Repeated roll-to-roll transfer of two-dimensional materials by electrochemical delamination.

Two-dimensional (2D) materials such as graphene (Gr), molybdenum disulfide and hexagonal boron nitride (hBN) hold great promise for low-cost and ubiquitous electronics for flexible displays, solar cells or smart sensors. To implement this vision, scalable production, transfer and patterning technologies of 2D materials are needed. Recently, roll-to-roll (R2R) processing, a technique that is widely used in industry and known to be cost-effective and scalable, was applied to continuously grow and transfer graphene. However, more work is needed to understand the possibilities and limitations of this technology to make R2R processing of 2D materials feasible. In this work, we fabricated a custom R2R transferring system that allows the accurate control of the process parameters. We employ continuous electrochemical delamination, known as "bubble transfer", to eliminate chemical etchant waste and enable the continuous transfer of 2D materials from metal foils. This also makes our transfer method a renewable and environmentally friendly process. We investigate the surface topology as well as the electrical parameters of roll-to-roll transferred graphene on polyethylene terephthalate (PET) coated with ethylene-vinyl acetate (EVA). Furthermore, we demonstrate for the first time the stacking of two layers of graphene or graphene on hBN by repeated lamination and delamination onto EVA/PET. These results are an important contribution to creating low-cost, large scale and flexible electronics based on 2D materials.

Brainstem melanomas presenting as a cavernous malformation.

BACKGROUND: Melanoma lesions in the brainstem can be difficult to distinguish radiographically and clinically from cavernous malformations. However, the treatment modalities and clinical course of these two diseases differ considerably. We report two cases of melanoma presenting as brainstem hemorrhages. CASE DESCRIPTION: A 69-year-old male was found to have a hemorrhagic lesion of the right dorsal midbrain. After a repeat hemorrhage, the lesion was resected and found to be hyperchromatic. Nonetheless, the patient suffered rebleeding and died 3 months later. A 62-year-old female was similarly found to have an acute pontine hemorrhage. After resection of the lesion, she underwent whole-brain radiation therapy but ultimately died 5.5 months later. The histopathology of both lesions was consistent with melanoma. CONCLUSIONS: Melanoma in the brainstem can mimic cavernous malformations. While management of these lesions includes stereotactic radiosurgery, whole-brain radiation, and surgical resection, metastatic brainstem melanoma follows an aggressive clinical course with a poor prognosis.

Interindividual variability in inhibition and induction of cytochrome P450 enzymes.

Drug interactions have always been a major concern in medicine for clinicians and patients. Inhibition and induction of cytochrome P450 (CYP) enzymes are probably the most common causes for documented drug interactions. Today, many pharmaceutical companies are predicting potential interactions of new drug candidates. Can in vivo drug interactions be predicted accurately from in vitro metabolic studies? Should the prediction be qualitative or quantitative? Although some scientists believe that quantitative prediction of drug interactions is possible, others are less optimistic and believe that quantitative prediction would be very difficult. There are many factors that contribute to our inability to quantitatively predict drug interactions. One of the major complicating factors is the large interindividual variability in response to enzyme inhibition and induction. This review examines the sources that are responsible for the interindividual variability in inhibition and induction of cytochrome P450 enzymes.

Human cytochrome P-450 3A4: in vitro drug-drug interaction patterns are substrate-dependent.

Testosterone, terfenadine, midazolam, and nifedipine, four commonly used substrates for human cytochrome P-450 3A4 (CYP3A4), were studied in pairs in human liver microsomes and in microsomes from cells containing recombinant human CYP3A4 and P-450 reductase, to investigate in vitro substrate-substrate interaction with CYP3A4. The interaction patterns between compounds with CYP3A4 were found to be substrate-dependent. Mutual inhibition, partial inhibition, and activation were observed in the testosterone-terfenadine, testosterone-midazolam, or terfenadine-midazolam interactions. However, the most unusual result was the interaction between testosterone and nifedipine. Although nifedipine inhibited testosterone 6beta-hydroxylation in a concentration-dependent manner, testosterone did not inhibit nifedipine oxidation. Furthermore, the effect of testosterone and 7,8-benzoflavone on midazolam 1'-hydroxylation and 4-hydroxylation demonstrated different regiospecificities. These results may be explained by a model in which multiple substrates or ligands can bind concurrently to the active site of a single CYP3A4 molecule. However, the contribution of separate allosteric sites and conformational heterogeneity to the atypical kinetics of CYP3A4 can not be ruled out in this model.

