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Direct capture of product from fermentation broth using a cell-repelling ion exchanger.

A new technique for treating anion exchangers has been proposed allowing direct capture of the fermentation product, shikimic acid directly from the cell-containing fermentation broth. A layer of hydrophilic polymer, poly(acrylic acid) (PAA) has been physically adsorbed on the anion exchanger followed by a covalent cross-linking of PAA. The PAA layer is penetrable for small molecules despite being negatively charged as PAA is, but the polymer layer repels large negatively charged structures like cell debris and cells preventing them from adsorption to the chromatographic matrix. The binding capacity for pure shikimic was about 81 mg/ml adsorbent for both cross-linked PAA-Amberlite and native Amberlite in the fluidized mode of column operation. Binding capacity dropped to 17 and 15 mg per ml adsorbent, respectively, when using filtrated fermentation broth and to about 10 mg/ml adsorbent for cross-linked PAA-Amberlite when using directly the fermentation broth containing cells. Native Amberlite cannot be used for the direct capture of shikimic acid due to the immediate clogging of the column and the collapse of the expanded bed. The cross-linked PAA-Amberlite was used repeatedly for the direct adsorption of shikimic acid from the industrial fermentation broth.

Acrylic Resins↗

Structure-gas chromatographic electron capture sensitivity relationships of some substituted 17alpha-acetoxyprogesterones.

Structure-electron capture sensitivity relationships were established for underivatized 17alpha-acetoxyprogesterones. While progesterone was very insensitive, 17alpha-acetoxyprogesterone had a response of 4.8 X 10(2) C/mole. Methyl groups in the A or B ring of 17alpha-acetoxyprogesterone had no effect. A keto group at C-6 was 25 times more sensitive (1.2 X 10(4) C/mole). A double bond at C-6,7 enhanced the sensitivity sevenfold (3.5 X 10(3) C/mole), but double bonds at C-1,2 or C-9,11 had only slight effect. Substitution at C-16 was important. A methyl group at C-16 had two and three times the sensitivity in the 3-keto delta4 and 3-keto delta4,6 series (1.1 X 10(3) and 1.1 X 10(4) C/mole), respectively. A methylene group at C-16, in contrast showed a six-and twofold greater sensitivity over the C-16 methyl in the two series (7 X 10(3) and 2.2 X 10(4) C/mole), respectively. The most sensitive compound was 6-dehydro-6methyl-16-methylene-17alpha-acetoxyprogesterone (melengestrol acetate). Its sensitivity was 2.2 X 10(4) C/MOL, Comparable to the most sensitive halo esters of steroid alcohols reported in the literature. Its electron capture coefficient was 3-7.6X 10(10) 1/mole. The coef-icient was independent of the detector temperature, indicating low activation energy for electron absorpiton.

Chemical Phenomena↗

Pentafluorobenzoic anhydride as a derivatizing agent for alcohols and hydroxy fatty acid methyl esters detected by electron capture in gas chromatography.

Pentafluorobenzoate derivatives of primary and secondary alcohols have been prepared using pentafluorobenzoic anhydride. The gas chromatographic properties of the homologous series of the methyl esters of 2-hydroxycarboxylic acids from C12 to C26 have been studied on the high-temperature stationary phase Poly-S 179. The suitability of determining trace amounts of these compounds using gas chromatography with an electron-capture detector and with electron-capture negative-ion chemical-ionization mass spectrometry is discussed.

Anhydrides↗

Detection of trace levels of trichothecene mycotoxins in environmental residues and foodstuffs using gas chromatography with mass spectrometric or electron-capture detection.

Methods are described for the simultaneous detection of a wide range of trichothecenes, including the most polar ones and some macrocyclics, using either gas chromatography-mass spectrometry with selected ion monitoring, or gas chromatography with electron-capture detection. Trichothecenes were extracted directly from the various matrices, or from Clin Elut columns, and cleaned up on Florisil Sep-Pak cartridges. Macrocyclics and neosolaniol were detected after hydrolysis to verrucarol and T-2 tetraol respectively. For optimum sensitivity (0.5-10 ng per sample) over the range, trichothecenes were detected, both before and after hydrolysis of ester groups, as their heptafluorobutyrate derivatives using a quadrupole mass spectrometer and negative ion chemical ionisation. The use of a magnetic sector instrument with electron-impact ionisation gave comparable sensitivity for most trichothecenes, but was less useful for the simultaneous detection of verrucarol in the presence of other trichothecenes. The methods were used to detect the presence of scirpentriol, nivalenol and 15-monoacetoxyscirpendiol in sorghum from Thailand. Trichothecenes in less complex matrices could be detected, after hydrolysis, using gas chromatography with electron-capture detection.

