Detection of tetrodotoxin by thin-layer chromatography/fast atom bombardment mass spectrometry

A new method for detection of tetrodotoxin (TTX) by thin-layer chromatography/fast atom bombardment (FAB) mass spectrometry was developed. TTX and/or related substances were separated by TLC on LHP-K high-performance precoated plates, with a solvent system of pyridine:ethyl acetate:acetic acid:water (15:5:3:4). The plates were subjected to positive FAB mass spectrometry, under scanning within a mass range from m/z 100 to 500. TTX was identified by selected ion-monitored chromatograms at m/z 320 (M + H)+ and 302 (M + H - H2O)+, along with full scan positive ion FAB mass spectrometry. The limit of detection for TTX was about 0.1 micrograms. TTX was also detected by cellulose acetate membrane electrophoresis/FAB mass spectrometry.     

Two-dimensional electrophoresis on the layers of cellulose acetate membrane

A new type of two-dimensional electrophoresis for analysis of protein using cellulose acetate membrane has been developed. Prior to the separation, proteins in a sample are concentrated to a narrow zone on a strip of cellulose acetate according to “steady-state stacking” of isotachophoresis. Electroendosmotic counterflow on cellulose acetate membranes is advantageous for the isotachophoretic concentration of large sample volumes. The concentrated protein zone is then subjected to electrophoretic separation on the same strip. This first-dimensional separation including the concentrating process is named “concentrating electrophoresis.” Iso-electric focusing on several layers of cellulose acetate membrane is performed in the second-dimensional step. Many kinds of detection methods can be applied to the layers among which proteins are distributed. The novel two-dimensional electrophoresis takes only 5 h to perform.     

The fractionation of adenosine-rich oligoribonucleotides on polyethyleneimine-cellulose

After combined pancreatic and T(1) ribonuclease treatment of RNA, a characteristic series of products of the type A(n)N are obtained, where N can be any of the four ribonucleosides. Depending on whether phosphatase treatment is used before the addition of labelled phosphate at the 5'-terminus with bacteriophage phosphokinase, the labelled oligonucleotides obtained may or may not possess a phosphate group at the 3'- as well as the 5'-end. The behaviour of these characteristic products after electrophoresis on cellulose acetate strips followed by chromatography on polyethyleneimine-cellulose thin-layer plates was examined.     

The separation of oligonucleotides of baker''s-yeast value transfer ribonucleic acid 2b by high-voltage electrophoresis on DEAE-paper and by thin-layer chromatography.

A modified procedure for the separation of oligoribonucleotides is described that is based on the combination of t.l.c. on cellulose and electrophoresis on DEAE-paper at 4000 V on a cooling plate. The technique is relatively rapid and allows the analysis of larger quantities than is possible by electrophoresis on cellulose acetate.     

Cellulose acetate as solid phase in ELISA for plague

Antigen from Yersinia pestis was adsorbed on cellulose acetate discs (0.5 cm of diameter) which were obtained from dialysis membrane by using a paper punch. ELISA for human plague diagnosis was carried out employing this matrix and was capable to detect amount of 1.3 microg of antigen, 3,200 times diluted positive serum using human anti-IgG conjugate diluted 1:4,000. No relevant antigen lixiviation from the cellulose acetate was observed even after washing the discs 15 times. The discs were impregnated by the coloured products from the ELISA development allowing its use in dot-ELISA. Furthermore, cellulose acetate showed a better performance than the conventional PVC plates.     

Application of white cellulose acetate sheets on fossil wood investigation

Generally, transparent cellulose acetate sheets as a peel technique material are used in the identification of fossils, whereas white cellulose acetate sheets as a biochemistry technique material are applied in serum protein electrophoresis (SPE). Here we report the application of white cellulose acetate sheets for identifying a polished fossil wood from the Upper Mesozoic of West Liaoning, China. Based on the characters of transverse, radial, and tangential sections, the fossil wood is ascribed to a taxon of Protoglyptostroboxylon sp. Compared with transparent cellulose acetate sheets, white cellulose acetate sheets not only provide the similar information as that of the former, but also are more easily acquired in the Chinese market than the former. Because peel technique supported by white cellulose acetate sheets has the advantages of simple, time-saving, safe, reliable, and practical operation with lower material loss, it is a very good choice for the polished fossil wood investigation in labs, museums, geological parks, and handicraft shops. It is also a convenient approach to training students to learn the anatomic structure of fossil wood.     

