Toluene dioxygenase-catalyzed cis-dihydroxylation of benzo[b]thiophenes and benzo[b]furans: synthesis of benzo[b]thiophene 2,3-oxide

Enzymatic cis-dihydroxylation of benzo[b]thiophene, benzo[b]furan and several methyl substituted derivatives was found to occur in both the carbocyclic and heterocyclic rings. Relative and absolute configurations and enantiopurities of the resulting dihydrodiols were determined. Hydrogenation of the alkene bond in carbocyclic cis-dihydrodiols and ring-opening epimerization/reduction reactions of heterocyclic cis/trans-dihydrodiols were also studied. The relatively stable heterocyclic dihydrodiols of benzo[b]thiophene and benzo[b]furan showed a strong preference for the trans configuration in aqueous solutions. The 2,3-dihydrodiol metabolite of benzo[b]thiophene was utilized as a precursor in the chemoenzymatic synthesis of the unstable arene oxide, benzo[b]thiophene 2,3-oxide.

NF-κB is a critical mediator of BRCA1-induced chemoresistance

BRCA1 mediates resistance to apoptosis in response to DNA-damaging agents, causing BRCA1 wild-type tumours to be significantly more resistant to DNA damage than their mutant counterparts. In this study, we demonstrate that following treatment with the DNA-damaging agents, etoposide or camptothecin, BRCA1 is required for the activation of nuclear factor-?B (NF-?B), and that BRCA1 and NF-?B cooperate to regulate the expression of the NF-?B antiapoptotic targets BCL2 and XIAP. We show that BRCA1 and the NF-?B subunit p65/RelA associate constitutively, whereas the p50 NF-?B subunit associates with BRCA1 only upon DNA damage treatment. Consistent with this BRCA1 and p65 are present constitutively on the promoters of BCL2 and XIAP, whereas p50 is recruited to these promoters only in damage treated cells. Importantly, we demonstrate that the recruitment of p50 onto the promoters of BCL2 and XIAP is dependent upon BRCA1, but independent of its NF-?B partner subunit p65. The functional relevance of NF-?B activation by BRCA1 in response to etoposide and camptothecin is demonstrated by the significantly reduced survival of BRCA1 wild-type cells upon NF-?B inhibition. This study identifies a novel BRCA1-p50 complex, and demonstrates for the first time that NF-?B is required for BRCA1-mediated resistance to DNA damage. It reveals a functional interdependence between BRCA1 and NF-?B, further elucidating the role played by NF-?B in mediating cellular resistance of BRCA1 wild-type tumours to DNA-damaging agents.

Attenuation of monocyte chemotaxis--a novel anti-inflammatory mechanism of action for the cardio-protective hormone B-type natriuretic peptide

<p>B-type natriuretic peptide (BNP) is a prognostic and diagnostic marker for heart failure (HF). An anti-inflammatory, cardio-protective role for BNP was proposed. In cardiovascular diseases including pressure overload-induced HF, perivascular inflammation and cardiac fibrosis are, in part, mediated by monocyte chemoattractant protein (MCP)1-driven monocyte migration. We aimed to determine the role of BNP in monocyte motility to MCP1. A functional BNP receptor, natriuretic peptide receptor-A (NPRA) was identified in human monocytes. BNP treatment inhibited MCP1-induced THP1 (monocytic leukemia cells) and primary monocyte chemotaxis (70 and 50 %, respectively). BNP did not interfere with MCP1 receptor expression or with calcium. BNP inhibited activation of the cytoskeletal protein RhoA in MCP1-stimulated THP1 (70 %). Finally, BNP failed to inhibit MCP1-directed motility of monocytes from patients with hypertension (n = 10) and HF (n = 6) suggesting attenuation of this anti-inflammatory mechanism in chronic heart disease. We provide novel evidence for a direct role of BNP/NPRA in opposing human monocyte migration and support a role for BNP as a cardio-protective hormone up-regulated as part of an adaptive compensatory response to combat excess inflammation.p>

BRCA1 and c-Myc Associate to Transcriptionally Repress Psoriasin, a DNA Damage-Inducible Gene

Evidence is accumulating to suggest that some of the diverse functions associated with BRCA1 may relate to its ability to transcriptionally regulate key downstream target genes. Here, we identify S100A7 (psoriasin), S100A8, and S100A9, members of the S100A family of calcium-binding proteins, as novel BRCA1-repressed targets. We show that functional BRCA1 is required for repression of these family members and that a BRCA1 disease–associated mutation abrogates BRCA1-mediated repression of psoriasin. Furthermore, we show that BRCA1 and c-Myc form a complex on the psoriasin promoter and that BRCA1-mediated repression of psoriasin is dependent on functional c-Myc. Finally, we show that psoriasin expression is induced by the topoisomerase IIA poison, etoposide, in the absence of functional BRCA1 and increased psoriasin expression enhances cellular sensitivity to this chemotherapeutic agent. Therefore, we identified a novel transcriptional mechanism that is likely to contribute to BRCA1-mediated resistance to etoposide.

