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PubMed1994

Analysis of aspartic acid and asparagine metabolism in Xenopus laevis oocytes using a simple and sensitive HPLC method.

The amino acids in methanol-soluble extracts of Xenopus oocytes were measured using a method involving precolumn derivatization with phenylisothiocyanate and reverse phase HPLC of the derivatized amino acids. This technique allows the estimation of asparagine and glutamine pools in oocytes, estimated as 70 and 283 pmoles per oocyte, respectively. The pool sizes of the other amino acids were similar to previously reported results obtained using conventional ion exchange chromatography and postcolumn derivatization with ninhydrin. The advantages of the method developed here include picomolar sensitivity and the enhanced resolution of asparagine and glutamine from other amino acids. The kinetics of aspartic acid and asparagine utilization were monitored following microinjection of oocytes with [3H]aspartic acid and [14C]asparagine. The aspartic acid pool turned over rapidly with a half-time of < 30 min. The asparagine pool was metabolized much more slowly and appeared to be utilized almost completely for protein synthesis. The absolute rate of protein synthesis in oocytes was calculated from the incorporation data and chemical pool measurements as approximately 25 ng/hr-oocyte. The methodology developed here may be useful in experimental situations involving limited amounts of biological material.

Amino Acids↗

PubMed2004

Brain GABA editing by localized in vivo (1)H magnetic resonance spectroscopy.

Editing of GABA by (1)H MRS in a specific brain area is a unique tool for in vivo non-invasive investigation of neurotransmission disorders. Selective GABA detection is achieved using sequences based on double quantum coherence (DQC). Our pulse sequence makes accurate measurements without artefacts due to spatial localization. The sequence was tested on a phantom solution. The effect of vigabatrin, a specific inhibitor of GABA transaminase, was measured in rat brain and GABA detection was performed in vivo in monkey brain using this procedure. Rats were split into two groups. In the control group, the rats had access to water and, in the other group (vigabatrin, VGB, rats), animals were allowed free access to drinking water containing vigabatrin. After 3 weeks of treatment, rats were anesthetized for in vivo NMR spectroscopy investigation. At the end of the experiment, brains were quickly removed, freeze-clamped and extracted with 4% perchloric acid. One part of the acid extract was used for GABA concentrations assessment by ion exchange chromatography with ninhydrin detection. The second was used for high-resolution NMR analysis. By chromatography measurements, the GABA concentration was 1.23+/-0.06 micromol/g for controls, while for vigabatrin-treated rats the GABA concentration was 4.89+/-1.60 micromol/g. The NMR in vivo results were closely correlated with the NMR ex vivo (r=0.99, p<0.01) and chromatography results (r=0.98, p<0.01). The correlation between ex vivo results and chromatography results was also high (r=0.99, p<0.001). This pulse sequence performed GABA editing from a 376 microl voxel located on the right basal ganglia area in a non-human primate brain. This in vivo GABA editing scheme can thus be proposed for accurate measurement of brain GABA concentrations.

Administration, Oral↗

PubMed2003

ESI-MS/MS analysis of underivatised amino acids: a new tool for the diagnosis of inherited disorders of amino acid metabolism. Fragmentation study of 79 molecules of biological interest in positive and negative ionisation mode.

The diagnosis of inherited disorders of amino acids (AA) metabolism is usually performed on automated analysers by ion-exchange chromatography and quantification after ninhydrin derivatisation of about 50 different AA. A single run liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for these molecules can be an alternative to this time-consuming technique. The first step of this development is the infusion study of the fragmentation of 79 molecules of biological interest in electrospray ionisation tandem mass spectrometry (ESI-MS/MS), in positive and in negative ionisation mode. Among them, three molecules can be detected only in negative ionisation mode, 38 only in positive mode and 38 in the two modes. All the most abundant fragmentations are presented, with optimisation of the MS/MS parameters. The positive ionisation mode was retained for the simultaneous analysis of 76 molecules. One sensitive and/or specific transition is proposed for the monitoring of each molecule. Improvement in sensitivity of detection was obtained with the use of an acidic mobile phase. Flow injection analysis studies led us to highlight a number of interferences-due to isobaric molecules, to in-source collision-induced dissociation, or to natural isotopic distribution of the elements-which are listed. For a reliable quantification method, these molecules have to be separated by LC before analysis in the tandem mass spectrometer. Ion-pairing reversed-phase liquid chromatography (RPLC) using perfluorinated carboxylic acids as ion-pairing agents has already been found suitable for analysis of AA in MS/MS positive ionisation mode and is under development.

