Solution 1H NMR of the active site of substrate-bound, cyanide-inhibited human heme oxygenase. comparison to the crystal structure of the water-ligated form

The majority of the active site residues of cyanide-inhibited, substrate-bound human heme oxygenase have been assigned on the basis of two-dimensional NMR using the crystal structure of the water-ligated substrate complex as a guide (Schuller, D. J., Wilks, A., Ortiz de Montellano, P. R., and Poulos, T. L. (1999) Nat. Struct. Biol. 6, 860-867). The proximal helix and the N-terminal portion of the distal helix are found to be identical to those in the crystal except that the heme for the major isomer ( approximately 75-80%) in solution is rotated 180 degrees about the alpha-gamma-meso axis relative to the unique orientation in the crystal. The central portion of the distal helix in solution is translated slightly over the heme toward the distal ligand, and a distal four-ring aromatic cluster has moved 1-2 A closer to the heme, which allows for strong hydrogen bonds between the hydroxyls of Tyr-58 and Tyr-137. These latter interactions are proposed to stabilize the closed pocket conducive to the high stereospecificity of the alpha-meso ring opening. The determination of the magnetic axes, for which the major axis is controlled by the Fe-CN orientation, reveals a approximately 20 degrees tilt of the distal ligand from the heme normal in the direction of the alpha-meso bridge, demonstrating that the close placement of the distal helix over the heme exerts control of stereospecificity by both blocking access to the beta, gamma, and delta-meso positions and tilting the axial ligand, a proposed peroxide, toward the alpha-meso position.     

Topically applied nitropyrenes are potent inducers of cutaneous and hepatic monooxygenases

The inducibility of skin and liver microsomal cytochrome P-450 dependent aryl hydrocarbon hydroxylase and other monooxygenases by a mixture of nitropyrenes was assessed and compared with the parent non-nitrated compound, pyrene. A single topical application of nitropyrenes to neonatal rats resulted in highly significant induction of aryl hydrocarbon hydroxylase, ethoxycoumarin O-de-ethylase, and ethoxyresorufin O-de-ethylase activities in skin and liver after 24 hours. Inducibility of the skin and liver enzymes was 3.9-5.7 fold and 1.8-10.3 fold respectively. On the other hand, aminopyrine N-demethylase, benzphetamine N-demethylase and epoxide hydrolase activities in the liver were unaffected by topically applied nitropyrenes. Furthermore, treatment with nitropyrenes produced a 1 nm shift to the blue region in the wavelength maximum of hepatic microsomal cytochrome P-450. Topically applied pyrene produced only marginal or no effects on cutaneous and hepatic enzyme activities. Our results suggest that nitration of pyrene, a relatively ineffective enzyme inducer, produces nitropyrenes which are potent inducers of hepatic and cutaneous monooxygenases and they resemble 3-methylcholanthrene in this inducing effect.     

Folic acid incorporated nitrogen-doped carbon dots as a turn-on fluorescence probe for homocysteine detection

This study describes the development of a low-cost fluorescence assay for detecting homocysteine (Hcy) without the interference of cysteine and glutathione using carbon quantum dots. Herein nitrogen-doped carbon quantum dots (NCDs) were synthesized from citric acid as the carbon source and urea as the dopant using a one-pot microwave-assisted method. The obtained NCDs were incorporated with folic acid (FA) by the direct ex situ addition method and were used as a fluorescence probe to detect Hcy. The probe exhibited a fluorescence turn-on response with increased Hcy concentration up to 50 μM with a limit of detection of 2.276 μM. The point of care detection of Hcy using the probe was also tested with a paper-based assay strip.     

Purification and characterization of β-glucuronidase from bull seminal plasma and its role in fertilization

Bull seminal plasma contains high levels of β-glucuronidase. The present study describes the isolation and characterization of β-glucuronidase, and its role in fertilization. β-glucuronidase was purified by ion exchange chromatography, saccharolactone-agarose affinity chromatography, and gel filtration. The specific activity of the purified enzyme was 4,414 μmoles/mg protein/min. The purified enzyme showed a single band on 7.5% PAGE. On SDS-PAGE, the enzyme appeared to consist of four identical subunits of M_r 75,000 each. The apparent K_m and V_max for β-glucuronidase were 0.4 mM and 5.7 μmol/min using phenolpthalein mono-β-glucuronic acid as the substrate. β-glucuronidase appeared to accelerate the cumulus dispersion in vitro. © 1994 Wiley-Liss, Inc.     

