Chromatographic separation and identification of nucleotides from animal tissues and yeast cells on a polyethyleneimine cellulose column.

A procedure has been developed for the separation and identification of nucleotides by anion-exchange chromatography. The method includes column chromatography on PEI-cellulose with an unbuffered exponential salt gradient. This easily handled procedure gives a high degree of resolution and reproducibility and demands no personal attendance during elution. Application of the method is demonstrated with a mouse liver extract and extracts of yeast cells (Saccharomyces cerevisiae).     

Chromatography of Cytokinins on a Neutral Polystyrene Resin: A Simple Procedure for the Separation of the cis and trans Isomers of Zeatin or Ribosylzeatin

A simple procedure for the separation of the cis and trans isomers of zeatin and ribosylzeatin by column chromatography on a neutral polystyrene resin, Porapak Q, in aqueous ethanol solutions is reported. The method has been used to examine the stereoisomer composition of ribosylzeatin isolated from wheat germ transfer RNA. Chromatographic data for several other cytokinins are also presented.     

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.     

Identification of proteins according to biological activity following separation by two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis: analysis of human exocrine pancreatic proteins

A novel procedure is described whereby proteins can be identified according to their biological activity after their separation in two dimensions using isoelectric focusing and polyacrylamide gel electrophoresis in sodium dodecyl sulfate (G. Scheele, 1975, J. Biol. Chem., 250, 5375–5385). This procedure includes an optimal staining method for the visualization of two-dimensional gel spots, which avoids the use of chemical fixatives, and a one-step method for elution and renaturation of proteins. Fifteen out of the twenty discrete proteins separated from human pancreatic juice by the two-dimensional gel method were successfully identified by this procedure.     

High-performance liquid chromatographic separation of human haemoglobins: Simultaneous quantitation of foetal and glycated haemoglobins

A high-performance liquid chromatographic system, which uses a weak cation exchanger (PolyCATA) together with Bis-Tris buffer (pH 6.47–7.0) and sodium acetate gradients, is described. Samples from adults and newborns were analysed and a clean separation of many minor and major normal and abnormal haemoglobin (Hb) variants was greatly improved. The method allows the separation of minor foetal haemoglobin (HbF) variants and the simultaneous quantitation of HbF and glycated HbA. HbF values correlated well with those obtained by the alkali denaturation method (r=0.997). The glycated haemoglobin (HbAr_Ic) levels measured in patients with high HbF concentrations correlated with the total glycated haemoglobin determined by bioaffinity chromatography (r=0.973). The procedure is useful for diagnostic applications and affords an effective and sensitive way of examining blood samples for haemoglobin abnormalities.     

Separation of long RNA by agarose–formaldehyde gel electrophoresis

We describe a method to facilitate electrophoretic separation of high-molecular-weight RNA species, such as ribosomal RNAs and their precursors, on agarose–formaldehyde gels. Two alternative “pK-matched” buffer systems were substituted for the traditionally used Mops-based conductive medium. The key advantages include shortened run times, a 5-fold reduction in formaldehyde concentration, a significantly improved resolution of long RNAs, and consistency in separation. The new procedure has a streamlined work flow that helps to minimize errors and is broadly applicable to agarose gel electrophoresis of RNA samples and their subsequent analysis by Northern blotting.     

Preparation and characterization of radioactive monoiodotyrosine and diiodotyrosine derivatives of parathyroid hormone

Highly purified native parathyroid hormone was iodinated by the enzymatic method and separated from unlabeled hormone by isocratic HPLC. The separation system used also resolved iodohistidine, monoiodotyrosine, and diiodotyrosine forms of the hormone from one another. A simplified procedure for direct bioassay of the carrier-free, high specific activity, mono- and diiodinated parathyroid hormone (PTH) by the renal membrane adenylyl cyclase method was also developed. Both labeled forms of the hormone are very potent in this assay, but the iodinated forms appeared to give a lower V_max than the native hormone. The methods for iodination, separation and biological characterization of this PTH tracer are exceptionally facile, inexpensive, and convenient.     

Use of High-Affinity Cell Wall-Binding Domains of Bacteriophage Endolysins for Immobilization and Separation of Bacterial Cells

Immobilization and magnetic separation for specific enrichment of microbial cells, such as the pathogen Listeria monocytogenes, depends on the availability of suitable affinity molecules. We report here a novel concept for the immobilization and separation of bacterial cells by replacing antibodies with cell wall-binding domains (CBDs) of bacteriophage-encoded peptidoglycan hydrolases (endolysins). These polypeptide modules very specifically recognize and bind to ligands on the gram-positive cell wall with high affinity. With paramagnetic beads coated with recombinant Listeria phage endolysin-derived CBD molecules, more than 90% of the viable L. monocytogenes cells could be immobilized and recovered from diluted suspensions within 20 to 40 min. Recovery rates were similar for different species and serovars of Listeria and were not affected by the presence of other microorganisms. The CBD-based magnetic separation (CBD-MS) procedure was evaluated for capture and detection of L. monocytogenes from artificially and naturally contaminated food samples. The CBD separation method was shown to be superior to the established standard procedures; it required less time (48 h versus 96 h) and was the more sensitive method. Furthermore, the generalizability of the CBD-MS approach was demonstrated by using specific phage-encoded CBDs specifically recognizing Bacillus cereus and Clostridium perfringens cells, respectively. Altogether, CBD polypeptides represent novel and innovative tools for the binding and capture of bacterial cells, with many possible applications in microbiology and diagnostics.     

