Iron overload induced by diphenylamine triggers reactive oxygen species and apoptosis in the spleen of pregnant rats and their fetuses.

Diphenylamine (DPA) is an aniline derivative, used widely as an industrial antioxidant, dye mordant, and agricultural fungicide. DPA was reported as hazardous to mammals both acutely and chronically, however little is known about the toxicity of DPA and its derivatives during pregnancy. This study aimed to evaluate and explain the possible mechanism of toxicity induced by DPA on blood and spleen, as a fundamental hematopoietic target organ, in pregnant rats and their fetuses. Pregnant rats were orally administrated distilled water, corn oil, and/or DPA (400 mg/kg b.wt) from the 5th to 19th day of gestation. DPA-induced spleen toxicity was mirrored by significant upregulation of programmed death-1 (PD-1) protein expression and an increase in the percentage of apoptotic cells and a decrease in their proliferating capacity. These results have been confirmed through marked G0/G1 cell-cycle arrest that was observed by flow cytometric analysis of spleen cells. Moreover, the contents of reactive oxygen species and iron in the spleen tissue were significantly higher than that of the control group. DPA resulted in severe anemia, decreased hemoglobin and hematocrit, thrombocytopenia and leukopenia in addition to significant changes in differential leukocytic count of both mothers and fetuses. Evidently, DPA triggered serious pathological changes in the spleen tissue of both mothers and fetuses and the histochemical examination revealed a significant increase in iron expression. In conclusion, these results implicate the hemato- and splenotoxicity of DPA and the possible role of oxidative stress and apoptosis in DPA-induced toxicity in the spleen of pregnant rats and their fetuses. This in-turn suggests the urgent need to reduce exposure to DPA as possible as it can.

CYP2C9 and 3A4 play opposing roles in bioactivation and detoxification of diphenylamine NSAIDs.

Diphenylamine NSAIDs are taken frequently for chronic pain conditions, yet their use may potentiate hepatotoxicity risks through poorly characterized metabolic mechanisms. Our previous work revealed that seven marketed or withdrawn diphenylamine NSAIDs undergo bioactivation into quinone-species metabolites, whose reaction specificities depended on halogenation and the type of acidic group on the diphenylamine. Herein, we identified cytochromes P450 responsible for those bioactivations, determined reaction specificities, and estimated relative contributions of enzymes to overall hepatic bioactivations and detoxifications. A qualitative activity screen revealed CYP2C8, 2C9, 2C19, and 3A4 played roles in drug bioactivation. Subsequent steady-state studies with recombinant CYPs recapitulated the importance of halogenation and acidic group type on bioactivations but importantly, showed patterns unique to each CYP. CYP2C9, 2C19 and 3A4 bioactivated all NSAIDs with CYP2C9 dominating all possible bioactivation pathways. For each CYP, specificities for overall oxidative metabolism were not impacted significantly by differences in NSAID structures but the values themselves differed among the enzymes such that CYP2C9 and 3A4 were more efficient than others. When considering hepatic CYP abundance, CYP2C9 almost exclusively accounted for diphenylamine NSAID bioactivations, whereas CYP3A4 provided a critical counterbalance favoring their overall detoxification. Preference for either outcome would depend on molecular structures favoring metabolism by the CYPs as well as the influence of clinical factors altering their expression and/or activity. While focused on NSAIDs, these findings have broader implications on bioactivation risks given the expansion of the diphenylamine scaffold to other drug classes such as targeted cancer therapeutics.

Impacts of diphenylamine NSAID halogenation on bioactivation risks.

