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At least 631 records · Page 35Linked to original sources
PubMed1996

Spindle cells isolated from Kaposi's sarcoma-like lesions of BKV/tat-transgenic mice co-express markers of different cell types.

OBJECTIVE: To characterize murine spindle cells isolated from Kaposi's sarcoma-like skin lesions developed in BK virus (BKV)/tat-transgenic mice. METHODS: Kaposi's sarcoma-like spindle cells isolated from the lesions were propagated in vitro, and their phenotype was investigated using a panel of antibodies against various cell markers and angiogenic factors. Immunofluorescence and Western blot techniques were used. RESULTS: We observed co-expression of antigens specific for endothelial, smooth muscle and antigen-presenting cells, suggesting that cells from the TTB cell line represent poorly differentiated vascular precursors. Since TTB cells were derived from highly vascularized skin lesions, it is noteworthy that they synthesize a complex mixture of angiogenic factors, including fibroblast growth factor-2, vascular endothelial growth factor, placental growth factor, and hepatocyte growth factor. Due to their role in invasiveness and angiogenesis, we also observed the expression of urokinase plasminogen activator (uPA), uPA receptor, and plasminogen activator inhibitor-type 1 by TTB cells. CONCLUSIONS: Our results suggest that TTB cells share several features with human Kaposi's sarcoma spindle cells and can be a useful in vitro system to study the molecular mechanisms involved in Kaposi's sarcoma pathogenesis. Moreover, they synthesize a complex mixture of angiogenic factors and are growth-inhibited by the anti-angiogenic drug AGM-1470.

Animals↗
PubMed2004

Relative hopane content confirming the mineral origin of hydrocarbons contaminating foods and human milk.

Hopanes, triterpenoid hydrocarbons formed under geological conditions, were analysed to confirm the mineral origin of the unresolved complex mixtures of hydrocarbons observed in the gas chromatography with flame ionization detection chromatograms of human milk and certain foodstuffs. The 'relative hopane content' (RHC) is introduced, i.e. it is the area ratio of the sum of the hopanes and the paraffins in the same segment of the chromatogram. The RHC in various mineral oil products (motor oils, hydraulic oils, lubricating oils, Vaseline) was 3.4%, with a relative standard deviation of 19%. The RHC determined in samples of vegetable oils, mussels and clams as well as of human milk containing an unresolved complex mixture of hydrocarbons was in the same range, confirming that these samples were contaminated by mineral oil material.

Animals↗
PubMed1980

Fingerprint analyses of plant and animal tissues with respect to the occurrence of foreign compounds.

Glass capillary chromatography can be used for the characterization of complex mixtures of plant and animal samples after clean-up by counter-current distribution. For good reproducibility of fingerprint patterns a special splitless injection system is used. This method prevents any loss at injection. In order to determine organochlorine substances also in trace amounts (a few pg) an electroncapture detector (ECD) was used. Each step in the analysis procedure was checked with the aid of radioactively labelled substances, e.g. the polar compound pentachlorophenol and the non-polar compound hexachlorobenzene. With the aid of capillary chromatography-mass spectrometry substances in complex mixtures in a concentration of 10 to 100 ng/microliters can be characterized. Suitable glass capillaries for these investigation were prepared in this laboratory. The results indicate that a standardization of the procedure is practicable.

Animals↗
PubMed1991

Biomedical applications of high-performance liquid chromatography-mass spectrometry with continuous-flow fast atom bombardment.

This report describes the application of high-performance liquid chromatography combined with continuous-flow fast atom bombardment mass spectrometry to analytical problems in the biomedical laboratory. Applications include the compound-specific detection of diagnostic acylcarnitines in human urine, the separation and analysis of acyl-coenzyme A thioesters, and qualitative studies on complex mixtures of modified peptides (dansyl and dinitrophenyl derivatives). For each of these applications standard analytical columns (3.9 mm I.D.) and 1 ml/min flow-rates were employed with post-column stream splitting (1:100) before mass spectrometry. Various mobile phase compositions and solvent gradients were employed. The addition of 1-5% glycerol to the mobile phase was shown to have little effect on the chromatography. For all compounds studied (acylcarnitines, acyl-coenzyme A thioesters, and derivatized peptides) molecular weight information was obtained and sufficient sensitivity was achieved to allow unambiguous identification of trace components in complex mixtures.

