Cyclooxygenase expression is elevated in retinoic acid-differentiated U937 cells

Cyclooxygenase (COX) is the rate-limiting enzyme for the biosynthesis of prostaglandins in monocytes/macrophages. The COX-1 is constitutively expressed in most tissues and may be involved in cellular homeostasis, whereas the COX-2 is an inducible enzyme that may play an important role in inflammation and mitogenesis. When U937 monocytic cells were incubated with retinoic acid (RA) for 48 h, cell differentiation took place with concomitant increases in prostaglandin E₂ (PGE₂) production and COX activity. In this study, the mechanism of RA (all-trans- or 9-cis-RA)-induced enhancement of PGE₂ biosynthesis in U937 cells was examined. Treatment of cells with all-trans- or 9-cis-RA up to 48 h caused an increase in PGE₂ production in a time- and dose-dependent manner. Both RA isomers caused the enhancement of PGE₂ production and the up-regulation of COX-1 expression at the protein and mRNA levels. The increase in COX-1 mRNA was found to precede the increase in COX-1 protein expression. Interestingly, the COX-2 protein and COX-2 mRNA were not detected in U937 cells, and their levels remained undetectable during the entire course of RA treatment. We conclude that treatment of U937 cells by RA for 48 h caused the initiation of cell differentiation, which was found to be concomitant with a significant increase in PGE₂ production mediated via the up-regulation of COX-1 mRNA and protein expression.     

Trifluoromethylated carboline compounds targeting DNA: Synthesis,binding and anti-proliferative effects on human cancer cell lines

Three sets of carboline derived compounds were prepared by Pictet-Spengler cyclization. These tetrahydro β- and γ-carbolines have CF₃ group with an additional amino alkyl chains (α- or δ-position) and guanidine alkyl chains (α-position), of varying length. Structure–activity relationship of these molecules with calf thymus DNA was emphasized by fluorescence, ITC, FTIR and viscosity. Binding with DNA resulted in dramatic enhancement and quenching in the fluorescence emission. Gamma-carboline analogs showed maximum DNA binding followed by beta-carboline compounds with amino alkyl chain and least with guanidine alkyl chain compounds. It decreased with increasing chain length. The bindings were entropically driven being more with guanidine alkyl chain analogs. Site preference and mode of binding with partial intercalation and external binding was supported by FTIR and viscosity. Cytotoxic potencies of the compounds were tested on seven different cancer cell lines. The smallest alkyl chain analog attached to gamma position, Comp3, showed maximum cytotoxicity with GI₅₀ 6.2 µM, against HCT-116 causing apoptosis, followed by the guanidine alkyl chain compounds, but amino alkyl chain compounds to beta position showed poor cytotoxicity.These results may be of prospective use in a framework to design novel carboline derivatives as antitumor drugs for improved therapeutic applications in future.     

Enzyme-bound early product of purified poly(ADP-ribose) polymerase.

Bovine thymus poly(ADP-ribose) polymerase with a purity of 99% on a SDS-poly-acrylamide gel electrophoresis was able to initiate poly(ADP-ribose) synthesis without adding any exogenous acceptor protein to the reaction system. Analyses of the early reaction product synthesized without exogenous acceptor protein revealed that the product was oligo(ADP-ribose) with a mean chain length of 2.6 and was bound tightly to the enzyme protein. When the radioactive early reaction product was chased by incubating further with cold NAD⁺, ADP-ribose unit was found to be added to the terminal AMP-residue of the oligo(ADP-ribose) attached to the enzyme. The stability of the early reaction product in high concentration of salt, strong acid, sodium dodecyl sulfate, and urea strongly suggests a covalent nature of the binding of oligo(ADP-ribose) to the enzyme.     

