Photoactivated zinc silicate in thin layer chromatography plates: A potential cause for error in liquid scintillation counting

Thin layer chromatography plates impregnated with fluorescent indicator permit easy identification of many compounds by exposure to ultraviolet light. Exposure of zinc silicate treated plates to ultraviolet light (254 nm) for 30 seconds induced photoluminescence which persisted at significant levels for longer than 24 hours. This artifact significantly interfered with radioactivity quantitation in three commercially available liquid scintillation solutions. Chromatography plates impregnated with calcium silicate demonstrated insignificant ultraviolet light induced photoluminescence. The use of ¹⁴C rather than ³H-radiolabeled compounds, protecting the plate with aluminum foil when visualizing reference compounds with ultraviolet light, and heating the photoactivated plate to accelerate disappearance of the photoluminescence minimized the error in liquid scintillation counting.     

Identification in TLC of fructose and fructosyl derivatives in levan and sugar mixtures with resorcinol and thiourea

Fructose and fructosyl derivatives were detected on TLC plates using a modification of the Roe-Papadopoulos method. A large increase in sensitivity was obtained using H₂SO₄ in the TLC assay. Fructose and fructosyl groups (sucrose, raffinose, and levan) appeared as purple spots after spraying the modified Roe-Papadopoulus reagent onto the plate and then heating with hot air for a few minutes. Reducing sugars, such as glucose and xylose, and nonreducing sugar trehalose were not detectable by this technique.     

Display Of Ribonuclease T1 On The Surface Of Bacteriophage M13

Abstract

In this work the expression of functional ribonuclease T1 on the surface of the filamentous Escherichia coli phage M13 is described. Ribonuclease T1 was fused to the phage coat protein pIII and functionally displayed on the phage surface as shown by a negative staining zymogram and RNase indicator plates.     

Differentiation of species of Terebratella (Brachiopoda: Terebratellinae)

Abstract

Terebratella sanguinea and T. incollSpicua have been differentiabed on foramen position and ornament. Further comparison shows that the valves of T. sanguinea are more strongly curved than those of T. inconspicua, a condition resulting in a longer pedicile, a larger beak, and hinge plates with greater elevation. The relationship between hinge plate ccndition and shell curvature is evident in other terebratellid genera.     

Histochemistry of acetylcholine receptors and acetylcholinesterase during the formation of neuromuscular junctions in the urodele Hynobius nigrescens

The development and structure of neuromuscular junctions (n-m-js) in stylopodia of forelimbs of larvae and adults of Hynobius nigrescens were histochemically investigated for acetylcholine receptors (AChRs) and acetylcholinesterase (AChE) activity. In larvae, the tetramethyl rhodamine-labelled α-bungarotoxin (TMR-αBT) positive areas appeared either as small fluorescent spots or fluorescent plates of various sizes. The mature fluorescent plate was found to be formed by the successive addition of spots, and the plates thus established were arranged linearly parallel to the axes of muscle fibers. AChE activity occurred almost exactly at TMR-αBT-positive sites. In adults, plate assemblies were often seen as a single dotted line (type A form) for both AChR binding and AChE reaction, in contrast to larval n-m-js in which AChE activity appeared as a continuous line. By applying the TMR-αBT method, two other forms of adult n-m-js were observed: type B, a long dotted line several plates wide; and type C, with a cluster of plates randomly dispersed over the whole width of the muscle fiber. It seems that protoforms of the latter two forms of n-m-js appear in the muscles just before and after metamorphosis.     

Immunostaining on thin-layer chromatograms of oligosaccharides released from gangliosides by endoglycoceramidase