Chemical Phenomena↗

Simple and rapid method for the determination of progesterone in rat plasma by gas-liquid chromatography with electron-capture detection.

A method for estimating progesterone in rat plasma using gas-liquid chromatography (GLC) with electron-capture detection is described. By means of a column chromatographic technique for the extraction of progesterone from plasma, it is possible to isolate the steroid in such a specific way that, contrary to previously published methods, no additional chromatographic purification and separation steps are needed prior to the final GLC analysis. As a derivative for electron-capture detection we used the 3,20-di-O-pentafluorobenzyloxime of progesterone. This derivative is superior to those used hitherto with regard to sensitivity and stability. The high overall recovery (mean value, 89.4%) makes this method especially convenient for research work when only little plasma is available, or when the concentration of progesterone is very low.

Animals↗

Preparation and characterization of prototypes for multi-modal separation media aimed for capture of negatively charged biomolecules at high salt conditions.

Several prototypes of multi-modal ligands suitable for the capture of negatively charged proteins from high conductivity (28 mS/cm) mobile phases were coupled to Sepharose 6 Fast Flow. These new prototypes of multi-modal anion-exchangers were found by screening a diverse library of multi-modal ligands and selecting anion-exchangers resulting in elution of test proteins at high ionic strength. Candidates were then tested with respect to breakthrough capacity of BSA in a buffer adjusted to a high conductivity (20 mM Piperazine and 0.25 M NaCl, pH 6.0). The recovery of BSA was also tested with a salt step (from 0.25 to 2.0 M NaCl using 20 mM Piperazine as buffer, pH 6.0) or with a pH-step to pH 4.0. We have found that non-aromatic multi-modal anion-exchange ligands based on primary or secondary amines (or both) are optimal for the capture of proteins at high salt conditions. Furthermore, these new multi-modal anion-exchange ligands have been designed to take advantage not only of electrostatic but also hydrogen bond interactions. This has been accomplished through modification of the ligands by the introduction of hydroxyl groups in the proximity of the ionic group. Experimental evidence on the importance of the relative position of the hydroxyl groups on the ligand in order to improve the breakthrough capacity of BSA has been found. Compared to strong anion-exchangers such as Q Sepharose Fast Flow the new multi-modal weak anion-exchangers have breakthrough capacities of BSA at mobile phases of 28 mS/cm and pH 6.0 that are 20-30 times higher. The new multi-modal anion-exchangers can also be used at normal anion-exchange conditions and with either a salt step or a pH-step to acidic pH can accomplish the elution of proteins. In addition, the functional performance of the new anion-exchangers was found to be intact after treatment in 1.0 M sodium hydroxide solution for 1 week. A number of multi-modal anion-exchange ligands based on aromatic amines exhibiting high breakthrough capacity of BSA have been found. With these ligands recovery was often found to be low due to strong non-electrostatic interactions. However, for phenol derived anion-exchange media the recovery can be improved by desorption at high pH.

Anion Exchange Resins↗

Atrial escape-capture bigeminy in dominant atrial rhythm with 2:1 exit block.

A 27-year-old woman with atrial bigeminy is reported in whom long PP intervals alternate with short PP intervals. All P waves are negative in lead II and all PR intervals measure 0.12 s. In the 12-lead electrocardiogram, however, these P waves were definitely different in configuration from each other, and were divided into two groups. Namely, these negative P waves are divided into those of dominant atrial rhythm J1 with 2:1 exit block, and those of atrial escape J2. Long J1-J2 intervals alternate with short J2-J1 intervals. These electrocardiographic findings show the presence of atrial escape-capture bigeminy. Such atrial escape-capture bigeminy in dominant atrial rhythm with 2:1 exit block has never been reported before.

Adult↗

Atrial escape capture bigeminy.

Atrial escape capture bigeminy is a rare electrocardiographic entity. A case of atrial escape sinus capture presenting as true atrial bigeminy is reported.

Aged↗

Prey capture in long-jawed butterflyfishes (Chaetodontidae): the functional basis of novel feeding habits.