Amino Acid Metabolism, Inborn Errors↗

PubMed2005

Ion-pairing reversed-phase liquid chromatography/electrospray ionization mass spectrometric analysis of 76 underivatized amino acids of biological interest: a new tool for the diagnosis of inherited disorders of amino acid metabolism.

Seventy-six molecules of biological interest for the diagnosis of inherited disorders of amino acids (AA) metabolism have previously been demonstrated to be detectable in electrospray ionization tandem mass spectrometry (ESI-MS/MS) positive mode without derivatization. Reversed-phase liquid chromatography (RPLC) separation on different C18 columns using various perfluorinated carboxylic acids as ion-pairing agents has been found suitable for coupling with MS/MS, and for the separation of AA. A new procedure was optimized in order to replace the usual ion-exchange chromatographic, post-column ninhydrin derivatization, time-consuming routine method. This procedure allowed an adequate separation of all the molecules from other known interfering compounds, and a throughput of two samples per hour. Quantification limits for each molecule were found to be compatible with their measurement in plasma and urine. We validated the qualitative part of the method by analyzing plasma and urine samples from patients affected with several inherited disorders of AA metabolism. We validated the quantification of 16 AA using their stable isotopes as internal standard. The calibration curves were linear over the range 0-3 mM. The quantitative results obtained with the new method on 105 plasma and 99 urine samples were in good agreement with those obtained by the established routine method. Spiking experiments and precision results were also satisfactory.

Amino Acid Metabolism, Inborn Errors↗

PubMed1993

N-acylphosphatidylethanolamine synthesis in plants: occurrence, molecular composition, and phospholipid origin.

We have identified a microsomal phospholipid as N-acylphosphatidylethanolamine (NAPE) that was radiolabeled following incubation of 1-day-old cotyledons of cotton seedlings with [14C]ethanolamine. Radiolabeled NAPE comigrated with commercially available L-alpha-dipalmitoyl phosphatidyl(N-palmitoyl)ethanolamine (std-NAPE) in one- and two-dimensional TLC. This NAPE comprised 7.2 +/- 1.0% (by weight) of microsomal phospholipids when hot isopropanol was used to inactivate endogenous phospholipases prior to extraction of lipids. In vitro degradation of putative cottonseed radiolabeled NAPE by Streptomyces chromofuscus phospholipase D resulted in production of a ninhydrin-reactive, radiolabeled lipid which comigrated with N-acylethanolamine (NAE) that was produced from a similar enzymatic cleavage of std-NAPE. Transmethylation of cottonseed radiolabeled NAE yielded radiolabeled ethanolamine and fatty acid methyl esters, nearly all of which were saturated (myristate, palmitate, and stearate together were 92% of the acyl components of cottonseed NAE). Positional analysis and relative abundance of the O-acyl groups of cottonseed microsomal NAPE were determined following a double enzymatic cleavage with Trimeresurus flavoviridis venom (phospholipase A2 activity) and S. chromofuscus phospholipase D. We substantiated our identification of cottonseed NAPE by 1H NMR spectroscopy and by mass spectrometry (fast-atom-bombardment ionization and tandem MS, FAB-MS/MS). Radiolabeled NAPE was synthesized in vivo in varying amounts from [14C]ethanolamine applied to maturing seeds of cotton and soybean, cotyledons of dark-grown cotton and soybean seedlings, cotyledons of light-grown okra, cotton and soybean seedlings, endosperm tissue of castor bean, and suspension cell cultures of rice. In pulse-chase radiolabeling experiments in cotyledons of 1-day-old cotton seedlings, radiolabeled NAPE increased and radiolabeled phosphatidylethanolamine (PE) decreased over a 12-h chase period (in the dark or light), suggesting that NAPE was synthesized from PE in vivo. In vitro, the synthesis of NAPE from PE (radiolabeled in vivo) proceeded in a linear fashion in microsomes of cotton cotyledons with or without 1 mM EGTA and with or without 1 mM CaCl2 for 90 min. NAPE was synthesized in vitro from PE synthesized by the exchange pathway (microsomes preincubated with [14C]++e+thanolamine) and from PE synthesized by the nucleotide pathway (microsomes preincubated with [14C]CDPethanolamine). Collectively, our data indicate that (a) NAPE is a widespread, natural phospholipid component of plants, (b) NAPE is synthesized in vivo under normal physiological growth conditions in cotyledons of cotton seedlings, (c) NAPE is localized and synthesized in cottonseed microsomes, and (d) NAPE is likely synthesized by a direct acylation of PE.