Diphenylamine NSAIDs are highly prescribed therapeutics for chronic pain despite causing symptomatic hepatotoxicity through mitochondrial damage in five percent of patients taking them. Differences in toxicity are attributed to structural modifications to the diphenylamine scaffold rather than its inherent toxicity. We hypothesize that marketed diphenylamine NSAID substituents affect preference and efficiency of bioactivation pathways and clearance. We parsed the FDA DILIrank hepatotoxicity database and modeled marketed drug bioactivation into quinone-species metabolites to identify a family of seven clinically relevant diphenylamine NSAIDs. These drugs fell into two subgroups, i.e., acetic acid and propionic acid diphenylamines, varying in hepatotoxicity risks and modeled bioactivation propensities. We carried out steady-state kinetic studies to assess bioactivation pathways by trapping quinone-species metabolites with dansyl glutathione. Analysis of the glutathione adducts by mass spectrometry characterized structures while dansyl fluorescence provided quantitative yields for their formation. Resulting kinetics identified four possible bioactivation pathways among the drugs, but reaction preference and efficiency depended upon structural modifications to the diphenylamine scaffold. Strikingly, diphenylamine dihalogenation promotes formation of quinone metabolites through four distinct metabolic pathways with high efficiency, whereas those without aromatic halogen atoms were metabolized less efficiently through two or fewer metabolic pathways. Overall metabolism of the drugs was comparable with bioactivation accounting for 4-13% of clearance. Lastly, we calculated daily bioload exposure of quinone-species metabolites based on bioactivation efficiency, bioavailability, and maximal daily dose. The results revealed stratification into the two subgroups; propionic acid diphenylamines had an average four-fold greater daily bioload compared to acetic acid diphenylamines. However, the lack of sufficient study on the hepatotoxicity for all drugs prevents further correlative analyses. These findings provide critical insights on the impact of diphenylamine bioactivation as a precursor to hepatotoxicity and thus, provide a foundation for better risk assessment in drug discovery and development.

Targeting phosphatidylethanolamine and phosphatidylserine for imaging apoptosis in cancer.

INTRODUCTION: Both phosphatidylethanolamine (PE) and phosphatidylserine (PS) can be externalized to the outer cell membrane in apoptosis. Thus the objective was to determine whether PE-targeting 18F-duramycin and PS-targeting 18F-Zn-DPA could be used for imaging apoptosis. METHODS: Duramycin and Zn-DPA were labeled with either 18F-Al or 18F-SFB. U937 cells were incubated with four different concentrations of camptothecin (CPT). For assessing the effect of incubation time on uptake, 37 MBq of radiotracer was added to cells incubated for 15, 30, 60, and 120 min at 37 °C. For blocking experiments, 150 µg duramycin and 40 µg Zn-DPA were added to cells for 15 min prior to the addition of either duramycin or Zn-DPA labeled with 18F. Apoptosis was measured by flow cytometry using an annexin-V/PI kit. Cells were co-stained with Hoechst, Cy5-duramycin, and PSVue480 (FITC-Zn-DPA) to localize fluorescent dye uptake in cells. RESULTS: Apoptosis in cells increased proportionally with CTP as confirmed by both flow cytometry and fluorescent staining. Both FITC-Zn-DPA and FITC-duramycin localized mainly on the cell membrane during early apoptosis and then translocated to the inside during late apoptosis. Uptake of FITC-duramycin, however, was higher than that of FITC-Zn-DPA. Cellular uptake of four different radiotracers was also proportional to the degree of apoptosis, increasing slightly over time and reaching a plateau at about 1 h. The blocking experiments demonstrated that uptake in all the control groups was predominantly non-specific, whereas the specific uptake in all the treated groups was at least 50% for both 18F labeled duramycin and Zn-DPA. CONCLUSION: Both PE-targeting 18F-duramycin and PS-targeting 18F-Zn-DPA could be considered as potential radiotracers for imaging cellular apoptosis. Advances in knowledge and implications for patient care: Cellular data support the further development of radiotracers targeting either PE or PS for imaging apoptosis, which can associate with clinical outcome for cancer patients.

Occurrence of substituted diphenylamine antioxidants and benzotriazole UV stabilizers in Arctic seabirds and seals.