Improved Method for HPLC Analysis of Polyamines, Agmatine and Aromatic Monoamines in Plant Tissue

The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucus carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues.     

Improvement of the Immunomagnetic Separation Method Selective for Escherichia coli O157 Strains

Immunomagnetic separation is a useful enrichment method selective for Escherichia coli O157 cells against non-O157 E. coli cells from a preenrichment culture. However, E. coli cells are adsorbed onto a solid surface nonspecifically. With the conventional immunomagnetic separation method, this nonspecific adsorption interfered with immunomagnetic separation. It was found that this interference could be reduced with a low-ionic-strength solution. When immunomagnetic separation was carried out with this solution, the proportion of E. coli O157 cells to non-O157 E. coli cells increased from 9.6 to 31.4 times compared to the proportion obtained by the conventional immunomagnetic separation method. The effectiveness of this solution was successfully evaluated by the use of E. coli O157-spiked samples.     

High-pressure liquid chromatographic determination of 13-cis-retinoic acid and all-trans-retinoic acid in human plasma

A simple and selective method is described for the determination of 13-cis-retinoic acid and all-trans-retinoic acid in plasma. The assay involves lyophilization of the sample, extraction of the residue with methanol, and separation of the retinoids by reverse-phase high-pressure liquid chromatography. The procedure can detect as little as 0.05 μg of either compound. It is capable of being readily modified to determine the amount of retinoic acid in other tissues as well as tissue concentrations of other retinoids. Using this assay, the absorption and disappearance from human plasma of an oral dose of 13-cis-retinoic acid have been followed.     

Approach to the large-scale preparation of highly pure phosphatidylserine from bovine brain

An approach to the large-scale preparation of highly pure phosphatidylserine from bovine brain is described in this paper. The method is based on (i) the separation of phosphatidylserine from phosphatidylinositol in bovine brain extract by preparative aminopropyl normal-phase high-performance liquid chromatography using methanol-1 1 M phosphoric acid (90:10, v/v) as mobile phase (ii)_further purification of phosphatidylserine by anion-exchange chromatography. The main advantage of this approach is that polyunsaturated acid-containing molecular species of brain phosphatidylserine are not lost in the preparation procedure.     

Microdetermination of unbound tryptophan in plasma by a combination of ultrafiltration and high-performance liquid chromatography

Previous methods for measuring unbound plasma tryptophan are not completely satisfactory, and therefore we have developed an improved method for this purpose. Unbound tryptophan is separated from bound tryptophan by centrifugation through Amicon ultrafiltration membrane cones in a short period (within 2 min). The first fraction of filtrate is obtained in 30 s by centrifugation at 3000g at controlled pH and is assayed with a high-performance liquid chromatography system. The first filtrate has a higher tryptophan concentration than that obtained by prolonged centrifugation. We propose that the tryptophan concentration in the first filtrate is the value nearest that of the plasma, since a change of equilibrium between bound and unbound tryptophan during the separation procedure is quite small. The method is also simple and convenient for clinical application.     

Agar gel chromatography of rat liver ribosomal ribonucleic acids.

Ribosomal RNA (28s rRNA) of rat liver is selectively retained in 2 or 4% squashed agar gels equilibrated at 24–25°C with a sodium dodecyl sulfate-Tris-EDTA buffer containing 0.7 m NaCl. The absorbed polynucleotide could be recovered by elution with 0.1 m NaCl in the same buffer. Agar-gel electrophoresis and nucleotide composition indicate that the separation is close to quantitative. Column beds of 100–200 ml were used in the range of 3–6 mg of RNA. The procedure is simple, rapid and reproducible and gives excellent separation of 28 and 18s rat liver rRNA.     

Purification of the glial fibrillary acidic protein by anion-exchange chromatography

A procedure for the isolation of assembly-competent glial fibrillary acidic (GFA) protein from 2 m urea extracts of bovine spinal cord by anion-exchange chromatography is reported. The tissue was previously extracted with low-ionic-strength buffer. The procedure allowed the separation of nondegraded GFA protein from GFA protein comprising degraded species. As previously reported for neurofilament preparations obtained from porcine spinal cord (N. Geisler and K. Weber, J. Mol. Biol., 151, 565–571 (1981)), the procedure also allowed the simultaneous separation of the three neurofilament polypeptides (200,000; 150,000; and 70,000 daltons) contained in the 2 m urea extract. Brain filament proteins sequentially eluted at increasing salt concentration (25–200 mm NaCl) according to their isoelectric point. Proteins with higher pI eluted first. Tubulin eluted between the 200,000- and 150,000-dalton neurofilament polypeptides.     