A highly sensitive fluorescence sensor based on lucigenin/chitosan/SiO2 composite nanoparticles for microRNA detection using magnetic separation

In this paper, a convenient reverse‐phase microemulsion method for the synthesis of SiO₂ nanoparticles (NPs) by simply introducing the chitosan and fluorescent dye of lucigenin during the formation reaction of SiO₂ NPs was proposed. Addition of chitosan can make the SiO₂ NPs porous, and increases lucigenin molecule incorporation into chitosan/SiO₂ NPs nanopores based on electrostatic interaction and supermolecular forces. Therefore, fluorescence quantum yield of the lucigenin/chitosan/SiO₂ composite nanoparticles was increased by introduction of chitosan and compared with lucigenin/SiO₂ NPs without chitosan. Because the number of negative charges carried when using single‐stranded DNA (ssDNA) was different from that of double‐stranded DNA (dsDNA), the numbers of lucigenin/chitosan/SiO₂ composite nanoparticles with positive charge adsorbed using ssDNA or dsDNA were different. Consequently, fluorescence intensity caused using ssDNA or dsDNA/miRNA was clearly discriminative. With increase in target DNA/miRNA concentration, the difference in fluorescence intensity also increased, resulting in a good linear relationship between fluorescence intensity sensitizing value and target miRNA concentrations. Therefore, a new fluorescence analysis method for direct detection of let‐7a in human gastric cancer cell samples without enzyme, label free and no immobilization was established using lucigenin/chitosan/SiO₂ composite nanoparticles as a DNA hybrid indicator. The proposed method had high sensitivity and selectivity, low cost and the detection limit was 10 fM (S/N = 3).     

Hydrolysis of a fluorescent phospholipid substrate by phospholipase A2 and lipoprotein lipase

The fluorescent phospholipid 1-acyl-2-[6-[(7-nitro-2,1,3benzoxadiazol-4-yl)amino]-caproyl]phosphatidylcholine (C₆-NBD-PC) was used as a substrate for porcine pancreatic phospholipase A₂ (PA₂) and bovine milk lipoprotein lipase (LpL). Hydrolysis of C₆-NBD-PC by either enzyme resulted in a greater than 50-fold fluorescence enhancement with no shift in the emission maximum at 540 nm; Ca⁺⁺ was required for PA₂ catalysis. Identification of the products of hydrolysis showed cleavage at the $\text{sn}$-1 and $\text{sn}$-2 positions for LpL and PA₂, respectively. For PA₂, but not for LpL, there was a marked enhancement of enzyme catalysis at lipid concentrations above the critical micellar concentration of the lipid. Furthermore, apolipoprotein C-II, the activator protein of LpL for long-chain fatty acyl substrates, did not enhance the rate of catalysis of the water-soluble fluorescent phospholipid for either enzyme.     

Conformational dynamics of the interaction of Escherichia coli endonuclease VIII with DNA substrates

Endonuclease VIII (Nei) from Escherichia coli is a DNA repair enzyme that removes a wide range of oxidized pyrimidine bases from DNA. As inferred from the crystal structures and biochemical studies, recognition of DNA lesions by Nei involves several conformational changes in both protein and DNA, such as DNA kinking, damaged base eversion into the enzyme's active site, and insertion of a loop of the enzyme into the void formed by the eversion. Excision of the damaged base by Nei also proceeds through several chemical steps: N-glycosidic bond breakage, β-elimination and δ-elimination of the phosphates flanking the lesion. We have used stopped-flow kinetics with fluorescence detection to follow conformational changes in the Nei molecule when the enzyme binds normal DNA, damaged but uncleavable DNA, or several cleavable damaged DNA substrates. Binding normal or damaged uncleavable DNA proceeded in two fluorescently discernible reversible stages, while processing of cleavable substrates involved three reversible stages followed by and irreversible stage and equilibrium with the reaction product. Individual rate constants were calculated for each reaction step. Based on the stopped-flow data, crystal structure, and a comparison with the stopped-flow kinetics of E. coli formamidopyrimidine-DNA glycosylase, a homolog of Nei, we propose the nature of some of the steps that may be involved into the recognition and excision of damaged bases by Nei.     

Purification and characterization of the messenger ribonucleoprotein-associated casein kinase II of Artemia salina cryptobiotic gastrulae

The mRNP-associated protein kinase is purified to near homogeneity by ion-exchange chromatography on phosphocellulose and affinity chromatography on casein-Sepharose 4B and ATP-agarose. The cyclic nucleotide-independent enzyme phosphorylates casein using either ATP or GTP. The enzyme exists in two forms composed of subunits with M_r 36 500 (α) and 28 000 (β) and of subunits with M_r 36 500 (α), 33 000 (α′) and 28 000 (β). The undegraded enzyme has an M_r of 136 000 ± 7000. The enzyme is inhibited by heparin and hemin and stimulated by spermine. The mRNP-associated protein kinase may be classified as a casein kinase II. Main mRNP protein phosphate acceptors have M_r values of 112 000, 72 000, 65 000, 53 000, 38 000, 28 000, 23 500 and 21 000. Phosphorylation of the M_r 38 000 poly(A)-binding protein resulted in the generation of different acidic ionic species. From the observed inhibition of the translational activity after phosphorylation by the mRNP-associated protein kinase a function in the repression of mRNP is proposed.     