Substituted diphenylamine antioxidants (SDPAs) and benzotriazole UV stabilizers (BZT-UVs) are contaminants of emerging environmental concern. However, little is known about the occurrence of these contaminants in the Arctic. In this study, we investigated the levels of 11 SDPAs and 6 BZT-UVs in livers and eggs of two seabird species, the black-legged kittiwake (Rissa tridactyla) and northern fulmar (Fulmarus glacialis), as well as the liver of ringed seals (Pusa hispida) from Canadian high- and sub-Arctic sites. The concentrations of ΣSDPAs in seabird livers (median 336 pg g-1, wet weight (ww)) were significantly higher than the eggs (median 24 pg g-1, ww) and the seal livers (median 38 pg g-1, ww), suggesting liver was a primary tissue of SDPA accumulation in seabirds and that seabirds were at greater risk of exposure to SDPAs than marine mammals in the Arctic. The predominant SDPA was monostyryl octyl-diphenylamine and this compound was detected in every seabird and seal sample, indicating the widespread distribution of this contaminant in Arctic food webs. Unlike SDPAs, the detection rate and concentrations of BZT-UVs in seals were higher than in seabirds. The compound 2-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol (UV329) or its isomer 2-(2H-benzotriazol-2-yl)-4-(tert-butyl)-6-(sec-butyl) phenol (UV350) was the predominant BZT-UVs in seals, with the concentrations of ΣBZT-UVs between

Impairment of cocaine-mediated behaviours in mice by clinically relevant Ras-ERK inhibitors.

Ras-ERK signalling in the brain plays a central role in drug addiction. However, to date, no clinically relevant inhibitor of this cascade has been tested in experimental models of addiction, a necessary step toward clinical trials. We designed two new cell-penetrating peptides - RB1 and RB3 - that penetrate the brain and, in the micromolar range, inhibit phosphorylation of ERK, histone H3 and S6 ribosomal protein in striatal slices. Furthermore, a screening of small therapeutics currently in clinical trials for cancer therapy revealed PD325901 as a brain-penetrating drug that blocks ERK signalling in the nanomolar range. All three compounds have an inhibitory effect on cocaine-induced ERK activation and reward in mice. In particular, PD325901 persistently blocks cocaine-induced place preference and accelerates extinction following cocaine self-administration. Thus, clinically relevant, systemically administered drugs that attenuate Ras-ERK signalling in the brain may be valuable tools for the treatment of cocaine addiction.

Molecular Engineering of Aqueous Soluble Triarylboron-Compound-Based Two-Photon Fluorescent Probe for Mitochondria H2S with Analyte-Induced Finite Aggregation and Excellent Membrane Permeability.

Hydrogen sulfide (H2S) is a multifunctional signaling molecule that participates in many important biological processes. Herein, by functionalizing triarylboron with cyclen and diphenylamine, we synthesized TAB-1, TAB-2, and TAB-3 for H2S recongnization by rational design of molecular structures. Among them, aqueous soluble TAB-2 possesses excellent properties, including large two-photon action cross section, membrane permeability and can effectively complex with Cu(2+). The complex of TAB-2-Cu(2+) can selectively detect H2S with an instant response and mitochondria targeted. Moreover, the H2S-induced finite aggregation of indicators enhances their photostability and causes variation of the fluorescence lifetime. TAB-2-Cu(2+) has also been successfully applied for the mitochondria H2S imaging in NIH/3T3 fibroblast cells by TPM and FLIM.

Modular Assembly of Allosteric MEK Inhibitor Structural Elements Unravels Potency and Feedback-Modulation Handles.

Having recently identified a so-far unexplored area adjacent to the known binding site of allosteric mitogen-activated protein kinase kinase (MEK) inhibitors, we now report an extension of these studies by combining our new side chains with different MEK inhibitor cores in a modular manner. Replacement of the amide headgroup with inverse sulfonamides resulted in the identification of new MEK inhibitors with at least 10-fold higher cellular potency against K-Ras-mutated tumor cells. A selected inhibitor from this new series retained the favorable pharmacokinetic profile of its predecessor in rodent and non-rodent species and displayed significant in vivo efficacy at once-daily oral doses of 0.25-1 mg kg(-1) in a K-Ras-mutated xenograft model. The brain penetration potential of this analogue was significantly attenuated relative to PD325901. In a second series, the central fluorophenyl core was replaced by a pyridine moiety which gave rise to a similar boost in cellular potency. Most notably, analogues from this second series do not show MEK feedback phosphorylation in K-Ras-mutated A549 cells. Our results complement recent reports on the structural intricacies of MEK-Raf feedback interactions.

