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PubMedDate not supplied

The covalent attachment of multiple fluorophores to DNA containing phosphorothioate diesters results in highly sensitive detection of single-stranded DNA.

DNA fragments containing multiple internucleotidic phosphorothioate diesters, prepared by either chemical or enzymatic syntheses, are amenable to labeling with the fluorophore monobromobimane. With the incorporation of phosphorothioate diesters at each internucleotidic site, multiple fluorophores, ideally one for each nucleotide residue, can be covalently attached to the DNA fragment. The presence of multiple labels can be expected to interfere with analysis techniques, such as polyacrylamide gel electrophoresis. To avoid such problems, the fluorophores are introduced in a "postassay" fashion, that is, while the fragments are still embedded within the gel matrix. The detection limit (to the naked eye) for multiply labeled single-stranded DNA containing hundreds of base residues is in the low femtomole range. DNA containing greater than 1000 base residues can be visualized in some cases in the subfemtomole range without the use of sophisticated electronic instrumentation.

Bridged Bicyclo Compounds↗
PubMed2000

Sensitivity enhancement in fluorescence correlation spectroscopy of multiple species using time-gated detection.

Fluorescence correlation spectroscopy (FCS) is a powerful technique to measure chemical reaction rates and diffusion coefficients of molecules in thermal equilibrium. The capabilities of FCS can be enhanced by measuring the energy, polarization, or delay time between absorption and emission of the collected fluorescence photons in addition to their arrival times. This information can be used to change the relative intensities of multiple fluorescent species in FCS measurements and, thus, the amplitude of the intensity autocorrelation function. Here we demonstrate this strategy using lifetime gating in FCS experiments. Using pulsed laser excitation and laser-synchronized gating in the detection channel, we suppress photons emitted within a certain time interval after excitation. Three applications of the gating technique are presented: suppression of background fluorescence, simplification of FCS reaction studies, and investigation of lifetime heterogeneity of fluorescently labeled biomolecules. The usefulness of this technique for measuring forward and backward rates of protein fluctuations in equilibrium and for distinguishing between static and dynamic heterogeneity makes it a promising tool in the investigation of chemical reactions and conformational fluctuations in biomolecules.

Diffusion↗
PubMed1995

The development and validation of a high performance liquid chromatography (HPLC)/tandem mass spectrometry assay for fenticonazole in human plasma and comparison with an HPLC-UV method.

A method is described for the determination of fenticonazole in human female plasma. The method utilizes high performance liquid chromatography coupled to atmospheric pressure positive-ion chemical ionization triple quadrupole mass spectrometry. Multiple reaction monitoring is employed for selectivity and sensitivity which enables quantification over the range 0.5-20 ng mL-1 with acceptable precision and accuracy. A comparison is made with an existing HPLC-UV assay and the utility of the technology of combined liquid chromatography and tandem mass spectometry for subnanogram per mL assays is discussed.

Antifungal Agents↗
PubMed2001

Polymorphisms in candidate asthma genes.

The triad of reversible airway obstruction, bronchial hyperresponsiveness, and airway inflammation characterizes asthma. The etiology of asthma is complex and involves the interaction of multiple genetic foci and a variety of environmental factors, such as protein allergens, chemical sensitizers, and viral or bacterial proteins. Candidate asthma genes have been identified that may be linked or associated with the asthmatic phenotype. Potential candidate asthma genes include cytokine genes, receptor genes, transcription factors, immune recognition genes, and genes regulating lipid mediator generation. Although polymorphisms within either the promoter or coding region of individual asthma candidate genes have been identified, the association between these genetic polymorphisms and the asthmatic phenotype remains incompletely defined. Furthermore, genetic polymorphisms mediating the asthmatic phenotype are rarely identified in individual patients. This manuscript reviews several of the specific mutations and polymorphisms that have been identified in candidate asthma genes, such as the high affinity IgE receptor, the beta2-adrenergic receptor, the interleukin-4 promoter and receptor, the tumor necrosis factor gene, and the 5-lipoxygenase promoter.