New anthracenedione derivatives: Interaction with DNA and biological effects

Two anthracenedione derivatives [1 - (ω - diethylaminopropylamido) - 4 - hydroxy - 9,10 - anthracenedione hydrochloride (I) and 1 - (ω - diethylamino-propylamido) - 2 - methoxy - 4 - hydroxy - 9,10 - anthracenedione hydrochloride (II)], having an electron-rich planar chromophore and an amino-substituted side chain, have been synthesized. Their binding ability to DNA was investigated by means of spectroscopic, equilibrium dialysis and fluorescence measurements. Their inhibition efficiency on nucleic acid synthesis was also evaluated both in mouse and human cells. Our results indicate that, in comparison with adriamycin, compound I shows a slightly weaker complexation ability to DNA, while compound II interacts with DNA at a substantially lower level. These data match quite well with the biological response on the inhibition of DNA and RNA synthesis exhibited by the above mentioned compounds; in fact compound I is slightly less efficient than adriamycin and about ten times more efficient than compound II. The close relationship between the results of physicochemical and biological studies is discussed.     

Interactions of beef heart mitochondrial adenosine triphosphatase and aurovertin.

Aurovertin forms a complex with soluble beef heart mitochondrial ATPase (F₁) while exhibiting a biphasic fluorence enhancement. The effect of substrate, activators and inhibitors of F₁¹ of the fluorescence of the aurovertin-F₁ complex is reported. The aurovertin-F₁ complex can exist in two different states, one showing low fluorescence and the other with high fluorescence. Transition into the low fluorescence state is induced by various nucleoside triphosphates (ATP ± Mg²⁺, ITP ± Mg²⁺, GIP + Gg²⁺, and AMP-P(NH)P ± Mg²⁺). The rate and extent of fluorescence decrease caused by nucleotide addition (except that caused by ATP) is dependent on the presence of added Mg²⁺. The inhibitors of ATPase activity (AMP-P(NH)P, GMP-P(NH)P and EDTA) at concentrations that inhibit hydrolysis of ATP did not prevent the ATP induced decrease of aurovertin fluorescence. EDTA at high concentration (>0.4 mM) enhanced the effect of ADP.The complex of aurovertin with F₁ that had previously been treated with butanedione loses sensitivity to ATP. Addition of ADP to the system containing butanedione-treated enzyme caused a 2-fold greater enhancement of fluorescence than the addition of ADP to the control system. In contrast to the butanedione-treated enzyme, the complex of aurovertin with F₁ previously treated at pH 5.6 loses sensitivity to ADP. Addition of ATP to this system lowered the fluorescence as in the system containing native enzyme.On the basis of the analyses of the aurovertin fluorescence changes and hydrolytic activity of F₁, the existence of several types of ligand binding sties with varying degrees of specificity are proposed. It is further proposed that these sites are important in control of the conformation and the catalytic properties of the ATPase molecule.     

Interaction between brain enzymes glutamate dehydrogenase and aspartate aminotransferase.

Fluorescence spectroscopic methods were used to study the interaction between aspartate aminotransferase and glutamate dehydrogenase isolated from pig brain. The conversion of the P-pyridoxal form of the aminotransferase to the P-pyridoxamine form of the enzyme is easily monitored by recording emission spectra upon excitation at 330 nm. Evidence for the interaction between the enzymes was obtained from fluroescence measurements conducted on aspartate aminotransferase label with a fluorescence probe (1-5-AEDANS) attached to one sH residue of the protein. The interaction of the aminotransferase (1μM) with glutamate dehydrogenase (2μM) brings about an enhancement as well as a blue shift in the band position of the fluorescence emitted by the dansyl chromophore. Polarization of fluorescence measurements conducted over a wide range of temperatures reveal that the rotational correlation time of aspartate aminotransferase (35 n.seconds) is increased to a value of 100 n.seconds upon addition of glutamate dehydrogenase.     