Optical detection of glucose and glycated hemoglobin using etched fiber Bragg gratings coated with functionalized reduced graphene oxide

An enhanced optical detection of D‐glucose and glycated hemoglobin (HbA_1c) has been established in this study using etched fiber Bragg gratings (eFBG) coated with aminophenylboronic acid (APBA)‐functionalized reduced graphene oxide (RGO). The read out, namely the shift in Bragg wavelength (Δλ_B) is highly sensitive to changes that occur due to the adsorption of glucose (or HbA_1c) molecules on the eFBG sensor coated with APBA–RGO complex through a five‐membered cyclic ester bond formation between glucose and APBA molecules. A limit of detection of 1 nM is achieved with a linear range of detection from 1 nM to 10 mM in the case of D‐glucose detection experiments. For HbA_1c, a linear range of detection varying from 86 nM to 0.23 mM is achieved. The observation of only 4 pm (picometer) change in Δλ_B even for the 10 mM lactose solution confirms the specificity of the APBA–RGO complex coated eFBG sensors to glucose molecules.

     

Molecular differences in the mitochondrial cytochrome oxidase I (mtCOI) gene and development of a species-specific marker for onion thrips, Thrips tabaci Lindeman, and melon thrips, T. palmi Karny (Thysanoptera: Thripidae), vectors of tospoviruses (Bunyaviridae)

A quick and developmental-stage non-limiting method of the identification of vectors of tospoviruses, such as Thrips tabaci and T. palmi, is important in the study of vector transmission, insecticide resistance, biological control, etc. Morphological identification of these thrips vectors is often a stumbling block in the absence of a specialist and limited by polymorphism, sex, stage of development, etc. Molecular identification, on the other hand, is not hampered by the above factors and can easily be followed by a non-specialist with a little training. The mitochondrial cytochrome oxidase I (mtCOI) exhibits reliable inter-species variations as compared to the other markers. In this communication, we present the differences in the mtCOI partial sequence of morphologically identified specimens of T. tabaci and T. palmi collected from onion and watermelon, respectively. Species-specific markers, identified in this study, could successfully determine T. tabaci and T. palmi, which corroborated the morphological identification. Phylogenetic analyses showed that both T. tabaci and T. palmi formed different clades as compared to the other NCBI accessions. The implication of these variations in vector efficiency has to be investigated further. The result of this investigation is useful in the quick identification of T. tabaci and T. palmi, a critical factor in understanding the epidemiology of the tospoviruses, their management and also in quarantine.     

Carcinogen metabolism in human skin grafted onto athymic nude mice: a model system for the study of human skin carcinogenesis

Human skin grafted onto athymic nude mice maintains its major histological features and may provide a useful system with which to assess the carcinogen interaction with human skin. Significant differences were observed in basal levels of cytochrome P-450 and cytochrome P-448-dependent monooxygenase activities between human grafted and nude mouse epidermis. Topical application of crude coal tar (CCT) to human skin transplanted onto nude mice resulted in 3.9 & 3.5; 3.2 & 2.9 and 1.1 & 1.2 fold increases in mouse and human epidermal aryl hydrocarbon hydroxylase (AHH), ethoxyresorufin deethylase (ERD) and ethoxycoumarin deethylase (ECD) activities, respectively. CCT applied topically to mouse skin resulted in 27.8 & 6.4; 12.8 & 3.3 and 1.7 & 2.6 fold increases in mouse and human epidermal AHH, ERD and ECD activities, respectively. Topical application of coal tar either onto human transplanted skin or to mouse skin also resulted in substantial induction of hepatic and pulmonary AHH and ERD activities. These studies indicate that human skin grafted onto nude mice preserves its metabolic capacity and offers a useful model system with which to assess the effects of polycyclic aromatic hydrocarbons and CCT on cutaneous xenobiotic metabolism in the human population.