Arachidonate 5-Lipoxygenase↗
PubMedDate not supplied

The presence of some yeasts species in occupational dermatitis.

The samples of skin lesions were collected from forearms, palms and phalanges of ten females working in a manufacturing section (clothes tailoring and assemblage) using imported textiles. The samples were examined for bacteriological and mycotic flora by using classical methods. The textile fragments of 1-2 square centimeters from four samples were investigated using the same classical methods. The gauze fragments were used as toxic negative control samples. In 6 out of 10 samples from cutaneous lesions, Candida albicans was isolated and in 5 samples Rhodotorula species was identified. The yeasts species were identified by cultural and biochemical characteristics. All C. albicans strains were positive for the germ tube test and produced spherical chlamydospores on corn meal agar. The Rhodotorula strains grew on Sabouraud agar pure orange-red pigmented and mucoid colonies of 2-3 mm diameter. The textile samples incubated on blood--agar (10% sheep blood) demonstrated a high toxic activity producing a surrounding beta-hemolysis area of 3-5 cm. This lysis of the sheep red cell was caused by an unidentified chemical agent. In our cases, dermatites were associated with chemical and yeasts agents. The chemical agents irritated and made the derma sensitive, favouring C. albicans and Rhodotorula species multiplication.

Candida albicans↗
PubMed1981

Issues relevant to the assessment of chemically induced chromosome damage in vivo and their relationship to chemical mutagenesis.

Rats have been exposed by different routes of administration (inhalation, orally and intraperitoneally) to known mutagens and their bone-marrow cells sampled at different times to determine the extent of chromosome damage. The mutagens investigated were ethyl methanesulphonate, mitomycin C, trimethylphosphate, benzene and vinyl chloride, at single and/or multiple doses (5 consecutive daily). Various categories of chromosome damage were observed in all cases. However, the extent of damage due to chromosome and chromatid gaps was greater than, and generally increased in parallel with, other categories of damage. It has been suggested tht chromosome and chromatid gaps are indicative of toxic phenomena but this study suggests that such aberrations could be useful and sensitive indicators of chemically induced genetic damage. In addition the study has also confirmed that single exposure is as effective as multiple exposure in producing chromosome damage and that the correct sampling time is necessary to detect this damage. Therefore for screening purposes a time course sampling after a single treatment regime would be suitable for detecting the mutagenic potential of a chemical.

Animals↗
PubMedDate not supplied

Evaluation of an ex vivo murine local lymph node assay: multiple endpoint comparison.

The local lymph node assay (LLNA) is used to assess the skin sensitization potential of chemicals. In the standard assay, mice are treated topically on the dorsum of both ears with test substance for 3 days. Following 2 days of rest, the initiation of the hypersensitivity response is evaluated by injecting (3)H-thymidine into a tail vein, and then measuring the levels of radioisotope incorporated into the DNA of lymph node cells draining the ears. In the current study, BALB/c mice were treated with the contact sensitizers hexylcinnamic aldehyde (HCA) and oxazolone, and the nonsensitizer methyl salicylate. The proliferative response of lymph node cells was evaluated in an ex vivo assay, in which isolated cells were cultured in vitro with (3)H-thymidine. Treatment of mice with HCA at 5-50% resulted in concentration-related increases in (3)H-thymidine incorporation, with stimulation indices ranging from 3 to 14. Low animal-to-animal variability was seen in three replicate assays testing HCA at 25%. As anticipated, the proliferative response induced by the potent sensitizer oxazolone at 0.25% was greater than HCA at all concentrations tested. Stimulation indices of 1.5 and 3 were seen in two independent experiments with methyl salicylate. These equivocal findings were likely due to the irritancy properties of the compound. Importantly, measuring ex vivo (3)H-thymidine incorporation was more sensitive than evaluating lymph node weight and cellularity, and in vitro bromodeoxyuridine incorporation. Furthermore, the results of the ex vivo LLNA were comparable to the standard assay. This study provided evidence that supports the use of an ex vivo LLNA for hazard assessment of contact hypersensitivity.