A method for immobilizing oligosaccharides on a TLC plate for immunostaining has been developed. N-Glycolylneuraminic acid (NeuGc)-containing oligosaccharides derived from II3NeuGc-LacCer, IV3NeuGc-nLcOse4Cer, II3NeuGc-GgOse3Cer, and II3(NeuGc)2-LacCer by digestion with our newly isolated endoglycoceramidase (Ito, M. & Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282) and sialyllactose were chromatographed on polyamide 11 TLC or NH2-HPTLC plates, and covalently linked to the plates by reductive amination with sodium cyanoborohydride (NaBH3-CN). The immobilized oligosaccharides were detected by enzyme-immunostaining using NeuGc-specific chicken anti-NeuGc-LacCer and horseradish peroxidase-conjugated rabbit anti-chicken IgG. II3NeuGc-nLcOse4 showed the highest reactivity with the antibody, followed by II3NeuGc-GgOse3. As little as 0.8 nmol of the NeuGc-containing oligosaccharides was detected. The polyamide 11 TLC aluminum plate was found to be more suitable for the immunostaining than the NH2-HPTLC plate under the conditions used. For binding of the oligosaccharides to the NH2-HPTLC plate, reductive amination was found to be superior to the heating method reported earlier.     

Biotin-Bearing pH-Sensitive Liposomes: High-Affinity Binding to Avidin Layer

Abstract

A procedure is described for the preparation of biotin-bearing pH-sensitive liposomes. Liposomes were composed of phosphatidylethanolamine/oleic acid/ cholesterol/biotinaminocaproyl phosphatidylethanolamine (molar ratio 7:3:3:0.1) and contained the fluorescent dye calcein in self-quenched concentrations. these liposomes rapidly destabilized in weakly acidic media (pH 5.7). Liposomes were also readily agglutinated by acetylated avidin. Addition of biotin blocked the agglutination process. Binding of liposomes to a plastic surface coated with acetylated avidin was studied. the high-affinity binding with an apparent K_D value up to 10^?11 M liposomes was demonstrated by Scatchard analysis of the binding curves. A method of efficiently coating polyvinyl chloride microELisA plates with acylated avidin is also described.     

Quinacrine fluorescence analysis of the chromosomes of Dipsacus fullonum L. (Dipsacaceae)

Abstract

Quinacrine fluorescence analysis of the chromosome complement of Dipsacus fullonum L. shows clear heterochromatic regions appearing as brightly fluorescent bands or clusters of fluorescent dots in most chromosomes. The resulting fluorescent pattern is chromosome specific.     

Development of a new doubly-labeled fluorescent ceramide probe for monitoring the metabolism of sphingolipids in living cells

A new ceramide analog, 1, containing two fluorescent dyes, NBD in the N-acyl part and KFL5 in the alkyl part, was synthesized. The fluorescence from both NBD and KFL5 was detected in living cells in a time-dependent manner. A multi-wavelength fluorescence detector was used to detect ceramide metabolites including sphingosine, sphingosine-1-phosphate, glucosylceramide, and sphingomyelin, which are connected to the fluorescent dyes, simultaneously in a single TLC plate.     

Evidence for sodium metasilicate receptors on the human osteoblast cell surface; spatial localization and binding properties

Abstract

We report details of the interaction of sodium metasilicate with osteoblast cellular membranes using Fluoresceinphosphatidylethanolamine (FPE) as a fluorescent indicator of membrane interactions. Fluorescence imaging studies of the FPE-based indicator system revealed areas of localized binding that would be consistent with the presence of a structure with ‘receptor-like’ properties. From these results, it seems unlikely that silica binds ‘non-specifically’ to the osteoblast surface. Moreover, the receptors are localized into membrane domains. Such regions of the cell membrane could well be structures such as ‘rafts’ or other such localized domains within the membrane. The binding profile of silica with the osteoblast cell surface takes place with all the characteristics of a receptor-mediated process best represented by a cooperativity (sigmoidal) binding model with a Hill coefficient of 3.6.     

Quantitative One Step Derivatization of Oligonucleotides by a Fluorescent Label Through Abasic Site Formation

Abstract

Reaction of abasic site-containing oligonucleotides with an oxyamino fluorescent label is described. The reaction represents an efficient method to functionalize oligonucleotides at preselected positions.     

