Improved Procedure For Isolation Research Articles

Characterization of VIM-1-Producing E. coli Isolated From a German Fattening Pig Farm by an Improved Isolation Procedure.

A few reports indicate that livestock might represent a new reservoir for carbapenemase-producing Enterobacteriaceae (CPE). In 2015, VIM-1-producing Escherichia coli were detected at slaughter in colon contents of animals from a German fattening pig farm within the national monitoring on ESBL-producing E. coli. In this study, pooled faces samples from pigs, as well as samples from the barn surrounding environment of this fattening farm were taken, to evaluate the dissemination of CPEs. Several modifications of the culture-dependent detection procedure were investigated for their potential to improve the sensitivity of the CPE isolation method. The current reference procedure was adapted by adding a real-time PCR pre-screening and additional enrichment steps. It was possible to isolate 32 VIM-1-producing E. coli from four fecal samples of three different barns using two serial enrichment steps in combination with real-time PCR and selective agar plates. By genetic typing, we confirmed the presence of two E. coli clonal lineages circulating on this particular farm: one was harboring the blaVIM–1 on an IncHI2 plasmid while the second lineage carried the gene on the chromosome. Despite its different locations, the blaVIM–1 gene was harbored on a class 1 integron in both clonal lineages. Whole-genome sequencing revealed that the VIM-1-carrying plasmids exhibited only slight variability in its compositions and sizes. We assume that the prevalence of CPEs in animal production in Germany and other European countries might be underestimated and there is a concern of further spread of VIM-1-producing bacteria in German livestock and food.

Improved isolation procedure for shikonin from the root of the Chinese medicinal plant Lithospermum erythrorhizon and its solubilization with cyclodextrins

In this study, we improved the method for the isolation and purification of the bioactive compound shikonin from the dried root of the traditional Chinese medicinal plant Lithospermum erythrorhizon without a tedious chromatographic separation. Furthermore, we also attempted the solubilization of shikonin in water with cyclodextrins (CDs) using a solid phase “high-speed vibration milling”. Among the various CDs, sulfobutylether-β-CD (SBEβ-CD) showed a highest solubilizing ability and 52.5% of shikonin was solubilized with equimolar amount of SBEβ-CD. In addition, the SBEβ-CD·shikonin complex showed higher apoptogenic activity than that of the same concentration of free shikonin against murine primary peritoneal macrophages. These results indicate that SBEβ-CD is a suitable excipient for clinical application of shikonin.

Novel MtCEP1 peptides produced in vivo differentially regulate root development in Medicago truncatula.

Small, post-translationally modified and secreted peptides regulate diverse plant developmental processes. Due to low natural abundance, it is difficult to isolate and identify these peptides. Using an improved peptide isolation protocol and Orbitrap mass spectrometry, nine 15-amino-acid CEP peptides were identified that corresponded to the two domains encoded by Medicago truncatula CEP1 (MtCEP1). Novel arabinosylated and hydroxylated peptides were identified in root cultures overexpressing MtCEP1. The five most abundant CEP peptides were hydroxylated and these species were detected also in low amounts in vector control samples. Synthetic peptides with different hydroxylation patterns differentially affected root development. Notably, the domain 1 peptide hydroxylated at Pro4 and Pro11 (D1:HyP4,11) imparted the strongest inhibition of lateral root emergence when grown with 5mM KNO3 and stimulated the highest increase in nodule number when grown with 0mM KNO3. Inhibition of lateral root emergence by D1:HyP4,11 was not alleviated by removing peptide exposure. In contrast, the domain 2 peptide hydroxylated at Pro11 (D2:HyP11) increased stage III-IV lateral root primordium numbers by 6-fold (P < 0.001) which failed to emerge. Auxin addition at levels which stimulated lateral root formation in wild-type plants had little or no ameliorating effect on CEP peptide-mediated inhibition of lateral root formation or emergence. Both peptides increased and altered the root staining pattern of the auxin-responsive reporter GH3:GUS suggesting CEPs alter auxin sensitivity or distribution. The results showed that CEP primary sequence and post-translational modifications influence peptide activities and the improved isolation procedure effectively and reproducibly identifies and characterises CEPs.

