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PubMed1987

Organ extraction of atrial natriuretic peptide (ANP) in man. Significance of sampling site.

Arterial plasma immunoreactivity of endogenous human alpha-atrial natriuretic peptide (ANP) underwent mean 54%, 28% and 40% extraction during one passage through the circulation in the kidney (n = 12), liver-intestine (n = 14) and lower limb (n = 8), respectively, in supine fasting subjects with no detectable disease or subjects with cardiovascular or hepatic disorders of minor degree undergoing a haemodynamic investigation. No extraction was identified across the lungs as evaluated by the same concentration of ANP in pulmonary and femoral arteries (n = 7). The concentration of ANP in a superficial arm vein relative to the femoral artery varied considerably and extractions from 0% up to 58% were identified (mean 18%). The results suggest a high degree of, but only to some extent selective, extraction of ANP, which may account for its proposed short plasma half-life. Due to the different concentrations of ANP in various vascular beds, sampling site should be thoroughly specified.

Adult↗

PubMed1997

Enantioseparation using apoenzymes immobilized in a porous polymeric membrane.

Chemical separations represent a large portion of the cost of bringing any new pharmaceutical product to the market. Membrane-based separation technologies, in which the target molecule is selectively extracted and transported across a membrane, are potentially more economical and easier to implement than competing separations methods; but membranes with higher transport selectivities are required. Here we describe an approach for preparing highly selective membranes which involves immobilizing apoenzymes within a microporous composite. The apoenzyme selectively recognizes its substrate molecule and transports it across the composite membrane, without effecting the unwanted chemical conversion of the substrate molecule to product. We demonstrate this approach using three different apoenzymes. Most importantly, it can be used to make enantioselective membranes for chiral separations, one of the most challenging and important problems in bioseparations technology. We are able to achieve a fivefold difference between the transport rates of D- and L-amino acids.

Alcohol Dehydrogenase↗

PubMed1981

A radioimmunoassay for ethinyl oestradiol in plasma incorporating an immunosorbent, pre-assay purification procedure.

A radioimmunoassay for plasma ethinyl oestradiol, featuring an immunosorbent extraction procedure, is described. Ethinyl oestradiol (EE2) was extracted using a non-specific, anti-oestrogen serum, raised to an oestradiol-17-hemisuccinate conjugate. The antiserum, coupled to microcrystalline cellulose, selectively extracted EE2 but not norethisterone ( NE), thus conferring specificity on a radioimmunoassay which has previously exhibited unacceptably high cross-reactivity with the synthetic progestagen, norethisterone, often used concomitantly with ethinyl oestradiol. This radioimmunoassay was shown to fulfil accepted assay validation criteria. Levels in subjects not receiving EE2 were less than 25 pmol/l. Circulating concentrations of EE2 could therefore be accurately determined in patients receiving low-dose combined preparations (EE2 35 micrograms; NE 500 micrograms).

Antigen-Antibody Complex↗

PubMed1983

Mouse skin tumor initiation-promotion and complete carcinogenesis bioassays: mechanisms and biological activities of emission samples.

Extracts of soots obtained from various sources were applied to the skin of mice in an effort to identify carcinogens in these mixtures and to link these materials to the etiology of human cancer. Samples of coal chimney soot, coke oven materials, industrial carbon black, oil shale soot, and gasoline vehicle exhaust materials have been examined by this method. The studies reported here have been constructed to compare the carcinogenic and tumorigenic potency of extracts from various particulate emissions: coke ovens, diesel and gasoline vehicles and a roofing tar pot. Automobile emission samples were obtained by collecting the diluted and cooled exhaust on Teflon-coated glass fiber filters. Coke oven and roofing tar samples were particulate emission samples collected by impaction and filtration. The organic components associated with each of the particles were extracted with dichloromethane and dermally applied to SENCAR mice. All agents were applied as tumor initiators by using a five-dose protocol. Selected extracts were also applied as complete carcinogens and as tumor promotors. Statistical analyses of the resulting tumor data were performed by using nonlinear Poisson and probit models. The results from these experiments provide a suitable data base for comparative potency estimation of complex mixtures.

