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PubMed2003

Analysis of preneoplastic and neoplastic renal lesions in Tsc2 mutant Long-Evans (Eker) rats following exposure to a mixture of drinking water disinfection by-products.

Disinfection of surface water for human consumption results in the generation of a complex mixture of chemicals in potable water. Cancer risk assessment methodology assumes additivity of carcinogenic effects in the regulation of mixtures. A rodent model of hereditary renal cancer was used to investigate the carcinogenic response to a mixture of drinking water disinfection by-products (DBPs). Rats carrying a mutation in the Tsc2 tumor suppressor gene (Eker rats) readily develop renal preneoplastic and neoplastic lesions, and are highly susceptible to the effects of renal carcinogens. Male and female Eker rats were exposed via drinking water to individual or a mixture of DBPs for 4 or 10 months. Potassium bromate, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), chloroform, and bromodichloromethane were administered at low concentrations of 0.02, 0.005, 0.4 and 0.07 g/l, respectively, and high concentrations of 0.4, 0.07, 1.8 and 0.7 g/l, respectively. Low and high dose mixture solutions were comprised of all four chemicals at either low concentrations or high concentrations, respectively, Following necropsy, each kidney was examined microscopically for preneoplastic lesions (atypical tubules and hyperplasias) and tumors. While some of the mixture responses observed in male rats did fall within the range expected for an additive response, especially at the high dose, predominantly antagonistic effects on renal lesions were observed in response to the low dose mixture in male rats and the high dose mixture in female rats. These data suggest that current default risk assessments assuming additivity may overstate the cancer risk associated with exposure to mixtures of DBPs at low concentrations.

Animals↗
PubMed2005

Analysis of neutral surfactants by non-aqueous capillary electrophoresis using an electroosmotic flow reversal.

The separation of KM 20, that is in fact a mixture of non-ionic surfactants, was carried out by non-aqueous capillary electrophoresis. This complex mixture resulting from the condensation of ethylene oxide with fatty alcohols does not have chromophoric moieties. So, we analysed it after derivatization by means of 3,5-dinitrobenzoyl chloride. The proposed approach is based both on the formation of complexes with alkaline or ammonium cations in methanol and on the utilisation of a positively charged capillary. From a comparative study on the capillary treatment procedure, we used hexadimethrine bromide as electroosmotic flow reverser in order to obtain both repeatable analyses and good resolutions of the largest KM 20 oligomers. Then, among the five cations used to form complexes with KM 20, we pointed out that ammonium cation led to the best resolutions. Moreover, we evidenced that the counter-ion of this cation had a great influence on resolution because it modified the magnitude of electroosmotic flow. Ion pair formation that is more or less strong between ammonium and its counter-ion was involved in this variation of electroosmotic flow. So, we calculated the association constants for various ammonium salts in methanol. Then, using ammonium chloride as background electrolyte, we optimised the concentration of this salt, in methanol, in order to reach the optimal separation of KM 20 oligomers. Thus, a baseline separation was obtained by using 6 x 10(-2) mol/L NH4Cl as running electrolyte. In these conditions, we separated, in about 30 min, more than 30 oligomers of KM 20. The distribution of these oligomers that was determined from the optimal separation, appeared consistent with that obtained from HPLC analyses. Indeed, we determined that the mean ethoxylation number was equal to 18 while its real value is equal to 20.

Electrophoresis, Capillary↗
PubMedDate not supplied

Aminonitriles: possible role in chemical evolution.

The formation of HCN, ammonium cyanide, alkylnitriles, aminoacetonitrile and its C- and N-methyl homologs was demonstrated earlier in a simulated Jovian atmosphere. The polymeric material resulting in these experiments was shown to give glycine, alanine, sarcosine, aspacrtic acid and some imino dibasic acids on acid hydrolysis suggesting thereby the participation of the monomeric nitriles into the formation of the polymeric product(s). Further examination of products resulting from semi-corona and arc discharge through a mixture of methane and ammonia has provided evidence for the formation of alkylaminopropionitriles as a complex mixture and also some pyridyl and pyrimidyl type heterocyclic compounds. A GC-MS examination of the heterocylics showed resemblance with those found in some carbonaceous chondrites. The significance of these findings in relation to chemical evolution will be discussed.

Ammonia↗
PubMed1994

Partial characterization of the protein and carbohydrate constituents of the egg shell of Hymenolepis diminuta (Cestoda).