Residual keratan sulfate in chondroitin sulfate formulations for oral administration

Chondroitin sulfate is a biomedical glycosaminoglycan (GAG) mostly used as a dietary supplement. We undertook analysis on some formulations of chondroitin sulfates available for oral administration. The analysis was based on agarose-gel electrophoresis, strong anion-exchange chromatography, digestibility with specific GAG lyases, uronic acid content, NMR spectroscopy, and size-exclusion chromatography. Keratan sulfate was detected in batches from shark cartilage, averaging ∼16% of the total GAG. Keratan sulfate is an inert material, and hazardous effects due to its presence in these formulations are unlikely to occur. However, its unexpected high percentage compromises the desired amounts of the real ingredient specified on the label claims, and forewarns the pharmacopeias to update their monographs. The techniques they recommended, especially cellulose acetate electrophoresis, are inefficient in detecting keratan sulfate in chondroitin sulfate formulations. In addition, this finding also alerts the manufacturers for improved isolation procedures as well as the supervisory agencies for better audits. Analysis based on strong anion-exchange chromatography is shown to be more reliable than the methods presently suggested by standard pharmacopeias.

Improved Isolation Procedure for Azaspiracids from Shellfish, Structural Elucidation of Azaspiracid-6, and Stability Studies

Azaspiracids are a group of lipophilic polyether toxins produced by the small dinoflagellate Azadinium spinosum. They may accumulate in shellfish and can result in illnesses when consumed by humans. Research into analytical methods, chemistry, metabolism, and toxicology of azaspiracids has been severely constrained by the scarcity of high-purity azaspiracids. Consequently, since their discovery in 1995, considerable efforts have been made to develop methods for the isolation of azaspiracids in sufficient amounts and purities for toxicological studies, in addition to the preparation of standard reference materials. A seven-step procedure was improved for the isolation of azaspiracids-1-3 (1, 2, and 3) increasing recoveries 2-fold as compared to previous methods and leading to isolation of sufficiently purified azaspiracid-6 (6) for structural determination by NMR spectroscopy. The procedure, which involved a series of partitioning and column chromatography steps, was performed on 500 g of Mytilus edulis hepatopancreas tissue containing ~14 mg of 1. Overall yields of 1 (52%), 2 (43%), 3 (43%), and 6 (38%) were good, and purities were confirmed by NMR spectroscopy. The structure of 6 was determined by one- and two-dimensional NMR spectroscopy and mass spectrometry. The stability of 6 relative to 1 was also assessed in three solvents in a short-term study that demonstrated the greatest stability in aqueous acetonitrile.

MRSA: why have we got it and can we do anything about it?

MRSA, first identified in 1960, became a major cause of healthcare-associated infection with the emergence of epidemic strains EMRSA 15 and 16 in the 1990 s. MRSA bacteraemia surveillance in England showed a peak of 7700 in 2003-2004. A target was set to halve MRSA bacteraemias by 2008 backed by a central improvement programme for infection prevention and control. Healthcare-associated infection is a patient safety issue with joint responsibility between: clinicians responsible for patient care; managers responsible for the organisation of services; and the government/Department of Health responsible for national strategy, prioritisation and performance management, together with introducing a statutory Code of Practice. By 2011, the number of MRSA bacteraemias had reduced by 80% to 1481. The key drivers of improvement were management responsibility, enhanced surveillance, adherence to clinical protocols and care bundles for invasive procedures, hand hygiene and environmental cleaning, and improved isolation procedures and antibiotic stewardship. The target has been translated into an ongoing MRSA objective, and further control of MRSA is supported by a screening programme aimed at all relevant hospital admissions. Sustaining the reduction will depend upon joint responsibility between management maintaining compliance assurance with policies and individual clinicians keeping it as a priority in patient safety.