Random DNA fragmentation with endonuclease V: application to DNA shuffling

The enzyme endonuclease V nicks uracil-containing DNA at the second or third phosphodiester bond 3′ to uracil sites. I applied the enzyme to random fragmentation of DNA to revise the complex DNA shuffling protocol. The merit of using endonuclease V is that cleavage occurs at random sites and the length of the fragments can easily be adjusted by varying the concentration of dUTP in the polymerase chain reaction. Unlike the conventional method using DNase I, no partial digestion or gel separation of fragments is required. Therefore, labor is dramatically reduced and reproducibility ensured. I applied this method to recombine two truncated green fluorescent protein (GFP) genes and demonstrated successful DNA shuffling by the appearance of the fluorescent full-length GFP genes.     

Ultrafast dynamics-driven biomolecular recognition where fast activities dictate slow events

In general, biological macromolecules require significant dynamical freedom to carry out their different functions, including signal transduction, metabolism, catalysis and gene regulation. Effectors (ligands, DNA and external milieu, etc) are considered to function in a purely dynamical manner by selectively stabilizing a specific dynamical state, thereby regulating biological function. In particular, proteins in presence of these effectors can exist in several dynamical states with distinct binding or enzymatic activity. Here, we have reviewed the efficacy of ultrafast fluorescence spectroscopy to monitor the dynamical flexibility of various proteins in presence of different effectors leading to their biological activity. Recent studies demonstrate the potency of a combined approach involving picosecond-resolved Förster resonance energy transfer, polarisation-gated fluorescence and time-dependent stokes shift for the exploration of ultrafast dynamics in biomolecular recognition of various protein molecules. The allosteric protein–protein recognition following differential protein–DNA interaction is shown to be a consequence of some ultrafast segmental motions at the C-terminal of Gal repressor protein dimer with DNA operator sequences O_E and O_I. Differential ultrafast dynamics at the C-terminal of λ-repressor protein with two different operator DNA sequences for the protein–protein interaction with different strengths is also reviewed. We have also systemically briefed the study on the role of ultrafast dynamics of water molecules on the functionality of enzyme proteins α-chymotrypsin and deoxyribonuclease I. The studies on the essential ultrafast dynamics at the active site of the enzyme α-chymotrypsin by using an anthraniloyl fluorescent extrinsic probe covalently attached to the serine-195 residue for the enzymatic activity at homeothermic condition has also been reviewed. Finally, we have highlighted the evidence that a photoinduced dynamical event dictates the molecular recognition of a photochromic ligand, dihydroindolizine with the serine protease α-chymotrypsin and with a liposome (L-α-phosphatidylcholine).     

Estimation of Förster's distance between two ends of Dps protein from mycobacteria: distance heterogeneity as a function of oligomerization and DNA binding

Dps protein (DNA binding Protein from Starved Cells) from Mycobacterium smegmatis (Ms-Dps) is known to undergo an in vitro irreversible oligomeric transition from trimer to dodecamer. This transition helps the protein to provide for bimodal protection to the bacterial DNA from the free radical and Fenton mediated damages in the stationary state. The protein exists as a stable trimer, when purified from E. coli cells transformed with an over-expression plasmid. Both trimer as well as dodecamer are known to exhibit ferroxidation activity, thus removing toxic hydroxyl radicals in vivo, whereas iron accumulation and non-sequence specific DNA binding activity are found in dodecamer only. This seems to be aided by the positively charged long C-terminal tail of the protein. We used frequency domain phase-modulation fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET) to monitor this oligomeric switch from a trimer to a dodecamer and to elucidate the structure of DNA–Dps dodecamer complex. As Ms-Dps is devoid of any Cysteine residues, a Serine is mutated to Cysteine (S169C) at a position adjacent to the putative DNA binding domain. This Cysteine is subsequently labeled with fluorescent probe and another probe is placed at the N-terminus, as crystal structure of the protein reveals several side-chain interactions between these two termini, and both are exposed towards the surface of the protein. Here, we report the Förster's distance distribution in the trimer and the dodecamer in the presence and absence of DNA. Through discrete lifetime analysis of the probes tagged at the respective regions in the macromolecule, coupled with Maximum Entropy Method (MEM) analysis, we show that the dodecamer, upon DNA binding shows conformational heterogeneity in overall structure, perhaps mediated by a non-specific DNA–protein interaction. On the other hand, the nature of DNA–Dps interaction is not known and several models exist in literature. We show here with the help of fluorescence anisotropy measurements of labeled DNA having different length and unlabeled native dodecameric protein that tandem occupation of DNA binding sites by a series of Dps molecules perhaps guide the tight packing of Dps over DNA backbone.     

