The human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) is irreversibly inhibited by inorganic (Hg) and organic mercury (CH3Hg): Mechanism and kinetics

Human arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine chemicals. We show here that biologically-relevant concentrations of inorganic (Hg²⁺) and organic (CH₃Hg⁺) mercury inhibit the biotransformation functions of NAT1. Both compounds react irreversibly with the active-site cysteine of NAT1 (half-maximal inhibitory concentration (IC₅₀) = 250 nM and k_inact = 1.4 × 10⁴ M⁻¹ s⁻¹ for Hg²⁺ and IC₅₀ = 1.4 μM and k_inact = 2 × 10² M⁻¹ s⁻¹ for CH₃Hg⁺). Exposure of lung epithelial cells led to the inhibition of cellular NAT1 (IC₅₀ = 3 and 20 μM for Hg²⁺ and CH₃Hg⁺, respectively). Our data suggest that exposure to mercury may affect the biotransformation of aromatic amines by NAT1.     

Sensitive cell viability assay for use in drug screens and for studying the mechanism of action of drugs in Dictyostelium discoideum

In order to increase the effectiveness of Dictyostelium discoideum as a lead genetic model for drug discovery, a luminescence-based assay has been adapted and standardized for sensitive and rapid cell viability measurements. The applicability of the assay was demonstrated by measuring the cytotoxicity of several drugs in wild-type and mutant cells. The robustness and ease of the assay demonstrate that it can be used in high-throughput applications such as drug or mutant screens. Conclusions from these studies are applicable to evaluating cell viability assays in other systems as well.     

S-phase arrest by reactive nitrogen species is bypassed by okadaic acid, an inhibitor of protein phosphatases PP1/PP2A

In mammalian cells DNA damage activates a checkpoint that halts progression through S phase. To determine the ability of nitrating agents to induce S-phase arrest, mouse C10 cells synchronized in S phase were treated with nitrogen dioxide (NO(2)) or SIN-1, a generator of reactive nitrogen species (RNS). SIN-1 or NO(2) induced S-phase arrest in a dose- and time-dependent manner. As for the positive controls adozelesin and cisplatin, arrest was accompanied by phosphorylation of ATM kinase; dephosphorylation of pRB; decreases in RF-C, cyclin D1, Cdc25A, and Cdc6; and increases in p21. Comet assays indicated that RNS induce minimal DNA damage. Moreover, in a cell-free replication system, nuclei from cells treated with RNS were able to support control levels of DNA synthesis when incubated in cytosolic extracts from untreated cells, whereas nuclei from cells treated with cisplatin were not. Induction of phosphatase activity may represent one mechanism of RNS-induced arrest, for the PP1/PP2A phosphatase inhibitor okadaic acid inhibited dephosphorylation of pRB; prevented decreases in the levels of RF-C, cyclin D1, Cdc6, and Cdc25A; and bypassed arrest by SIN-1 or NO(2), but not cisplatin or adozelesin. Our studies suggest that RNS may induce S-phase arrest through mechanisms that differ from those elicited by classical DNA-damaging agents.     

C/EBPβ regulates TNF induced MnSOD expression and protection against apoptosis

The CCAAT enhancer binding protein-β (C/EBPβ) is a critical regulator of many cellular processes. Exposure of C/EBPβ-deficient fibroblasts to tumor necrosis factor-α (TNF) resulted in their death due to apoptosis. While, the expression of Bad, Bcl-2, Bcl-x, CAS, and hILP/XIAP, as well as the nuclear translocation of NF-κB was normal in C/EBPβ-deficient cells, induction of manganous superoxide dismutase (MnSOD) gene did not occur. Ectopic expression of C/EBPβ in C/EBPβ–deficient fibroblasts prevented TNF-induced apoptosis. C/EBPβ complemented cells were able to induce MnSOD in response to TNF, ruling out the possibilities that C/EBPβ could render protection by regulating early apoptotic gene expression and/or NF-κB p65 expression. Moreover, C/EBPβ-deficient cells stably transfected with an MnSOD expression vector bypassed the requirement of C/EBPβ in protection against TNF-induced cell death, suggesting that C/EBPβ protects TNF-induced apoptotic cell death through its role in activating MnSOD expression. Mechanistically, C/EBPβ was required for induced NF-κB p65 binding to MnSOD’s intronic TNF response element and indispensable for histone acetylation of the element in response to TNF. These results suggest a role for C/EBPβ in MnSOD regulation through remodeling of local chromatin structure. This work was supported by a grant from the National Institutes of Health, CA96810.     