Acylation↗

PubMed1993

Synthesis and characterization of N-octanoyl-beta-D-glucosylamine, a new surfactant for membrane studies.

The new nonionic glycosidic surfactant N-octanoyl-beta-D-glucosylamine (NOGA, molar mass 305.37 g) was synthesized through an easy and efficient two-step procedure. Specifically, beta-D-glucosylamine was obtained by the replacement of the anomeric hydroxyl of D-glucose by an amino group which was then selectively acylated. NOGA was finally purified by silica gel column chromatography and recrystallization. This compound is stable and soluble in water and usual buffers up to 80 mM at 4 degrees C and up to 0.2 M at 37 degrees C. NOGA solutions are also characterized by a low ultraviolet light absorbance above 250 nm (epsilon 280 approximately 1.5 M-1 cm-1). Due to its very high critical micelle concentration (CMC = 80 mM, as determined by spectrofluorimetry), this surfactant may easily be removed from samples by dialysis or, to a lesser extent, by adsorption onto hydrophobic beads. Furthermore, NOGA is colorimetrically titrable by the ninhydrin method and its weak interference in protein determination by the bicinchoninic acid method is easy to overcome. This surfactant exhibits a good solubilizing power toward membrane proteins, with a marked selectivity for spiralin, a bacterial surface antigen. Protein extraction started below the CMC, but was much more effective above this concentration threshold. NADH oxidase activity, ligand binding by the glycine betaine-binding protein, and antigenicity of more than 20 membrane or soluble proteins were not altered by NOGA. Thus, owing to its extraction efficacy and mildness toward protein structure and activity, NOGA should prove useful for membrane studies and offers the additional advantage of being easy to synthesize at low cost.

Adsorption↗

PubMed1998

Determination of proteins, phosphatidylethanolamine, and phosphatidylserine in organic solvent extracts of tissue material by analysis of phenylthiocarbamyl derivatives.

Amino acid analysis of organic solvent extracts of tissue material has been evaluated for determination of protein content. Conventional ninhydrin-based analysis does not allow determination of a large number of lipid-rich samples. Therefore, the hydrolyzed samples were treated with phenylisothiocyanate and the phenylthiocarbamyl (PTC) derivatives obtained were separated by reverse-phase HPLC. With this method, analysis of many lipid-rich samples is feasible. In addition, phosphatidylethanolamine and phosphatidylserine can then be determined together with the amino acid constituents. The PTC/reverse-phase HPLC method was used for analysis of chloroform/methanol extracts of spinal cord, lung, and bile after chromatography on Lipidex 5000 in methanol/ethylene chloride, 4:1 (v/v). The chromatography profiles show that in all tissue samples the proteins elute before the phospholipids. Consequently, a single step of Lipidex 5000 chromatography can be used to purify polypeptides present in organic solvent extracts. Using pulmonary surfactant extracts (with about 98% phospholipids and 1-2% proteins), we find that individual contents of surfactant proteins B and C can be determined by amino acid analysis.

Amino Acids↗

PubMed1999

A single-sample method for determination of carbohydrate and protein contents glycoprotein bands separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis.

A method is described for determination of carbohydrate and protein contents of glycoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electroblotted onto polyvinylidene difluoride (PVDF) membranes. Blots were stained, and appropriate pieces of PVDF membranes were excised, destained, and subjected to sequential hydrolysis with 0.2 M trifluoroacetic acid (TFA) for 1 h at 80 degrees C, then with 2 M TFA for 4 h at 100 degrees C, and finally with 6 M HCl at 100 degrees C for 24 h to release sialic acids, neutral sugars with hexosamines, and amino acids, respectively. In some instances preliminary methanolysis was used. Carbohydrates including sialic acids were quantitated by high pH anion exchange chromatography with pulsed amperometric detection. Protein content of the bands was determined as amino acids by the fluorescamine or ninhydrin method. In the calculation of results proper adjustments were made for small amounts of fucose released by hydrolysis with 0.2 M TFA at 80 degrees C, and for partial degradation of protein during hydrolysis with 2 M TFA at 100 degrees C. Recoveries of amino acids from hydrolysates of glycoproteins that had been electroblotted onto PVDF membranes equaled those of carbohydrates. This was possible because of preliminary hydrolysis of glycoproteins with TFA, as well as washing of wet, instead of dried, PVDF membranes after hydrolysis with 6 M HCl. The two modifications increased yields of amino acids by about 30%. The method was successfully applied to the determination of molar and weight percentage composition of human transferrin, band 3 protein, glycophorin A, and alpha(1)-acid glycoprotein. In each case the results obtained for directly hydrolyzed and electrophoresed/electroblotted glycoproteins were practically identical. We also determined the glucosamine content of band 4.1 protein of erythrocytes.