Thin Layer Chromatography of Radiolabeled Oligonucleotide Analogues: A Rapid and Sensitive Purity Assay

Abstract

Oligonucleotide analogues are being developed for use in cell culture, animals and for therapy. Prior to use it is important to have an indication of the oligomers' purity. Thin layer chromatography (TLC) has been applied to analyze hosphoromonothioate and phosphorodithioate oligonucleotides radiolabeled with either ³²P Or ¹⁴C. TLC coupled with radioactivity has been compared to Polyacrylamide Gel Electrophoresis (PAGE) and High Pressure Liquid Chromatography (HPLC). TLC is a rapid and sensitive alternative to these methods and is particularly suited for chemically modified oligonucleotides.     

An easy ratiometric compensation for the extracellular Ca indicator-caused fluorescence artifact

Measurement of intracellular Ca²⁺ dynamics is one of the most central real-time assays for cellular signaling. Ratiometric methods reduce the need for internal calibration and also effectively compensate for most artifacts when used in imaging. However, ratiometric calculation cannot compensate for extracellularly leaked (and fluorescent) Ca²⁺ indicator and will instead indicate erroneous Ca²⁺ concentration. This frequently occurs in systems where extracellular indicator is accumulated such as fluorescence spectrophotometers and plate readers. Here I present a method that, for the first time, fully compensates for this phenomenon. The method uses a single-step internal calibration together with a predefined ratiometric calibration protocol.     

Modulation of the cardiac membrane-bound cyclic nucleotide phosphodiesterase: inhibition due to a contamination of TLC purified S-adenosyl-L-[methyl-3H] methionine by Zn++ ions

Cardiac membranes pretreated with S-Adenosyl-L-[methyl-3H] methionine([3H] SAM) purified on TLC silica gel 60 F254 plates exhibited a marked decrease in cyclic AMP and cyclic GMP phosphodiesterase activity. However, this inhibition did not appear when membranes were incubated with either [14C] SAM or unlabelled SAM. We showed that, during the TLC purification of [3H] SAM, which involved an acidic elution step, minute amounts of the fluorescent indicator F254 (Zn sulfur) were eluted. The contaminating Zn++ ions strongly inhibited cyclic nucleotide phosphodiesterase activity and phospholipid methylation with I50 values in the micromolar range.     

Enantiomeric resolution of amino acids by thin-layer chromatography

A simple and rapid method of separating optical isomers of amino acids on a reversed-phase TLC plate, without using impregnated plates or a chiral mobile phase, is described. Amino acids derivatized with 1-fluoro-2,4-dinitrophenyl-5- -alanine amide were spotted on a reversed phase pre-coated TLC plate. Enantiomers of glutamate and aspartate were separated most effectively with solvent consisting of 25% acetonitrile in triethylamine-phosphate buffer (50 mM, pH 5.5) (v/v). Separation of - and -serine was achieved with 30% of acetonitrile solvent. The enantiomers of threonine, proline and alanine were separated with 35% of acetonitrile solvent, and those of methionine, valine, phenylalanine and leucine with 40% of acetonitrile solvent. The possibility of using TLC for quantitative determination of amino acid enantiomers was shown by the quantitative recovery of - and -alanine from the TLC plate in the range of 0.56–4.48 nmol.     

Book Reviews

Plant Life in West Africa. G. W. LAWSON. 8 × 5¼ in. pp. ix + 150 with 34 plates. Oxford: University Press, 1966. 22s. 6d. Reviewed by R. W. J. Keay

Wild Life and the Countryside: Nos. 255–262. Wildlife Publications, 10 Bouverie St., London, E.C.4. 16s./p.a. (9 copies). Reviewed by Ernest Neal

Vertebrate Palaeontology. A. S. ROMER. 3rd edition, pp. 468, 443 figs. Chicago and London: University of Chicago Press, 1967, 72s. Reviewed by C. B. Cox

Sensory Mechanisms. J. CASE. Pp. 113. Macmillan, N.Y.: Current Concepts in Biology Series, 1966. Reviewed by J. D. Carthy

Wealth from the Oceans. TONY LOFTAS. Pp. 69, 20 plates. London: Phoenix House (Progress of Science Series), 1967. 12s. 6d. Reviewed by J. Bossanyi

Poisonous Plants and Fungi in Colour. P. M. NORTH. Pp. 161, 133 plates, 7 figs. London: Blandford Press Ltd., 1967. 21s. Od. Reviewed by C. T. Prime     