Acyl Coenzyme A↗
PubMed1996

Influence of matrix solution conditions on the MALDI-MS analysis of peptides and proteins.

Sample-matrix preparation procedures are shown to greatly influence the quality of the matrix-assisted laser desorption/ionization (MALDI) mass spectra of peptides and proteins. In particular, dramatic mass discrimination effects are observed when the matrix 4-hydroxy-alpha-cyanocinnamic acid is used for analyzing complex mixtures of peptides and proteins. The discrimination effects are found to be strongly dependent on the sample-matrix solution composition, pH, and the rates at which the sample-matrix cocrystals are grown. These findings demonstrate the need to exercise great care in performing and interpreting the MALDI analysis of biological samples. The results also indicate that there is a reverse-phase chromatographic-like dimension in the sample-matrix preparation procedures that can be exploited to optimize the analysis. The present work describes the conditions under which the majority of components of a complex mixture of peptides and proteins can be successfully measured.

Amino Acid Sequence↗
PubMed2006

The blot rolling assay: a method for identifying adhesion molecules mediating binding under shear conditions.

Adhesive interactions of cells with blood vessel walls under flow conditions are critical to a variety of processes, including hemostasis, leukocyte trafficking, tumor metastasis, and atherosclerosis. We have developed a new technique for the observation of binding interactions under shear, which we have termed the "blot rolling assay." In this method, molecules in a complex mixture are resolved by gel electrophoresis and transferred to a membrane. This membrane can be rendered semitransparent and incorporated into a parallel-plate flow chamber apparatus. Cells or particles bearing adhesion proteins of interest are then introduced into the chamber under controlled flow, and their interactions with individual components of the immobilized substrates can be visualized in real time. The substrate molecules can be identified by staining with specific antibodies or by excising the relevant band(s) and performing mass spectrometry or microsequencing of the isolated material. Thus, this method allows for the identification, within a complex mixture and without previous isolation or purification, of both known and novel adhesion molecules capable of binding under shear conditions.

Animals↗
PubMed2002

Cell bioassays for detection of aryl hydrocarbon (AhR) and estrogen receptor (ER) mediated activity in environmental samples.

In vitro cell bioassays are useful techniques for the determination of receptor-mediated activities in environmental samples containing complex mixtures of contaminants. The cell bioassays determine contamination by pollutants that act through specific modes of action. This article presents strategies for the evaluation of aryl hydrocarbon receptor (hereafter referred as dioxin-like) or estrogen receptor mediated activities of potential endocrine disrupting compounds in complex environmental mixtures. Extracts from various types of environmental or food matrices can be tested by this technique to evaluate their 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents or estrogenic equivalents and to identify contaminated samples that need further investigation using resource-intensive instrumental analyses. Fractionation of sample extracts exhibiting significant activities, and subsequent reanalysis with the bioassays can identify important classes of contaminants that are responsible for the observed activity. Effect-directed chemical analysis is performed only for the active fractions to determine the responsible compounds. Potency-balance estimates of all major compounds contributing to the observed effects can be calculated to determine if all of the activity has been identified, and to assess the potential for interactions such as synergism or antagonism among contaminants present in the complex mixtures. The bioassay approach is an efficient (fast and cost effective) screening system to identify the samples of interest and to provide basic information for further analysis and risk evaluation.

Biological Assay↗
PubMedDate not supplied

Error propagation as a factor in selection of measurement intervals for the determination of polycyclic aromatic hydrocarbons by second-derivative spectrofluorimetry.

The most suitable wavelength intervals were selected for the determination of 4 polycyclic aromatic hydrocarbons (PAHs; benzo[g,h,i]perylene, dibenzo[a,h]anthracene, pyrene, and triphenylene) in very complex mixtures of 11 PAHs: anthracene, benz[a]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[g,h,i]perylene, benzo[k]fluoranthene, chrysene, dibenz[a,h]anthracene, phenanthrene, pyrene, and triphenylene. The multiple linear regression algorithm was applied to measurements made in several wavelength intervals previously selected on the basis of sensitivity and minimum number of interfering compounds. Of the different models obtained, those displaying minimum error propagation in the analytical result were selected. By applying the models proposed in this study, we precisely and accurately determined benzo[g,h,i]perylene, dibenz[a,h]anthracene, pyrene, and triphenylene in complex mixtures--a feat that could not be achieved by the use of constant-wavelength spectrofluorimetry in combination with second-derivative techniques.