The egg shell of Hymenolepis diminuta is composed of 31.7% protein and 2.9% carbohydrate (by weight), as determined using the Lowry and phenol-sulfuric acid assays and bovine serum albumin and glucose, respectively, as standards. Amino acid analyses of egg shells demonstrated the presence of 16 naturally occurring amino acids and six unidentified residues. Histidine accounted for > 22% of the amino acid residues. Hypochlorite-solubilized egg shells were fractionated using a combination of gel, hydroxyapatite and anion-exchange chromatography, and the fractions were analyzed by isoelectric focusing and gel chromatography. The results demonstrate that the shell consists of a complex mixture of proteins (almost all of which have acidic isoelectric points), glycoproteins and possibly free complex carbohydrates.

Amino Acids↗
PubMed2006

Sulphate efflorescent minerals from El Jaroso Ravine, Sierra Almagrera--An SEM and Raman spectroscopic study.

Two sulphate efflorescent evaporite mineral samples from Jaroso, Spain have been studied by scanning electron microscopy and Raman spectroscopy. SEM by comparison with known minerals shows the evaporite mineral is a mixture of halotrichite and jarosite, whilst the oxidised mineral is predominantly jarosite. SEM characterises the halotrichite as long narrow crystals and the jarosite as distorted rhombohedral crystals. Raman spectra of the sulphates of K, Mg, Fe(II), Fe(III) are compared with the spectra of halotrichite, jarosite and the two sulphate efflorescent samples. The efflorescent sample was proven by Raman spectroscopy to be a mixture of halotrichite and jarosite and the oxidised efflorescent sample to be jarosite and a complex mixture of sulphates.

Alum Compounds↗
PubMed2006

Review of the toxicity of chemical mixtures containing at least one organochlorine.

An analysis of current research on mixture toxicity was conducted by critically reviewing published journal articles. The scope was limited to complex mixtures (more than two components) where at least one component was a chlorinated organic chemical. Although the basics of dose-response are widely accepted for mixtures, a number of technical issues, including dose metrics and the unquantified influence of toxicity modifying factors, confound data interpretation and restrict the ability to establish reliable determinations of the presence, nature, and extent of additivity. Lack of knowledge about dose level influences and species-specific variations contribute further interpretational limitations. Within this context, available data indicates that most tested mixtures are near or below simple dose/concentration additivity. Exceptions (both positive and negative) tend to occur when tested mixtures have only a few components or where sensitive whole organism or sub-organismal changes are used as the response metric. Available information does not routinely identify the presence of chlorine as a marker either of a particular type of toxicity or consistently greater potency. The most profound difficulty is the problem of clearly defining when and why similarity and dissimilarity of toxic action is expected for a particular mixture. This impediment largely results from the lack of a generally accepted, technical classification for mode/mechanism of toxic action coupled with the lack of a generally accepted classification scheme for mode/mechanism of toxicity interactions.

Animals↗
PubMed1999

NMR tools for biotechnology.

Recent developments in NMR spectroscopy verify that NMR continues to be an exciting area of research. These advances can be placed into three general categories: new hardware; new techniques; and novel applications. The hardware developments include many advances in the area of flow NMR and some new probe designs. The new techniques include several ways to edit the NMR spectra of mixtures without using chromatographic separation. These new NMR tools are now allowing us to analyze complex mixtures, combinatorial-chemistry libraries, bound drugs, unstable compounds, very small samples, and heterogeneous samples.

Biotechnology↗
PubMed2003

Oxidation of resveratrol catalyzed by soybean lipoxygenase.

In this work the oxidative degradation of resveratrol catalyzed by lipoxygenase-1 (LOX-1) has been studied. The process has been characterized by spectroscopic and polarographic measurements. The oxidation of resveratrol was dependent on the concentration of resveratrol and the enzyme. When resveratrol was incubated in the presence of lipoxygenase at pH 9.0, the reaction displayed a k(M) value of 18.6 x 10(-)(6) M and a catalytic efficiency (k(cat)/k(M)) of 4.3 x 10(4) s(-)(1) M(-)(1). These values are close to those shown by the enzyme when linoleic acid is used as the substrate. The effect of lipoxygenase inhibitors on the lipoxygenase-catalyzed resveratrol oxidation was also evaluated. The rate of resveratrol oxidation was markedly decreased by the presence of NDGA in the incubation mixture. From HPLC measurements, it can be deduced that resveratrol is oxidatively decomposed to a complex mixture of products similar to those obtained when the molecule is oxidized by hydrogen peroxide.

Chromatography, High Pressure Liquid↗
PubMed1989

The identification of antigens in antibody/antigen complexes using high performance liquid chromatography.