Inhibitory anti-CYP3A4 peptide antibody: mapping of inhibitory epitope and specificity toward other CYP3A isoforms.

An antipeptide antibody has been produced that recognizes CYP3A4 and exhibits greater than 90-95% inhibition on CYP3A4-mediated reactions [Wang RW and Lu AYH (1997) Drug Metab Dispos 25:762-767]. The inhibitory epitope of the 21-amino acid peptide, corresponding to residues 253 to 273 of CYP3A4, has been identified to reside in a 7-amino acid sequence (LEDTQKH: residues 261-267 of CYP3A4). This conclusion was based on the reversal of antibody inhibition of testosterone 6beta-hydroxylation when peptides with overlapping sequence in this region were preincubated with the antibody. In immunoblotting analysis, this antibody did not recognize CYP3A5 or CYP3A7 in microsomes prepared from baculovirus-infected cells containing these two expressed isoforms. In addition, the antipeptide antibody did not inhibit testosterone 6beta-hydroxylation or midazolam 1'- and 4-hydroxylation in microsomes containing expressed CYP3A5 and CYP3A7. Because the corresponding sequence in CYP3A5 (LNDKQKH) and CYP3A7 (LKETQKH) differs from CYP3A4 by only two amino acids, six peptides with either one or two amino acid changes were used to determine which amino acid is essential for antibody-antigen interaction. Our data indicate that Glu, Asp, and Thr in the 7-amino acid sequence of CYP3A4 are critical determinants of selectivity among CYP3A isoforms.

Drug-metabolism research challenges in the new millennium: individual variability in drug therapy and drug safety.

One of the most challenging research areas in pharmacology in the new millennium is to understand why individuals respond differently to drug therapy and to what extent that individual variability in disposition is responsible for the observed differences in therapeutic efficacy and adverse reactions. To answer these complex questions, drug-metabolism research will rely on multidisciplinary approaches more than ever to investigate the many components involved in drug metabolism and disposition. Major research challenges include the following: (1) the genetic variation of drug targets (receptors, enzymes, etc.), drug transporters (multispecific organic anion transporter, P-glycoprotein, alpha-1-acid glycoprotein, etc.), and drug-metabolizing enzymes (cytochrome P450s and other enzymes); (2) the structure and function of all genetic variants of drug receptors, transporters, and metabolizing enzymes; (3) the induction, repression, and inhibition of all components involved in drug disposition; (4) the development of noninvasive in vivo methods to determine the physiological significance of various components in the handling of specific therapeutic agents in humans; (5) the mechanism of idiosyncratic adverse drug reactions; and (6) the pharmacokinetic and pharmacodynamic relationships to explain the individual differences in therapeutic efficacy and drug safety. Thus successful drug-metabolism research in the new millennium must integrate receptor biology, enzymology, recombinant DNA technology, biochemical toxicology, and drug disposition into study design and conduct balanced in vitro and in vivo experiments to allow a full understanding of the mechanisms of individual variability in drug therapy and drug safety.

Inhibition and induction of cytochrome P450 and the clinical implications.

