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PubMed1987

Antibody for detection and quantitation of membrane-associated folate-binding protein from Lactobacillus casei.

Lactobacillus casei cells contain a 25 kDa, membrane-associated, folate-binding protein (fbp), which is a component of the folate transport system. Polyclonal antibody to fbp (anti-fbp) has been prepared, and conditions have been established for detection and quantitation of the protein. Anti-fbp did not block [3H]folate transport or binding in L. casei cells. As judged by Western blots, the antibody reacted only with fbp on sodium dodecyl sulfate electrophoretograms of Triton X-100 extracts of L. casei membranes. Anti-fbp showed no cross-reactivity with L. casei dihydrofolate reductase, L. casei 5,10-methenyltetrahydrofolate synthetase, L1210 dihydrofolate reductase, rat liver dihydrofolate reductase, or L1210 folate-binding protein. Enzyme-linked immunosorbent assay measurements indicated the presence of an fbp in membranes of Lactobacillus salivarius and two transport-defective sublines of L. casei. Anti-fbp was used to demonstrate selective extraction, with n-butanol, of fbp from a mixture of Triton-solubilized L. casei membrane proteins; repression of fbp in membranes of L. casei cells grown on high levels of folate; and localization of fbp by electron microscopy, using anti-fbp in conjunction with goat anti-rabbit IgG gold conjugate, in L. casei membranes.

Animals↗
PubMed1991

Selective assay of protein kinase C with a specific peptide substrate.

Protein kinase C is a family of multifunctional protein serine/threonine kinase and generally accepted to be involved in a wide variety of cellular signal transduction. Biochemical and immunochemical studies as well as sequence analysis of its cDNA clones have revealed the existence of multiple subspecies of this enzyme with obvious tissue-specific expression. Enzymatic properties of type I, II, and III protein kinase C subspecies, which are encoded by gamma-, beta I- and beta II, and alpha-cDNA, respectively, are well characterized. Many proteins and peptides are reported as phosphate acceptors of these protein kinase C subspecies. In this study, it is shown that a synthetic peptide, Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu, which corresponds to amino acid residues 4-14 of bovine myelin basic protein, is the most specific and convenient substrate for selective assay of protein kinase C among various phosphate acceptor proteins and peptides. This peptide is phosphorylated at Ser-8, but not Ser-11 by protein kinase C subspecies in a manner dependent on Ca2+, phosphatidylserine, and diacylglycerol. This peptide is not phosphorylated by other protein serine/threonine kinases such as cyclic AMP-dependent protein kinase. Thus, it is possible to assay protein kinase C activity in the crude tissue extracts selectively using this peptide as a phosphate acceptor.

Amino Acid Sequence↗
PubMed1999

Comparative study of methodologies for the analysis of PCDDs and PDCFs in powdered full-fat milk. PCB, PCDD and PCDF levels in commercial samples from Spain.

Feasibility of two different extraction methods involving solid-liquid extraction and Soxhlet extraction, previously used for the determination of lipid contents and PCB levels on powdered full-fat milk, are now examined for simultaneous PCDD/F analysis. The results of this study are consistent with those found for PCBs. The solid-liquid procedure provides the most efficient extraction of both labelled spiked and endogenous PCDD/Fs with the lowest variability. The average recoveries were 101% (R.S.D. = 6.9%) for 13C12-2,3,7,8-TCDD and 95% (R.S.D. = 11%) for 13C12-1,2,3,4,7,8-HxCDD. The R.S.D.s for endogenous 2,3,7,8-PCDD/Fs were in the 9.3-25% range. In addition, this methodology simplified the subsequent clean-up step as it allowed a semi-selective extraction of the apolar lipids from the milk. The proposed method was applied to simultaneous PCB and PCDD/F analyses in different brands of powdered full-fat milks commercially available in Spain. In all cases, very similar PCB and PCDD/F levels were found. Differences among batches from the same manufacturer were even lower. The total i-TEQ average for PCDDs and PCDFs in the 22 powdered full-fat milk samples analysed was 1.87 pg/g fat basis. This value was similar to those found in cow's milk from other European countries.