An Improved Isolation Procedure for Adult Mouse Cardiomyocytes

Isolated adult mouse cardiomyocytes are an important tool in cardiovascular research, but are challenging to prepare. Because the energy supply determines cell function and viability, we compared total creatine ([Cr]) and [ATP] in isolated cardiomyocytes with the intact mouse heart. Isolated myocytes suffered severe losses of Cr (-70%) and ATP (-53%). Myocytes were not able to replete [Cr] during a 5h incubation period in medium supplemented with 1mM Cr. In contrast, adding 20mM Cr to the digestion buffers was sufficient to maintain normal [Cr]. Supplementing buffers with 5mM of inosine (Ino) and adenosine (Ado) to prevent loss of cellular nucleosides partially protected against loss of ATP. To test whether maintaining [ATP] and [Cr] improves contractile function, myocytes were challenged by varying pacing rate from 0.5 to 10Hz and by adding isoproterenol (Iso) at 5 and 10Hz. All groups performed well up to 5Hz, showing a positive cell shortening-frequency relationship; however, only 16% of myocytes isolated under standard conditions were able to sustain pacing with Iso challenge at 10Hz. In contrast, 30-50% of the myocytes with normal Cr levels were able to contract and maintain low diastolic [Ca(2+)]. Cell yield also improved in Cr and the Cr/Ino/Ado-treated groups (85-90% vs. 70-75% rod shaped in untreated myocytes). These data suggest that viability and performance of isolated myocytes are improved when they are protected from the severe loss of Cr and ATP during the isolation, making them an even better research tool.

Intracellular, time-resolved speciation and quantification of arsenic compounds in human urothelial and hepatoma cells

In contrast to the temporary administration of arsenic in cancer therapy (e.g. in the form of arsenic trioxide), chronic exposure to low doses can cause bladder cancer and other cancers. Especially the trivalent arsenic species MMA(III) (monomethylarsonous acid) and DMA(III) (dimethylarsinous acid) are known to be highly toxic. In the present study we analysed the soluble, intracellular biotransformation products of MMA(III) in methylating HepG2 (hepatocytes) and non-methylating UROtsa cells (urothelial cells) after various times of exposure. As most of the intracellular arsenic is bound to cellular structures and proteins the soluble arsenic metabolites can hardly be speciated and even less quantified. Using an improved isolation procedure and HPLC-ICP/MS, we investigated the time-resolved biotransformation of MMA(III) and detected and quantified MMA(V) (monomethylarsonic acid) as an oxidation product of MMA(III) and, to a minor degree, DMA(V) (dimethylarsenic acid) as a methylation and oxidation product of MMA(III) in the lysates of HepG2 cells. In contrast, only MMA(V) but no DMA(V) was detected in the lysates of UROtsa cells. We conclude from our study that MMA(III) is taken up by HepG2 and UROtsa cells and immediately oxidized to MMA(V). Only in HepG2 cells MMA(V) is finally methylated to DMA(V) over time. The new method might help to advance the analysis of metabolic pathways of arsenic in mammalian cells.

Isolation of specific cranberry flavonoids for biological activity assessment

Abstract Characterisation of cranberry compound biological activity is constrained by limitations in isolation methodology. A single rapid procedure for polyphenolic isolation was developed using semi-preparative-HPLC. Non-flavonoid compounds were removed by pre-purification procedures prior to semi-preparative-HPLC. Fractions were analysed to ascertain purity (99%) with HPLC and ESI mass spectrometric detection in negative ion mode and on-line diode array ultraviolet–visible spectroscopy. Isolated cranberry flavonols included quercetin-3-galactoside, quercetin-3-glucoside, quercetin-3-rhamnoside, myricetin-3-galactoside, myricetin-3-arabinofuranoside, and quercetin-3-O-(6″-p-benzoyl)-β-galactoside. Proanthocyanidin isolates contained monomers, dimers, trimers, and larger polymers. Anthocyanins consisted largely of galactoside and arabinoside conjugates of cyanidin and peonidin. Identities were confirmed using 1D-NMR- and 2D-NMR-spectrometry as well as reference standards. Flavonol fractions exhibited the highest antioxidant activity in a dosedependent manner, while the anthocyanin fraction exhibited the least activity. Biological activity studies of cranberry phenolics will benefit from the improved isolation procedures described in this study and the confirmation of antioxidant activities of various cranberry constituents.