Spectroscopic investigation of lipase from Pseudomonas cepacia solubilized in 1,4‐dioxane by non‐covalent complexation with methoxypoly(ethylene glycol)

Lipase from Pseudomonas cepacia was made soluble in 1,4‐dioxane by lyophilization of the enzyme from aqueous solutions containing methoxypoly(ethylene glycol) (PEG). The solubility of the enzyme–PEG complex depended both on protein concentration and PEG protein ratio. Intrinsic protein fluorescence and far‐ and near‐UV circular dichroism revealed that not only did the enzyme not unfold in the organic solvent, but rather became more compact. This was seen by the slight quenching of fluorescence intensity and by the enhancement of the near‐UV circular dichroism negative signals, which are indicative of stronger interactions of tryptophanyl and/or tyrosyl residues among themselves or with other parts of the enzyme molecule. The specific activity of the lipase–PEG complex in the organic solvent was at least 2 orders of magnitude higher than that of the enzyme powder. This can be attributed both to the maintenance of native conformation and to enzyme dissolution in the reaction medium which should minimize possible limitations to enzyme–substrate interactions. © 1999 John Wiley & Sons, Inc., Biotechnol Bioeng 64: 624–629, 1999.     

Entropic Enhancement of Protein-DNA Affinity by Oxygen-to-Sulfur Substitution in DNA Phosphate

Dithioation of DNA phosphate is known to enhance binding affinities, at least for some proteins. We mechanistically characterized this phenomenon for the Antennapedia homeodomain-DNA complex by integrated use of fluorescence, isothermal titration calorimetry, NMR spectroscopy, and x-ray crystallography. By fluorescence and isothermal titration calorimetry, we found that this affinity enhancement is entropy driven. By NMR, we investigated the ionic hydrogen bonds and internal motions of lysine side-chain NH₃⁺ groups involved in ion pairs with DNA. By x-ray crystallography, we compared the structures of the complexes with and without dithioation of the phosphate. Our NMR and x-ray data show that the lysine side chain in contact with the DNA phosphate becomes more dynamic upon dithioation. Our thermodynamic, structural, and dynamic investigations collectively suggest that the affinity enhancement by the oxygen-to-sulfur substitution in DNA phosphate is largely due to an entropic gain arising from mobilization of the intermolecular ion pair at the protein-DNA interface.     

Compact form of DNA induced by DNA-binding protein HU.

Interaction of DNA-binding protein HU from Bacillus stearothermophilus (HUBst) with coliphage T2 DNA was investigated by means of a single-duplex DNA chain visualization method using fluorescence microscopy. Fluorescence microscopic images of coliphage T2 DNA molecules were observed as a function of HUBst concentration. The average fluorescence image size of T2 DNA decreased with increase in HUBst concentration to a size comparable to that of a DNA globule induced by polyethylene glycol (PEG) and multivalent cation (MVC). The change to globule-like DNA proceeded gradually and monotonously, in contrast to the coil-globule transition of DNA induced by PEG and MVC. The histogram of the fluorescence image length was essentially a single-modal one throughout the process of conformational change. These results indicate that the process of shrinking of DNA from a random coil to a globule-like one is not of a transitional nature. The interaction of HUBst with DNA and the mechanism of shrinkage are concluded to be different from those of PEG-induced and MVC-induced coil-globule transition of DNA.     

