Crystal structure of the anthrax drug target, Bacillus anthracis dihydrofolate reductase

Spores of Bacillus anthracis are the infectious agent of anthrax. Current antibiotic treatments are limited due to resistance and patient age restrictions; thus, additional targets for therapeutic intervention are needed. One possible candidate is dihydrofolate reductase (DHFR), a biosynthetic enzyme necessary for anthrax pathogenicity. We determined the crystal structure of DHFR from B. anthracis (baDHFR) in complex with methotrexate (MTX; 1) at 2.4 Angstrom resolution. The structure reveals the crucial interactions required for MTX binding and a putative molecular basis for how baDHFR has natural resistance to trimethoprim (TMP; 2). The structure also allows insights for designing selective baDHFR inhibitors that will have weak affinities for the human enzyme. Additionally, we have found that 5-nitro-6-methylamino-isocytosine (MANIC; 3), which inhibits another B. anthracis folate synthesis enzyme, dihydropteroate synthase (DHPS), can also inhibit baDHFR. This provides a starting point for designing multi-target inhibitors that are less likely to induce drug resistance.     

Correlation of Levels and Patterns of Genomic Instability With Histological Grading of DCIS

BACKGROUND: Histological grading of ductal carcinoma-in-situ (DCIS) lesions separates DCIS into three subgroups (well-, moderately, or poorly differentiated). It is unclear, however, whether breast disease progresses along a histological continuum or whether each grade represents a separate disease. In this study, levels and patterns of allelic imbalance (AI) were examined in DCIS lesions to develop molecular models that can distinguish pathological classifications of DCIS. METHODS: Laser microdissected DNA samples were collected from DCIS lesions characterized by a single pathologist including well- (n = 18), moderately (n = 35), and poorly differentiated (n = 47) lesions. A panel of 52 microsatellite markers representing 26 chromosomal regions commonly altered in breast cancer was used to assess patterns of AI. RESULTS: The overall frequency of AI increased significantly (P < .001) with increasing grade (well differentiated, 12%; moderately differentiated, 17%; poorly differentiated, 26%). Levels of AI were not significantly different between well- and moderately differentiated grades of disease but were significantly higher (P < .0001) in poorly differentiated compared with well- or moderately differentiated disease. No statistically significant differences in patterns of AI were detected between well- and moderately differentiated disease; however, AI occurred significantly more frequently (P < .05) in high-grade lesions at chromosomes 6q25-q27, 8q24, 9p21, 13q14, and 17p13.1, and significantly more frequently in low-grade lesions at chromosome 16q22.3-q24.3. CONCLUSIONS: The inability to discriminate DCIS at the genetic level suggests that grades 1 and 2 DCIS may represent a single, non-high-grade form of DCIS, whereas poorly differentiated DCIS seems to be a genetically more advanced disease that may represent a discrete disease entity, characterized by a unique spectrum of genetic alterations.     

Cigarette smoke components inhibited intercellular communication and differentiation in human pancreatic ductal epithelial cells

Smoking is a well-documented risk factor for the development of pancreatic adenocarcinoma. Although the most abundant polycyclic aromatic hydrocarbons (PAHs) in cigarette smoke are methylated anthracenes and phenanthrenes, the epigenetic toxicity of these compounds has not been extensively studied. We previously showed that methylanthracenes, which possess a bay-like structure, affect epigenetic events such as an induced release of arachidonic acid, inhibition of gap junctional intercellular communication (GJIC) and induction of mitogen-activated protein kinases in a pluripotent rat liver epithelial stem cell line. Anthracenes with no bay-like structures were inactive. These biological effects are all molecular events associated with the promotional phase of cancer. A human immortalized, nontumorigenic pancreatic ductal epithelial cell line, H6c7, was examined to study the epigenetic toxicity of PAHs related to pancreatic cancer by using scrape-loading dye transfer, immunostaining, RT-PCR and telomerase assay methods. H6c7 cells were GJIC-incompetent and exhibited high telomerase activity when grown in growth factor and hormone-supplemented medium. In the presence of the cAMP elevating drugs (forskolin and IBMX) the cells became GJIC competent and expressed connexins. Telomerase activity was also decreased by cAMP elevating drug treatment. After induction of cAMP, 1-methylanthracene with bay-like structures inhibited GJIC, whereas the 2-methylanthracene lacking a bay-like structure had no effect on GJIC. Telomerase activity remained high in 1-methylanthracene treatment but not with 2-methylanthracene. These results indicate that a prominent component of cigarette smoke, namely methylanthracenes with distinct structural configurations, could be a potential etiological agent contributing to the epigenetic events of pancreatic cancer.     

Safety of drotrecogin alfa (activated) in severe sepsis: Data from adult clinical trials and observational studies

Drotrecogin alfa (activated) (DrotAA), or recombinant human activated protein C, represents the only Food and Drug Administration-approved therapy for mortality reduction in adult patients with severe sepsis. Drotrecogin alfa (activated) has properties that address microvascular injury in severe sepsis through its direct effects on endothelial cells and leukocytes while also having antithrombotic and indirect profibrinolytic properties. Sepsis bundle and guideline implementation has been associated with improved survival and includes DrotAA administration in appropriate patients. Several DrotAA postapproval clinical studies have yielded additional outcome and safety data, better defining its benefit/risk profile. Bleeding is more common in DrotAA-treated patients; therefore, a careful assessment of bleeding risk and an understanding of the safety profile is required. This summary provides a detailed review of safety data and outcomes of patients treated with DrotAA in recent clinical studies enrolling more than 7000 adult patients.