The Pk-3 gene determines both the heart,M1, and the kidney,M2, pyruvate kinase isozymes in the mouse; and a simple electrophoretic method for separating phosphoglucomutase-3

We have found that in mice carrying Pk-3r, an allele leading to loss or activity of kidney pyruvate kinase, the activity of heart pyruvate kinase is also diminished. Electrophoretic studies on tissues from mice carrying Pk-3r and/or Pk-3b, an allele determining an electrophoretically detectable variant, show that Pk-3 affects the expression of both the heart, M1, and the kidney, M2, pyruvate kinase isozymes. These results, together with linkage data, indicate that both isozymes are determined by the same structural gene, Pk-3. We also report a simple method for separating phosphoglucomutase-3 (PGM-3) by electrophoresis on cellulose acetate plates.     

Die Bestimmung der sauren Erythrocytenphosphatase(SEP)-Isoenzyme mit der Celluloseacetatfolien-Elektrophorese

Zusammenfassung In der vorliegenden Arbeit wird eine schnelle und einfache Methode zur Bestimmung der sauren Erythrocytenphosphatase(SEP)-Isoenzyme mit Hilfe der Celluloseacetatfolien-Elektrophorese beschrieben.

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Determination of acid phosphatase isoenzymes by cellulose acetate electrophoresis

Summary This paper describes a new method of investigating red cell acid phosphatase isoenzymes by cellulose acetate electrophoresis.

     

Studies on phospholipase A in Trimeresurus flaoviridis venom. III. Purification and some properties of phospholipase A inhibitor in Habu serum.

Phospholipase A [EC 3.1.1.4] inhibitor was purified from Habu (Trimeresurus flavivurudls) serum by gel filtration on Sephadex G-200, chromatography on DE-23 cellulose and affinity chromatography on a Sepharose 4B-phospholipase A column. By these procedures, a 31-fold increase in specific activity was attained with a yield of 15%. The purified material was homogeneous as judged by cellulose acetate and polyacrylamide gel electrophoresis. It had an apparent molecular weight of 100,000 as measured by gel filtration on Sephadex G-200. The purified inhibitor was stable for 20 min at 80 degrees and was unstable below pH 6. It migrated before albumin in cellulose acetate electrophoresis and did not form any precipitin line with the crude venom or with purified phospholipase A in immunodiffusin tests. An 8-fold excess of the purified inhibitor by weight was required to inhibit completely both the egg yolk clearing action and the hemolytic action of phospholipase A.     

Cellulose acetate impregnated with polyacrylamide: a novel medium for electrophoresis

Cross-linked polyacrylamide gel containing a low proportion of methylenebis-acrylamide has been incorporated into cellulose acetate membranes. Unlike on cellulose acetate itself, electrophoresis on these modified membranes enables molecular sieving of proteins under a wide range of conditions. By modifying only part of the membrane, samples can be loaded in the normal way and sharpen as they migrate across the cellulose acetate-polyacrylamide boundary. The thin membranes retain their general ease of handling and speed of staining and destaining.     

Adaptation of mesophilic anaerobic sewage fermentor populations to thermophilic temperatures

Thermophilic (50 degrees C) and obligately thermophilic (60 degrees C) anaerobic carbohydrate- and protein-digesting and methanogenic bacterial populations were enumerated in a mesophilic (35 degrees C) fermentor anaerobically digesting municipal primary sludge. Of the total bacterial population in the mesophilic fementor, 9% were thermophiles (36 x 10(6)/ml) and 1% were obligate thermophiles (4.5 x 10(6)/ml). Of these 10%, the percentages of bacteria (thermophiles and obligate thermophiles, respectively) able to use specific substrates were further enumerated as follows: bacteria able to digest albumin, casein, starch, and mono- and disaccharides, 30 and 10%; pectin degraders, 10 and 0.2%; cellulose degraders, 2 and 0.06%; methanogens that grow with H2 and CO2, methanol, and dimethylamine, 9 and 1%; methanogens that grow with formate, 8 and 5%; and methanogens that grow with acetate, 25 and less than 0.8%. Shortly after the temperature was elevated from 35 to 50 or 60 degrees C, the digestion of albumin, casein, starch, and mono- and disaccharides was detected, and methane was produced from H2 and CO2. Methane produced from acetate was not delayed at 50 degrees C, but was delayed by 29 days at 60 degrees C. Methane produced from formate was delayed by 3 days, from methanol by 7 days, and from dimethylamine by 5 days at 50 and 60 degrees C. A 10- and 20-day acclimation period was required for hydrolysis of pectin and cellulose, respectively, at 50 degrees C. Digestion of pectin required 20 days and cellulose longer than 85 days when the temperature was elevated abruptly from 35 to 60 degrees C. The acclimation period for the digestion of pectin and cellulose at 60 degrees C was shortened to 3 and 15 days, respectively, by seeding with a small amount of a culture acclimated to 50 degrees C. The data suggest that enrichment of cellulolytic, pectinolytic, and acetate-utilizing bacteria is crucial for the digestion of sewage sludge at 60 degrees C.     