Carbohydrates↗

PubMed2000

Amino acid analysis by capillary electrophoresis after phenylthiocarbamylation.

Capillary electrophoresis using SDS in phosphate buffer provides high resolution and short separation time for peptide and protein hydrolysate amino acids after derivatization with phenylisothiocyanate. The phenylthiocarbamyl derivatives are quantified in the picomole and femtomole range at signal-to-noise ratios better than 3:1 (for 50 fmol) and with a linearity correlation coefficient averaging 0.9938. The migration time and peak area variabilities were on average 1.1 and 2.7%, respectively. Complete separation of all the 18 amino acids normally found in polypeptide hydrolysates is achieved in less than 30 min using 75-microm capillaries while 50-microm capillaries require less than 15 min. Analysis of peptide and protein hydrolysates in the range 10-600 residues revealed excellent agreement with the known compositions at sensitivities better by large factors than the corresponding HPLC methodology (about 20-fold) and conventional ninhydrin-based analysis (about 1000-fold).

Amino Acids↗

PubMed2001

Incomplete dialysis of protein samples containing 3-[(3-chlolamidopropyl)dimethylammonio]-1-propanesulfonate may lead to erroneous estimation of histidine content on amino acid analysis.

The zwitterionic detergents Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and its 2-hydroxyl derivative Chapso have been widely used in the biomembrane field as well as other fields for their nondenaturing character. Due to their structure containing an amide bond in the molecules center they are prone to hydrolysis with 6 N HCl into cholic acid and 3-aminopropyldimethylammonio-1-propanesulfonate (APS) and its 2-hydroxyl derivative (APSO), respectively. On amino acid analyses with the ninhydrin detection system, His, APS, and APSO were eluted at 24.42, 24.55, and 24.85 min, respectively, and APS was identified as His due to their close retention times. Moreover, the mixture of His and APS coeluted as a single peak at 24.51 min leading to an erroneous His content for Chaps-contaminated protein samples. For full separation of APS, APSO, and His the elution program was improved by an additional 5-min elution with Buffer 7, i.e., 1:1 mixture of Buffers 3 and 4, before elution with Buffer 4, enabling complete separation of the 18 amino acids, APS, and APSO in a single run.

Cholic Acids↗

PubMed2001

A high-performance liquid chromatography method for the detection of diaminopimelic acid in urine.

We describe a quantitative assay for diaminopimelic acid (DAP) in urine. It involves (i) hydrolysis of urine samples, (ii) purification by several different liquid chromatography steps, and (iii) analysis by high-performance liquid chromatography on a reversed-phase C18 column. Tritiated-DAP, the internal standard, allows one to precisely follow the DAP-containing fractions and to determine the yield during purification. Sensitive and relatively accurate quantification of DAP, with a threshold of 50 fmol, is based on ion-pairing properties of eluants and ortho-phthaldialdehyde derivatization. The presence of DAP in relevant fractions was confirmed by combined gas chromatography and mass spectrometry. The DAP concentration in adult human urine pooled over 24 h ranges from 0.69 to 2.01 microM, a result in fair agreement with previously published values obtained by ninhydrin derivatization or gas chromatography.

Adult↗

PubMed1978

Inhibition of lysinoalanine synthesis by protein acylation.

Treating wheat gluten, soy protein, and lactalbumin under alkaline conditions at 65 degree C for various times destroys part of the threonine, cystine, lysine, tyrosine, and arginine residues in these proteins. The losses were accompanied by the appearance of lysinoalanine and unidentified ninhydrin-positive substances. Treating wool under somewhat milder alkaline conditions destroys part of the cystine and lysine residues, but not the other amino acids cited. In this case, loss of cystine and lysine was accompanied by the appearance of lanthionine and lysinoalanine. Amino acid analysis of alkali-treated acylated proteins revealed that acylation by acetic and succinic anhydrides prevents or minimizes destruction of lysine residues and the formation of lysinoalanine in wheat gluten and soy protein and, in wool, minimizes destruction of cystine and lysine residues and the formation of lanthionine lysinoalanine. Mechanisms are proposed to explain the observed inhibiting effects of protein acylation and oertain additives on lysinoalanine formation.