Several species of butterflyfishes (Chaetodontidae) possess extremely elongate jaws, and feed mostly by probing the benthos and biting off pieces of attached invertebrates. In contrast, Forcipiger longirostris, the longest-jawed chaetodontid, exhibits a novel pattern of prey use, feeding almost exclusively on small caridean shrimp, a mobile and highly elusive prey type that lives within the structure of coral reefs. We explored the functional basis of this novel pattern of prey use by comparing prey capture kinematics in this and four other butterflyfish species, including two other species that possess elongate jaws. High speed video recordings of feeding events on live adult brine shrimp were analyzed from individuals of five species: Forcipiger longirostris, F. flavissimus, Chelmon rostratus, Heniochus acuminatus, and Chaetodon xanthurus. We focused on a comparison among species of the relative contribution of "suction", measured as the amount of movement of the prey toward the predator's mouth, and "ram", measured as the distance moved by the predator toward the prey during the strike. All five species utilized a combination of suction and ram while feeding on brine shrimp. The contribution of suction did not differ significantly among species. However, F. longirostris exhibited a ram contribution to the strike that was more than twice that seen in any of the other species, permitting this species to initiate strikes from the greatest initial predator-prey distance. F. longirostris is known to possess a major structural novelty in the feeding mechanism that permits anterior movement of the entire jaw apparatus. The ability of this species to feed successfully on elusive prey appears to be related to exceptional jaw protrusion, resulting in greater use of ram during prey capture. This ability to protrude long, slender jaws toward the prey may allow it to move the jaws without detection within close enough proximity of the prey to then permit the effective use of suction. The use of extensive ram in this manner by small-mouthed fishes may be more widespread than previously thought.

Journal Article↗

Detection of biotinylated cell surface receptors and MHC molecules in a capture ELISA: a rapid assay to measure endocytosis.

Cell surface receptors and antigens, such as TfR and MHC molecules, are endocytosed and subsequently redisplayed on the plasma membrane. The internalization and recycling of MHC molecules is thought to play an important role in antigen presentation, but studying this process has been hindered due to the lack of a rapid and easily quantitated assay. The combination of a cleavable biotin reagent to label surface molecules and a capture ELISA to detect these molecules of interest, allows for the quantitation of their cell surface expression, endocytosis and recycling. The endocytosis of TfR and MHC II molecules was readily quantitated in B cell lines using this procedure with results nearly identical to previously published data using more laborious radioactive methods. Evidence for the recycling of class II antigens and TfR back to the plasma membrane was obtained by monitoring the exit of these molecules from endosomes. Exposing cells to hypertonic media blocks clathrin-dependent endocytosis and was found to inhibit the internalization of MHC class II proteins on B cells. This flexible assay to capture and quantitate the cell surface expression and endocytosis of MHC molecules and other surface antigens offers a sensitive and non-radioactive alternative to study the intracellular trafficking of diverse membrane proteins.

Antigen Presentation↗

Production of glutathione-coated microtitre plates for capturing recombinant glutathione S-transferase fusion proteins as antigens in immunoassays.

Glutathione S-transferase (GST) is commonly used as a fusion partner in producing recombinant proteins and this technology is increasingly being used to produce antigens for use in immunoassays to measure antibodies. To circumvent the requirement to purify such antigens before use, we developed a method for coupling glutathione to microtitre plates so that GST-containing recombinant proteins could be purified and immobilised in one step in a suitable state for immunoassays. This procedure involves covalent linkage (using the heterobifunctional cross-linker sulphosuccinimidyl 4-(p-maleimidophenyl)butyrate) of reduced glutathione through its sulphydryl group to lysine residues of haemoglobin previously immobilised on microtitre plates. Haemoglobin was superior over other proteins tested in giving the lowest non-specific binding; in this regard it was also important to limit the amount of cross-linker used to 0.1 mM. Using glutamic acid decarboxylase as a model antigen, the new affinity capture assay was at least as good as the two-step procedure involving direct adsorption to plates of previously purified antigen; it may have the additional advantage of preserving the antigen in a more native conformation than direct adsorption. The new assay also performed as well as an assay using anti-GST antibodies adsorbed onto plates; glutathione plates, unlike anti-GST plates, will only capture recombinant proteins containing functional GST--a significant point for some recombinant expression systems in which a large proportion of the protein product is insoluble because of incorrect folding.

Autoantibodies↗

Quantitation of human haptoglobin: comparative ELISA studies using adsorption and capture methods.