Isolation of a diphenylamine-degrading bacterium and characterization of its metabolic capacities, bioremediation and bioaugmentation potential.

The antioxidant diphenylamine (DPA) is used in fruit-packaging plants for the control of the physiological disorder apple scald. Its use results in the production of DPA-contaminated wastewater which should be treated before finally discharged. Biological treatment systems using tailored-made microbial inocula with specific catabolic activities comprise an appealing and sustainable solution. This study aimed to isolate DPA-degrading bacteria, identify the metabolic pathway of DPA and evaluate their potential for future implementation in bioremediation and biodepuration applications. A Pseudomonas putida strain named DPA1 able to rapidly degrade and utilize DPA as the sole C and N source was enriched from a DPA-contaminated soil. The isolated strain degraded spillage-level concentrations of DPA in liquid culture (2000 mg L(-1)) and in contaminated soil (1000 mg kg(-1)) and metabolized DPA via the transient formation of aniline and catechol. Further evidence for the bioremediation and biodepuration potential of the P. putida strain DPA1 was provided by its capacity to degrade the post-harvest fungicide ortho-phenylphenol (OPP), concurrently used by the fruit-packaging plants, although at slower rates and DPA in a wide range of pH (4.5-9) and temperatures (15-37 °C). These findings revealed the high potential of the P. putida strain DPA1 for use in future soil bioremediation strategies and/or as start-up inocula in wastewater biodepuration systems.

The transient dermal exposure II: post-exposure absorption and evaporation of volatile compounds.

The transient dermal exposure is one where the skin is exposed to chemical for a finite duration, after which the chemical is removed and no residue remains on the skin's surface. Chemical within the skin at the end of the exposure period can still enter the systemic circulation. If it has some volatility, a portion of it will evaporate from the surface before it has a chance to be absorbed by the body. The fate of this post-exposure "skin depot" is the focus of this theoretical study. Laplace domain solutions for concentration distribution, flux, and cumulative mass absorption and evaporation are presented, and time domain results are obtained through numerical inversion. The Final Value Theorem is applied to obtain the analytical solutions for the total fractional absorption by the body and evaporation from skin at infinite time following a transient exposure. The solutions depend on two dimensionless variables: χ, the ratio of evaporation rate to steady-state dermal permeation rate; and the ratio of exposure time to membrane lag time. Simple closed form algebraic equations are presented that closely approximate the complete analytical solutions. Applications of the theory to the dermal risk assessment of pharmaceutical, occupational, and environmental exposures are presented for four example chemicals.

Distribution of colored carotenoids between light-harvesting complexes in the process of recovering carotenoid biosynthesis in Ectothiorhodospira haloalkaliphila cells.

The processes of recovering colored-carotenoid (Car) biosynthesis in Car-less cells of the purple sulfur bacterium Ectothiorhodospira haloalkaliphila grown with diphenylamine (DPA-cells) have been studied. It has been found that (1) the rate of recovering colored-Car biosynthesis in the lag-phase is far ahead of the growth rate of the cells themselves; (2) several Cars (ζ-carotene, neurosporene etc.) act as intermediates in Car biosynthesis; (3) because filling the "empty" Car pockets in the LH1-RC complexes is faster than in LH2, available spirilloxanthin is preferentially incorporated into the nascent LH1-RC core particles; (4) as a consequence of the resulting lack of spirilloxanthin availability, the biosynthetic intermediates (anhydrorhodovibrin, rhodopin and lycopene) fill the empty nascent LH2 Car pockets. In the present report, we further discuss the process of colored Car incorporation into LH complexes during the recovery of Car biosynthesis in the DPA-cells of Ect.haloalkaliphila.

Relative stability of G-quadruplex structures: Interactions between the human Bcl2 promoter region and derivatives of carbazole and diphenylamine.