Air Pollutants↗

PubMed1985

Use of digitonin extraction to distinguish between autophagic-lysosomal sequestration and mitochondrial uptake of [14C]sucrose in hepatocytes.

In isolated rat hepatocytes, electroinjected [14C]sucrose is sequestered both by mitochondria and by autophagosomes/lysosomes. Radioactivity can be selectively extracted from the latter organelles by low concentrations of digitonin, thereby providing a specific bioassay for autophagic sequestration. By including a digitonin extraction step in the assay procedure, autophagic [14C]sucrose sequestration could be shown to be virtually completely (greater than 90%) suppressed by the autophagy inhibitor 3-methyladenine (10 mM), whereas mitochondrial sugar uptake was unaffected. An amino acid mixture likewise suppressed autophagic sequestration very strongly, while having no detectable effect on the mitochondria.

Adenine↗

PubMed1981

Preparation and characterization of antisera to the myelin-associated glycoprotein.

Rabbits were immunized with the myelin-associated glycoprotein (MAG) that had been purified from isolated rat brain myelin by selective extraction with lithium diiodosalicylate (LIS) and phenol followed by preparative SDS gel electrophoresis. Antibodies to MAG were detected qualitatively by immunodiffusion and quantitatively by a double antibody assay utilizing [3H]fucose-labeled MAG as antigen. The antisera were capable of precipitating between 300 and 500 micrograms of MAG/ml of serum under the conditions of the assay. Preincubation of the anti-MAG serum with other glycoproteins or glycolipids did not inhibit the precipitation of labeled MAG. Similarly, preincubation of the antiserum with LIS-phenol extracts of non-neural tissues did not inhibit the immune precipitation of MAG. The specificity of the antiserum was also indicated by the selective double antibody precipitation of MAG from solubilized whole myelin that contained a heterogeneous mixture of [3H]fucose-labeled glycoproteins. The antibodies to MAG were not effectively absorbed by whole brain homogenate or purified myelin, indicating that the antigenic site(s) is not accessible in the intact membranes, but can be exposed by treatment with detergent or partial purification. Low levels of antibodies reacting with MAG were detected in three rabbits with experimental allergic encephalomyelitis induced by injection of purified myelin in complete Freund's adjuvant.

Animals↗

PubMed2005

Down-modulation of nitric oxide production in murine macrophages treated with crude plant extracts from the Brazilian Cerrado.

Several plant species from the Cerrado biome in Brazil are popularly used as herbal medicines for its reputed analgesic, anti-acid, anti-microbial, anti-inflammatory and anti-tumoral properties, among others. It has been reported that some plant extracts interfere in the production of nitric oxide (NO), an important inflammatory mediator. In the present study, we investigated the effect of hexanic and ethanolic extracts from three plant species on NO production by LPS/IFN-gamma-activated J774 macrophages based on traditional use. The cytotoxic effect of the crude extracts was determined by the thiazolyl blue test (MTT) to measure cell viability. Serjania lethalis stem extracts and Cupania vernalis leaf extracts significantly inhibited NO production, while extracts from Casearia sylvestris var. lingua were inactive or showed low activity on NO production, or were very cytotoxic. The ethanolic stem bark and leaf extracts of Serjania lethalis and Cupania vernalis, respectively, almost completely inhibited the production of NO by J774 macrophages. It can be concluded that the selected extracts are potential sources of active compounds that might be used as anti-inflammatory agents.

Animals↗

PubMed2003

Extraction of 15 microcystins and nodularin using immunoaffinity columns.

