and Prevention in collaboration with county and city health officials conducted a study of exposure and health effects among residents. We administered 254 household and 747 individual questionnaires; urine analysis for p-nitrophenol (PNP, a metabolite of methyl parathion) was available for 626 participants. We also reviewed medical records of 49 people who were hospitalized or died after their homes were sprayed. People living in homes sprayed <180 days previously were most likely to have the highest PNP levels (22.9% > 100 ppb PNP), but even people living in homes sprayed more than a year previously appeared to be highly exposed (8.5% > 100 ppb PNP). The National Health and Nutrition Examination Survey reference range is 0-63 ppb. Median detectable PNP levels among children younger than 3 years of age were 93.9 ppb compared with 41.6 ppb among people older than 3 years. Younger children appeared to be at greatest risk of exposure. In none of the medical records that we reviewed did a health care provider consider pesticide poisoning as a potential etiology.

Methyl parathion in residential properties: relocation and decontamination methodology.

In November 1994 methyl parathion (MP), a restricted agricultural pesticide, was discovered to have been illegally sprayed within hundreds of residences in Lorain County, Ohio. Surface levels and air concentrations of MP revealed detectable levels of the pesticide 3 years after spraying. Because of the high toxicity of MP (lethal dose to 50% of rats tested [LD50] = 15 mg/kg) and long half-life indoors, risk-based relocation and decontamination criteria were created. Relocation criteria were derived based on levels of p-nitrophenol in urine, a metabolic byproduct of MP exposure. In Ohio, concentrations of MP on surfaces and in the air were also used to trigger relocations. The criteria applied in Ohio underwent refinement as cases of MP misuse were found in Mississippi and then in several other states. The MP investigation (1994-1997) was the largest pesticide misuse case in the nation, ultimately involving the sampling of 9,000 residences and the decontamination of 1,000 properties. This article describes the methodology used for relocation of residents and decontamination of properties having MP.