The bcl2 promoter region forms a G-quadruplex structure, which is a crucial target for anticancer drug development. In this study, we provide theoretical predictions of the stability of different G-quadruplex folds of the 23-mer bcl2 promoter region and G-quadruplex ligand. We take into account the whole G-quadruplex structure, including bound-cations and solvent effects, in order to compute the ligand binding free energy using molecular dynamics simulation. Two series of the carbazole and diphenylamine derivatives are used to screen for the most potent drug in terms of stabilization. The energy analysis identifies the predominant energy components affecting the stability of the various different G-quadruplex folds. The energy associated with the stability of the G-quadruplex-K(+) structures obtained displays good correlation with experimental Tm measurements. We found that loop orientation has an intrinsic influence on G-quadruplex stability and that the basket structure is the most stable. Furthermore, parallel loops are the most effective drug binding site. Our studies also demonstrate that rigidity and planarity are the key structural elements of a drug that stabilizes the G-quadruplex structure. BMVC-4 is the most potential G-quadruplex ligand. This approach demonstrates significant promise and should benefit drug design.

Germline transmission of an embryonic stem cell line derived from BALB/c cataract mice.

Mice embryonic stem (ES) cells have enabled the generation of mouse strains with defined mutation(s) in their genome for putative disease loci analysis. In the study of cataract, the complex genetic background of this disease and lack of long-term self-renewal ES cells have hampered the functional researches of cataract-related genes. In this study, we aimed to establish ES cells from inherited cataract mice (BALB/CCat/Cat). Embryos of cataract mice were cultured in chemical-defined N2B27 medium with the presence of two small molecules PD0325901 and CHIR99021 (2i) and an ES cell line (named EH-BES) was successfully established. EH-BES showed long-term self-renewal in 2i medium and maintained capacity of germline transmission. Most importantly, the produced chimera and offspring developed congenital cataract as well. Flow cytometry assay revealed that EH-BES are homogeneous in expression of Oct4 and Rex1in 2i medium, which may account for their self-renewal ability. With long-term self-renewal ability and germline-competent, EH-BES cell line can facilitate genetic and functional researches of cataract-related genes and better address mechanisms of cataract.

A simplified diphenylamine colorimetric method for growth quantification.

Cell growth needs to be monitored in biological studies and bioprocess optimization. In special circumstances, such as microbial fermentations in media containing insoluble particles, accurate cell growth quantification is a challenge with current methods. Only the Burton method is applicable in such circumstances. The original Burton method was previously simplified by adopting a two-step sample pretreatment in perchloric acid procedure to eliminate the need for DNA extraction. Here, we further simplified the Burton method by replacing the previous two-step perchloric acid pretreatment with a new and one-step diphenylamine reagent pretreatment. The reliability and accuracy of this simplified method were assessed by measuring the biomass of four model microorganisms: Escherichia coli, Streptomyces clavuligerus, Saccharomyces cerevisiae, and Trichoderma reesei grown in normal media or those containing solid particles. The results demonstrate that this new simplified method performs comparably to the conventional methods, such as OD600 or the previously modified Burton method, and is much more sensitive than the dry weight method. Overall, the new method is simple, reliable, easy to perform, and generally applicable in most circumstances, and it reduces the operation time from more than 12 h (for the previously simplified Burton method) to about 2 h.

Carbon and nitrogen isotope effects associated with the dioxygenation of aniline and diphenylamine.

Dioxygenation of aromatic rings is frequently the initial step of biodegradation of organic subsurface pollutants. This process can be tracked by compound-specific isotope analysis to assess the extent of contaminant transformation, but the corresponding isotope effects, especially for dioxygenation of N-substituted, aromatic contaminants, are not well understood. We investigated the C and N isotope fractionation associated with the biodegradation of aniline and diphenylamine using pure cultures of Burkholderia sp. strain JS667, which can biodegrade both compounds, each by a distinct dioxygenase enzyme. For diphenylamine, the C and N isotope enrichment was normal with ε(C)- and ε(N)-values of -0.6 ± 0.1‰ and -1.0 ± 0.1‰, respectively. In contrast, N isotopes of aniline were subject to substantial inverse fractionation (ε(N) of +13 ± 0.5‰), whereas the ε(C)-value was identical to that of diphenylamine. A comparison of the apparent kinetic isotope effects for aniline and diphenylamine dioxygenation with those from abiotic oxidation by manganese oxide (MnO(2)) suggest that the oxidation of a diarylamine system leads to distinct C-N bonding changes compared to aniline regardless of reaction mechanism and oxidant involved. Combined evaluation of the C and N isotope signatures of the contaminants reveals characteristic Δδ(15)N/Δδ(13)C-trends for the identification of diphenylamine and aniline oxidation in contaminated subsurfaces and for the distinction of aniline oxidation from its formation by microbial and/or abiotic reduction of nitrobenzene.