Microcystins (MCYSTs) were isolated from surface water using reusable immunoaffinity columns. Individual MCYST were determined by high performance liquid chromatography equipped with a photo-diode array detector (HPLC-PDA, 200-300 nm). Subsequent analysis of the samples by liquid chromatography-electrospray ionization mass spectrometry (LC-ESMS) provided molecular weight information, which was used to tentatively identify individual MCYST variants for which standards were not available. Results obtained using immunoaffinity columns (IAC)-HPLC-PDA were compared to those obtained using solid phase extraction (SPE) Oasis HLB-HPLC-PDA. This is the first report of the extraction of 15 microcystins and nodularin using immunoaffinity columns. Whereas previous reports demonstrates the use of IAC for four microcystins, we found that IAC selectively extracted the following microcystins: MCYST-RR, [D-Asp3]MCYST-RR, MCYST-YR, MCYST-LR, 3 MCYST-LR variants, MCYST-AR, MCYST-FR, MCYST-WR, MCYST-LA, MCYST-LA variant, the less polar microcystins such as MCYST-LF, MCYST-LW and nodularin. The IAC extracts were free of interferences which enabled better detection and identification of MCYSTs. Based on the amount loaded to the cartridges, the method detection limit was 10-14 ng when using IAC and 25 ng for SPE of each MCYST-RR, MCYST-YR and MCYST-LR. Reproducibilities expressed as relative standard deviation were 6-10% for SPE and 4-17% for IAC.

Antibodies↗

PubMed1983

Advances in fused-silica column technology for the analysis of underivatized drugs.

The chemical durability of a cross-linked 5% phenyl methylsilicone stationary phase is investigated for the analysis of underivatized drugs. Column performance is tested with respect to quantitative reproducibility, solvent extractability, selectivity for various drug families, and capacity for the polar solute. Data are presented showing the effects of a polar solvent and a serum extract on column lifetime with splitless and cool on-column injection. Examples of a human drug screen and the forensic analysis of street heroin are provided.

Alkaloids↗

PubMed2005

StarD10, a START domain protein overexpressed in breast cancer, functions as a phospholipid transfer protein.

We originally identified StarD10 as a protein overexpressed in breast cancer that cooperates with the ErbB family of receptor tyrosine kinases in cellular transformation. StarD10 contains a steroidogenic acute regulatory protein (StAR/StarD1)-related lipid transfer (START) domain that is thought to mediate binding of lipids. We now provide evidence that StarD10 interacts with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by electron spin resonance measurement. Interaction with these phospholipids was verified in a fluorescence resonance energy transfer-based assay with 7-nitro-2,1,3-benzoxadiazol-4-yl-labeled lipids. Binding was not restricted to lipid analogs since StarD10 selectively extracted PC and PE from small unilamellar vesicles prepared with endogenous radiolabeled lipids from Vero monkey kidney cells. Mass spectrometry revealed that StarD10 preferentially selects lipid species containing a palmitoyl or stearoyl chain on the sn-1 and an unsaturated fatty acyl chain (18:1 or 18:2) on the sn-2 position. StarD10 was further shown to bind lipids in vivo by cross-linking of protein expressed in transfected HEK-293T cells with photoactivable phosphatidylcholine. In addition to a lipid binding function, StarD10 transferred PC and PE between membranes. Interestingly, these lipid binding and transfer specificities distinguish StarD10 from the related START domain proteins Pctp and CERT, suggesting a distinct biological function.

Animals↗

PubMedDate not supplied

Application of supercritical fluid extraction in biotechnology.

In the present paper recent investigations on the applications of supercritical fluid extraction (SCE) from post fermentation biomass or in situ extraction of inhibitory fermentation products as a promising method for increasing the yield of extraction have been reviewed. Although supercritical CO2 (SC-CO2) is unfriendly, or even toxic, for some living cells and precludes direct fermentation in dense CO2, it does not rule out other useful applications for in situ extraction of inhibitory fermentation products and fractional extraction of biomass constituents. This technique is a highly desirable method for fractional extraction of biomass constituents, and intracellular metabolites due to the potential of system modification by physical parameters and addition of co-solvents to selectively extract compounds of different polarity, volatility and hydrophilicity without any contamination.

Biopolymers↗

PubMed1992

A differential location of phosphoinositide kinases, diacylglycerol kinase, and phospholipase C in the nuclear matrix.

