Marine Icing Sensor with Phase Discrimination.

In this paper, a novel approach is presented to the measurement of marine icing phenomena under the presence of a two-phase condition. We have developed a sensor consisting of an electrostatic array and a signal processing based on a decision tree method. A three-element electrostatic array is employed to derive signals having linearly decoupled characteristics from which two key parameters, ice and water accretion layer dimension, can be determined for the purpose of environmental monitoring. The quantified characteristics revealed a correlation with the ice layer thickness in spite of the strong influence from the top water phase layer. The decision tree model established a relationship between the signal characteristics and the two accretion thickness parameters of water and ice layer. Through experimental verification, it has been observed that our sensor array in combination with the decision tree model based signal processing provides a simple practical solution to the challenging field of a two phase composition measurement such as in the marine icing considered in this study.

Metallacarborane Sulfamides: Unconventional, Specific, and Highly Selective Inhibitors of Carbonic Anhydrase IX.

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme that regulates pH in hypoxic tumors and promotes tumor cell survival. Its expression is associated with the occurrence of metastases and poor prognosis. Here, we present nine derivatives of the cobalt bis(dicarbollide)(1-) anion substituted at the boron or carbon sites by alkysulfamide group(s) as highly specific and selective inhibitors of CAIX. Interactions of these compounds with the active site of CAIX were explored on the atomic level using protein crystallography. Two selected derivatives display subnanomolar or picomolar inhibition constants and high selectivity for the tumor-specific CAIX over cytosolic isoform CAII. Both derivatives had a time-dependent effect on the growth of multicellular spheroids of HT-29 and HCT116 colorectal cancer cells, facilitated penetration and/or accumulation of doxorubicin into spheroids, and displayed low toxicity and showed promising pharmacokinetics and a significant inhibitory effect on tumor growth in syngenic breast 4T1 and colorectal HT-29 cancer xenotransplants.

Spectral counterstaining in luminescence-enhanced biological Raman microscopy.

Cell imaging heavily depends on fluorescent labels typically incompatible with Raman microscopy. The europium(iii) complex based on dipicolinic acid (DPA) presented here is an exception from this rule. Although its luminescence bands are very narrow, their intensity is comparable to the background Raman bands. This makes it complementary to less luminous compounds referred to as Raman tags. Through several examples we show that the complex provides a morphological context in otherwise unstained cells, thus acting as a spectral-counterstaining agent.

Detection of Circularly Polarized Luminescence of a Cs-Eu(III) Complex in Raman Optical Activity Experiments.

Circularly polarized luminescence (CPL) spectra are extremely sensitive to molecular structure. However, conventional CPL measurements are difficult and require expensive instrumentation. As an alternative, we explore CPL using Raman scattering and Raman optical activity (ROA) spectroscopy. The cesium tetrakis(3-heptafluoro-butylryl-(+)-camphorato) europium(III) complex was chosen as a model as it is known to exhibit very large CPL dissymmetry ratio. The fluorescent bands could be discriminated from true Raman signals by comparison of spectra acquired with different laser excitation wavelengths. Furthermore, the ROA technique enables fluorescence identification by measuring the degree of circularity. The CPL dissymmetry ratio was measured as the ROA circular intensity difference of 0.71, the largest one ever reported. The alternative CPL measurement enhances applications of lanthanides in analytical chemistry and chemical imaging of biological objects.

Interaction of selected platinum(II) complexes containing roscovitine-based CDK inhibitors as ligands with human liver microsomal cytochrome P450.

BACKGROUND: We studied the interaction of oxaliplatin derivatives involving cytotoxic adenine-based cyclin-dependent kinase inhibitors, with human liver microsomal cytochrome P450. METHODS AND RESULTS: The activities of 9 human liver microsomal CYP forms (CYPs 1A2, 7-ethoxyresorufin O-deethylation; 2A6, coumarin 7-hydroxylation; 2B6, 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation; 2C8, luciferin-6´ methyl ether demethylation; 2C9, diclofenac 4´-hydroxylation, 6´-deoxyluciferin hydroxylation; 2C19, (S)-mephenytoin 4´-hydroxylation; 2D6, bufuralol 1´-hydroxylation, 2E1, chlorzoxazone 6-hydroxylation; 3A4, testosterone 6ß-hydroxylation, luciferin-6´ benzyl ether debenzylation) were tested using HPLC, fluorescence and luminescence product detection. At 100 µM platinum(II) oxalato complex concentration, CYP inhibition was in general 25%-50%, except for the CYP3A4 form which showed roughly twice the inhibition (72%-95%). At low complex concentration (10 µM), the difference in inhibition of CYP3A4 and other forms was even more pronounced. Dixon and Lineweaver-Burk plots indicated a partially noncompetitive mechanism of CYP3A4 inhibition. CONCLUSIONS: The tested complexes significantly inhibit human liver microsomal CYP3A4 activity even at clinically relevant concentrations. This could be a serious drawback for the use of these compounds in clinical practice.