A method for the identification of antigens in a complex mixture of proteins is described. It exploits the effect which addition of a monoclonal antibody and resultant formation of antibody/antigen complexes has on the apparent retention time of the antigen on a size exclusion column. An antigen in the mixture can be identified as that component whose retention time is decreased by addition of its antibody. The principle of the method is demonstrated using an HPLC size exclusion column (DuPont GF450) and three MoAbs, PD7/26, DA6 147 and W6/32 which are directed against three antigens of the lymphocyte surface, the leucocyte-common antigen, class II MHC and class I MHC respectively.

Antibodies, Monoclonal↗
PubMed2004

Comparative mixture effects of JP-8(100) additives on the dermal absorption and disposition of jet fuel hydrocarbons in different membrane model systems.

Jet fuel are complex mixtures of hydrocarbon fuel components and performance additives. Three different membrane systems, silastic, porcine skin and the isolated perfused porcine skin flap (IPPSF) were used to gain insight into the possible mechanism for additive interactions on hydrocarbon component absorption. Influence of JP-8(100) additives on the dermal kinetics of 14C-naphthalene and 14C/3H-dodecane as markers of hydrocarbon absorption, were evaluated using analysis of means (ANOM) and analysis of variance (ANOVA). This study indicated that the naphthalene absorption through silastic membrane was significantly different with JP-8 plus individual additives as compared to controls, i.e. JP-8 and JP-8(100). The porcine skin data indicated that neither individual nor combinations of additives affected naphthalene absorption. The third membrane system (IPPSF) showed that only MDA and BHT were important additives altering naphthalene absorption. MDA was a significant suppressor while BHT was a significant enhancer of naphthalene absorption. MDA significantly decreased dodecane absorption in skin flaps. All individual and combinations of two additives with JP-8 affected naphthalene and dodecane surface retention in silastic membrane. The IPPSF indicated that only 8Q405 is a significant modulator of surface retention for both marker hydrocarbons. The 8Q405 significantly reduced naphthalene contents in dosed silastic and skin indicating a direct interaction between additive and marker hydrocarbons. The MDA and BHT, which significantly retained naphthalene in the stratum corneum of porcine skin individually, led to a statistical decrease in its retention in the stratum corneum when in combination (MDA + BHT) suggesting a potential biological interaction. These observations demonstrate that the single membrane system may not be suitable for the final prediction of complex additive interactions in jet fuels. Rather a combination of different membrane systems may provide the insight to elucidate the possible mechanism for additive interactions. Finally, it is important to assess all components of a chemical mixture since the effects of single components administered alone or as pairs may be confounded when all are present in the complete mixture.

Administration, Topical↗
PubMedDate not supplied

Numerical and chemical classification of Streptosporangium and some related actinomycetes.

One hundred and seventeen streptosporangia from soil were compared with marker strains of the family Streptosporangiaceae for many phenotypic properties. The data were examined using the Jaccard, pattern and simple matching coefficients with clustering achieved using average, complete and single linkage algorithms. Particular confidence was placed in the product of the pattern, average linkage analysis given the sharp definition of aggregate groups and clusters and a combination of low test error and high cophenetic correlation values. The test strains were assigned to five aggregate groups that were equated with the genera Streptosporangium (group A), Microbispora (group B), Planobispora and Planomonospora (Group C), Kutzneria (neé Streptosporangium viridogriseum (group D), and Microtetraspora (group E). The streptosporangia, both isolates and marker strains, were assigned to 5 major, 7 minor and 18 single membered clusters. Representative streptosporangia examined for chemical markers were characterised by the presence of meso-diaminopimelic acid in whole-organism hydrolysates, complex mixtures of straight- and branched chain fatty acids, di- and tetrahydrogenated menaquinones as predominant isoprenologues, and complex polar lipid patterns containing diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and uncharacterised components. The chemical and numerical data support the taxonomic integrity of the validly described species of Streptosporangium and suggest that the genus is markedly underspeciated.

Actinomycetales↗
PubMed2007

Quantification of diacylglycerol species from cellular extracts by electrospray ionization mass spectrometry using a linear regression algorithm.