Acrolein↗
PubMed2002

Necessity to establish new risk assessment and risk communication for human fetal exposure to multiple endocrine disruptors in Japan.

Our recent study clearly shows that fetuses are exposed to multiple chemicals including endocrine disruptors in Japan. Although the embryo and fetus stages are the most sensitive period to chemicals in humans' life cycle, the health effects of the chemicals such as endocrine disruptors to them are largely unknown. The conventional risk assessment method cannot assess the risk to fetuses precisely. Now we need a new risk assessment, in which the target is fetuses and not the adults, in addition to the conventional risk assessment. At the same time, we also need a new strategy to practically eliminate the risk for the future generations. To make the strategy effective, we suggest a new approach to reduce the risk and avoid the possible adverse health effects, using primary, secondary and tertiary preventions as they are used in public health. We also suggest a new concept of "pre-primary prevention" to reduce the risk for fetuses. Furthermore, to make this method even more practical, we suggest a new risk communication method. In this paper, we present a framework of risk avoidance of multiple chemical exposure to fetuses.

Congenital Abnormalities↗
PubMed2000

Transient-state kinetic analysis of Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase reveals that a step after chemical transformation is rate limiting.

MyristoylCoA:protein N-myristoyltransferase is a member of the superfamily of GCN5-related N-acetyltransferases and catalyzes the covalent attachment of myristate to the N-terminal Gly residue of proteins with diverse functions. Saccharomyces cerevisiae Nmt1p is a monomeric protein with an ordered bi-bi reaction mechanism: myristoylCoA is bound prior to peptide substrate; after catalysis, CoA is released followed by myristoylpeptide. Analysis of the X-ray structure of Nmt1p with bound substrate analogues indicates that the active site contains an oxyanion hole and a catalytic base and that catalysis proceeds through the nucleophilic addition-elimination mechanism. To determine the rate-limiting step in the enzyme reaction, pre-steady-state kinetic analyses were performed using a new, sensitive nonradioactive assay that detects CoA. Multiple turnover quenched flow studies disclosed that a step after the chemical transformation limits the overall rate of the reaction. Multiple and single turnover analyses revealed that the rate for the chemical transformation step is 13.8+/-0.6 s(-1) while the slower steady-state phase is 0.10+/-0.01 s(-1). Stopped flow kinetic studies of substrate acquisition indicated that binding of myristoylCoA to the apo-enzyme occurs through at least a two-step process, with a fast phase rate of 3.2 x 10(8) M(-1) s(-1) and a slow phase rate of 23+/-2 s(-1) (defined at 5 degrees C). Binding of an octapeptide substrate, representing the N-terminal sequence of a known yeast N-myristoylprotein (Cnb1p), to a binary complex composed of Nmt1p and a nonhydrolyzable myristoylCoA analogue (S-(2-oxo)pentadecylCoA) has a second-order rate constant of 2.1+/-0.3 x 10(6) M(-1) s(-1) and a dissociation rate of 26+/-15 s(-1) (defined at 10 degrees C). These results are interpreted in light of the X-ray structures of this enzyme.

Acyl Coenzyme A↗
PubMed2000

The role of gastrointestinal vagal afferents in the control of food intake: current prospects.

Meals are the functional units of food intake in humans and mammals, and physiologic approaches to understanding the controls of meal size have demonstrated that the presence of food in the upper gastrointestinal tract plays a critical role in determining meal size. The vagus nerve is the primary neuroanatomic substrate in the gut-brain axis, transmitting meal-related signals elicited by nutrient contact with the gastrointestinal tract to sites in the central nervous system that mediate ingestive behavior. This article describes progress in examining the role of the vagal gut-brain axis in the negative-feedback control of meal size from four perspectives: neuroanatomic, neurophysiologic, molecular, and behavioral. Vagal afferents are strategically localized to be sensitive to meal-related stimuli, and their central projections are organized viscerotopically in the caudal brainstem. Vagal afferents are sensitive to mechanical, chemical, and gut and peptide meal-related stimuli and can integrate multiple such modalities. Meal-elicited gastrointestinal stimuli activate distinct patterns of c-fos neural activation within caudal brainstem sites, where gut vagal afferents terminate. Results of selective chemical and surgical vagal deafferentation studies have refined our understanding of the sites and types of critical gastrointestinal feedback signals in the control of meal size. Recent behavioral, molecular, and neurophysiologic data have demonstrated brainstem sites where centrally acting neuropeptides may modulate the processing of gut vagal afferent meal-related signals to alter feeding. Investigations of the structure and function of splanchnic visceral afferents and enterics and characterization of the integrative capacities of the hindbrain and forebrain components of the gut-brain axis are critical next steps in this analysis.