Several enzymes involved in the phosphoinositide metabolism have been shown to be present in nuclei of rat liver and Friend cells. In this paper we demonstrate that nuclear matrices of mouse NIH 3T3-fibroblasts and rat liver cells, isolated by nuclease treatment and high salt extraction, contain phosphatidylinositol 4-kinase (PdtIns 4-kinase), phosphatidylinositol 4-phosphate 5-kinase (PtdIns(4)P 5-kinase), diacylglycerol kinase, and phospholipase C. By a selective extraction the nucleus can be dissected in the peripheral matrix (lamina-pore complex) and the internal matrix as shown by using marker antibodies. Surprisingly, PtdIns 4-kinase was found exclusively in the peripheral nuclear matrix, whereas PtdIns(4)P 5-kinase was found to be associated to internal matrix structures. Diacylglycerol kinase and phospholipase C activities were also preferentially detected in the internal matrix. These data demonstrate a differential localization of the phosphoinositide kinases in the nucleus and suggest that the phosphoinositide metabolism may play a specific role in the nucleus.

1-Phosphatidylinositol 4-Kinase↗

PubMed1982

Basic dyes for the staining of DNA in mammalian tissues and absorption spectra of stained nuclei in the visible light.

This communication presents informations on the staining of DNA with basic dyes, such as ethyl violet, Janus green, a new red dye obtained from Janus green, and Giemsa, belonging to different chemical groups. It has been found that DNA of tissue sections from which RNA has been extracted selectively with cold concentrated phosphoric acid can be stained with aqueous solutions of these dyes. Further, DNA of tissue sections from which RNA has been extracted and the sections then hydrolysed in 6 N HCl at 28 degrees C for 15 min can also be stained with aqueous solutions of ethyl violet, Janus green, and Giemsa. Moreover, tissue sections that have been hydrolysed in 6 N HCl and then stained with aqueous solutions of these dyes, including the new red dye obtained from Janus green, reveal well-stained nuclei. The absorption spectra of nuclei stained with aqueous solutions of ethyl violet, Janus green and Giemsa, following the above stated three procedures of staining have been presented. Absorption data of nuclei stained with the new red dye after Feulgen hydrolysis of tissue sections as well as those of nuclei in tissue sections from which RNA has been extracted have also been presented herein. The implications of these findings have been discussed.

Animals↗

PubMed2006

A general approach for the quantitative analysis of bisphosphonates in human serum and urine by high-performance liquid chromatography/tandem mass spectrometry.

Bisphosphonates are extremely hydrophilic and structurally similar to many endogenous phosphorylated compounds, making their selective extraction from serum or urine very challenging. Many bisphosphonates lack strong chromophores for sensitive UV or fluorescence detection. We report here the first general approach to enable sensitive and selective quantitation of N-containing bisphosphonates by liquid chromatography/tandem mass spectrometry (LC/MS/MS) following derivatization with diazomethane. The novelty of the strategy lies in performing the derivatization on silica-based anion-exchange sorbents as an integrated step in the sample purification by solid-phase extraction (SPE). The 'on-cartridge' reaction with diazomethane not only led to higher efficiency of derivatization, but also enabled a more discriminatory recovery of the drug's derivatives. The derivatized bisphosphonates demonstrated improved chromatographic separation and increased sensitivity of the detection. The general applicability of the approach was demonstrated by validation of bioanalytical methods for risedronate and alendronate in human serum and urine. Sensitivity was achieved at the pg/mL level with merely 100-200 microL of sample.

Alendronate↗

PubMed1975

Immunologic properties of bacterial lipopolysaccharide (LPS). II. The unresponsiveness of C3H/HeJ Mouse spleen cells to LPS-induced mitogenesis is dependent on the method used to extract LPS.