Purification and proteomic characterization of plastids from Brassica napus developing embryos

Plastids are functionally and structurally diverse organelles responsible for numerous biosynthetic reactions within the plant cell. Plastids from embryos have a range of properties depending upon the plant source but compared to other plastid types are poorly understood and therefore, we term them embryoplasts. Isolating intact plastids from developing embryos is challenging due to large starch granules within the stroma and the prevalence of nonplastid, storage organelles (oil bodies and protein storage vacuoles) which compromise plastid integrity and purity, respectively. To characterize rapeseed embryoplasts it was necessary to develop an improved isolation procedure. A new method is presented for the isolation of intact plastids from developing embryos of Brassica napus seeds. Intactness and purity of embryoplast preparations was determined using phase-contrast and transmission electron microscopy, immunoblotting, and multidimensional protein identification technology (MudPIT) MS/MS. Eighty nonredundant proteins were identified by MudPIT analysis of embryoplast preparations. Approximately 53% of these proteins were components of photosystem, light harvesting, cytochrome b/f, and ATP synthase complexes, suggesting ATP and NADPH production are important functions for this plastid type.

Quantification of 3-aminopropionamide in cocoa, coffee and cereal products

Based on recent results confirming 3-aminopropionamide (3-APA) as a very effective precursor of acrylamide in the absence of further “catalysts”, this compound was quantified for the first time in cocoa masses, cocoa beans, coffee and cereal products by LC–MS–MS after derivatisation with dansyl chloride. Cocoa masses contained >3000 μg/kg of 3-APA, but varied significantly in its concentration. For the quantification of acrylamide (AA) in cocoa and coffee, an improved isolation procedure using charcoal was developed. In various samples of unroasted and roasted cocoa beans, the concentrations of AA were by a factor of >5 lower than those of 3-APA, but the concentrations of 3-APA and AA were more closely correlated as compared to the concentrations of AA and Asparagine. Experiments on authentic cocoa beans from Ghana and Sulawesi indicated that the thermal generation of 3-APA during roasting was much more pronounced as compared to its biochemical formation. By administering fermented cocoa beans with [13C4 15N2]-asparagine before roasting, 3-APA was confirmed as transient intermediate in AA formation during cocoa roasting. Among the cereal products analysed, in particular popcorn contained quite high amounts of 3-APA, which were also well correlated with the AA concentration. Contrary, in coffee products, 3-APA was always lower than AA.

An evaluated improvement of the extinction dilution method for isolation of ammonia-oxidizing bacteria

An improved method for isolation of ammonia-oxidizing bacteria (AOB) by the extinction dilution technique is described. It is important to prevent the growth of heterotrophic organisms, which may easily outnumber the AOB in mixed cultures. This was achieved by careful elimination of C sources in the medium and by sealing the cultures from contact with the atmosphere, thus excluding air-borne, volatile compounds which support growth of heterotrophs. The sealing of the cultures reduced the number of heterotrophs by a factor of 10, thus grossly increasing the chances of obtaining pure AOB cultures. Another important factor is to use actively growing ‘late log’ cultures during the final isolation step. This was achieved by adjusting the buffer capacity to ensure a clearly visible pH indicator shift at a stage when one-third to one-half of the ammonia had been oxidized. By this improved isolation procedure, AOB were isolated from three different locations: an arable soil, a lead-contaminated soil and an animal house. For an unknown reason, several attempts to isolate pure cultures from a forest soil were unsuccessful, despite the presence of AOB in the primary extinction dilution cultures. The isolates from soils were all Nitrosospira spp. For isolation of AOB from the animal house, two growth media were used, one containing ammonium sulfate, and one containing urea. From the cultures with ammonium sulfate, Nitrosomonas spp. were isolated, whereas Nitrosospira spp. were isolated from the cultures with urea as the main ammonia source. The identifications of all isolates are based on morphology and 16S rDNA sequences.

Expression of activation and costimulatory elements by human intestinal intraepithelial lymphocytes.