Microbial keratinases and their prospective applications: an overview

Microbial keratinases have become biotechnologically important since they target the hydrolysis of highly rigid, strongly cross-linked structural polypeptide “keratin” recalcitrant to the commonly known proteolytic enzymes trypsin, pepsin and papain. These enzymes are largely produced in the presence of keratinous substrates in the form of hair, feather, wool, nail, horn etc. during their degradation. The complex mechanism of keratinolysis involves cooperative action of sulfitolytic and proteolytic systems. Keratinases are robust enzymes with a wide temperature and pH activity range and are largely serine or metallo proteases. Sequence homologies of keratinases indicate their relatedness to subtilisin family of serine proteases. They stand out among proteases since they attack the keratin residues and hence find application in developing cost-effective feather by-products for feed and fertilizers. Their application can also be extended to detergent and leather industries where they serve as specialty enzymes. Besides, they also find application in wool and silk cleaning; in the leather industry, better dehairing potential of these enzymes has led to the development of greener hair-saving dehairing technology and personal care products. Further, their prospective application in the challenging field of prion degradation would revolutionize the protease world in the near future.     

Small interfering RNA screens reveal enhanced cisplatin cytotoxicity in tumor cells having both BRCA network and TP53 disruptions

RNA interference technology allows the systematic genetic analysis of the molecular alterations in cancer cells and how these alterations affect response to therapies. Here we used small interfering RNA (siRNA) screens to identify genes that enhance the cytotoxicity (enhancers) of established anticancer chemotherapeutics. Hits identified in drug enhancer screens of cisplatin, gemcitabine, and paclitaxel were largely unique to the drug being tested and could be linked to the drug's mechanism of action. Hits identified by screening of a genome-scale siRNA library for cisplatin enhancers in TP53-deficient HeLa cells were significantly enriched for genes with annotated functions in DNA damage repair as well as poorly characterized genes likely having novel functions in this process. We followed up on a subset of the hits from the cisplatin enhancer screen and validated a number of enhancers whose products interact with BRCA1 and/or BRCA2. TP53(+/-) matched-pair cell lines were used to determine if knockdown of BRCA1, BRCA2, or validated hits that associate with BRCA1 and BRCA2 selectively enhances cisplatin cytotoxicity in TP53-deficient cells. Silencing of BRCA1, BRCA2, or BRCA1/2-associated genes enhanced cisplatin cytotoxicity approximately 4- to 7-fold more in TP53-deficient cells than in matched TP53 wild-type cells. Thus, tumor cells having disruptions in BRCA1/2 network genes and TP53 together are more sensitive to cisplatin than cells with either disruption alone.     

Antitumor agents. Part 226: synthesis and cytotoxicity of 2-phenyl-4-quinolone acetic acids and their esters

2-Phenyl-4-quinolone acetic acids and their esters were synthesized and evaluated for interaction with tubulin and for cytotoxicity against a panel of human tumor cell lines. 2-Phenyl- and 2-(2'-fluorophenyl)-4-quinolone-8-acetic acids (11 and 12) displayed potent cytotoxicity with ED(50) values at nanomolar concentrations, but had minimal activity against tubulin polymerization. 2-(2'-Fluorophenyl)-4-quinolone-6-acetic acid (3) and 2-(2'-fluorophenyl)-4-quinolone-8-acetic acid methyl ester (10) moderately inhibited tubulin polymerization.     

Large scale international replication and meta-analysis study confirms association of the 15q14 locus with myopia. The CREAM consortium

Myopia is a complex genetic disorder and a common cause of visual impairment among working age adults. Genome-wide association studies have identified susceptibility loci on chromosomes 15q14 and 15q25 in Caucasian populations of European ancestry. Here, we present a confirmation and meta-analysis study in which we assessed whether these two loci are also associated with myopia in other populations. The study population comprised 31 cohorts from the Consortium of Refractive Error and Myopia (CREAM) representing 4 different continents with 55,177 individuals; 42,845 Caucasians and 12,332 Asians. We performed a meta-analysis of 14 single nucleotide polymorphisms (SNPs) on 15q14 and 5 SNPs on 15q25 using linear regression analysis with spherical equivalent as a quantitative outcome, adjusted for age and sex. We calculated the odds ratio (OR) of myopia versus hyperopia for carriers of the top-SNP alleles using a fixed effects meta-analysis. At locus 15q14, all SNPs were significantly replicated, with the lowest P value 3.87?×?10(-12) for SNP rs634990 in Caucasians, and 9.65?×?10(-4) for rs8032019 in Asians. The overall meta-analysis provided P value 9.20?×?10(-23) for the top SNP rs634990. The risk of myopia versus hyperopia was OR 1.88 (95?% CI 1.64, 2.16, P?相似文献