O-(fluoresceinylmethyl)hydroxylamine (OFMHA): a reagent for the preparation of fluorescent O-(fluoresceinylmethyl)oxime (FMO)-steroid conjugates.

The 5 and 6-isomers of O-(fluoresceinylmethyl)hydroxylamine reacted with a representative sample of oxo-steroids (6-oxoestradiol, estrone, norethindrone, cortisol, progesterone, and digitoxin-dialdehyde) to produce O-(fluoresceinylmethyl)oxime conjugates in a single step in 24-84% yield after preparative high performance liquid chromotography.     

Preparative separation of the saponin lancemaside a from Codonopsis lanceolata by centrifugal partition chromatography

Introduction. Lancemaside A is a saponin that inhibits decreases in blood testosterone level and thus prevents or ameliorates symptoms associated with male climacteric disorder. Our initial attempt to preparative isolation of lancemaside A from the saponin fraction of Codonopsis lanceolata roots by a preparative HPLC did not give a clear result. Objective. To develop a simple and efficient method for the preparative isolation of lancemaside A from the hot water extract of C. lanceolata roots using centrifugal partition chromatography (CPC). Methodology. The saponin fraction obtained from the hot water extract of C. lanceolata roots was used as the sample for preparative‐scale separation of lancemasides by CPC using n‐hexane:n‐butanol:methanol:0.1% aqueous formic acid (3:4:1:6, v/v) as the two‐phase solvent system. The upper phase (organic phase) of the two‐phase solvent system was used as the mobile phase, and 0.5 g of saponin fraction was applied for separation by CPC. Each fraction that was separated by CPC was analysed by HPLC, and the fractions containing each of the separated compounds were pooled together, and then were purified by simple preparative HPLC. Results. The demonstrated separation sequence, hot water extraction, DIAION HP‐20 column chromatography, CPC and preparative HPLC, yielded lancemaside A, foetidissimoside A and astersaponin Hb in their pure forms. Conclusion. The simple and efficient method for the preparative isolation of lancemaside A along with two other saponins, foetidissimoside A and astersaponin Hb, from the saponin fraction of C. lanceolata was established using CPC.     

Preparation and diastereomeric separation of an (S)- and (R)-1-(methoxycarbonyl)tridec-10-yl glucoside derivative,a precursor for a monosaccharide constituent of resin glycosides

The 6-O-mesyl derivative of phenyl 1-thio-beta-D-glucopyranoside was prepared from D-glucose as a synthetic equivalent of a 6-deoxy-hexosyl donor. Racemic methyl 11-hydroxytetradecanoate (methyl convolvulinolate) was synthesized by Grignard reaction of propylmagnesium bromide with 10-undecenal followed by hydroboration. Both intermediates were coupled by NIS-TfOH-promoted glycosidation to give a mixture of two diasteromeric glucopyranosides, which were separated on a preparative scale by medium pressure chromatography. One of the products was identified as having the natural (S)-configuration by comparison of its 1H NMR spectrum with an authentic sample prepared from the corresponding chiral hydroxyfatty acid.     

O-(Acridinium)hydroxylamine (AHA): a reagent for the preparation of chemiluminescent acridinium oxime (AO)-steroid conjugates

O-(Acridinium)hydroxylamine (AHA) reacted with a representative sample of oxo-steroids (6-oxoestradiol, estrone, norethindrone, cortisol, progesterone, digoxin dialdehyde, and digitoxin dialdehyde) to produce chemiluminescent acridinium oxime (AO) conjugates in a single step in 37-68% yield after preparative HPLC.     

Fractionation and characterization of a Polysaccharide from the sclerotia of Pleurotus tuber-regium by preparative size-exclusion chromatography

A kind of regenerated cellulose gel (RCG) particles were treated by toluene to obtain particles with smaller mean pore size, then was mixed with the cellulose gel with pore size of 370 and 525 nm. A preparative size-exclusion chromatography (SEC) column (700 x 20 mm) packed with three gel particles was used to fractionate water-soluble polysaccharide (WEP) extracted from the sclerotia of Pleurotus tuber-regium by aqueous solution. The exclusion limit and fractionation range of the stationary phase of the preparative SEC were molecular mass 8 x 10(5) and 5 x 10(3) to 8 x 10(5), respectively. The calibration curve of the preparative SEC was represented as: log M=13.96-0.53 Ve. The WEP sample (weight-average molecular mass M(w)=2.2 x 10(4), polydispersity=2.4) was divided into three fractions with M(w) ranging from 1.4 x 10(4) to 3.4 x 10(4) by the preparative SEC column, and the fractions were characterized by gas chromatography GC, SEC combined with laser light scattering (SEC-LLS) and viscometry. The unfractionated WEP exhibited triple peaks due to different molecular mass, but each fraction exhibited single peak with the polydispersity of 1.1-1.8 in the SEC patterns. The results indicated that the preparative SEC was efficient for fractionation of polysaccharides having low molecular weight and for determination of their molecular mass.     