Reproducible preparation of spheroids of pancreatic hormone positive cells from human iPS cells: An in vitro study

BackgroundTransplantation of islets of Langerhans is regarded as a promising therapy for type 1 diabetes. A large number of β-cells are required for the treatment of human type 1 diabetes. Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, have been considered as new sources for cell replacement therapy.MethodsCell aggregates were prepared from human iPS cells using agarose microwell plates and differentiated into pancreatic endocrine cells by changing the culture media with different additives.ResultsAfter 20 days of culture, approximately 30% of cells in aggregates were positive for C-peptide. After another 14 days in culture, the cells gained an ability to alter C-peptide release in response to changes in the glucose concentration.ConclusionsUniform aggregates of human iPSCs were easily prepared on agarose microwell plates and efficiently differentiated into the pancreatic endocrine lineage. Thus, aggregate culture is a suitable method for preparing islet-like aggregates from human iPSCs.General significanceOur results indicate that the microwell plate is suitable for scaling up the preparation of pancreatic endocrine cells from human iPS cells in a robotic system.     

Analysis of tamoxifen-induced DNA adducts by 32P-postlabelling assay using different chromatographic techniques

DNA isolated from livers of rats receiving tamoxifen was analysed by the ³²P-postlabelling method. The postlabelled DNA hydrolysis mixture was analysed both by reversed-phase HPLC with ³²P on-line detection and by TLC on polyethyleneimine plates followed by autoradiography. Using the HPLC method, five well separated adduct peaks could be detected, while by the TLC method, two groups of adduct spots were observed. The detection limit of the TLC assay was lower (0.5 adducts/10¹⁰ nucleotides) than that of the HPLC assay (3 adducts/10¹⁰ nucleotides). Thus, the TLC assay is more sensitive but also more laborious. The advantages of the HPLC assay were, in addition to better resolution, the ease of quantification and operation.     

SEM survey on mediterranean species of Podolampas

Abstract

In this work we have considered the features of thecal plates of some mediterranean species of Podolampas. The thecal plates have pores different in size and distribution according to their position and to the species considered. We have found some constant structures: kind of perforations of the apical, preequatorial and postequatorial thecal plates. The antiapical thecal plates have both constant and distinctive specific features.     

A TLC bioautographic method for the detection of α‐ and β‐glucosidase inhibitors in plant extracts

Introduction – Bioautographic assays using TLC play an important role in the search for active compounds from plants. A TLC assay has previously been established for the detection of β‐glucosidase inhibitors but not for α‐glucosidase. Nonetheless, α‐glucosidase inhibition is an important target for therapeutic agents against of type 2 diabetes and anti‐viral infections. Objective – To develop a TLC bioautographic method to detect α‐ and β‐glucosidase inhibitors in plant extracts. Methodology – The enzymes α‐ and β‐d ‐glucosidase were dissolved in sodium acetate buffer. After migration of the samples, the TLC plate was sprayed with enzyme solution and incubated at room temperature for 60 min in the case of α‐d ‐glucosidase, and 37°C for 20 min in the case of β‐d ‐glucosidase. For detection of the active enzyme, solutions of 2‐naphthyl‐α‐D‐glucopyranoside or 2‐naphthyl‐β‐D‐glucopyranoside and Fast Blue Salt were mixed at a ratio of 1 : 1 (for α‐d ‐glucosidase) or 1 : 4 (for β‐d ‐glucosidase) and sprayed onto the plate to give a purple background colouration after 2–5 min. Results – Enzyme inhibitors were visualised as white spots on the TLC plates. Conduritol B epoxide inhibited α‐d ‐glucosidase and β‐d ‐glucosidase down to 0.1 µg. Methanol extracts of Tussilago farfara and Urtica dioica after migration on TLC gave enzymatic inhibition when applied in amounts of 100 µg for α‐glucosidase and 50 µg for β‐glucosidase. Conclusion – The screening test was able to detect inhibition of α‐ and β‐glucosidases by pure reference substances and by compounds present in complex matrices, such as plant extracts. Copyright © 2009 John Wiley & Sons, Ltd.