Diacylglycerols (DAGs) play significant roles in both intermediate metabolism and signal transduction. These lipid species are second messengers involved in modulating a plethora of cellular processes. Evaluation of DAG species concentrations has been hampered by the lack of a reliable method for molecular species analysis within a complex mixture of cellular lipids. We describe a new method for quantitative analysis of DAG species from complex biological extracts based on positive mode electrospray ionization mass spectrometry without prior derivatization. Quantification is achieved using internal standards and calibration curves constructed by spiking cell extracts with different concentrations of DAG species containing various acyl chain lengths and degrees of unsaturation. The new mass spectral data processing algorithm incorporates a multiple linear regression model including a factor accountable for possible interactions between experimental preparations and the slope of the curve for the standards, allowing the examinations of the effects of sample origin conditions (such as cell types, phenotypes, etc.) and instrument variability on this slope. Internal standards provide a basis for quantification of 28 DAG molecular species detected in RAW 264.7 cells after stimulation of a G-protein coupled receptor with platelet activating factor. This method displays excellent reproducibility over the established range of concentrations with variations of < or =10% and is highly sensitive with a detection limit of 0.1-0.4 pmol/microL depending upon acyl chain composition. We have shown differential effects on various DAGs in response to a ligand which illustrates the importance of examining lipids at the molecular species level rather than as a single homogeneous entity.

Algorithms↗
PubMed2005

Hexameric and pentameric slipped-cofacial dimers: toward an artificial light-harvesting complex.

[structure: see text] We prepared a zinc complex of bis(1-methylimidazolyl)-m-gable porphyrin. The automatically assembled coordinate species showed a complex mixture of wide molecular weight distributions accompanied with assemblies of specific assembly numbers. When this zinc complex was once dissociated by the addition of methanol and reorganized again by elimination of the methanol under high-dilution conditions in chloroform to facilitate intramolecular coordinate structure formation, two convergent assemblies were obtained through analysis by gel permeation chromatography. These assemblies gave round-shaped particles on solid substrates by probe microscopies (atomic force and scanning tunneling microscopies). These two components were separated by GPC and evidence from small-angle X-ray scattering measurements in solution with synchrotron radiation was consistent with hexameric and pentameric macrorings of the gable porphyrin. The porphyrin macrorings did not show any fluorescence quenching by assembly formation, and we anticipate that the macrorings of gable porphyrins represent a good model for an artificial light-harvesting complex.

Journal Article↗
PubMed2006

Quantitative analysis of acrylamide labeled serum proteins by LC-MS/MS.

Isotopic labeling of cysteine residues with acrylamide was previously utilized for relative quantitation of proteins by MALDI-TOF. Here, we explored and compared the application of deuterated and (13)C isotopes of acrylamide for quantitative proteomic analysis using LC-MS/MS and high-resolution FTICR mass spectrometry. The method was applied to human serum samples that were immunodepleted of abundant proteins. Our results show reliable quantitation of proteins across an abundance range that spans 5 orders of magnitude based on ion intensities and known protein concentration in plasma. The use of (13)C isotope of acrylamide had a slightly greater advantage relative to deuterated acrylamide, because of shifts in elution of deuterated acrylamide relative to its corresponding nondeuterated compound by reversed-phase chromatography. Overall, the use of acrylamide for differentially labeling intact proteins in complex mixtures, in combination with LC-MS/MS provides a robust method for quantitative analysis of complex proteomes.

Acrylamides↗
PubMed2005

Simultaneous detection of Escherichia coli O157:H7 and Salmonella Typhimurium using quantum dots as fluorescence labels.

In this study, we explored the use of semiconductor quantum dots (QDs) as fluorescence labels in immunoassays for simultaneous detection of two species of foodborne pathogenic bacteria, Escherichia coli O157:H7 and Salmonella Typhimurium. QDs with different sizes can be excited with a single wavelength of light, resulting in different emission peaks that can be measured simultaneously. Highly fluorescent semiconductor quantum dots with different emission wavelengths (525 nm and 705 nm) were conjugated to anti-E. coli O157 and anti-Salmonella antibodies, respectively. Target bacteria were separated from samples by using specific antibody coated magnetic beads. The bead-cell complexes reacted with QD-antibody conjugates to form bead-cell-QD complexes. Fluorescent microscopic images of QD labeled E. coli and Salmonella cells demonstrated that QD-antibody conjugates could evenly and completely attach to the surface of bacterial cells, indicating that the conjugated QD molecules still retain their effective fluorescence, while the conjugated antibody molecules remain active and are able to recognize their specific target bacteria in a complex mixture. The intensities of fluorescence emission peaks at 525 nm and 705 nm of the final complexes were measured for quantitative detection of E. coli O157:H7 and S. Typhimurium simultaneously. The fluorescence intensity (FI) as a function of cell number (N) was found for Salmonella and E. coli, respectively. The regression models can be expressed as: FI = 60.6 log N- 250.9 with R(2) = 0.97 for S. Typhimurium, and FI = 77.8 log N- 245.2 with R(2) = 0.91 for E. coli O157:H7 in the range of cell numbers from 10(4) to 10(7) cfu ml(-1). The detection limit of this method was 10(4) cfu ml(-1). The detection could be completed within 2 hours. The principle of this method could be extended to detect multiple species of bacteria (3-4 species) simultaneously, depending on the availability of each type of QD-antibody conjugates with a unique emission peak and the antibody coated magnetic beads specific to each species of bacteria.