Acylation↗

PubMed1995

Deamination of 3-hydroxykynurenine in bovine lenses: a possible mechanism of cataract formation in general.

BACKGROUND: 3-Hydroxykynurenine, a metabolite of tryptophan, acts as UV filter in the human lens. In this study, we looked for this substance and its metabolites in young and old bovine lenses, because of their possible role in the formation of cataract. METHODS: The substances were detected by HPLC analysis. The fluorescent substance formed from 3-hydroxykynurenine was characterized by thin-layer chromatography followed by reaction with ninhydrin, UV and fluorescence spectrum analysis, and atom bombardment for molecular mass determination. The kynurenine aminotransferase activity was determined by the method of Tobes. RESULTS: 3-Hydroxykynurenine was detected at concentrations of 0.07, 0.19, and 1.14 micrograms/g of tissue in the bovine iris/ciliary body, retina, and transparent bovine lenses respectively. 3-Hydroxykynurenine was deaminated in old bovine eyes but not in calf eyes. In old eyes, kynurenine aminotransferase activity was 2.7, 3.5, and 9.6 mumol/g of tissue per h in retina, iris/ciliary body, and lens respectively. CONCLUSION: The deamination of 3-hydroxykynurenine resulted in the formation of a fluorescent substance which was identified as oxidized xanthurenic acid. This substance, accumulating in the bovine lens and interacting with lens proteins, could induce cataract formation.

Animals↗

PubMed1984

Detection of epsilon(gamma-glutamyl) lysine.

Detection of epsilon(gamma-glutamyl) lysine crosslinks is not only necessary for establishing the importance of the dipeptide as a post-translational modification of proteins, but provides information as to the importance of the transglutaminase enzyme in a biological system. The crosslink may be detected using both indirect and direct methodology. Indirect methods for its detection include measurement of 'masked lysines' within a protein, detection of polymer formation by gel-electrophoresis and the inhibition of crosslinking by the incorporation of small molecular weight amines into the substrate protein. Direct methods for the detection of epsilon(gamma-glutamyl) lysine require the actual isolation of the dipeptide following its release from the sample protein by exhaustive proteolytic digestion. Separation of the dipeptide from other components of the digest may be achieved by either ion-exchange chromatography or gel filtration and its qualitative identification achieved by techniques such as paper-electrophoresis or thin layer chromatography. Quantitative estimation of epsilon(gamma-glutamyl) lysine normally involves its further separation by ion-exchange chromatography and its post-column detection following derivatisation with ninhydrin. More recent techniques include pre-column derivatisation of the dipeptide with fluorogenic reagents such as sigma-pthalaldehyde and separation by reverse phase HPLC. With the recent advances in liquid chromatography resulting in the improved resolution of amino acids, increased sensitivity, rapid analysis times, and small sample sizes, it appears likely that direct quantitation of epsilon(gamma-glutamyl) lysine will be the preferred method for the future.

Acyltransferases↗

PubMed1983

Cytoplasmic 3H-arginine polypeptides of Ehrlich ascites tumour cells.

Low molecular weight ninhydrin positive peptide fractions of the Ehrlich tumour cell cytoplasm were isolated and characterized. After preliminary gel filtration of the cytoplasm on Sephadex G-25 column, the peptide mixture was fractionated on cationic exchanger SP-Sephadex C-25 column and eluted with increasing pH gradient. Five peaks were obtained. Only the first peak contained sugar component. All five peptides were studied with respect to molecular weight, isoelectric point and electrophoretic homogeneity. The cytoplasm of Ehrlich tumour cells contains one peptide of acidic (pI-5.0), two slightly basic (pI-7.7 and pI-7.7) and two strongly basic nature (pI-8.7 and pI-8.9). Molecular weights varied from 8 500 to 18 500 daltons. The origin of these peptides is briefly discussed.

Amino Acids↗

PubMed1990

Biological monitoring of isocyanates and related amines. I. Determination of 1,6-hexamethylene diamine (HDA) in hydrolysed human urine after oral administration of HDA.