Animals↗
PubMedDate not supplied

A novel technique for the pre-concentration and extraction of inositol hexakisphosphate from soil extracts with determination by phosphorus-31 nuclear magnetic resonance.

Inositol hexakisphosphate (IP6) is often the dominant form of soil organic phosphorus (P), but is rarely investigated because of the analytical difficulties encountered in its extraction, separation, and detection in environmental samples. In particular, recent advances in the study of soil organic P with 31P nuclear magnetic resonance (NMR) have been of limited use for the study of IP6, because the technique does not discriminate between IP6 and other forms of P. This was addressed by developing a novel analytical procedure using the retentive properties of gel-filtration gels for IP6, which allows the combined selective extraction and pre-concentration of IP6 from soil extracts with determination by 31P NMR. While the technique is still in the developmental stage, the results demonstrate that the gel does not interfere with 31P NMR analysis and retains IP6 to concentrations well above those required to give clear spectral signals. The technique has considerable potential for application to the study of IP6 in soil extracts and water samples and, with development, could help to answer fundamental questions regarding the dynamics of organic P in the environment.

Carrier Proteins↗
PubMed1961

Ultrastructural cytochemistry. Enzyme and acid hydrolysis of nucleic acids and protein.

Selective extraction of specific cell components by enzyme or acid hydrolysis is possible from ultrathin sections for electron microscopy and parallel 2 micro sections for light microscopy of tissues fixed in formalin and embedded in a water-soluble polyepoxide, product X133/2097. Normal rat tissues fixed 15 minutes in formalin at 3 degrees C are more rapidly digested by proteinases than those fixed for the same length of time at 20 degrees C. Trypsin selectively attacks the nuclear chromatin and the ribonucleoprotein particles of the ergastroplasm, whereas mitochondria and zymogen granules resist tryptic digestion. Pepsin rapidly attacks the mitochondria and zymogen granules. The ergastoplasm and nucleus at first resist peptic digestion, but in time the entire cytoplasm and interchromatinic portion of the nucleus are attacked. Ribonuclease abolishes cytoplasmic basophilia in 2 micro sections, but parallel ultra-thin sections, stained with uranyl acetate and examined in the electron microscope, show no change in the ribonucleoprotein particles of the ergastoplasm. Desoxyribonuclease alone had no effect, but after pretreatment of the sections with pepsin or hydrochloric acid, desoxyribonuclease specifically attacked the nuclear chromatin. Nucleic acid-containing structures in the sections are gradually disintegrated by perchloric acid or hydrochloric acid.

Animals↗
PubMed2002

Sol-gel glass immunosorbent-based determination of s-triazines in water and soil samples using gas chromatography with a nitrogen phosphorus detection system.

A rapid and efficient method for the selective extraction of s-triazine herbicides in environmental samples was developed using an immunosorbent of monoclonal antiatrazine antibodies, which were encapsulated in a sol-gel glass matrix. The cross-reactivity of the antibody for analytes structurally related with atrazine enabled the simultaneous extraction of several s-triazine herbicides (atrazine, propazine, terbuthylazine, cyanazine, desethyl atrazine). After trace enrichment on the immunoextraction column, the s-triazines were desorbed by means of an acidic buffer (pH 2.5) and further extracted with ethyl acetate before being injected into the GC. Compared to liquid-liquid extraction and solid-phase extraction with a hydrophobic SDB-L support, the GC-NPD chromatograms obtained after immunoaffinity enrichment of surface water (river water) samples or soil extracts and analysis were free from matrix interferences. Nonspecific adsorption of humic acids was not observed. The method allows for the determination of the herbicides in linear ranges up to 1.5 microg/L with correlation coefficients higherthan 0.99 and relative standard deviations between 4% and 7% (n = 5). The LODs for 50-mL water samples were in the range 0.02 microg/L (atrazine, propazine) to 0.1 microg/L (desethyl atrazine) (S/N = 3). In addition to its high selectivity, the immunosorbent proved to be reusable for a significant number of preconcentration runs. However, the composition of samples may influence the lifetime of the column.

Chromatography, Gas↗
PubMed2005

Cholesterol-dependent hemolytic activity of Passiflora quadrangularis leaves.