The cytochrome P450s (CYPs) constitute a superfamily of isoforms that play an important role in the oxidative metabolism of drugs. Each CYP isoform possesses a characteristic broad spectrum of catalytic activities of substrates. Whenever 2 or more drugs are administered concurrently, the possibility of drug interactions exists. The ability of a single CYP to metabolise multiple substrates is responsible for a large number of documented drug interactions associated with CYP inhibition. In addition, drug interactions can also occur as a result of the induction of several human CYPs following long term drug treatment. The mechanisms of CYP inhibition can be divided into 3 categories: (a) reversible inhibition; (b) quasi-irreversible inhibition; and (c) irreversible inhibition. In mechanistic terms, reversible interactions arise as a result of competition at the CYP active site and probably involve only the first step of the CYP catalytic cycle. On the other hand, drugs that act during and subsequent to the oxygen transfer step are generally irreversible or quasi-irreversible inhibitors. Irreversible and quasi-irreversible inhibition require at least one cycle of the CYP catalytic process. Because human liver samples and recombinant human CYPs are now readily available, in vitro systems have been used as screening tools to predict the potential for in vivo drug interaction. Although it is easy to determine in vitro metabolic drug interactions, the proper interpretation and extrapolation of in vitro interaction data to in vivo situations require a good understanding of pharmacokinetic principles. From the viewpoint of drug therapy, to avoid potential drug-drug interactions, it is desirable to develop a new drug candidate that is not a potent CYP inhibitor or inducer and the metabolism of which is not readily inhibited by other drugs. In reality, drug interaction by mutual inhibition between drugs is almost inevitable, because CYP-mediated metabolism represents a major route of elimination of many drugs, which can compete for the same CYP enzyme. The clinical significance of a metabolic drug interaction depends on the magnitude of the change in the concentration of active species (parent drug and/or active metabolites) at the site of pharmacological action and the therapeutic index of the drug. The smaller the difference between toxic and effective concentration, the greater the likelihood that a drug interaction will have serious clinical consequences. Thus, careful evaluation of potential drug interactions of a new drug candidate during the early stage of drug development is essential.

Inhibitory anti-peptide antibody against human CYP3A4.

An inhibitory anti-peptide antibody was raised against a 21-amino acid peptide (VKRMKESRLEDTQKHRVDFLQ) corresponding to residues 253-273 of human cytochrome P450 3A4. High titer antibodies were produced by rabbits immunized with this peptide coupled to keyhole limpet hemocyanin, as judged by ELISA. Anti-peptide antibody recognized a single protein band in microsomes prepared from cells expressing recombinant human CYP3A4 in immunoblotting analysis. No immunodetectable proteins were found in microsomes containing other cytochrome P450 isoforms. In addition, the antibody did not recognize CYP3A5, a closely related isoform in the CYP3A family. In human liver microsomes, only one protein band which comigrated with human CYP3A4 was recognized by this antibody and the relative blotting intensity of this protein band correlated significantly with human CYP3A4-catalyzed testosterone 6 beta-hydroxylase activities (r = 0.96). More importantly, this antibody exhibited greater than 90-95% inhibition of testosterone 6 beta-hydroxylation, while other cytochrome P450-mediated reactions in human liver microsomes were not inhibited. Because of its specificity and inhibitory potency, this anti-peptide antibody should be a valuable tool in evaluating the role of CYP3A in mediating in vitro metabolism of therapeutic agents.

Human cytochrome P450 3A4-catalyzed testosterone 6 beta-hydroxylation and erythromycin N-demethylation. Competition during catalysis.

Cytochrome P450 3A4 is known to catalyze the metabolism of both endogenous substrates (such as the 6 beta-hydroxylation of testosterone) and many important therapeutic agents, including the N-demethylation of erythromycin. However, erythromycin and testosterone have been reported to have little or no effect on the metabolism of each other by recombinant CYP3A4. In an effort to understand the basis of these observations, we studied the N-demethylation of erythromycin and the 6 beta-hydroxylation of testosterone in human liver microsomes and in microsomes from cells containing recombinant human CYP3A4 and P450 reductase under a variety of experimental conditions. In both human liver microsomal and recombinant CYP3A4 systems, erythromycin inhibited testosterone 6 beta-hydroxylation in a concentration dependent manner, and vice versa. However, the inhibition mechanism was complex. At low substrate concentrations, testosterone and erythromycin acted as competitive inhibitors to each other. Under these experimental conditions, an apparent competitive inhibition of testosterone 6 beta-hydroxylation by erythromycin was observed, with Ki values similar to that of the K(m) values for erythromycin. When the rates of testosterone 6 beta-hydroxylation and erythromycin N-demethylation were determined in microsomal incubations containing both substrates at lower concentrations, the observed rates for each reaction were in good agreement with the calculated rates based on the rate equation describing simultaneous metabolism of two substrates by a single enzyme. However, at high substrate concentrations, the kinetic results could be best explained by a mechanism involving partial competitive inhibition. We conclude from these studies that testosterone and erythromycin mutually inhibit the metabolism of each other, consistent with the fact that CYP 3A4 catalyzes the metabolism of both substrates.