DNA sequence analysis: a formula to predict electrophoretic mobilities of oligonucleotides on cellulose acetate

A simple method has recently become available for sequence analysis of large oligonucleotide fragments. Sequences are derived from the characteristic mobility shifts of the sequential partial degradation products of the oligonucleotide on two dimensional homochromatography. We have now developed an empirical formula for predicting the relative mobilities of each of the partial products in the first dimension (electrophoresis on cellulose acetate gel). The formula allows a more precise interpretation of the sequence of the oligonucleotide. It eliminates the ambiguities present in the method previously reported for sequence analysis by simple inspection of the mobility shifts. In order to amplify the mobility shifts so that they may be more easily and accurately measured, methods have been developed for preparing and fractionating the oligonucleotides on 40 × 40 cm DEAE-cellulose plates. Both improvements have proven valuable for direct sequence analysis by mapping.     

Adaptation of mesophilic anaerobic sewage fermentor populations to thermophilic temperatures.

Thermophilic (50 degrees C) and obligately thermophilic (60 degrees C) anaerobic carbohydrate- and protein-digesting and methanogenic bacterial populations were enumerated in a mesophilic (35 degrees C) fermentor anaerobically digesting municipal primary sludge. Of the total bacterial population in the mesophilic fementor, 9% were thermophiles (36 x 10(6)/ml) and 1% were obligate thermophiles (4.5 x 10(6)/ml). Of these 10%, the percentages of bacteria (thermophiles and obligate thermophiles, respectively) able to use specific substrates were further enumerated as follows: bacteria able to digest albumin, casein, starch, and mono- and disaccharides, 30 and 10%; pectin degraders, 10 and 0.2%; cellulose degraders, 2 and 0.06%; methanogens that grow with H2 and CO2, methanol, and dimethylamine, 9 and 1%; methanogens that grow with formate, 8 and 5%; and methanogens that grow with acetate, 25 and less than 0.8%. Shortly after the temperature was elevated from 35 to 50 or 60 degrees C, the digestion of albumin, casein, starch, and mono- and disaccharides was detected, and methane was produced from H2 and CO2. Methane produced from acetate was not delayed at 50 degrees C, but was delayed by 29 days at 60 degrees C. Methane produced from formate was delayed by 3 days, from methanol by 7 days, and from dimethylamine by 5 days at 50 and 60 degrees C. A 10- and 20-day acclimation period was required for hydrolysis of pectin and cellulose, respectively, at 50 degrees C. Digestion of pectin required 20 days and cellulose longer than 85 days when the temperature was elevated abruptly from 35 to 60 degrees C. The acclimation period for the digestion of pectin and cellulose at 60 degrees C was shortened to 3 and 15 days, respectively, by seeding with a small amount of a culture acclimated to 50 degrees C. The data suggest that enrichment of cellulolytic, pectinolytic, and acetate-utilizing bacteria is crucial for the digestion of sewage sludge at 60 degrees C.     