Plants used in traditional medicine are rich sources of hemolysins and cytolysins, which are potential bactericidal and anticancer drugs. The present study demonstrates for the first time the presence of a hemolysin in the leaves of Passiflora quadrangularis L. This hemolysin is heat stable, resistant to trypsin treatment, has the capacity to froth, and acts very rapidly. The hemolysin activity is dose-dependent, with a slope greater than 1 in a double-logarithmic plot. Polyethylene glycols of high molecular weight were able to reduce the rate of hemolysis, while liposomes containing cholesterol completely inhibited it. In contrast, liposomes containing phosphatidylcholine were ineffective. The Passiflora hemolysin markedly increased the conductance of planar lipid bilayers containing cholesterol but was ineffective in cholesterol-free bilayers. Successive extraction of the crude hemolysin with n-hexane, chloroform, ethyl acetate, and n-butanol resulted in a 10-fold purification, with the hemolytic activity being recovered in the n-butanol fraction. The data suggest that membrane cholesterol is the primary target for this hemolysin and that several hemolysin molecules form a large transmembrane water pore. The properties of the Passiflora hemolysin, such as its frothing ability, positive color reaction with vanillin, selective extraction with n-butanol, HPLC profile, cholesterol-dependent membrane susceptibility, formation of a stable complex with cholesterol, and rapid erythrocyte lysis kinetics indicate that it is probably a saponin.

Animals↗
PubMed1985

Selective removal of plasma constituents potential application in the treatment of renal disease.

Perfectioning of the currently available techniques of selective extraction of plasma constituents by chemical, immunological and physical means either on-line or off-line is likely to have an impact on the current practice of plasmapheresis in renal disease. Because of its intrinsic advantages and the resemblance to standard dialysis techniques, hemoperfusion provides a model for future developments. Likely to find an application in the therapy of renal diseases are techniques of cascade filtration, cryogelation and filtration as well as more selective techniques such as removal of specific antibodies, G gammaglobulins, immune complexes and DNA antibodies.

Amino Acids↗
PubMed1986

Differential extraction of proteoglycans from cartilage tissue matrix compartments in isotonic buffer salt solutions and commercial tissue-culture media.

Small tissue blocks of native rat growth plate cartilage were incubated for short periods in one of several generally used isotonic buffer salt solutions or commercial tissue-culture media. The total percentage (approximately 12) of [35S]-labeled proteoglycans (PG) extracted from cartilage matrix under these conditions was not significantly influenced by either the chemical composition of the medium or the presence of a protease inhibitor. Morphological examination of incubated tissue after fixation in the presence of ruthenium hexamine trichloride (RHT) (included to preserve PG in situ) revealed, however, that the PG staining profiles across cartilage matrix varied with the composition of the incubation medium used. The various susceptibilities exhibited by PG within the different matrix compartments to selective extraction was estimated semi-quantitatively. The observed effects may prove useful in extracting these molecules differentially from cartilage matrix compartments.

Animals↗
PubMed2001

Solid-phase precipitation and extraction, a new separation process applied to the isolation of synthetic peptides.

A new method for separation and purification is described. The process, referred to as solid-phase precipitation and extraction (SPPE), was developed and applied to postcleavage isolation of synthetic peptides. The technique uses normal approaches of chromatography and solid-phase extraction sorbents with a precipitation or drying procedure so that the sorbent becomes a support matrix for thin-film deposition of the compounds of interest. This procedure causes precipitated compounds of interest to be trapped on the large surface area or in the pores of the matrix so that by-products and impurities can be removed by strong wash solvents. In application to solid-phase peptide synthesis chemistry, by-products from the cleavage and deprotection are selectively extracted from the crude sample mixture under mild conditions. In comparison to the ether precipitation method used in peptide chemistry, the SPPE process provides isolated products that are 14-17% (w/w) higher purity.

Amino Acid Sequence↗
PubMed1985

Purification of Legionella pneumophila major outer membrane protein and demonstration that it is a porin.