Ligand effects on the fluorescence properties of tyrosine-9 in alpha 1-1 glutathione S-transferase.

A conserved tyrosine plays a critical role in catalysis by mammalian glutathione S-transferases (GSTs) of the alpha-, mu-, and pi-classes, by forming a hydrogen bond to and stabilizing the thiolate form of glutathione. The hydrogen bonding properties of this tyrosine in the rat A1-1 GST (Tyr-9), in the absence and presence of ligands, have been studied by steady state and time-resolved fluorescence spectroscopy. In order to achieve this, the single tryptophan (Trp 21) found in the rat A1-1 GST has been replaced with the fluorometrically silent phenylalanine (W21F). Additionally, a double mutant lacking this tryptophan and the catalytic tyrosine (W21F:Y9F) has been constructed, and these mutants have been used as probes of ligand effects at Tyr-9. A comparison of the correlated excitation--emission spectra of the W21F mutant and the W21F-Y9F indicates that a red-shifted emission component is contributed by Tyr-9 with excitation bands at 255 and 300 nm, in the ligand-free enzyme. The pH-dependence of the intensity of these spectral cross-peaks is consistent with an active site tyrosine with a pKa of 8.1-8.3. Upon addition of GSH, the red-shifted component is quenched. Multifrequency phase/modulation fluorescence experiments qualitatively demonstrate that GSH causes a decrease in the average excited state lifetime on the red-edge of the spectrum of W21F but not of the W21F:Y9F spectrum. Steady state correlated difference spectra (W21F-W21F:Y9F) have been used to obtain a model for the excitation-emission correlated spectrum of Tyr-9, which indicates that Tyr-9 is heterogeneous at pH 7.5, with properties of both tyrosinate and "normal tyrosine". The tyrosinate fraction is eliminated, and the blue-shifted component becomes more intense upon addition of GSH conjugates, indicating that the weak hydrogen bond between Tyr-9 and thioethers has little charge-transfer character. The S-methyl GSH yields an "anomalous" spectrum at pH 7.5, which retains cross-peaks consistent with ionized tyrosinate. These results indicate that, in the absence of ligand, Tyr-9 forms a strongly polarized hydrogen bond or a fraction of the phenolic hydroxyl group is partially deprotonated. However, when a GSH conjugate with a sufficiently large hydrophobic group occupies the H-site, Tyr-9 is fully protonated, with little charge-transfer character.

Roles of histidine-194, aspartate-163, and a glycine-rich sequence of NAD(P)H:quinone oxidoreductase in the interaction with nicotinamide coenzymes.

NAD(P)H:(quinone-acceptor) oxidoreductase (NQOR, EC 1.6.99.2), an enzyme catalyzing the obligatory two electron reduction of quinones, can utilize both NADH and NADPH as electron donors at similar efficiencies. Based on site-directed mutagenesis studies, we previously suggested that the glycine-rich region of rat liver NQOR is important for the binding of NAD(P)H (Ma et al., J. Biol. Chem. 267, 22298-22304, 1992). However, the mode of interactions between the active site and NADH or NADPH is not clearly known. In this study, we conducted site-directed mutagenesis experiments and identified H194 and D163 of NQOR as key residues affecting the Km of NADPH. Steady-state kinetic analysis for the reduction of dichloroindophenol (DCIP) showed that Km(NADPH) values of purified mutant proteins H194D, H194A, and D163V were 288-, 14-, and 96-fold higher, respectively, than that of NQOR; but the Km(NADH) values were only slightly higher. The kcat(NADPH) values were almost the same as that of NQOR in the reduction of DCIP at the respective pH optima which were affected by the mutations. The kcat(NADH) values of these mutant enzymes were 30 to 60% that of NQOR. In the reduction of menadione, the mutations also caused much larger increases in km(NADPH) than Km(NADH). The results suggest that H194 and D163 are important for the interaction with the 2'-phosphate group of NADPH. NAD(P)H analogues, N-methyldihydronicotinamide and dihydronicotinamide mononucleotide, can also serve as electron donors for NQOR, but the Km values were 4.5- and 495-fold higher, respectively, than that with NADH. Mutations at H194 and D163 and at the glycine-rich region of NQOR, which increased Km(NADH) and Km(NADPH), did not substantially affect the Km values of these two analogues. This result is consistent with the suggested roles of these amino acid residues in the interaction with nicotinamide coenzymes. Based on these results, a model of the NAD(P)H binding site is proposed showing the interaction of the pyrophosphate group with the glycine-rich region and the interaction of 2'-phosphate group with H194 and D163.