Distribution of scytonemin in endolithic microbial communities from halite crusts in the hyperarid zone of the Atacama Desert, Chile.

Scytonemin, a UV-screening molecule produced by certain Cyanobacteria to protect against harmful UV radiation, was studied in endolithic cyanobacterial colonies in the halite crust from one of the driest places on Earth - the hyperarid zone of the Atacama Desert. The distribution of the pigment within the evaporitic crust was studied in detail by various independent analytical methods: Raman spectroscopy (including Raman imaging); advanced microscopic observations (fluorescence microscopy, confocal laser scanning microscopy, low-temperature scanning electron microscopy); and spectrophotometric analyses. The differences in scytonemin biosynthesis were mapped within the colonized interior layers, which can be divided into scytonemin-rich and scytonemin-poor zones. A 532 nm laser for excitation proved to be an ideal excitation source with which to observe the relative content of scytonemin within a particular cell aggregate, as well as between different cell aggregates; based on the scytonemin/carotenoid Raman signal intensity ratio of selected corroborative bands for these two compounds. Significantly, scytonemin was found to accumulate within a decayed biomass in the surface portions of the halite crust. These were found to be highly enriched in both the absolute scytonemin content (as documented by UV/VIS spectrophotometry) and its content relative to other pigments associated with the cyanobacterial cells (e.g. carotenoids and chlorophyll).

Binding of quinidine radically increases the stability and decreases the flexibility of the cytochrome P450 2D6 active site.

Human cytochrome P450 2D6 (CYP2D6) is an enzyme of the CYP superfamily responsible for biotransformation of about 20% of drugs of known metabolism containing a basic nitrogen and a planar aromatic ring. Here, we present a combined experimental and computational study on the compressibility and flexibility of unliganded and quinidine-bound CYP2D6. Experimentally, high-pressure induced Soret band shifts of the enzyme were measured by UV/VIS spectroscopy, while 100 ns all atomic molecular dynamics (MD) simulations in explicit water were used in the computational analysis. We identified sharp differences between ligand-free and quinidine-bound CYP2D6 forms in compressibility, flexibility parameters and active site solvation. While the unliganded CYP2D6 is compressible, quinidine binding significantly rigidifies the CYP2D6 active site. In addition, MD simulations show that quinidine binding results in pronounced reductions in active site flexibility and solvation.

Interaction of N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin conjugates with human liver microsomal cytochromes P450: comparison with free doxorubicin.

Interaction of nine forms of human hepatic cytochromes P450 (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) with two N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-based doxorubicin (DOX) conjugates designed for passive tumor targeting was studied using pooled human microsomes. The compounds used in this study were two high-molecular-weight HPMA copolymers bearing doxorubicin attached to the polymeric carrier by 1) hydrazone bond enabling intracellular pH-controlled drug release or 2) amide bond through enzymatically cleavable tetrapeptide GlyPheLeuGly spacer. Both polymeric conjugates differing in mechanism of their antitumor activity and the free doxorubicin as the control were tested for potential inhibition activity. Among nine cytochrome P450 forms studied, no HPMA copolymer with bound DOX caused an inhibition of potential clinical significance. The extent of inhibition of enzymatic activities of the cytochrome P450 forms studied was negligible with the exception of CYP2B6 and was apparently caused by DOX as no inhibition was observed with polymers alone, and the extent of inhibition by the complex corresponded to this of the free DOX at the same concentration. In conclusion, the polymers and their conjugates with DOX seem to be relatively safe, at least in this respect, i.e., of inhibition of the liver microsomal drug-metabolizing enzymes.

Interaction of antitumor platinum complexes with human liver microsomal cytochromes P450.

Interaction of nine human hepatic cytochromes P450 (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) with six platinum complexes was studied using pooled human microsomes. The compounds used were cisplatin, oxaliplatin, carboplatin, transplatin, and trans-[PtCl2(NH3) (Am)], where Am=2-methylbutylamine or sec-butylamine. No significant inhibition of all CYP activities by carboplatin was observed. With cisplatin and oxaliplatin, a minor inhibition of CYP2C9 enzyme (75% of control at 400 miromol/l of these complexes) was seen; cisplatin also inhibited slightly the CYP2B6 activity (85% of control). With respect to plasma levels of cisplatin obtained in clinical applications, these effects are probably not important. In contrast, clinically ineffective transplatin, inhibited the CYP2B6 as well as CYP2C9 activities significantly (to 50-35% of control at 100 micromol/l); also, an inhibition of CYP2E1 activity was found here (to 70% at 100 micromol/l). Two other derivatives of transplatin (new antitumor agents with trans geometry), inhibited CYP activities more strongly reaching nearly a complete inhibition of the respective CYP activities at concentration of 200 micromol/l. Half maximal inhibitory concentration values were found in the range of tens of micromol/l indicating that there is a possibility of potential interactions of these compounds with drugs metabolized by CYP3A4, CYP2E1, CYP2D6, CYP2C19, CYP2B6, CYP2A6, and CYP1A2. Interestingly, clinically non-significant inhibition was found with the CYP2C9 and CYP2C8 indicating low probability of interactions with, for example, warfarin. The results document that the new antitumor agents based on the transplatin should be more thoroughly tested for interactions with liver microsomal drug-metabolizing cytochromes P450.