Fully activated MEK1 exhibits compromised affinity for binding of allosteric inhibitors U0126 and PD0325901.

Kinases catalyze the transfer of γ-phosphate from ATP to substrate protein residues triggering signaling pathways responsible for a plethora of cellular events. Isolation and production of homogeneous preparations of kinases in their fully active forms is important for accurate in vitro measurements of activity, stability, and ligand binding properties of these proteins. Previous studies have shown that MEK1 can be produced in its active phosphorylated form by coexpression with RAF1 in insect cells. In this study, using activated MEK1 produced by in vitro activation by RAF1 (pMEK1(in vitro)), we demonstrate that the simultaneous expression of RAF1 for production of activated MEK1 does not result in stoichiometric phosphorylation of MEK1. The pMEK1(in vitro) showed higher specific activity toward ERK2 protein substrate compared to the pMEK1 that was activated via coexpression with RAF1 (pMEK1(in situ)). The two pMEK1 preparations showed quantitative differences in the phosphorylation of T-loop residue serine 222 by Western blotting and mass spectrometry. Finally, pMEK1(in vitro) showed marked differences in the ligand binding properties compared to pMEK1(in situ). Contrary to previous findings, pMEK1(in vitro) bound allosteric inhibitors U0126 and PD0325901 with a significantly lower affinity than pMEK1(in situ) as well as its unphosphorylated counterpart (npMEK1) as demonstrated by thermal-shift, AS-MS, and calorimetric studies. The differences in inhibitor binding affinity provide direct evidence that unphosphorylated and RAF1-phosphorylated MEK1 form distinct inhibitor sites.

Crystal structure of a trypanocidal 4,4'-bis(imidazolinylamino)diphenylamine bound to DNA.

The pursuit of small molecules that bind to DNA has led to the discovery of selective and potent antitrypanosomal agents, specifically 4,4'-bis(imidazolinylamino)- and 4,4'-bis(guanidino)diphenylamine compounds, CD27 and CD25, respectively. Although the antitrypanosomal properties of these compounds have been characterized, further development of this series of compounds requires assessment of their DNA site selectivities and affinities. Toward this end, both compounds have been analyzed and found to selectively bind AT sequences. However, CD27 was found to bind with higher affinity to 5'-AATT than 5'-ATAT while CD25 bound more weakly but equally well to either sequence. To detail the nature of its interactions with DNA, the crystal structure of CD27, bound to its preferred DNA-binding site 5'-AATT within a self-complementary oligonucleotide, 5'-d(CTTAATTCGAATTAAG), was determined at 1.75 A using a host-guest approach. Although CD27 is predicted to be highly twisted in its energy-minimized state, it adopts a more planar crescent shape when bound in the minor groove of the DNA. Interactions of CD27 with 5'-AATT include bifurcated hydrogen bonds, providing a basis for selectivity of this site, and favorable van der Waals interactions in a slightly widened minor groove. Thus, an induced fit results from conformational changes in both the ligand and the DNA. Our studies suggest a basis for understanding the mechanism of the antitrypanosomal activity of these symmetric diphenylamine compounds.

Pathway and evolutionary implications of diphenylamine biodegradation by Burkholderia sp. strain JS667.