Algorithms↗
PubMed2001

Proteomics of glycoproteins based on affinity selection of glycopeptides from tryptic digests.

Identification of glycoproteins in complex mixtures derived from either human blood serum or a cancer cell line was achieved in a process involving the steps of (1) reduction and alkylation, (2) proteolysis of all proteins in the mixture with trypsin, (3) affinity chromatographic selection of the glycopeptides with an immobilized lectin, (4) direct transfer of the glycopeptide fraction to a reversed-phase liquid chromatography (RPLC) column and further fractionation by gradient elution, (5) matrix-assisted laser desorption ionization mass spectrometry of individual fractions collected from the RPLC column, and (6) peptide identification based on a database search. The types of glycoproteins analyzed were; (1) N-type glycoproteins of known primary structure, (2) N-type glycoproteins of unknown structure, and (3) O-type glycoproteins glycosylated with a single N-acetylglucosamine. Identification of peptides from complex mixtures was greatly facilitated by either C-terminal sequencing with a carboxypeptidase mixture or by comparing chromatographic behavior and mass to standards, as in the case of a known protein. In addition, deglycosylation of peptides with N glycosidase F was necessary to identify N-type glycoproteins of unknown structure. The strength of this approach is that it is fast and targets specific molecular species or classes of glycoproteins for identification. The weakness is that it does not discriminate between glycoforms.

Amino Acid Sequence↗
PubMed1994

Use of quantitative epidemiologic data in regulatory approaches to air pollution.

Ambient air is a complex mixture containing a variety of substances, some of which are known to be carcinogenic. To develop a homogeneous approach for regulating the emission of these compounds, their individual carcinogenic potential needs to be placed on a comparable scale. The unit risk may be considered as an appropriate measure that condensates dose-response analyses of epidemiologic data into a single, easily interpretable estimate. Given the information on the carcinogenic potency of single compounds, more information on the occurrence of the components and the relation of emissions to specific emittents needs to be considered. In Germany, an approach has been developed that combines different assumptions on complex mixtures for the regulation of the overall risk. This paper outlines some of the principal aspects of the underlying concepts.

Air Pollution↗
PubMed2001

Multiple separations facilitate identification of protein variants by mass spectrometry.

Identification of variant proteins from complex biological samples promises to contribute much to our understanding the etiology of pathological states. Characterization of variants, either due to genetic mutations in protein sequences or to post-translational modifications, is considerably more difficult than the simple protein identifications typical of most current proteomic investigations. Identification of a few peptides by database retrieval is not adequate when the goal is to have a complete understanding of the modifications of the protein. Although one advantage of mass spectrometry is its ability to obtain specific responses to several components, the complexity of biological samples is often overwhelming, resulting in spectra lacking useful information. For complex mixtures, isolation procedures before mass spectrometric analysis may need to include a variety of chromatographic and electrophoretic separation techniques. In this report, we illustrate how several preparative steps were essential for obtaining information about modified human lens beta-crystallins. The preparative techniques prior to mass spectrometry included size exclusion chromatography, reversed phase chromatography, two-dimensional gel electrophoresis, in situ digestion of the proteins and peptide trapping and washing before a final reversed phase high performance liquid chromatographic separation on-line to the mass spectrometer. This approach for isolation and analysis, when customized for other proteins, should find application in many studies where protein variants of complex mixtures are to be identified.

Chromatography, Gel↗
PubMed2005

Interplay between the maillard reaction and lipid peroxidation in biochemical systems.

The Maillard reaction and lipid peroxidation are two of the most important chemical reactions that take place in biochemical systems. Both include a whole network of different reactions in which an extraordinarily complex mixture of compounds is produced in very different amounts, with both positive and negative consequences. In addition, both reactions are intimately interrelated, and the products of each reaction influence the other. Furthermore, there are common intermediates and products in both pathways; these products are usually known as advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs). Moreover, other AGE/ALEs are analogous and participate similarly in both amino acid degradation and amino phospholipid/protein polymerization by identical mechanisms. All these data suggest that the Maillard reaction and lipid peroxidation are so closely interrelated that both reactions should be considered simultaneously to understand the reaction mechanisms, kinetics, and products in the complex mixtures of carbohydrates, lipids, and proteins occurring in biochemical systems. In these systems, lipids and carbohydrates are competing in the chemical modification of amino phospholipids and proteins. Therefore, although there are significant differences between the Maillard reaction and lipid peroxidation, many aspects of both reactions can be better understood if they are included in only one general carbonyl pathway that can be initiated by both lipids and carbohydrates.