Domain organization and biochemical features of Sulfolobus solfataricus DNA polymerase

DNA polymerase from Sulfolobus solfataricus, strain MT4 (Sso DNA pol), was one of the first archaeal DNA polymerases to be isolated and characterized. Its encoding gene was cloned and sequenced, indicating that Sso DNA pol belongs to family B of DNA polymerases. By limited proteolysis experiments carried out on the recombinant homogeneous protein, we were able to demonstrate that the enzyme has a modular organization of its associated catalytic functions (DNA polymerase and 3′-5′ exonuclease). Indeed, the synthetic function was ascribed to the enzyme C-terminal portion, whereas the N-terminal half was found to be responsible for the exonucleolytic activity. In addition, partial proteolysis studies were utilized to map conformational changes on DNA binding by comparing the cleavage map in the absence or presence of nucleic acid ligands. This analysis allowed us to identify two segments of the Sso DNA pol amino acid chain affected by structural modifications following nucleic acid binding: region 1 and region 2, in the middle and at the C-terminal end of the protein chain, respectively. Site-directed mutagenesis studies will be performed to better investigate the role of these two protein segments in DNA substrate interaction. Received: January 22, 1998 / Accepted: February 16, 1998     

Fluorescence melting curve analysis using self-quenching dual-labeled peptide nucleic acid probes for simultaneously identifying multiple DNA sequences

Previous fluorescence melting curve analysis (FMCA) used intercalating dyes, and this method has restricted application. Therefore, FMCA methods such as probe-based FMCA and molecular beacons were studied. However, the usual dual-labeled probes do not possess adequate fluorescence quenching ability and sufficient specificity, and molecular beacons with the necessary stem structures are hard to design. Therefore, we have developed a peptide nucleic acid (PNA)-based FMCA method. PNA oligonucleotide can have a much higher melting temperature (T_m) value than DNA. Therefore, short PNA probes can have adequate T_m values for FMCA, and short probes can have higher specificity and accuracy in FMCA. Moreover, dual-labeled PNA probes have self-quenching ability via single-strand base stacking, which makes PNA more favorable. In addition, this method can facilitate simultaneous identification of multiple DNA templates. In conventional real-time polymerase chain reaction (PCR), one fluorescence channel can identify only one DNA template. However, this method uses two fluorescence channels to detect three types of DNA. Experiments were performed with one to three different DNA sequences mixed in a single tube. This method can be used to identify multiple DNA sequences in a single tube with high specificity and high clarity.     

Properties of 3-methyladenine-DNA glycosylase from Escherichia coli.

An Escherichia coli enzyme that releases 3-methyladenine and 3-ethyladenine in free form from alkylated DNA has been purified 2800-fold in 7% yield. The enzyme does not liberate several other alkylation products from DNA, including 7-methylguanine,O6-methylguanine, 7-methyladenine, N6-methyladenine, 7-ethylguanine, O6-ethylguanine, and the arylalkylated purine derivatives obtained by treatment of DNA with 7-bromomethyl-12-methylbenz[a]anthracene. The reaction of the enzyme with alkylated DNA leads to the introduction of apurinic sites but no chain breaks (less than one incision per ten apurinic sites), and there is no detectable nuclease activity with native DNA, depurinated DNA, ultraviolet-irradiated DNA, or X-irradiated DNA as potential substrates. The enzyme is termed 3-methyladenine-DNA glycosylase. It is a small protein, Mr = 19 000, that does not require divalent metal ions, phosphate, or other cofactors in order to cleave base-sugar bonds in alkylated DNA.     

The microsomal activation of aflatoxin B1 and 2-(N-ethylcarbamoyloxymethyl)furan in vitro using a novel diffusion apparatus

The activation by rat liver microsomal systems in vitro of a naturally occurring and a synthetic furan-containing toxin, aflatoxin B₁ and 2-(N-ethylcarbamoyloxymethyl)furan (CMF) has been examined. Both compounds are metabolised to form products which bind covalently to DNA and microsomal protein, Using a specially designed two-chamber diffusion apparatus it has been demonstrated that the active metabolite of CMF is able to bind covalently to DNA separated by a membrane barrier from the microsomal site of activation. In the case of aflatoxin B₁ the DNA must be in physical contact with the microsomal system for the active metabolite of aflatoxin B₁ to bind covalently. Differences between the activation of the two compounds have also been found with regard to their relative efficiencies in binding to DNA and also the effects of the nucleophile GSH. These results have suggested that if the molecular mechanisms of activation of the two compounds be similar, other factors, for example differences in lipid solubility, may play important roles in determining the relative biological activaties of the compounds. The results suggested that the subcellular site of activation of aflatoxin B₁, unlike that of CMF, may need to be adjacent to the target DNA. It is proposed that this site might be the outer nuclear membrane. Alternatively a carrier molecular might exist for the activated aflatoxin B₁ metabolite in vivo.