Afferent Pathways↗
PubMed2000

Discriminating duplex and hairpin oligonucleotides using chemical shifts: application to the anticodon stem-loop of Escherichia coli tRNA(Phe).

A sensitive NMR spectroscopic method for detection of duplex forms of self-complementary nucleic acid sequences has been implemented. The G.U wobble base pair formed between a (15)N-labeled strand and an unlabeled probe strand is used to identify the duplex. The guanine imino resonance, with its characteristic chemical shift, is detected using a 2D (15)N-(1)H heteronuclear multiple quantum coherence (HMQC) spectrum and provides a sensitive and unambiguous route to hairpin-duplex discrimination. The method has been used to identify the duplex and hairpin forms of an RNA oligonucleotide at concentrations of approximately 20 microM. This method has also been used to rule out possible duplex formation of an RNA oligonucleotide corresponding to the unmodified anticodon stem-loop of Escherichia coli tRNA(Phe) and suggests that this hairpin has a 3 nt loop.

Anticodon↗
PubMed1982

Mutagenic responses of five independent genetic loci in CHO cells to a variety of mutagens. Development and characteristics of a mutagen screening system based on selection for multiple drug-resistant markers.

With the aim of developing a sensitive mutagen screening system, the responses of 15 different chemical mutagens at 5 independent genetic loci in Chinese hamster ovary (CHO) cells have been determined. The genetic markers which have been employed include resistance to thioguanine (Thgr), ouabain (OuaR), the protein synthesis inhibitor emetine (Emtr), the polyamine synthesis inhibitor methylglyoxal bisguanylhydrazone (Mbgr) and the nucleoside analog 5,6-dichlororibofuranosyl benzimidazole (DrbR). The optimal selection conditions for all of these genetic markers in CHO cells have been described. The chemicals whose response was investigated in these studies include direct-acting alkylating agents (ethyl methane-sulfonate, methyl methanesulfonate, beta-propiolactone, ethyleneimine, N-nitrosomethylurea and 4-nitroquinoline-N-oxide), DNA intercalating and cross-linking agents (ICR-170, acridine orange, ethidium bromide, mitomycin C and actinomycin D), polycyclic hydrocarbons (benzo[a]pyrene (B(a)P) and 7,12-dimethylbenz[a]anthracene (DMBA)) and aromatic amines (benzidine and beta-naphthylamine). Simultaneous examination of the response of the set of genetic markers to these chemicals revealed that although all of these chemicals caused a dose-dependent increase in the frequency of mutations at many of the above genetic loci, the magnitude of the mutagenic response at different genetic loci varied greatly depending upon the chemical. Of the genetic loci examined, no one single locus showed higher response to all of the above chemicals, instead, depending upon the chemical, specific loci were found to be more responsive than others. The polycyclic hydrocarbons and aromatic amines were weakly mutagenic in this system at several genetic loci even without any exogenous microsomal activation, although in the presence of a rat liver S9 fraction similar toxic and mutagenic effects of B(a)P and DMBA were observed at 5-20-fold lower concentrations. These results indicate that CHO cells may possess significant capacity for the metabolic activation of many procarcinogens, and also underscore the merits of measuring the mutagenic response at multiple genetic loci in mutagen screening studies.

Animals↗
PubMedDate not supplied

Skin sensitization: modeling based on skin metabolism simulation and formation of protein conjugates.