Biochemistry↗
PubMed1978

Comparative study of the glycosphingolipids of chicken bursa of Fabricius and of chicken, rat and human thymus.

1. The glycosphingolipid compositions of the thymus and bursa of Fabricius of young male chickens were compared. The two tissues were found to contain complex mixtures of both neutral glycosphingolipids and gangliosides. Both tissues contained mono-, di-, tri-, tetra- and penta-glycosylceramides; the pentaglycosylceramide displayed a reaction of identity with authentic Forssman antigen when tested against a specific anti-(Forssman antigen) serum. The ganglioside G(m3) containing N-acetylneuraminic acid was the principle ganglioside of both tissues. 2. The thymus contained appreciable amounts of the simple ganglioside N-acetylneuraminylgalactosylceramide, a compound not found in the bursa. The ganglioside G(d3) (disialohaematoside) was detected in both tissues. 3. Rat and human thymus, like sheep thymus (Narasimhan, Hay, Greaves & Murray (1976) Biochim, Biophys. Acta 431, 578-591), both contained a tetraglycosylceramide species as their most complex neutral glycosphingolipid and possessed little or no Forssman antigen. They also contained a complex mixture of gangliosides. 4. The possible significance of these results is briefly discussed.

Animals↗
PubMed1980

The separation of sterol intermediates in cholesterol biosynthesis by high pressure liquid chromatography.

A three-step procedure has been developed for the separation of complex mixtures of sterol intermediates in cholesterol biosynthesis. The method has been applied to the separation of sterol intermediates formed from [14C]mevalonate by normal rat hepatocyte culture cells. In Step 1, a short gravity-flow silicic acid column (1.2 x 6.5 cm) separates the incubation products into four classes consisting of A) squalene + squalene oxide, B) methyl sterol precursors, C) C27 sterols, and D) polar compounds. In Step 2, the components of classes B) and C) are further resolved by reverse-phase high pressure liquid chromatography (HPLC) on a microBondapak-C18 column. In Step 3, (after acetylation) high pressure liquid chromatography on a microPorasil column of peaks obtained from Step 2 is conducted. This last step resolves mixtures which may be present in peaks resulting from Step 2. The relative retention times of unknown radioactive sterols are compared with authentic co-chromatographed reference sterols in both Steps 2 and 3. Relative retention time factors for several functional groups encountered in sterol intermediates in cholesterol biosynthesis have been determined for both reverse-phase and silicic acid HPLC systems. The use of these functional group factors allows one to calculate a predicted relative retention time for a variety of structural possibilities. The HPLC techniques described utilize single columns, isocratic solvent systems, and comparatively short (< 30 min) elution times, and the three-step procedure is capable of resolving complex mixtures of sterol intermediates.

Cholesterol↗
PubMed1991

Mutagenicity spectra in bacterial strains of airborne and engine exhaust particulate extracts.

The mutagenicity spectra of the organic extracts of both airborne particulate matter and diesel and gasoline soot particles were determined using a battery of 9 bacterial strains of different genetic specificity. The assays with crude extracts and with fractionated acidic, neutral and basic components revealed striking differences in the patterns of mutagenic responses produced by each of the complex mixtures investigated. The mutagenicity of air particulate matter was shown to depend mainly on direct-acting acidic and neutral compounds, with a lesser contribution of basic promutagens which required exogenous metabolic activation by liver S9. The assays with a diesel soot extract indicated the prevailing contribution of direct-acting acidic and neutral compounds, and suggested an important role also for nitro derivatives other than nitropyrenes. The gasoline exhaust was characterized by powerful promutagenic compounds, belonging to either the acidic, neutral or basic fractions. The implications of these results are discussed with respect to the contribution of engine exhausts to air pollution, and the possible use of mutagenicity spectra in the analysis of environmental complex mixtures.

Air Pollutants↗
PubMed1989

Exposure assessment of oxidant gases and acidic aerosols.