The C3H/HeJ mouse strain, previously shown to be a nonresponder to bacterial lipopolysaccharide (LPS)-induced mitogenesis in vitro, was demonstrated by the present studies to be competent to respond mitogenically to LPS, but only to LPS preparations obtained by selected extraction methods. These preparations appear to be confined to LPS isolated by mild extraction techniques, such as TCA or butanol. In contrast, those obtained by techniques utilizing phenol were only weakly stimulatory or completely nonstimulatory for spleen cells from the C3H/HeJ. All LPS preparations tested, on the other hand, were highly stimulatory for cells from another mouse strain, namely the C3H/St. The critical importance of the method of extraction of LPS on its mitogenic activity for C3H/HeJ cells was stressed by experiments in which LPS was prepared from Escherichia coli K235 using either of two procedures. In these experiments, phenol-extracted LPS, although mitogenic in the C3H/St, was completely nonstimulatory in the C3H/HeJ; whereas, butanol-extracted LPS was highly stimulatory in both strains of mice. This striking difference was attributed to a destructive effect of phenol on LPS, as demonstrated by the fact that treatment of butanol LPS with phenol resulted in a total loss of its mitogenic activity in the C3H/HeJ, but in only a partial loss in the C3H/St. In general, the mitogenic response observed with selected LPS preparations in the C3H/HeJ was quantitatively lower and more transient than that seen with the C3H/St, although qualitatively these responses appeared to be similar. This was evidenced by the observation that in both mouse strains LPS was a specific mitogen for B cells, a property which was also attributed in both strains to the same distinct structural region of the LPS molecule, that is lipid A. A preparation of LPS that failed to stimulate B cells from the C3H/HeJ nonetheless had the capacity to block activation of these B cells by a stimulatory preparation of LPS. These results strongly suggest that mitogenic stimulation of B cells by LPS is a function of the structural integrity of both the LPS molecule and putative B-cell receptors for LPS.

Animals↗

PubMed1990

UDP-glucuronyltransferase activity toward harmol in human liver and human fetal liver cells in culture.

This paper presents a fast HPLC assay for measuring UDP-glucuronyltransferase (UDPGT) activity in extracts of adult human liver and human fetal liver cells in culture. Harmol glucuronide formed was quantitated directly without prior hydrolysis after a simple step of selective extraction of harmol. The method is applicable to crude liver homogenates as well as to partially fractionated preparations. It is several fold more sensitive than the conventional detection of harmol glucuronide by TLC, making it possible to distinguish the low and high affinity forms of UDPGT of adult human liver and to detect the low affinity form in fetal cells. The possible participation of both forms of GT in adults under different conditions and the apparent lack of the high affinity form in the fetal liver is discussed.

Alkaloids↗

PubMed1976

Selective effects of nonionic detergent and salt solutions in dissolving nuclear envelope protein.?20U.

Protein has been selectively extracted from isolated chicken erythrocyte nuclear envelope by (1) dilute MgCl2/Triton X-100 followed by (2) concentrated MgCl2/Triton X-100 solutions. Certain proteins appear to be selectively dissolved in the first solvent and may occur in the nuclear envelope primarily as lipoproteins. Among the proteins insoluble in the low MgCl2/Triton X-100 wash, as well as in 500 mM MgCl2 without Triton previously used in the preparation of the envelope fraction, the quantitatively major polypeptides dissolve in a combination of high MgCl2 and Triton X-100. Further, much of this dissolved protein precipitates when the MgCl2 concentration is lowered by dialysis. The insolubility of these proteins appears to result from a combination of ionic and hydrophobic interactions and may explain the resistance of nuclei to various manipulative procedures including nonionic detergent washes. The procedures described provide a route for gently and selectively dissolving representative proteins from the nuclear envelope lipoprotein matrix and from the envelope "residual" protein.

Animals↗

PubMed1997

Analytical extraction of regulatory peptides from rat lung tissue.

We evaluated protocols for the extraction of calcitonin gene-related peptide, neuropeptide Y, substance P, peptide YY and beta-endorphin from rat lung tissue for subsequent radioimmunoassay. The effects of varying acidity of the extraction solution and repeating extraction on the recovery of peptide immunoreactivity and non-specific tracer-binding were compared by analysis of variance. Moreover, variability of immunoreactivity was quantified for comparison. Considering all three criteria, the optimal acidity for extraction was: 0.1 M or 1 M acetic acid for CGRP and beta-endorphin, 0.1 M acetic acid for NPY, 1 M acetic acid for substance P and phosphate buffer for peptide YY. Double or combined extraction unambiguously improved assay results only for substance P. Reversed-phase high-performance liquid chromatography of CGRP-, NPY- and SP-immunoreactivity obtained from selected extracts suggested that differences in recovery of these peptides are not explainable by differential peptide fragmentation during extraction.

Animals↗