Rapid separation and quantification of abscisic Acid from plant tissues using high performance liquid chromatography

Abscisic acid (ABA) was purified from soybean (Glycine max [L.]) seed extract using a preparative high performance liquid chromatography (HPLC) procedure. The preparative procedure was rapid (70 minutes per sample), required no prior partitioning for purification and was quantitative as demonstrated with an internal standard of [2-¹⁴C]ABA, of which 98.9% was recovered.     

A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder

A preparative procedure for obtaining highly purified plasmid DNA from bacterial cells is described. The method is adapted from our earlier procedure, which gave partially purified plasmid in a form suitable for rapid screening of a large number of samples. In the present method, all detectable RNA, chromosomal DNA, and protein are removed without the use of enzymes, phenol extraction, dialysis, or equilibrium centrifugation. Binding of plasmid DNA to glass powder in the presence of 6 m sodium perchlorate is used for the final purification step.     

Stereospecificity of Pseudomonas fluorescens kynureninase for diastereomers of beta-methylkynurenine.

The diastereomers of beta-methyl-L-kynurenine were prepared by preparative ozonolysis of the respective diastereomers of beta-methyl-L-tryptophan. A practical method for preparative enzymatic resolution of the diastereomers of beta-methyltryptophan was developed using carboxypeptidase A digestion of the N-trifluoroacetyl derivatives. The stereochemical assignment was confirmed by X-ray crystal structure determination of (2S, 3R)-threo-beta-methyl-L-tryptophan. (2S,3S)-erythro-beta-Methyl-L-kynurenine is a slow substrate for kynureninase from Pseudomonas fluorescens (k(cat)/K(m) = 0.1% that of L-kynurenine), producing anthranilic acid, while (2S,3R)-threo-L-kynurenine is about 390-fold less reactive than erythro. Rapid-scanning stopped-flow measurements show that beta-methyl substitution affects the rate of alpha-deprotonation of the L-kynurenine-pyridoxal-5'-phosphate Schiffs base. This is consistent with the stereoelectronic requirements of the reaction. These results are the first demonstration that beta-substituted kynurenines can be substrates for kynureninase, and may be useful in the design of mechanism-based inhibitors.     

Determination of deoxynivalenol (DON) in blood, bile, urine and excrement samples from swine using immunoaffinity chromatography and LC-UV-detection

A work up procedure is described by which DON concentrations in blood, bile, urine and excrements from swine can be quantified by HPLC and UV- detection at λ = 220 nm. The central step thereby is the purification and concentration of DON by means of an immunoaffinity column. While, in our experiments, the quantification of DON in blood and urine was straightforward an additional purification step by a preparative HPLC run prior to immunoaffinity chromatography was needed when bile and excrements were investigated. However, when low DON concentrations in blood and urine are expected, a preparative HPLC run prior to immunoaffinity chromatography is recommended as well, because larger amounts of sample materials should be analyzed and more impurities interfere with the column proteins. In our study, using spiked samples, recoveries ranged from 75—90% and limits of detection were 0.01 to 0.02 μg/ml.     

Effect of organic contamination upon microbial distributions and heterotrophic uptake in a Cape Cod, Mass., aquifer.

Bacterial abundance, distribution, and heterotrophic uptake in a freshwater aquifer contaminated by treated sewage were determined from analyses of groundwater and sediment-core samples. The number of free-living (unattached) bacteria in contaminated groundwater declined steadily with increasing distance from the source of sewage infiltration, from 1.94 (+/- 0.20) X 10(6) ml-1 at 0.21 km to 0.25 (+/- 0.02) X 10(6) ml-1 at 0.97 km. Bacterial abundance in groundwater sampled at 0.31 km correlated strongly with specific conductance and increased sharply from 4.0 (+/- 0.3) X 10(4) ml-1 at a depth of 6 m to 1.58 (+/- 0.12) X 10(6) ml-1 at 14 m, then declined at 20 and 31 m to 1.29 (+/- 0.12) X 10(6) and 0.96 (+/- 0.12) X 10(6) ml-1, respectively. A majority of the bacteria in contaminated and uncontaminated zones of the aquifer were bound to the surfaces of particulates, less than 60 micron in diameter. The glucose uptake rate, assayed at in situ and 5 microM concentrations, declined steadily in contaminated groundwater sampled along a transect. A preparative wet-sieving technique for use in processing core samples for bacterial enumeration is described and evaluated.     