We have purified the major outer membrane protein (MOMP) of Legionella pneumophila, determined that it is associated with peptidoglycan, and characterized it as a porin. To purify the MOMP, we used a simple, rapid, three-step procedure that gave us the protein in high yield. The first step of the purification procedure involved selectively extracting the MOMP from whole bacterial cells with calcium and zwitterionic detergent. The second and third steps achieved purification by ion-exchange and molecular-sieve chromatography. The dissociation of the MOMP into monomers was dependent upon the presence of a reducing agent and was enhanced by treatment at 100 degrees C. To study the relationship of the MOMP to peptidoglycan, we extracted the protein by a modification of the Rosenbusch procedure. Like the Escherichia coli porins, the MOMP was peptidoglycan associated. The MOMP was at least partially dissociated from peptidoglycan in sodium dodecyl sulfate and a high salt concentration. To study the ion channel-forming properties of the MOMP, we reconstituted the MOMP in planar lipid membranes. The MOMP formed ion-permeable channels with a single-channel conductance size of 100 picoSiemens. The MOMP channels exhibited a fourfold selectivity for cations over anions and voltage-independent gating. These findings demonstrate that the MOMP is a porin with properties similar to those of E. coli porins.

Bacterial Outer Membrane Proteins↗
PubMed1998

Solid-phase microextraction with pH adjustment for the determination of aromatic acids and bases in water.

Adjusting the pH of water samples before performing solid-phase microextraction (SPME) analysis can be used to selectively extract organic acids (at pH 2) and bases (at pH 12). Sorption behavior of test organics is predictable based on the acid dissociation constant in water. In general, polyacrylate (PA) and Carbowax-divinylbenzene (CW-DVB) show substantially higher fiber/water sorption coefficients (Kd values) than a polydimethylsiloxane (PDMS) coated fiber. Gas chromatography-flame ionization detection (GC-FID) detection limits with the CW-DVB sorbent are approximately 0.5 to 10 ng/ml in a 2-ml water sample for a variety of aromatic amines, phenols, and chlorinated phenols, and are approximately 1 to 50 ng/ml for the same solutes using the PA sorbent. However, the PA fiber is more selective (depending on the water pH) for the acid or base components than the CW-DVB fiber. With proper pH adjustment, the recovery of spiked aromatic amines and phenols from a surface wetlands water ranged from 73 to 118% of the known values, with a precision (R.S.D.) of approximately 5 to 20%. SPME quantitation of phenols in a coal gasification wastewater using a PA fiber also gave excellent agreement with conventional methylene chloride extraction, although continued use of a single fiber with this wastewater led to poorer precision.

Acids↗
PubMed2005

Transport of organic solutes through amorphous teflon AF films.

Fluorous media have great potential for selective extraction (e.g., as applied to organic synthesis). Fluorous polymer films would have significant advantages in fluorous separations. Stable films of Teflon AF 2400 were cast from solution. Films appear defect-free (SEM; AFM). Rigid aromatic solutes are transported (from chloroform solution to chloroform receiving phase) in a size-dependent manner (log permeability is proportional to -0.0067 times critical volume). Benzene's permeability is about 2 orders of magnitude higher than in comparable gas-phase experiments. The films show selectivity for fluorinated solutes in comparison to the hydrogen-containing control. Transport rates are dependent on the solvent making up the source and receiving phases. The effect of solvent is, interestingly, not due to changes in partition ratio, but rather it is due to changes in the solute diffusion coefficient in the film. Solvents plasticize the films. A less volatile compound, -COOH-terminated poly(hexafluoropropylene oxide) (4), plasticizes the films (T(g) = -40 degrees C). Permeabilities are decreased in comparison to 4-free films apparently because of decreased diffusivity of solutes. The slope of dependence of log permeability on critical volume is not changed, however.

Benzene↗
PubMed1977

The surface of Neisseria gonorrhoeae: isolation of the major components of the outer membrane.

Outer membranes were isolated from several strains of Neisseria gonorrhoeae by extraction of whole cells with aqueous lithium acetate. The preparations contained a limited number of components including lipopolysaccharide and two major proteins. One protein was present in all strains examined; the second, which showed anomalous behaviour on sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was absent from some. Fluorescent labelling of intact N. gonorrhoeae showed that the two proteins and pili were the major surface proteins. Each of the three major outer membrane components was isolated in a homogeneous form by selective extraction followed by gel filtration.