A quantitative structure-activity relationship (QSAR) system for estimating skin sensitization potency has been developed that incorporates skin metabolism and considers the potential of parent chemicals and/or their activated metabolites to react with skin proteins. A training set of diverse chemicals was compiled and their skin sensitization potency assigned to one of three classes. These three classes were, significant, weak, or nonsensitizing. Because skin sensitization potential depends upon the ability of chemicals to react with skin proteins either directly or after appropriate metabolism, a metabolic simulator was constructed to mimic the enzyme activation of chemicals in the skin. This simulator contains 203 hierarchically ordered spontaneous and enzyme controlled reactions. Phase I and phase II metabolism were simulated by using 102 and 9 principal transformations, respectively. The covalent interactions of chemicals and their metabolites with skin proteins were described by 83 reactions that fall within 39 alerting groups. The SAR/QSAR system developed was able to correctly classify about 80% of the chemicals with significant sensitizing effect and 72% of nonsensitizing chemicals. For some alerting groups, three-dimensional (3D)-QSARs were developed to describe the multiplicity of physicochemical, steric, and electronic parameters. These 3D-QSARs, so-called pattern recognition-type models, were applied each time a latent alerting group was identified in a parent chemical or its generated metabolite(s). The concept of the mutual influence amongst atoms in a molecule was used to define the structural domain of the skin sensitization model. The utility of the structural model domain and the predictability of the model were evaluated using sensitization potency data for 96 chemicals not used in the model building. The TIssue MEtabolism Simulator (TIMES) software was used to integrate a skin metabolism simulator and 3D-QSARs to evaluate the reactivity of chemicals thus predicting their likely skin sensitization potency.

Animals↗
PubMed2003

New insight into the peroxidase-hydroxamic acid interaction revealed by the combination of spectroscopic and crystallographic studies.

Aromatic hydroxamic acids, such as salicylhydroxamic (SHA) and benzohydroxamic (BHA) acids, are commonly used as probes for studying the active sites of peroxidases. In this paper, we have extended the study of the complexes of Arthromyces ramosus peroxidase (ARP/CIP) with BHA and SHA by analyzing their Raman spectra in solution and in single crystals. The experiments were carried out under various conditions to identify the best experimental conditions, and hence, avoid artifacts deriving from the preparation of the samples or collection of the spectra. The analysis of the data takes also into account the characteristic of the electronic absorption spectra in solution and the crystal structures of the complexes. The results showed small differences between the solution and the crystal phases even though the coordination state can be dramatically affected by the physical or chemical conditions. The greater sensitivity of the spectroscopic technique enabled us to establish the existence of multiple species upon complexation of the protein with the hydroxamic acids that could not be detected by ordinary X-ray crystallography. Furthermore, SHA titration experiments and singular value decomposition analysis of the absorption spectra indicated the presence of two binding sites in the protein, one with a high affinity (K(d) = 1.7 mM), which should correspond to the SHA bound protein as determined by X-ray, and the other with a very low affinity (K(d) > 80 mM) probably located in a non-heme site. This suggests that the heterogeneous titration line shape involves ligand binding to a non-heme site in competition with the canonical heme site. In contrast, the titration profile obtained with the BHA ligand is monophasic, in agreement with all the peroxidases so far studied.

Binding Sites↗
PubMed1988

Disulfide bonds and thermal stability in T4 lysozyme.