Interspecies comparison of the glucuronidation processes in the man, monkey, pig, dog and rat.

OBJECTIVES: The study of interspecies differences in glucuronidation processes in the man, monkey, pig, dog and rat using liver microsomal fraction. The study is focused on determination of the enzyme activity of UGT1A6 (having also a toxicological importance) in microsomes of different species. METHODS: For determination of glucuronides formed, an HPLC method with UV detection and LC-MS characterization was used. p-Nitrophenol and 4-methylumbelliferon and silybin were chosen as model substrates. RESULTS: The data presented in this paper show an overall similarity in kinetic parameters of the UGT1A6 with p-nitrophenol and 4-methylumbelliferon for man, pig and monkey. The pattern of silybin glucuronides formed in monkey and dog samples are relatively close to this of the man. CONCLUSIONS: For studies of glucuronidation of xenobiotics where the role UGT1A6 is expected, the use of pig and monkey microsomes should be considered. As an optimal model for study of silybin glucuronidation, both the rhesus monkey and dog (Beagle) seem to be the best models. To elucidate the role of the UGT forms involved in metabolism of silybin, the experiments with recombinant UGT enzymes are needed.

Mechanism-based inhibitors of cytokinin oxidase/dehydrogenase attack FAD cofactor.

Cytokinin oxidases/dehydrogenases (CKOs) mediate catabolic regulation of cytokinin levels in plants. Several substrate analogs containing an unsaturated side chain were studied for their possible inhibitory effect on maize CKO (ZmCKO1) by use of various bioanalytical methods. Two allenic derivatives, N(6)-(buta-2,3-dienyl)adenine (HA-8) and N(6)-(penta-2,3-dienyl)adenine (HA-1), were identified as strong mechanism-based inhibitors of the enzyme. Despite exhaustive dialysis, the enzyme remained inhibited. Conversely, substrate analogs with a triple bond in the side chain were much weaker inactivators. The crystal structures of recombinant ZmCKO1 complexed with HA-1 or HA-8 were solved to 1.95 A resolution. Together with Raman spectra of the inactivated enzyme, it was revealed that reactive imine intermediates generated by oxidation of the allenic inhibitors covalently bind to the flavin adenine dinucleotide (FAD) cofactor. The binding occurs at the C4a atom of the isoalloxazine ring of FAD, the planarity of which is consequently disrupted. All the compounds under study were also analyzed for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4 in a bacterial receptor assay and for cytokinin activity in the Amaranthus bioassay. HA-1 and HA-8 were found to be good receptor ligands with a significant cytokinin activity. Nevertheless, due to their ability to inactivate CKO in the desired time intervals or developmental stages, they both represent attractive compounds for physiological studies, as the inhibition mechanism of HA-1 and HA-8 is mainly FAD dependent.

Raman spectroscopy of DNA modified by intrastrand cross-links of antitumor cisplatin.

Raman spectroscopy was employed to characterize the perturbations to DNA conformation induced in DNA by two different intrastrand adducts of antitumor cis-diamminedichloroplatinum(II) (cisplatin), namely by its 1,2-GG or 1,3-GTG intrastrand cross-links. We examined short deoxyribooligonucleotide duplexes containing single, site-specific cross-link by Raman spectroscopy and assigned the spectral alterations to conformational changes induced in DNA by 1,2-GG or 1,3-GTG intrastrand CLs determined earlier by other biochemical and biophysical methods. The results confirmed significant perturbations to the B-form DNA backbone due to the intrastrand lesions and that several nucleotides changed their conformation from C2'-endo to C3'-endo. Evidence for a partial transition from B- to A-form was found in several regions of the Raman spectra as well. The spectra also confirmed the different and more extensive distortion induced in B-DNA by 1,3-GTG in comparison with 1,2-GG intrastrand CLs, consistent with their already known high resolution structures. The results of the present work demonstrate that Raman spectroscopy represents a suitable tool to provide insights into structural factors involved in the mechanisms underlying antitumor effects of platinum drugs.

Comparison of "high throughput" micromethods for determination of cytochrome P450 activities with classical methods using HPLC for product identification.

Enzyme activities of the CYP enzymes (CYP3A4, CYP2C9 and CYP2A6) were determined using classical substrates (testosterone, diclofenac and coumarin, respectively) as well as with luminogenic or fluorogenic substrates in micromethod arrangement. The luciferin-based luminogenic substrates for CYP3A4 and CYP2C9 as well as coumarin in micromethod for assay of CYP2A6 activity gave results well comparable with the classical methods with determination of reaction products by the HPLC.