Preparative fractionation of protein, RNA, and plasmid DNA using centrifugal precipitation chromatography with tubular dialysis membrane inside a convoluted tubing as separation channel

Fractionation of clarified E. coli lysate components in bench-scale and preparative-scale centrifugal precipitation chromatography (CPC), using a solution of cationic surfactant cetyltrimethylammonium bromide (CTAB) containing 0.5 M NaCl as precipitant, are compared here. Step gradient of CTAB from 0.50% to 0.16% (w/v) gave a successful fractionation in bench-scale CPC; however, a linear gradient of lower CTAB concentration, 0.20-0% (w/v), was used in the preparative scale and resulted in similar fractionation. The preparative-scale CPC has a superior sample loading capacity by the use of tubular dialysis membrane inside convoluted tubing as the separation channel. In this study, the quantity of the sample loaded into the preparative CPC was about 15 times more than that in the bench scale, and in a single run the preparative CPC could prepare approximately 3 mg of plasmid DNA with about 96% of RNA removed. The higher surface area per length of the separation channel in the preparative CPC was believed to benefit mass transfer of CTAB across the membrane, leading to less CTAB being required in the process.     

Visual problems in the elderly population and implications for services.

OBJECTIVE--To determine the prevalence of visual disability and common eye disease among elderly people in inner London. DESIGN--Cross sectional random sample survey. SETTING--Inner London health centre. SUBJECTS--Random sample of people aged 65 and over taken from practice''s computerised age-sex register. MAIN OUTCOME MEASURES--Presenting binocular Snellen 6 m distance acuity and best monocular 3 m Sonksen-Silver acuity to classify prevalence of blindness by World Health Organisation criteria (less than 3/60 in better eye) and American criteria for legal blindness (better eye equal to 6/60 or less) and of low vision by WHO criteria (best acuity 6/18) and visual impairment by American criteria (less than 6/12 or 20/40 but greater than 6/60 or 20/200 in better eye). Principal cause of visual loss by diagnosis, referral indication by cause to hospital eye service, and proportion of cases known to primary care. RESULTS--207 of 288 (72%) eligible people were examined. 17 (8%) housebound subjects were examined at home. The prevalence of blindness was 1% by WHO criteria and 3.9% by American criteria. The prevalence of low vision (WHO criteria) was 7.7%. The prevalence of visual impairment (American criteria) was 10.6%. Cataract accounted for 75% of cases of low vision. Only eight out of 16 patients with low vision were known by their general practitioner to have an eye problem. 56 subjects (27%) would probably have benefited from refraction. Comparisons with studies in the United States and Finland suggested higher rates in this sample, mainly due to the prevalence of disabling cataract. CONCLUSION--There seems to be a considerable amount of undetected ocular disease in elderly people in the community.     

Laboratory database to manage electrophoresis and chromatography separations and the associated samples

A database was developed to store, organize, and retrieve the data associated with electrophoresis and chromatography separations. It allows laboratories to store extensive data on separation techniques (analytical and preparative). The data for gel electrophoresis includes gel composition, staining methods, electric fields, analysis, and samples loaded. The database stores data on chromatography conditions, the samples used, and the fractions collected. The data structure of this database was designed to maintain the link between samples (including fractions) from chromatography separations and their analysis by gel electrophoresis. The database will allow laboratories to organize and maintain a large amount of separation and sample data in a uniform data environment. It will facilitate the retrieval of the separation history of important samples and the separation conditions used.     

Solid-phase synthesis and biological activities of gastrointestinal hormones: Secretin and motilin

Many successful solid-phase syntheses of peptide chains in the region of 20–40 amino acid residues have now been routinely reported. Utilizing standard solid-phase synthetic methodologies but, particularly, new and powerful purification techniques we have been developing rapid and efficient preparative routes for the numerous neuro-gastrointestinal peptides. In the present study, secretin and motilin were obtained in 16% and 10% yields, respectively, after simplified two-step purification of hydrogen fluoride-cleaved peptides by gel filtration followed by preparative high performance liquid chromatography. Peptides were essentially homogeneous by TLC and analytical high performance liquid chromatography. Secretin was found to have full biological activity when tested against a standard sample of natural material for effects on pancreatic secretion in the dog. Motilin exhibited full biological activity on interdigestive motility in the dog. Secretin has been reported to undergo rearrangement with loss of bioactivity during purification and prolonged storage. We observed no obvious problems during our abbreviated purification schedule and have found no loss of purity of peptide which has been kept for 6 months as powder lyophilized from dilute acetic acid.     

Some properties and applications of Superose 6B

Applications of a new agarose-based medium for high-performance preparative gel filtration is described. The 30-micron bead size, high pore volume, and simple experimental technique to prepare highly efficient columns make Superose 6B very suitable for preparative purifications of protein mixtures in the molar mass range of 10(3) to 4 X 10(6). Sample load exceeds 100 mg/cm2, which indicates that gram quantities may be purified on K 50/60 columns. The scale-up procedure from analytical to preparative columns is demonstrated by the separation of serum samples and the industrial purification of an enzyme-antibody conjugate.     