It is unclear whether human intestinal intraepithelial T lymphocytes (iIEL) are resting or activated cells. To address this question, an improved isolation procedure was developed for small bowel iIEL, which were analysed by two-colour flow cytometry and compared with resting and mitogen-activated peripheral blood lymphocytes. iIEL expression of CD44 isoforms, Bcl-2 and Ki67 antigen was also determined in tissue sections. iIEL expressed CD69 at levels comparable with 48-72 h phytohaemagglutinin blasts, but did not express CD25 or CD95. iIEL were Bcl-2+ but not Ki67+. alphaEbeta7 and alpha4B7 expression was relatively high, whereas alphaLbeta2, CD5 and CD28 were expressed at low density. Isolated iIEL expressed CD44 (core epitopes) at lower levels than peripheral blood lymphocytes, although almost all CD44 contained splice variant 6 (CD44v6). Peripheral blood lymphocytes expressed CD44 at very high density, but little CD44v6, even after activation. However, in tissue sections, iIEL showed differential labelling with CD44 core epitope antibodies and no detectable CD44v6, implying CD44 receptor occupancy or epitope masking in situ. Thus, normal iIEL express a quasi-activated phenotype with unusual patterns of adhesion receptors, which may act as costimulatory elements. These may permit iIEL to assume effector functions, with absence of CD25 preventing entry into the cell cycle, thereby maintaining an apoptosis-resistant phenotype.

Reactions involving fluoride ion. Part 45 . An approach to surface treatment using perfluoro-(isopropyl)- s-triazines

Perfluoro-(isopropyl)- s-triazines 3– 5 have been synthesised by two methods, both involving fluoride ion induced reaction with hexafluoropropene. In reactions initiated by potassium fluoride, extraction of the perfluoroalkylated products from the polar aprotic solvent can be achieved using a perfluorocarbon fluid offering an improved isolation procedure. Perfluoroalkylation initiated by triethylamine in the absence of solvent provides alternative methodology. Both methods are much improved processes for scale-up. Perfluoro-(isopropyl)- s-triazines 3 and 4 react with a range of oxygen nucleophiles and application of this chemistry to the surface treatment of cellulose is described.

Purification and characterization of rat hippocampal CA3-dendritic spines associated with mossy fiber terminals

We report a revised and improved isolation procedure for CA3-dendritic spines, most of them still in association with mossy fiber terminals resulting in a 7.5-fold enrichment over nuclei and a 29-fold enrichment over myelin. Additionally, red blood cells, medullated fibers, mitochondria and small synaptosomes were significantly depleted. We show by high resolution electron microscopy that this subcellular fraction contains numerous dendritic spines with a rich ultrastructure, e.g. an intact spine apparatus, membranous organelles, free and membrane-bound polyribosomes, endocytic structures and mitochondria. This improved experimental system will allow us to study aspects of post-synaptic functions at the biochemical and molecular level.

Structural organization of the major subunits in cyanobacterial photosystem 1. Localization of subunits PsaC, -D, -E, -F, and -J.

Based on an improved isolation procedure using perfusion chromatography, trimeric Photosystem 1 (PS1) complexes have been isolated from various deletion mutants of the mesophilic cyanobacterium Synechocystis PCC 6803. These mutants are only deficient in the deleted subunits, which was carefully checked by high resolution gel electrophoresis in combination with immunoblotting. These highly purified and well characterized PS1 particles were then examined by electron microscopy, followed by computer-aided image processing with single particle averaging techniques as described earlier (Kruip, J., Boekema, E. J., Bald, D., Boonstra, A. F., and Rögner, M. (1993) J. Biol. Chem. 268, 23353-23360). This precise methodological approach allowed a confident localization of the PS1 subunits PsaC, -D, -E, -F, and -J; it also shows shape and size of these subunits once integrated in the PS1 complex. Subunits PsaC, -D, and -E form a ridge on the stromal site, with PsaE toward the edge of each monomer within the trimer and PsaD extending toward the trimeric center, leaving PsaC in between. PsaF (near PsaE) and PsaJ are close together on the outer edge of each monomer; their proximity is also supported by chemical cross-linking, using the zero-length cross-linker EDC. This localization of PsaF contradicts the position suggested by the published low resolution x-ray analysis and shows for the first time the existence of at least one transmembrane alpha-helix for PsaF. A topographic three-dimensional map has been drawn from this set of results showing the location of the major PS1 subunits (besides PsaA and PsaB). These data also led to the assignment of electron density in the recent medium resolution x-ray structure for PS1 (Krauss, N., Schubert, W.-D., Klukas, O., Fromme, P., Witt, H. T., Saenger, W. (1996) Nat. Struct. Biol. 3, 965-973).