Subunit functional studies of NAD(P)H:quinone oxidoreductase with a heterodimer approach.

NAD(P)H:quinone oxidoreductase (NQOR; EC 1.6.99.2) is a homodimeric enzyme which catalyzes the reduction of quinones, azo dyes, and other electron acceptors by NADPH or NADH. To pursue subunit functional studies, we expressed a wild-type/mutant heterodimer of NQOR in Escherichia coli. The wild-type subunit of the heterodimer was tagged with polyhistidine and the other subunit contained a His-194-->Ala mutation (H194A), a change known to dramatically increase the Km for NADPH. This approach enabled us to efficiently purify the heterodimer (H194A/HNQOR) from the homodimers by stepwise elution with imidazole from a nickel nitrilotriacetate column under nondenaturing conditions. The composition of the purified heterodimer was confirmed by SDS and nondenaturing polyacrylamide gel electrophoresis and immunoblot analysis. The enzyme kinetics of the purified heterodimer were studied with two two-electron acceptors, 2,6-dichloroindophenol and menadione, and a four-electron acceptor, methyl red, as the substrates. With two-electron acceptors, the Km(NADPH) and Km(NADH) values of the heterodimer H194A/HNQOR were virtually identical to those of the wild-type homodimer, but the kcat-(NADPH) and kcat(NADH) values were only about 50% those of the wild-type homodimer. With the four-electron acceptor, the Km and kcat values of H194A/HNQOR for NADPH and NADH were similar to those of the low-efficiency mutant homodimer. These results suggest that the subunits of NQOR function independently with two-electron acceptors, but dependently with a four-electron acceptor. This heterodimer approach may have general applications for studying the functional and structural relationships of subunits in dimeric or oligomeric proteins.

Cytochrome P450 inhibitors. Evaluation of specificities in the in vitrometabolism of therapeutic agents by human liver microsomes.

Identifying selective inhibitors of cytochrome P450 isoforms is a useful tool in defining the role of individual cytochrome P450s in the metabolism process. In this study, nine chemical inhibitors were selected based on literature data and were examined for their specificity toward cytochrome P450-mediated reactions in human liver microsomes. Furafylline was a potent, mechanism-based inhibitor for CYP1A2-mediated phenacetin O-deethylation. The probes sulfaphenazole (CYP2C9) and quinidine (CYP2D6) selectively inhibited tolbutamide methylhydroxylation and bufuralol 1'-hydroxylation, respectively. Additionally, the CYP2E1-catalyzed chlorzoxazone 6-hydroxylation was significantly inhibited by diethyldithiocarbamate. Of the CYP3A4 inhibitor probes used, troleandomycin proved to be the most specific for testosterone 6 beta-hydroxylation.

Fluorescence characterization of Trp 21 in rat glutathione S-transferase 1-1: microconformational changes induced by S-hexyl glutathione.

The glutathione S-transferase (GST) isoenzyme A1-1 from rat contains a single tryptophan, Trp 21, which is expected to lie within alpha-helix 1 based on comparison with the X-ray crystal structures of the pi- and mu-class enzymes. Steady-state and multifrequency phase/modulation fluorescence studies have been performed in order to characterize the fluorescence parameters of this tryptophan and to document ligand-induced conformational changes in this region of the protein. Addition of S-hexyl glutathione to GST isoenzyme A1-1 causes an increase in the steady-state fluorescence intensity, whereas addition of the substrate glutathione has no effect. Frequency-domain excited-state lifetime measurements indicate that Trp 21 exhibits three exponential decays in substrate-free GST. In the presence of S-hexyl glutathione, the data are also best described by the sum of three exponential decays, but the recovered lifetime values change. For the substrate-free protein, the short lifetime component contributes 9-16% of the total intensity at four wavelengths spanning the emission. The fractional intensity of this lifetime component is decreased to less than 3% in the presence of S-hexyl glutathione. Steady-state quenching experiments indicate that Trp 21 is insensitive to quenching by iodide, but it is readily quenched by acrylamide. Acrylamide-quenching experiments at several emission wavelengths indicate that the long-wavelength components become quenched more easily in the presence of S-hexyl glutathione. Differential fluorescence polarization measurements also have been performed, and the data describe the sum of two anisotropy decay rates. The recovered rotational correlation times for this model are 26 ns and 0.81 ns, which can be attributed to global motion of the protein dimer, and fast local motion of the tryptophan side chain. These results demonstrate that regions of GST that are not in direct contact with bound substrates are mobile and undergo microconformational rearrangement when the "H-site" is occupied.