High-performance liquid chromatographic method for quantitating plasma levels of amiloride and its analogues

An assay for amiloride was devised for efficient use with the wide variety of analogues available. Amiloride was extracted from 1-ml plasma samples by elution from a C₈ preparative column with 6% acetonitrile—45% methanol—5.4% acetic acid, adjusted to pH 4.0 with trimethylamine. Samples were lyophilized, resuspended in 50% methanol, filtered through 0.22-μm Spin-X cartridges, applied to a reversed-phase C₁₈ column, and eluted in a 0–50% acetonitrile gradient in 0.4% acetic acid, pH 4.5 (1.2 ml/min). Detection by ultraviolet absorbance at 360 nm was linear from 1 to 1000 ng. Versatility of the method was demonstrated with the analogues benzamil, 6-hydro-, 6-iodo-, 5-hexamethylene-, and 5-chlorobenzyl-2',4'-dimethylbenzylamiloride.     

Application of continuous zone electrophoresis to preparative separation of proteins

A comprehensive study of the application of continuous zone electrophoresis to preparative separation of proteins in free solution is presented. First, the influence of electric field strength, buffer residence time in the chamber, sample flow rate, and sample concentration on separation resolution and throughput were studied. Using multiple injections of sample into the electrophoresis chamber, a throughput of 500 mg protein/h was achieved for partially purified model proteins. Experiments on Escherichia coli crude extracts yielded a fivefold purification of beta-galactosidase along with a simultaneous separation of proteins from cell debris in a single step. Experiments correlating the electrophoretic mobility in continuous electrophoresis with the elution behavior in ion-exchange chromatography were performed on more than a dozen proteins which conclusively showed that separation of proteins in continuous zone electrophoresis is governed by net surface charge. Based on these results, the fraction numbers in which the proteins eluted could be correctly predicted. Proteins and enzymes with differences >0.5 M elution molarities in ion-exchange chromatography were separated by continuous zone electrophoresis on a preparative scale (mg/h or g/h) with >90% recovery. This corresponds to a preparative scale separation of proteins and enzymes which differ in apparent electrophoretic mobility by only 0.70 x 10(-5) cm(2)/V . s. (c) 1993 John Wiley & Sons, Inc.     

Hyperlipoproteinemia in fasting ponies

Ponies fasted for up to 8 days showed, both by agarose electrophoresis and preparative ultracentrifugation, the appearance of a pre-beta-migrating, very low density lipoprotein fraction in plasma. This lipoprotein differs from the very low density lipoprotein found in humans and rats in that it contains a relatively smaller amount of total cholesterol, 85% of which is present in the unesterified form. By the 8th day of fasting, plasma triglyceride concentrations had increased from a prefasting level of 20 mg/dl to as high as 1000 mg/dl. The increase in plasma lipid concentrations as a result of fasting was highly variable. Accumulation of plasma cholesterol and triglyceride after injection of Triton WR 1339 was not related to the degree of fasting hyperlipidemia. This suggests that the hyperlipoproteinemia of fasting may result from an impaired utilization of very low density lipoproteins.     

DigesTip: a new device for a rapid and efficient in-solution protein digestion

DigesTip is a new device for in-solution protein digestion, based on a patent pending technology, able to immobilize enzymes (trypsin, in this case) on a solid surface, keeping their activity preserved. DigesTip is a standard pipette tip, usable both by human and by robots. Its main performances are: very short digestion time (1 min) and usability with low protein sample concentrations (5 microg/mL). DigesTip obtains a clear signal in MS measurements and its usage rules out several preparative steps.     

Improved molecular weight-based processing of intact proteins for interrogation by quadrupole-enhanced FT MS/MS

Complete coverage of protein primary structure is demonstrated for 37 yeast protein forms between 6 and 30 kDa in an improved platform for Top Down mass spectrometry (MS). Tandem mass spectrometry (MS/MS) for protein identification with 100% sequence coverage is achieved in a highly automated fashion with 15-300-fold less sample amounts than an initial report of a proteome fractionation approach employing preparative gel electrophoresis with an acid-labile surfactant to facilitate reversed phase separation in a second dimension. Using a quadrupole-enhanced Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FTICRMS) improves the dynamic range for protein detection by approximately 50-fold and MS/MS by approximately 30-fold. The technology development illustrated here typifies an accelerating effort to detect whole proteins in a more general and higher throughput fashion for improved biomarker identification and detection of diverse post-translational modifications. Capillary RPLC is used in both off-line and on-line modes, with one on-line LC/FTMS sample providing 25 observed protein forms from 11 to 22 kDa.