COVASIAM: an Image Analysis Method That Allows Detection of Confluent Microbial Colonies and Colonies of Various Sizes for Automated Counting

In this work we introduce the confluent and various sizes image analysis method (COVASIAM), an automated colony count technique that uses digital imaging technology for detection and separation of confluent microbial colonies and colonies of various sizes growing on petri dishes. The proposed method takes advantage of the optical properties of the surfaces of most microbial colonies. Colonies in the petri dish are epi-illuminated in order to direct the reflection of concentrated light coming from a halogen lamp towards an image-sensing device. In conjunction, a multilevel threshold algorithm is proposed for colony separation and counting. These procedures improved the quantification of colonies showing confluence or differences in size. We tested COVASIAM with a sample set of microorganisms that form colonies with contrasting physical properties: Saccharomyces cerevisiae, Aspergillus nidulans, Escherichia coli, Azotobacter vinelandii, Pseudomonas aeruginosa, and Rhizobium etli. These physical properties range from smooth to hairy, from bright to opaque, and from high to low convexities. COVASIAM estimated an average of 95.47% (ς = 8.55%) of the manually counted colonies, while an automated method based on a single-threshold segmentation procedure estimated an average of 76% (ς = 16.27) of the manually counted colonies. This method can be easily transposed to almost every image-processing analyzer since the procedures to compile it are generically standard.     

Immunomagnetic Separation Combined with Polymerase Chain Reaction for the Detection of Alicyclobacillus acidoterrestris in Apple Juice

A combination of immunomagnetic separation (IMS) and polymerase chain reaction (PCR) was used to detect Alicyclobacillus acidoterrestris (A. acidoterrestris) in apple juice. The optimum technological parameters of the IMS system were investigated. The results indicated that the immunocapture reactions could be finished in 60 min and the quantity of IMPs used for IMS was 2.5 mg/mL. Then the combined IMS-PCR procedure was assessed by detecting A. acidoterrestris in apple juice samples. The agarose gel electrophoresis results of 20 different strains showed that the IMS-PCR procedure presented high specificity to the A. acidoterrestris. The sensitivity of the IMS-PCR was 2×10¹ CFU/mL and the total detection time was 3 to 4 h. Of the 78 naturally contaminated apple juice samples examined, the sensitivity, specificity and accuracy of IMS-PCR compared with the standardized pour plate method were 90.9%, 97.0% and 96.2%, respectively. The results exhibited that the developed IMS-PCR method will be a valuable tool for detecting A. acidoterrestris and improving food quality in juice samples.     

Chromatographic analysis of griseofulvin and metabolites in biological fluids

A simple and accurate assay for the determination of griseofulvin and its metabolites in biological fluids using high-performance liquid chromatography is described. Using a reversed phase column and a mobile phase solvent of 45% acetonitrile in 0.1 M acetic acid, baseline separation of griseofulvin and several analogues was obtained. The described method allows one to quantitatively determine griseofulvin, 6-demethylgriseofulvin, and griseofulvic acid, a newly identified metabolite in man, in urine and plasma samples. Treatment of plasma samples prior to the analysis is simply made by deproteinizing the samples with an equal volume of acetonitrile. For urine samples, the procedure involves diethyl ether extraction with subsequent evaporation to dryness and reconstitution with the mobile phase solvent.     

Determination of putrescine N-methyltransferase by high performance liquid chromatography

A novel procedure is described for the chemical synthesis of N-methylputrescine, the product of the title enzyme. This is obtained from putrescine by formylation followed by the reduction of the monoformylputrescine intermediate with LiA1H₄. An assay method for putrescine N-methyltransferase was developed which depends on the determination of N-methylputrescine in the presence of an excess of putrescine. This method, which makes use of a radiolabeled substrate unnecessary, is based on dansylation of the product followed by HPLC separation on a reversed-phase column. The enzyme activity of the protein peak extracted from plant material was measured after treatment by gel filtration on prepacked disposable PD 10 columns. The specific enzyme activities determined in the extract from the roots of Nicotiana tabacum and Datura stramonium plants, and from a root culture of D. stramonium, are reported. With an enzyme preparation from the last root culture, K_m values for putrescine and S-adenosylmethionine (SAM) were determined as 0.88 mM and 0.15 mM, respectively.     

Assay for human erythrocyte pyridoxine kinase

An accurate and rapid method for the assay of pyridoxine kinase in human erythrocytes has been developed. The procedure involves the separation of the radioactive product from the substrate with Dowex 50 resin in a test tube. Using the assay designed, we found that human red blood cells have a pyridoxine kinase activity of 1.381 nmole/min/g of hemoglobin (n = 25, SE = 0.051), and the enzyme has a K_m of approximately 1.72 × 10^?6m for pyridoxine. Pyridoxine phosphate was identified as the main product of the assay reaction catalyzed by human erythrocyte pyridoxine kinase in crude hemolysates.