BRCA1 Regulates IFN-γ Signaling through a Mechanism Involving the Type I IFNs

<p>BRCA1 encodes a tumor suppressor gene that is mutated in the germ line of women with a genetic predisposition to breast and ovarian cancer. BRCA1 has been implicated in a number of important cellular functions including DNA damage repair, transcriptional regulation, cell cycle control, and ubiquitination. Using an Affymetrix U95A microarray, IRF-7 was identified as a BRCA1 transcriptional target and was also shown to be synergistically up-regulated by BRCA1 specifically in the presence of IFN-gamma, coincident with the synergistic induction of apoptosis. We show that BRCA1, signal transducer and activator of transcription (STAT)-1, and STAT2 are all required for the induction of IRF-7 following stimulation with IFN-gamma. We also show that the induction of IRF-7 by BRCA1 and IFN-gamma is dependent on the type I IFNs, IFN-alpha and IFN-beta. We show that BRCA1 is required for the up-regulation of STAT1, STAT2, and the type I IFNs in response to IFN-gamma. We show that BRCA1 is localized at the promoters of the molecules involved in type I IFN signaling leading to their up-regulation. Blocking this intermediary type I IFN step using specific antisera shows the requirement for IFN-alpha and IFN-beta in the induction of IRF-7 and apoptosis. Finally, we outline a mechanism for the BRCA1/IFN-gamma regulation of target genes involved in the innate immune response, which is dependent on type I IFN signaling.p>

The visual processing of motion-defined transparency

Our understanding of how the visual system processes motion transparency, the phenomenon by which multiple directions of motion are perceived to co-exist in the same spatial region, has grown considerably in the past decade. There is compelling evidence that the process is driven by global-motion mechanisms. Consequently, although transparently moving surfaces are readily segmented over an extended space, the visual system cannot separate two motion signals that co-exist in the same local region. A related issue is whether the visual system can detect transparently moving surfaces simultaneously, or whether the component signals encounter a serial â??bottleneckâ?? during their processing? Our initial results show that, at sufficiently short stimulus durations, observers cannot accurately detect two superimposed directions; yet they have no difficulty in detecting one pattern direction in noise, supporting the serial-bottleneck scenario. However, in a second experiment, the difference in performance between the two tasks disappears when the component patterns are segregated. This discrepancy between the processing of transparent and non-overlapping patterns may be a consequence of suppressed activity of global-motion mechanisms when the transparent surfaces are presented in the same depth plane. To test this explanation, we repeated our initial experiment while separating the motion components in depth. The marked improvement in performance leads us to conclude that transparent motion signals are represented simultaneously.

Inactivation of Human beta-Defensins 2 and 3 by Elastolytic Cathepsins

beta-Defensins are antimicrobial peptides that contribute to the innate immune responses of eukaryotes. At least three defensins, human beta-defensins 1, 2, and 3 (HBD-1, -2, and -3), are produced by epithelial cells lining the respiratory tract and are active toward Gram-positive (HBD-3) and Gram-negative (HBD-1, -2, and -3) bacteria. It has been postulated that the antimicrobial activity of defensins is compromised by changes in airway surface liquid composition in lungs of patients with cystic fibrosis (CF), therefore contributing to the bacterial colonization of the lung by Pseudomonas and other bacteria in CF. In this report we demonstrate that HBD-2 and HBD-3 are susceptible to degradation and inactivation by the cysteine proteases cathepsins B, L, and S. In addition, we show that all three cathepsins are present and active in CF bronchoalveolar lavage. Incubation of HBD-2 and -3 with CF bronchoalveolar lavage leads to their degradation, which can be completely (HBD-2) or partially (HBD-3) inhibited by a cathepsin inhibitor. These results suggest that beta-defensins are susceptible to degradation and inactivation by host proteases, which may be important in the regulation of beta-defensin activity. In chronic lung diseases associated with infection, overexpression of cathepsins may lead to increased degradation of HBD-2 and -3, thereby favoring bacterial infection and colonization.

Molecular basis for estrogen receptor alpha deficiency in BRCA1-linked breast cancer

<p>Background BRCA1-mutant breast tumors are typically estrogen receptor alpha (ER alpha) negative, whereas most sporadic tumors express wild-type BRCA1 and are ER alpha positive. We examined a possible mechanism for the observed ER alpha-negative phenotype of BRCA1-mutant tumors.p><p>Methods We used a breast cancer disease-specific microarray to identify transcripts that were differentially expressed between paraffin-embedded samples of 17 BRCA1-mutant and 14 sporadic breast tumors. We measured the mRNA levels of estrogen receptor 1 (ESR1) ( the gene encoding ER alpha), which was differentially expressed in the tumor samples, by quantitative polymerase chain reaction. Regulation of ESR1 mRNA and ER alpha protein expression was assessed in human breast cancer HCC1937 cells that were stably reconstituted with wild-type BRCA1 expression construct and in human breast cancer T47D and MCF-7 cells transiently transfected with BRCA1-specific short-interfering RNA ( siRNA). Chromatin immunoprecipitation assays were performed to determine if BRCA1 binds the ESR1 promoter and to identify other interacting proteins. Sensitivity to the antiestrogen drug fulvestrant was examined in T47D and MCF-7 cells transfected with BRCA1-specific siRNA. All statistical tests were two-sided.p><p>Results Mean ESR1 gene expression was 5.4-fold lower in BRCA1-mutant tumors than in sporadic tumors ( 95% confidence interval [CI]=2.6-fold to 40.1-fold, P =.0019). The transcription factor Oct-1 recruited BRCA1 to the ESR1 promoter, and both BRCA1 and Oct-1 were required for ER alpha expression. BRCA1-depleted breast cancer cells expressing exogenous ER alpha were more sensitive to fulvestrant than BRCA1-depleted cells transfected with empty vector ( T47D cells, the mean concentration of fulvestrant that inhibited the growth of 40% of the cells [IC40] for empty vector versus ER alpha: > 10(-5) versus 8.0 x 10(-9) M [ 95% CI=3.1x10(-10) to 3.2 x 10(-6) M]; MCF-7 cells, mean IC40 for empty vector versus ER alpha : > 10(-5) versus 4.9 x 10(-8) M [ 95% CI=2.0 x 10(-9) to 3.9 x 10(-6) M]).p><p>Conclusions BRCA1 alters the response of breast cancer cells to antiestrogen therapy by directly modulating ER alpha expression.p>