Clearly the presence of high ozone and acidic species in North America is primarily dependent upon photochemical air pollution. Evidence shows, however, that high acid exposures may occur in specific types of areas of high sulfur fuel use during the winter. At the present time, our concerns about exposure to local populations and regional populations should be directed primarily toward the outdoor activity patterns of individuals in the summer, and how those activity patterns relate to the location, duration, and concentrations of ozone and acid aerosol in photochemical air pollution episodes. Lioy & Dyba (23) and Mage et al (33) have examined the activity patterns of children in summer camps. Because they spend more time outside than the normal population, these children form an important group of exercising individuals subject to photochemical pollution exposures. The dose of ozone inhaled by the children in the two camps was within 50% (23) and 25% (33) of the dose inhaled by adults in controlled clinical situations that produced clinically significant decrements in pulmonary function and increased the symptoms after 6.6 hr exposure in a given day (12, 30). The chamber studies have used only ozone, whereas in the environment this effect may be enhanced by the presence of a complex mixture. The work of Lioy et al (27) in Mendham, New Jersey found that hydrogen ion seemed to play a role in the inability of the children to return immediately to their normal peak expiratory flow rate after exposure. The camp health study conducted in Dunsville, Ontario suggested that children participating in a summer camp where moderate levels of ozone (100 ppb) but high levels of acid (46 micrograms/m3) occurred during an episode had a similar response. Thus, for children or exercising adults who are outdoors for at least one hour or more during a given day, the presence and persistence of oxidants in the environment are of particular concern. Lippmann (19, 30) has suggested that the complex mixture of pollutants in the atmosphere increased the decrements in pulmonary function of children in camps, since their decrements were similar to those observed at higher ozone concentrations in the clinical studies (30). This phenomenon must be investigated in future research studies since there appears to be a potential for enhanced exposure and effects. Ozone is present for many hours during the day in summer during episodes of photochemical air pollution.(ABSTRACT TRUNCATED AT 400 WORDS)

Acids↗
PubMed2005

Evaluation of the DotMap algorithm for locating analytes of interest based on mass spectral similarity in data collected using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry.

Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC x GC-TOF-MS) is a highly selective technique ideal for the analysis of complex mixtures. The instrument yields an abundance of data, with complete mass spectral scans at every time point in the GC x GC separation space. The development and application of appropriate tools for data mining is essential in making sense of the wealth of information available. An algorithm for locating analytes of interest based on mass spectral similarity in GC x GC-TOF-MS data, called DotMap, has been previously reported and is rigorously evaluated herein. A thorough investigation into the performance characteristics of DotMap, including the performance near the limit of detection and dynamic range of the algorithm as well as the capacity of the algorithm to deal with peak overlap, is investigated using jet fuel as a complex sample matrix. For instance, the algorithm can successfully identify a spiked compound at the single microg/ml level in a jet fuel sample with an overlapping interferent. The performance of the DotMap algorithm in situations with very limited mass spectral selectivity, specifically in the evaluation of spectra from isomer compounds, as well as the ability to tune DotMap results to provide the location of a specific analyte or of a class of compounds is demonstrated. The DotMap algorithm is demonstrated to be a sensitive tool that is useful in the analysis of complex mixtures and which possesses the capacity to be easily "tuned" to discern the location of analytes of interest.

Algorithms↗
PubMed2004

Screening for N-glycosylated proteins by liquid chromatography mass spectrometry.

In the last few years mass spectrometry has become the method of choice for characterization of post-translationally modified proteins. Whereas most protein chemical modifications are binary in the sense that only one change can be associated with a given residue, many different oligosaccharides can be attached to a glycosylation site residue. The detailed characterization of glycoproteins in complex biological samples is extremely challenging. However, information on N-glycosylation can be gained at an intermediary level. Here we demonstrate a procedure for mapping N-glycosylation sites in complex mixtures by reducing sample complexity and enriching glycoprotein content. Glycosylated proteins are selected by an initial lectin chromatography step and digested with endoproteinase Lys-C. Glycosylated peptides are then selected from the digest mixture by a second lectin chromatography step. The glycan components are removed with N-glycosidase F and the peptides digested with trypsin before analysis by on-line reversed-phase liquid chromatography mass spectrometry. Using two different lectins, concanavalin A and wheat germ agglutinin, this procedure was applied to human serum and a total of 86 N-glycosylation sites in 77 proteins were identified.

Amino Acid Sequence↗