Animals↗
PubMed1991

Branch capture reactions: displacers derived from asymmetric PCR.

Branch capture reactions (BCR) contain three DNA species: (i) a recipient restriction fragment terminating in an overhang, (ii) a displacer strand containing two adjacent sequences, with one complementary to the overhang and to contiguous nucleotides within the recipient duplex and (iii) a linker which is complementary to the second displacer sequence. Branched complexes containing all three species may be captured by ligation of the linker to the recipient overhang. The use of 5-MedC in the displacer facilitates BCR. High temperature ligation with a thermostable enzyme increased specificity for ligation to the correct recipient in a complex mixture of restriction fragments. Displacer synthesis by PCR permitted separate reactions of formation of stable displacement complexes and of high-temperature ligation. Ethylene glycol-containing buffer permitted PCR with 5-MedCTP or high G + C products using thermostable polymerases. BCR may be used to modify the ends of one recipient DNA duplex in a population of duplex DNA fragments. Modification of the recipient could be used to facilitate detection, affinity chromatography or cloning. By using PCR to obtain a BCR displacer, the sequence non-homologous to the recipient duplex may be expanded to include the sequence of a selectable marker, thus facilitating chromosome walking.

Base Sequence↗
PubMed2002

Assessment of dermal absorption and penetration of components of a fuel mixture (JP-8).

Occupational and environmental multi-chemical exposures are extremely common. Methods for assessment of the risks from dermal exposures to complex mixtures vary depending on the information available. The composition of a volatile mixture (such as JP-8 jet fuel) can change radically, depending on the phase of the mixture - vapor, liquid or aerosol. Assessing the absorption (into the skin) and penetration (through the skin) of components of the mixture can reduce uncertainty in the risk assessment process. Permeability coefficients of the 12 individual components that could be detected to penetrate the skin could be used to assess the toxicity of each individual component in the JP-8. The penetration of each of these components is related to and can be predicted from molecular weight and octanol water partition coefficients of that component. The composition of the components that penetrate the skin would be different from the composition of JP-8 because the permeability of the components differs by two orders of magnitude. Concentrations of the aliphatic chemicals found in the skin correlated well with carbon number. The JP-8 jet fuel is used as an example of how component data on absorption and penetration can be integrated into an assessment (McDougal et al., Toxicol Sci 2000; 55: 247-255). The component approach shows promise for estimating systemic toxicity of mixtures. Local toxicity (irritation, sensitization, etc.) may be better understood in the future when quantitative information becomes available about the duration and magnitude of chemical exposures required to cause local effects.

Absorption↗
PubMed2003

Evaluation of fish early life-stage toxicity models of chronic embryonic exposures to complex polycyclic aromatic hydrocarbon mixtures.

Polycyclic aromatic hydrocarbons (PAHs) can cause a variety of effects in early life-stages of fish that have been chronically exposed as embryos, including mortality, deformities, and edemas. Mechanistic models of the chronic toxicity of complex mixtures of PAHs in fish have not been reported, with the exception of a previously untested model based on the lipids of fish as the site of action and toxicity caused through a narcosis mechanism. Four mechanism-based models of the chronic toxicity of embryonic exposures to complex mixtures of petrogenic PAHs in two species of fish, Pacific herring and pink salmon, were evaluated using a toxic-units approach: narcosis, aryl hydrocarbon receptor (AhR) agonism, alkyl phenanthrene toxicity, and combined toxicity. Alkyl phenanthrenes were the predominant PAH constituent determining early life-stage toxicity in both herring and salmon. The alkyl phenanthrene model had 67 to 80% accuracy in predicting the absence or presence of significant early life-stage toxicity, compared with a 40 to 50% accuracy and general underprediction of toxicity with the narcosis model. PAHs with high relative AhR affinity did not appear to contribute substantially to the observed early life-stage toxicity because of low concentrations of the most potent AhR agonists. Narcosis appeared to primarily contribute to embryo mortality and to be predominantly controlled by the concentration of naphthalenes. Except for the highest PAH exposure to herring, the primary toxic unit contribution to the combined toxicity model was alkyl phenanthrene toxicity to both herring and salmon. We recommend the continued use of total PAHs as a metric of exposure until mechanistic models have been further evaluated.

Animals↗