Bacterial Proteins↗
PubMed1992

A high-mobility-group protein and its cDNAs from Drosophila melanogaster.

We have identified, purified, and characterized a high-mobility-group (HMG) protein and its cDNAs from Drosophila melanogaster. This protein, HMG D, shares most of the characteristics of vertebrate HMG proteins; it is extractable from nuclei with 0.35 M NaCl, is soluble in 5% perchloric acid, is relatively small (molecular weight of 12,000), has both a high basic (24%) and high acidic (24%) amino acid content, and is a DNA-binding protein. HMG D exhibits characteristics of both the vertebrate HMG 1 and 2 class and the HMG 14 and 17 class of proteins. Its amino acid sequence is similar (36% amino acid identity) to that of HMG1, while its size and selective extraction with ethidium bromide are similar to properties of the HMG 14 and 17 class of proteins. HMG D is encoded by a single-copy gene that maps to 57F8-11 on the right arm of chromosome 2. Two transcripts are observed during embryogenesis; the protein is relatively stable throughout development. By the biochemical criteria of size, solubility, and amino acid content, HMG D appears to be the major HMG protein of D. melanogaster.

Amino Acid Sequence↗
PubMed1982

Analyses of nicotine and cotinine in tissues by capillary gas chromatography and gas chromatography-mass spectrometry.

Selective extraction and chromatographic techniques have been developed to measure low nanogram quantities of nicotine and cotinine in tissues. Analyses were performed by capillary column gas chromatography with a specific nitrogen-phosphorus detector and by gas chromatography-mass spectrometry. With close structural analogues for internal standards, high quantitative accuracy and precision were demonstrated for the range 5-1000 ng per g of tissue. The sensitivity limit was 2-3 ng/g for both compounds. The advantage of these techniques compared to previously published methods is increased selectivity; the other methods were developed for analysis of biological fluids and are not readily adaptable to more complex biological matrices such as tissue homogenates. With the newly developed techniques, we were able to perform a pharmacokinetic study of nicotine and cotinine in mouse liver following a single intraperitoneal injection of nicotine.

Animals↗
PubMed2005

High-performance thin-layer chromatographic determination of lamotrigine in serum.

A simple and rapid high-performance thin-layer chromatographic (HPTLC) determination of lamotrigine (LTG) in serum is reported. The method involves extraction of the drug by ethyl acetate followed by separation on TLC silica plates using a mixture of toluene-acetone-ammonia (7:3:0.5), as eluting solvent. Densitometric analysis was carried out at 312 nm with lamotrigine being detected at Rf of 0.54. The analytical method has excellent linearity (r=0.998) in the range of 20-300 ng/spot. This assay range is adequate for analyzing human serum, as it corresponds to lamotrigine concentrations measured in human serum from epileptic patients. The method was validated for sensitivity, selectivity, extraction efficiency, accuracy and intra and inter-day reproducibility. The limit of detection and limit of quantification were found to be 6.4 and 10.2 ng, respectively. Good accuracy is reported in the range of 92.06-97.12% and high precision with %CV in range of 0.53-2.59. The method was applied for determination of serum lamotrigine levels in epileptic patients and in pharmacokinetic study of lamotrigine administered orally to rabbits.

Chromatography, High Pressure Liquid↗
PubMed1985

The 3' splice site of pre-messenger RNA is recognized by a small nuclear ribonucleoprotein.

A component present in splicing extracts selectively binds the 3' splice site of a precursor messenger RNA (pre-mRNA) transcript of a human beta-globin gene. Since this component can be immunoprecipitated by either autoantibodies of the Sm class or antibodies specifically directed against trimethylguanosine, it is a small nuclear ribonucleoprotein (snRNP). Its interaction with the 3' splice site occurs rapidly even at 0 degrees C, does not require adenosine triphosphate, and is altered by certain mutations in the 3' splice site region. Binding is surprisingly insensitive to treatment of the extract with micrococcal nuclease. The U5 particle is the only abundant Sm snRNP with a capped 5' end that is equally resistant to micrococcal nuclease. This suggests that, in addition to the U1 and U2 snRNP's, U5 snRNP's participate in pre-mRNA splicing.

Base Sequence↗