Sensitivity in Detection of Monoclonal Immunoglobulins by Thin-Layer Isoelectric Focusing and Certain Electrophoreses in Laboratory Use

By applying sera containing various myeloma proteins to thin-layer isoelectric focusing at various pH ranges, we found that the best range of pH gradient for the detection of the band group specific for monoclonal immunoglobulin was from 5 to 8. The lowest concentration of monoclonal immunoglobulin detected by this method was approximately 0.1 mg/ml which was 8 to 32 times lower than the concentration detectable by cellulose acetate membrane electrophoresis or immunoelectrophoresis in routine laboratory use. Monoclonal protein concentration from 0.1 to 20 mg/ml was determined quantitatively. Blind tests on sera resulted in a disagreement between the concentration of monoclonal immunoglobulin assumed from the results of cellulose acetate membrane electrophoresis and immunoelectrophoresis and that determined by isoelectric focusing. These results suggest that isoelectric focusing is useful for the surveillance of monoclonal immunoglobulinemia provided that the technique and equipment are improved for laboratory tests.     

An anti-B specific haemagglutinin from the seeds of Mucuna flagellipes

Seed fractionation in neutral media produced a globulin fraction which agglutinated B-blood group specifically. Combined application of cellulose acetate electrophoresis and gel chromatography showed two protein bands in this fraction.     

An electrophoretic method for distinguishing the young fry of salmon Salmo salar (L.) from those of trout Salmo trutta (L.)

Young fry of salmon were distinguished from those of trout using electrophoresis of whole fry extracts on cellulose acetate sheets. The separation patterns were revealed by general protein staining. The method was found helpful in resolving the mixed salmon and trout fry of an upland stream.     

Electrophoretic characterization of acidic and neutral amylo 1-4-glucosidase (acid maltase) in human tissues and evidence for two electrophoretic variants in acid maltase deficiency

Acidic and neutral amylo 1–4-glucosidase activities were clearly separated by cellulose acetate electrophoresis. An extra isozyme was found in kidney extracts. Two variants, one fast and one slow, were detected in two cases of amylo glucosidase deficient patients.     

Ethanol Production by Thermophilic Bacteria: Fermentation of Cellulosic Substrates by Cocultures of Clostridium thermocellum and Clostridium thermohydrosulfuricum

The fermentation of various saccharides derived from cellulosic biomass to ethanol was examined in mono- and cocultures of Clostridium thermocellum strain LQRI and C. thermohydrosulfuricum strain 39E. C. thermohydrosulfuricum fermented glucose, cellobiose, and xylose, but not cellulose or xylan, and yielded ethanol/acetate ratios of >7.0. C. thermocellum fermented a variety of cellulosic substrates, glucose, and cellobiose, but not xylan or xylose, and yielded ethanol/acetate ratios of ~1.0. At nonlimiting cellulosic substrate concentrations (~1%), C. thermocellum cellulase hydrolysis products accumulated during monoculture fermentation of Solka Floc cellulose and included glucose, cellobiose, xylose, and xylobiose. A stable coculture that contained nearly equal numbers of C. thermocellum and C. thermohydrosulfuricum was established that fermented a variety of cellulosic substrates, and the ethanol yield observed was twofold higher than in C. thermocellum monoculture fermentations. The metabolic basis for the enhanced fermentation effectiveness of the coculture on Solka Floc cellulose included: the ability of C. thermocellum cellulase to hydrolyze α-cellulose and hemicellulose; the enhanced utilization of mono- and disaccharides by C. thermohydrosulfuricum; increased cellulose consumption; threefold increase in the ethanol production rate; and twofold decrease in the acetate production rate. The coculture actively fermented MN300 cellulose, Avicel, Solka Floc, SO₂-treated wood, and steam-exploded wood. The highest ethanol yield obtained was 1.8 mol of ethanol per mol of anhydroglucose unit in MN300 cellulose.     

Detection of acyl esterase activity on electrophoresis membranes and separation from hyaluronidase.

A method is described for the detection of acyl esterase activity on cellulose acetate membranes following electrophoresis of the enzyme. It uses the indigogenic substrate, indoxyl acetate, which directly forms the colored product visualized in the test. This substrate also detects activity of acetyl cholinesterase and pseudocholinesterase. With this method, bovine testicular hyaluronidase is shown to contain acyl esterase activity. By electrophoresis of hyaluronidase preparations at pH 6.8, esterase and hyaluronidase activities are separated, further assuring the specificity of the method for hyaluronidase.     

Rapid Detection of Viral Antibody by Cellulose Acetate Electrophoresis

An immunoelectrophoretic procedure utilizing microprecipitation and cellulose acetate electrophoresis was developed for detection of antibody to specific virus. A model system of tobacco mosaic virus and homologous rabbit antiserum is described.