1,6-Hexamethylene diamine (HDA), used as raw material in industrial manufacturing operations, was orally administered to six healthy volunteers. After acid hydrolysis of the urine by hydrochloric acid, HDA and the metabolite 6-aminohexanoic acid were quantified. HDA was determined as an ethyl-chloroformate derivative by capillary gas chromatography using thermionic specific detection (TSD), and 6-aminohexanoic acid was quantified by ion chromatography using the ninhydrin reaction. In nonhydrolysed urine, monoacetylated HDA (N-acetyl-1,6-hexamethylene diamine) and HDA, were verified as heptafluorobutyric anhydride derivatives by gas chromatography-mass spectrometry (GC-MS), in a chemical ionization mode using isobutane and ammonia as reagent gases. In hydrolysed urine, a mean of 0.28 mg (range 1-6%) of the administered dose (8.2 mg) was recovered as HDA, and a mean of 0.8 mg (range less than 1-27%) as 6-aminohexanoic acid. The urinary excretion of both the determined compounds was rapid, and the principal part (greater than 90%) of the elimination was completed within 10 h. There was a considerable inter-individual variation in the excreted amounts, but the intra-individual variation in the excretion of HDA was limited. The subjects N-acetylator phenotype was determined by a dapsone test. Three slow acetylators excreted lower amounts (mean 2% of given dose) of HDA than three rapid ones (mean 5%).

Acetylation↗

PubMed1985

Aflatoxin-induced alterations in Glycine max, cv. 'Essex' root cell membrane protein content.

Aflatoxins (AFTs) are hepatocarcinogens, mutagens, teratogens and toxins. Isolates of AFTs-producing strains of Aspergillus flavus can grow upon both autoclaved soybeans and to a lesser extent field-grown beans. Besides inhibiting the elongation of excised, in vitro cultured soybean roots, the AFTs can impair the roots' ability to remove [14C]-leucine from a culture medium and to incorporate the amino acid into acid-insoluble, cytoplasmic protein. In this connection, exogenous AFTs once taken-up and compartmentalized by the excised roots can reduce the acid-insoluble protein content of what appears to be a non-enriched plasmalemma fraction. Here, we report a combined biochemical and microscopical attempt to determine whether the protein reduction occurs throughout the excised, in vitro-cultured root and whether the plasmalemma proteins which are affected by AFTs can be both solubilized and characterized. Histochemistry of Carnoy-fixed roots revealed a reduction in Ninhydrin-Schiff-stainability throughout the AFTs-treated root. Transmission electron microscopy of 80 000 X g pellets derived from homogenates of both non-treated (NT) and treated (T) roots provided further evidence to our previously reported marker enzyme analyses for the occurrence of the plasmalemma in the 80 000 X g pellet. Treatment of the latter with the Laemmli SDS procedure released greater than 85% of the protein associated with the pellets obtained from homogenates of either NT or T roots. Gel permeation chromatography on Biogel-100 of released proteins from 80 000 X g pellets of both NT and T roots yielded both void volume and retarded peaks. Both the amplitude and the quantities of protein recovered within the void volume peak were less within the void volume of the t than the NT root. Polyacrylamide tube gel electrophoresis of G-100 void volume peaks of chromatographed, SDS-released proteins from the pellets revealed quantitative but not qualitative differences in Coomassie Blue-gel staining patterns between T and NT roots. These results suggest that the SDS methods which we employed could solubilize the 80 000 X g pellet proteins but that combined gel permeation chromatography and tube gel electrophoresis were not sensitive enough to reveal whether AFTs could alter the types of proteins associated with the 80 000 X g pellet (purported plasmalemma).

Aflatoxins↗

PubMed1982

A fluorescence cytophotometer operated under computer control for multi-parameter cell analysis.

A new type of fluorescence cytophotometer has been developed for multi-parameter cell analysis (Olympus BH2-QRFL). For multi-color fluorescence cytophotometry, this instrument is equipped with four sets of interchangeable filters, each consisting of an excitation filter, a dichroic mirror with a barrier filter, and a measuring filter. For permitting automatic operation of the filter sets, the cytophotometer is connected on line with a personal computer (HP 85F). A desired sequence of filter sets can be memorized in the software and multiple cellular constituents can be rapidly and consecutively determined on a single cell basis. All data are stored in the same computer and can be retrieved for further statistical analysis and display either in tabular form, or as histogram, correlation histograms, two-dimensional scatter plot, or two-dimensional frequency distribution histogram, on the CRT (cathode ray tube) with simultaneous hard copy. As an example of multiparameter cell analysis, combined protein and DNA measurements were performed on normal, border-line, and cancerous gynecological cytology specimens by using the ninhydrin-Schiff and Feulgen techniques.

Cell Separation↗