Disulfide bonds are thought to serve a stabilizing role in extracellular globular proteins, but little is known about the modes of stabilization or their mechanisms. Thermodynamic data presented here demonstrate that an engineered 3-97 disulfide bond previously shown to stabilize T4 lysozyme in vitro against irreversible thermal inactivation also stabilizes the molecule against reversible thermal unfolding. In this paper, we explore the relationship between the disulfide's thermodynamic contribution to protein folding and its role in providing resistance to irreversible thermal inactivation. In T4 lysozyme (C54V/C97S), a non-crosslinked mutant lacking the two cysteines found in the wild type, sensitivity toward irreversible thermal inactivation increases dramatically at temperatures above the melting temperature of the molecule. In addition, most of the lost activity can be restored by denaturation/renaturation with guanidine hydrochloride. In contrast, the crosslinked mutant T4 lysozyme (13C-97C/C54V) inactivates relatively slowly, even above its melting temperature, and the lost activity is not restored by denaturation/renaturation. These observations suggest that the predominant inactivation pathways for non-crosslinked T4 lysozymes are conformation related, while those for the crosslinked variant are insensitive to the conformational route and thus are susceptible only to slower processes of a chemical nature. We also show that multiple mutants, constructed to contain the 3-97 disulfide plus a temperature-sensitive lesion, are more stable than the wild type to irreversible inactivation even though they are less stable to reversible thermal unfolding. These findings together suggest that the 3-97 disulfide provides stability to irreversible inactivation primarily via a pathway that is independent of its thermodynamic contribution. The 3-97 disulfide may stabilize T4 lysozyme by restricting the unfolded state to a class of more compact structures with less exposed hydrophobic surface, compared to the unfolded states of non-crosslinked T4 lysozymes. The results have implications both for the use of the stabilizing potential of disulfide bonds in protein engineering and for their roles in protein function and evolution.

Disulfides↗
PubMed1987

Hepatobiliary MR contrast agents: 5-substituted iron-EHPG derivatives.

A series of iron(III) ethylenebis(2-hydroxyphenyl)glycine [Fe(EHPG)]- derivatives have been examined for their role as paramagnetic hepatobiliary contrast agents for magnetic resonance (MR) imaging. The 5-substituted complexes, Fe(5-Me-EHPG)-, Fe(5-Cl-EHPG)-, and Fe(5-Br-EHPG)-, have been compared to the parent compound in rat biodistribution and MR imaging studies; correlative in vitro parameters for the complexes, including octanol-buffer partition coefficients and albumin binding affinity, have also been obtained. The three new derivatives exhibited higher degrees of lipophilicity and albumin binding affinity and varying degrees of improvement in liver-to-blood and bile-to-liver concentration ratios measured at 30 min postinjection. The 5-Cl complex had the best overall performance in terms of these tissue ratios as well as in terms of total biliary excretion. Sequential MR images of rats after administration of the complexes revealed subtle pharmacokinetic differences among the derivatives and, in general, correlated well with and complemented the biodistribution results. This study points to the sensitivity of hepatocellular uptake and excretion to simple chemical modifications and, moreover, demonstrates the importance of screening multiple derivatives to select optimal hepatobiliary MR imaging agents.

Animals↗
PubMed2001

A sensitive fluorometric assay for tissue transglutaminase.

We have devised a highly sensitive fluorometric well plate assay for tissue transglutaminase that is suitable for multiple kinetic analyses/high-throughput screening of chemical inventories for inhibitors of this enzyme. The procedure measures the rate of fluorescence enhancement (lambda(exc) 260 nm, lambda(em) 538 nm) when 1-N-(carbobenzoxy-l-glutaminylglycyl)-5-N-(5'N'N'-dimethylaminonaphthalenesulfonyl)diamidopentane (glutaminyl substrate) is cross-linked to dansyl cadaverine (amine substrate). The assay procedure can be used to measure the activity of as little as 60 microU of purified guinea pig liver tissue transglutaminase (4.2 ng or 54 fmol of enzyme).

Animals↗
PubMedDate not supplied

A technique for the in vivo study of multiple stable isotope-labeled essential fatty acids.

A novel tracer technique is presented for the simultaneous and independent measurement of multiple stable isotopically labeled essential fatty acids. Gas chromatography/negative chemical ionization mass spectrometry was employed for high sensitivity detection of the following isotopes: deuterium-labeled-linolenate, carbon-13-U-labeled-eicosapentaenoate, carbon-13-U-labeled-linoleate, and deuterium-labeled-dihomo-gamma-linolenate. These isotope-labeled fatty acids in vehicle oil were given to rats either singly or together as a single oral dose. Rat blood was collected after dosing and the isotopomers of the precursors and their main metabolites, including those containing both(13) C and (2)H, were detected simultaneously with good resolution and without interference from other isotopes due to differences in mass and chromatographic retention.

Administration, Oral↗