Three ELISA methods for the quantitation of haptoglobin (Hp) in plasma and albumin are described: a polystyrene direct adsorption method and capture methods with antibody and hemoglobin. Hp aggregates generated by 60 degrees C heating showed as much as a hundred-fold higher response by polystyrene adsorption compared to the two capture methods, while unheated Hp showed comparable responses by the three methods.

Adsorption↗

An improved method for the immunological detection of mineral bound protein using hydrofluoric acid and direct capture.

Immunological detection of proteins adsorbed to mineral and ceramic surfaces has proved not only difficult but controversial. Unlike the immunological detection of proteins associated with carbonate or phosphate minerals (e.g. shells and bones) proteins adsorbed to siliceous minerals cannot readily be removed by dissolution of the mineral phase. We have previously examined alternative extraction methodologies which claim to bring the protein into solution, but found none of these to be effective. Here we report a novel strategy for immuno-detection of proteins adsorbed to siliceous minerals, the Digestion and Capture Immunoassay (DACIA). The method involves the use of cold, concentrated (4M) hydrofluoric acid (HF) with the simultaneous capture of liberated protein onto a solid phase. The combination of low temperatures and surface stabilisation enables us to detect epitopes from even partially degraded proteins. The method may have a wide application in forensic, archaeological, soil and earth sciences.

Adsorption↗

Particulate emboli capture by an intra-aortic filter device during cardiac surgery.

OBJECTIVE: Particulate embolization is associated with neurologic morbidity after cardiac surgery. Crossclamp manipulation has been identified as the single most significant cause of particulate emboli release during cardiac surgery. A new intra-aortic filtration method has been assessed with regard to its safety and its ability to capture particulate emboli before they enter the central circulation. METHODS: Patients undergoing cardiac surgery with cardiopulmonary bypass through standard median sternotomy were selected for emboli management by means of intra-aortic filtration. A novel intra-aortic filter device was inserted through a modified 24F arterial cannula immediately before releasing the crossclamp in 77 patients. Filters remained in the aorta until cardiopulmonary bypass was discontinued and the heart was fully ejecting. The procedure was assessed for facility, safety, and effect on routine cardiopulmonary bypass operation and function. RESULTS: The insertion and removal of the intra-aortic filter were safe, easy, and uneventful in most patients. Patient hemodynamics and bypass flow rates remained normal throughout the filter dwell period. No strokes or gross neurologic defects were noted. Electron microscopic analysis of 12 filters revealed an insignificant degree of platelet adhesion on filter surfaces. Histology samples (n = 44) were examined, and 66% (n = 29) showed evidence of atheromatous material, 36% (n = 16) with platelet-fibrin, 25% (n = 11) with true thrombus and/or blood clot, 7% (n = 3) with normal vessel wall, and 2% (n = 1) with aggregates of cholesterol or grumous portion of atheromatous plaque. CONCLUSION: The intra-aortic filter can be safely deployed and captures particulate emboli, the predominant origin of which is atheromatous. The beneficial effects of this device on neurologic outcomes have yet to be determined.

Adult↗

Lipoprotein lipase-enhanced binding of lipoprotein(a) [Lp(a)] to heparan sulfate is improved by apolipoprotein E (apoE) saturation: secretion-capture process of apoE is a possible route for the catabolism of Lp(a).

Recently, it has been recognized that cell-bound heparan sulfate (HS) proteoglycans (HSPG) are able to bind and subsequently initiate degradation of lipoproteins. Two mediators of lipoprotein catabolism, both with HS binding capacity, lipoprotein lipase (LPL) and apolipoprotein E (apoE), are involved in this process. This mechanism is known as the secretion-capture process of apoE. Lipoprotein(a) [Lp(a)] was shown to have a strong binding capacity to cell-associated HSPG. This binding capacity was increased by LPL addition. We investigated the effects of recombinant apoE (r-apoE) enrichment of Lp(a) on the binding to HS. Lp(a), isolated by ultracentrifugation and gel filtration, was incubated with r-apoE and reisolated by ultracentrifugation, resulting in r-apoE-enriched Lp(a). ApoE-enriched Lp(a) and control Lp(a) were coated to microtiter plates. The capacity to bind biotin-conjugated HS (b-HS) in the presence or absence of inactivated bovine LPL was studied. R-apoE-enriched Lp(a) showed increased b-HS binding capacity versus control Lp(a). Addition of LPL resulted in an increased b-HS binding capacity of both control and r-apoE-enriched Lp(a). To investigate whether binding of Lp(a) to endothelial cell HSPG occurred in vivo, 39 volunteers were injected with heparin (50 U/kg) and plasma lipid and Lp(a) levels were determined before and 20 minutes after heparin injection. No significant increase in plasma Lp(a) concentrations was found. The results showed that Lp(a) can be enriched with apoE and that this resulted in increased LPL-enhanced binding to HSPG. From the in vitro studies, it can be concluded that the secretion-capture process of apoE is a possible catabolic route for Lp(a). However, whether this also occurs in vivo remains to be confirmed.