Site-directed mutagenesis of glutathione S-transferase YaYa. Mapping the glutathione-binding site.

Previous studies from our laboratory have shown that aspartic acid 101 plays an important role in glutathione interaction to rat glutathione S-transferase YaYa, while tyrosine 9 is directly involved in catalysis. Based on the available structural information, site-directed mutagenesis was conducted to examine the function of arginine, lysine, glutamine, and proline residues surrounding the GSH binding pocket. Arginine mutants R13K, R15K, R20K, and R20I retained partial enzymatic activities, while R13I and R15I lost most of their activities. Kinetic studies showed a marked increase in Km toward GSH for R15I suggesting that arginine 15 contributes significantly to the binding of GSH in the active site of glutathione S-transferase YaYa. A drastic decrease in enzymatic activities for R13I suggested the importance of the charged group of arginine 13 either in maintaining the structural integrity of the enzyme or in serving a vital role in enzymatic function. Replacement of glutamine 54 and 67 with glutamic acid or asparagine resulted in decreased enzymatic activities. Moreover, an 11-, 17-, and 9-fold increase in Km values toward GSH for mutant Q54E, Q54N, and Q67N was observed, respectively. These results suggested that glutamine 54 and 67 also contributed significantly to the binding of GSH. Proline at position 56 appears to be important for maintaining the structural integrity of the enzyme since mutants P56A and P56F were much less active and extremely less stable than that of the wild type enzyme. Both lysine mutants, K45R and K45I, exhibited substantially higher catalytic efficiencies toward both 1-chloro-2,4-dinitrobenzene and GSH than the wild type enzyme. Our data clearly show that lysine 45 is not an essential residue for catalysis nor for GSH binding in glutathione S-transferase YaYa.

The catalytic mechanism of glutathione S-transferase (GST). Spectroscopic determination of the pKa of Tyr-9 in rat alpha 1-1 GST.

The rat alpha 1-1 glutathione S-transferase (GST) contains a single, non-essential tryptophan and only 8 tyrosines in each subunit. One of these tyrosines, Tyr-9, hydrogen bonds to the substrate glutathione and stabilizes the nucleophilic thiolate anion. Two mutant proteins that allow for the spectrocopic determination of the pKa of this catalytic residue have been constructed. The W21F mutant provides a fully active GST with no tryptophans, and the double mutant W21F/Y9F lacks both tryptophan and the active site tyrosine. The intrinsic fluorescence and absorbance properties of these mutants are dominated by tyrosine. Fluorescence emission, fluorescence excitation, and absorbance spectral changes of samples containing the W21F mutant at several pH values in the range 6.8-9.0 reveal a pH-dependent increase in the contribution of tyrosinate. No spectral changes are observed with the W21F/Y9F protein in this pH range. At pH 12.5, both proteins exhibit complete deprotonation of all tyrosines. The pKa of Tyr-9 determined from these spectroscopic changes is 8.3-8.5. The changes in absorbance at 250 and 295 nm correspond to titration of 0.95 +/- 0.29 tyrosines/subunit in the W21F protein between pH 6.9 and 9.3. Moreover, addition of the inhibitor S-hexylglutathione results in an apparent increase in the pKa of Tyr-9. Together, these results indicate that the catalytically active Tyr of GSTs has a pKa value that is 1.8-2.0 pKa units below tyrosine in solution. It is likely that this decrease in the pKa of Tyr-9 contributes to catalysis by altering the equilibrium position of the proton shared between Tyr-9 and GSH, and this active site residue may function as a general base catalyst in addition to a hydrogen bond donor.