Isolation of Chloroflexus aurantiacus and related thermophilic phototrophic bacteria from Japanese hot springs using an improved isolation procedure.

The multicellular filamentous green bacteria, Chloroflexus species, inhabit natural hot springs. For the isolation of these bacteria, a new isolation method was developed using an organic medium and culture under alternating aerobic and anaerobic conditions. The improved method reliably provided a high rate of isolation of the thermophilic photoheterotrophs. Ten strains that may belong to this genus were isolated from Japanese hot springs in various areas. All these new isolates were thermophilic photosynthetic bacteria that were morphologically similar to Chloroflexus aurantiacus. The isolates were also similar to C. aurantiacus in photosynthetic pigments and guanine plus cytosine (G+C) contents. The results of DNA-DNA hybridization and quinone composition analysis indicated that eight of the new strains belong to C. aurantiacus, while two strains (MD-66 and YI-9) which have the ability to form bacterial mat-like dense aggregates, were significantly different from C. aurantiacus.

Algal carotenoids 47–NMR study of all‐trans‐peridinin, including complete 1H and 13C NMR assignments

AbstractThe allenic C37 skeletal carotenoid all‐trans‐(3S, 5R, 6R, 3′S, 5′R, 6′S)‐peridinin, isolated from Amphidinium carterae by an improved isolation procedure, was subjected to a detailed 1H and 13C NMR spectroscopic analysis. Complete assignments of the 500.13 MHz 1H and 125.76 MHz 13C spectra, including determination of coupling constants and relative stereochemistry, were obtained by ID and 2D techniques. These included 1H1H chemical shift correlated spectroscopy (2D COSY), phase‐sensitive double‐quantum COSY, difference 1D NOE experiments, 1D and 2D total correlation spectroscopy (HOHAHA, TOCSY) and 1D and 2D rotating frame nuclear Overhauser spectroscopy (ROESY, CAMELSPIN). The 13C NMR assignments were obtained through editing sequences (DEPT) and heterocorrelated 2D experiments. In particular, the 1H detected multiple bond 1H13C correlated 2D experiment proved valuable for assignments of all 13 quatenary carbons together with 13 methine carbons in peridinin [C37H47O6(COCH3)]. Detailed analysis of the proton coupling constants supported the conformations and relative stereochemistry of the two end‐groups. Spectral simulation of protons in the conjugated chain was necessary in order to ascertain more accurate chemical shifts for some protons in the strongly overlapping olefinic region. Variation in the trans double bond 3J(H, H) coupling was noted along the conjugated chain.

The Application of Solid Phase Extraction to the Analysis of Tobacco-Specific Nitrosamines

A new isolation and separation method has been developed for the alkaloid-related nitrosamines in tobacco, called tobacco-specific nitrosamines (TSNA). The improved isolation procedure involves the extraction by sonication of TSNA from cured tobacco with toluene and an aqueous citrate buffer solution, while purification is achieved by solid phase extraction on small silica cartridges. Interfering extract materials are eluted from the silica cartridges with the toluene and a chloroform-methylene chloride solvent, while subsequent elution with chloroform yields the TSNA in a purified fraction. Gas chromatographic analyses on an SE-54-coated glass capillary column and detection by a nitrogen-phosphorous detector produce very good data on the four TSNAs, which include N-nitrosanatabine (NATB), N-nitrosoanabasine (NAB), 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (called NNK), and N-nitrosonornicotine (NNN). The method is shown to be quantitative, reproducible, and applicable to the determination of TSNA in various tobacco samples, such as stems and lamina of cured tobacco leaves, as well as to smokeless tobacco.

C1: molecular interactions with activating systems

The molecular events controlling complement activation have been gradually unravelled over the past three decades, stimulated by improved isolation procedures and a better understanding o f the roles o f individual proteins. In this review, Bob Sim and Ken Reid examine the interactions between Clq and its numerous ligands in the initiation o f the classical pathway cascade.