Animals↗

Development of an immunomagnetic antigen capture system for detecting leptospires in bovine urine.

A magnetic bead antigen capture system which combined the use of two evolving techniques - immunomagnetic separation (IMS) and time-resolved fluoroimmunoassay (TR-FIA) - was developed to detect Leptospira borgpetersenii serovar hardjo in bovine urine. The assay utilised monoclonal antibody coated magnetic beads to capture leptospiral antigen which was in turn detected using another monoclonal antibody (Indicator) labelled with biotin. Signal was generated by the binding of europium labelled streptavidin to indicator antibody. The sensitivity of the assay was improved from 10(3) to 10(2) leptospires per ml by using an ethanol precipitation procedure to treat each sample. The assay detected only 31 of 56 (55 per cent) urine specimens culture-positive for hardjo, but seven of 24 urine samples culture-negative for hardjo were identified as positive by the assay. These seven samples were from animals which were culture positive on at least one other occasion. These results suggest that this system should be further investigated as a complementary test to culture for the identification of hardjo carrier animals.

Animals↗

"Phase capture" in the perception of interpolated shape: cue combination and the influence function.

This study was concerned with what stimulus information observers use to judge the shape of simple objects. We used a string of four Gabor patches to define a contour. A fifth, center patch served as a test pattern. The observers' task was to judge the location of the test pattern relative to the contour. The contour was either a straight line, or an arc with positive or negative curvature (the radius of curvature was either 2 or 6 deg). We asked whether phase shifts in the inner or outer pairs of patches distributed along the contour influence the perceived shape. That is, we measured the phase shift influence function. We found that shifting the inner patches of the string by 0.25 cycle results in almost complete phase capture (attraction) at the smallest separation (2 lambda), and the capture effect falls off rapidly with separation. A 0.25 cycle shift of the outer pair of patches has a much smaller effect, in the opposite direction (repulsion). In our experiments, the contour is defined by two cues--the cue provided by the Gabor carrier (the 'feature' cue) and that defined by the Gaussian envelope (the 'envelope' cue). Our phase shift influence function can be thought of as a cue combination task. An ideal observer would weight the cues by the inverse variance of the two cues. The variance in each of these cues predicts the main features of our results quite accurately.

Contrast Sensitivity↗

Merger of laser capture microdissection and mass spectrometry: a window into the amyloid plaque proteome.

The occurrence of protein accumulation and aggregation in the brain is one of the pathological hallmarks of neurodegenerative diseases such as Alzheimer's disease (AD). Although it is instructive to analyze the aggregated proteins in the brain, biochemical purification and identification of these proteins have been challenging. Recent developments in laser capture microdissection (LCM) and mass spectrometry (MS) enable large-scale protein profiling of captured tissue samples. We present here the method of analyzing senile plaques from postmortem AD brains by coupling LCM and highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS). First, the senile plaques were stained with thioflavin-S and precisely isolated by adjusted laser beams under a microscope. Total proteins in the isolated tissues were extracted and resolved on an SDS gel. To identify all proteins in the samples, the gel was excised into multiple pieces followed by trypsin digestion. The resulting peptides were further separated by reverse-phase chromatography and analyzed by tandem mass spectrometry. A database search of acquired MS/MS spectra allowed the identification of hundreds to thousands of peptides/proteins in the original samples. Moreover, quantitative comparison of protein composites in different LCM samples could be achieved by MS strategies. For instance, the comparison between plaques and surrounding nonplaque tissues from the same specimen revealed tens of proteins specifically enriched in the plaques. Finally, the data were corroborated by independent experiments using the approach of immunohistochemistry. Taken together, the merger of LCM and MS is a powerful tool to probe the proteome of any given pathological lesions.

Animals↗