Diphenylamine (DPA) is a common contaminant at munitions-contaminated sites as well as at aniline manufacturing sites. Little is known about the biodegradation of the compound, and bacteria able to use DPA as the growth substrate have not been reported. Burkholderia sp. strain JS667 and Ralstonia sp. strain JS668 were isolated by selective enrichment from DPA-contaminated sediment. The isolates grew aerobically with DPA as the sole carbon, nitrogen, and energy source. During induction of DPA degradation, stoichiometric amounts of aniline accumulated and then disappeared, which suggested that aniline is on the DPA degradation pathway. Genes encoding the enzymes that catalyze the initial steps in DPA degradation were cloned from the genomic DNA of strain JS667. The Escherichia coli clone catalyzed stoichiometric transformation of DPA to aniline and catechol. Transposon mutagenesis, the sequence similarity of putative open reading frames to those of well-characterized dioxygenases, and (18)O(2) experiments support the conclusion that the initial reaction in DPA degradation is catalyzed by a multicomponent ring-hydroxylating dioxygenase. DPA is converted to aniline and catechol via dioxygenation at the 1,2 position of the aromatic ring and spontaneous rearomatization. Aniline and catechol are further biodegraded by the well-established aniline degradation pathway. Genes that encode the complete aniline degradation pathway were found 12 kb downstream of the genes that encode the initial dioxygenase. Expression of the relevant dioxygenases was confirmed by reverse transcription-PCR analysis. Both the sequence similarity and the gene organization suggest that the DPA degradation pathway evolved recently by the recruitment of two gene clusters that encode the DPA dioxygenase and aniline degradation pathway.

APRIL is overexpressed in cancer: link with tumor progression.

BACKGROUND: BAFF and APRIL share two receptors - TACI and BCMA - and BAFF binds to a third receptor, BAFF-R. Increased expression of BAFF and APRIL is noted in hematological malignancies. BAFF and APRIL are essential for the survival of normal and malignant B lymphocytes, and altered expression of BAFF or APRIL or of their receptors (BCMA, TACI, or BAFF-R) have been reported in various B-cell malignancies including B-cell non-Hodgkin's lymphoma, chronic lymphocytic leukemia, Hodgkin's lymphoma, multiple myeloma, and Waldenstrom's macroglobulinemia. METHODS: We compared the expression of BAFF, APRIL, TACI and BAFF-R gene expression in 40 human tumor types - brain, epithelial, lymphoid, germ cells - to that of their normal tissue counterparts using publicly available gene expression data, including the Oncomine Cancer Microarray database. RESULTS: We found significant overexpression of TACI in multiple myeloma and thyroid carcinoma and an association between TACI expression and prognosis in lymphoma. Furthermore, BAFF and APRIL are overexpressed in many cancers and we show that APRIL expression is associated with tumor progression. We also found overexpression of at least one proteoglycan with heparan sulfate chains (HS), which are coreceptors for APRIL and TACI, in tumors where APRIL is either overexpressed or is a prognostic factor. APRIL could induce survival or proliferation directly through HS proteoglycans. CONCLUSION: Taken together, these data suggest that APRIL is a potential prognostic factor for a large array of malignancies.

Laccase-catalyzed removal of diphenylamine from synthetic wastewater.

The priority pollutant lists of both the U.S. Environmental Protection Agency (U.S. EPA) and the European Union (EU) include diphenylamine (DPA), a contaminant found in wastewater of various industries. This work demonstrates the potential of using enzymatic treatment to remove DPA from buffered synthetic wastewater. This treatment method includes oxidative polymerization of DPA using laccase from Trametes villosa, followed by removal of those polymers via adsorptive micellar flocculation (AMF) using sodium lauryl sulfate (SDS) and alum. Researchers investigated the effects of pH, laccase concentration, molecular mass, and concentration of polyethylene glycol (PEG) in continuously stirred batch reactors to achieve 95% substrate conversion in three hours. Treatment of 0.19 mM DPA was best at pH 7 and an enzyme concentration from 0.0025 to 0.0075 standard activity unit/mL. Except for PEG400 optimum enzyme and PEG concentrations decreased with an increase in PEG molecular mass. Optimum AMF conditions were pH 3.0 to 6.5, 200 mg/L of SDS, and 150 mg/L of alum.