Publications

The list below presents a selection of publications based on scientific work where JustTLC has been evaluated, reviewed or used.

3’ -UTR-dependent degradation of the aceA mRNA encoding the glyoxylate cycle enzyme isocitrate lyase by RNase E/G in Corynebacterium glutamicum
Tomoya Maeda, Masaaki Wachi
Applied and Environmental Microbiology, Published ahead of print, October 2012

Abstract

We previously reported that the Corynebacterium glutamicum RNase E/G encoded by the rneG gene (NCgl2281) is required for the 5’ maturation of 5S rRNA. In the search for the intracellular target RNAs of RNase E/G other than the 5S rRNA precursor, we detected that the amount of isocitrate lyase, an enzyme of the glyoxylate cycle, increased in rneG knockout mutant cells grown on sodium acetate as the sole carbon source. Rifampicin chase experiments showed that the half-life of the aceA mRNA was about 4 times longer in the rneG knockout mutant than in the wild type. Quantitative real-time PCR analysis also confirmed that the level of aceA mRNA was approximately 3-fold higher in the rneG knockout mutant strain than in the wild type. Such differences were not observed in other mRNAs encoding enzymes involved in acetate metabolism. Analysis by 3’ rapid amplification of cDNA ends suggested that RNase E/G cleaves the aceA mRNA at a single-stranded AU-rich region in the 3’ untranslated region (3’ -UTR). The lacZ fusion assay showed that the 3’ -UTR rendered lacZ mRNA RNase E/G dependent. These findings indicate that RNase E/G is a novel regulator of the glyoxylate cycle in C. glutamicum.

Full article at: 10.1128/​AEM.02304-12

Distribution measurements of 3,4-methylenedioxymethamphetamine and its metabolites in organs by matrix-assisted laser desorption/ ionization imaging mass spectrometry using an automatic matrix spraying system with an air brush and a turntable
Kenji Kuwayama, Kenji Tsujikawa, Hajime Miyaguchi, et al.
Analytical and Bioanalytical Chemistry, Volume 404, Issue 6-7, October 2012, Pages 1823–1830

Abstract

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI/IMS) is a useful tool for measuring drug distributions. To obtain reproducible analytical results with MALDI/IMS, it is essential to apply a homogeneous matrix coating onto sample surfaces. A simple and inexpensive automatic matrix spraying system (AMSS) with good reproducibility was developed in this study. In addition, drug distributions in organs were measured by MALDI/IMS using the AMSS for forensic toxicology applications. The AMSS was constructed from simple components, including an air brush, a turntable, and a microscope. Organ slices placed onto conductive sheets were attached to the turntable. The trigger of the air brush was held with a clamp to ensure that it sprayed continuously onto a defined area of the table. Periodic spraying of the matrix solution and evaporation of solvent were performed by rotating the turntable. The droplets and crystals on the sample surfaces were observed under a microscope attached to the turntable. The droplet size, rotation rate of the turntable, and the formulation of the matrix solution were optimized. The homogeneity of the matrix coating was evaluated using the coefficients of variation (CV) obtained by quantifying the color density of the sheet surface. The AMSS enabled more homogeneous matrix coating (intersheet CV05.4 %) than manual spraying (intersheet CV016.7 %) when 10 mL of 0.5 % aqueous trifluoroacetic acid/acetonitrile (1:3, v/v) containing 10 mg/mL α- cyano-4-hydroxycinnamic acid were sprayed as droplets less than 50 μm in diameter onto a turntable rotating at 30 rpm. The distributions of 3,4-methylenedioxymethamphetamine (MDMA) and its main metabolites in the brain, liver, and kidney of a mouse that died from an MDMA overdose (58 mg/kg i.p.) were visualized by MALDI/IMS using the AMSS. The ion intensities of MDMAobtained from the same regions on three sequential kidney slices showed acceptable variations (CV02.9–8.8 % for five different regions), implying repeatable measurements with MALDI/IMS using the AMSS. It was revealed that MDMAwas particularly concentrated around the brain stem and the major calix of the kidney. The AMSS would be suitable for preparing samples for measuring the distributions of drugs in organs at toxic dose levels in forensic toxicological applications.

Full article at: 10.1007/s00216-012-6279-x

β-Galactosidase Treatment Is a Common First-stage Modification of the Three Major Subtypes of Gc Protein to GcMAF
Yoshihiro Uto, Syota Yamamoto, Hirotaka Mukai, et al.
Anticancer Research, Volume 32, Issue 6, June 2012, Pages 2359-2364

Abstract

The 1f1f subtype of the group-specific component (Gc) protein is converted into Gc protein-derived macrophage-activating factor (GcMAF) by enzymatic processing with β-galactosidase and sialidase. We previously demonstrated that preGc(1f1f)MAF, a full Gc(1f1f) protein otherwise lacking a galactosyl moiety, can be converted to GcMAF by treatment with mouse peritoneal fluid. Here, we investigated the effects of the β-galactosidase-treated 1s1s and 22 subtypes of Gc protein (preGc(1s1s)MAF and preGc(22)MAF) on the phagocytic activation of mouse peritoneal macrophages.

Full article at: PMID 22641675

Effect of Growth Medium pH of Aeropyrum pernix on Structural Properties and Fluidity of Archaeosomes
Ajda Ota, Dejan Gmajner, Marjeta Sentjurc, et al.
Archaea, Volume 2012, Article ID 285152, April 2012, 9 Pages

Abstract

The influence of pH (6.0; 7.0; 8.0) of the growth medium of Aeropyrum pernix K1 on the structural organization and fluidity of archaeosomes prepared from a polar-lipid methanol fraction (PLMF) was investigated using fluorescence anisotropy and electron paramagnetic resonance (EPR) spectroscopy. Fluorescence anisotropy of the lipophilic fluorofore 1,6-diphenyl-1,3,5- hexatriene and empirical correlation time of the spin probe methylester of 5-doxylpalmitate revealed gradual changes with increasing temperature for the pH. A similar effect has been observed by using the trimethylammonium-6-diphenyl-1,3,5- hexatriene, although the temperature changes were much smaller. As the fluorescence steady-state anisotropy and the empirical correlation time obtained directly from the EPR spectra alone did not provide detailed structural information, the EPR spectra were analysed by computer simulation. This analysis showed that the archaeosome membranes are heterogeneous and composed of several regions with different modes of spin-probe motion at temperatures below 70◦C. At higher temperatures, these membranes become more homogeneous and can be described by only one spectral component. Both methods indicate that the pH of the growth medium of A. pernix does not significantly influence its average membrane fluidity. These results are in accordance with TLC analysis of isolated lipids, which show no significant differences between PLMF isolated from A. pernix grown in medium with different pH.

Full article at: 10.1155/2012/285152

Engineering the cytokinin-glucoside specificity of the maize b-D-glucosidase Zm-p60.1 using site-directed random mutagenesis
Tomáš Filipia, Pavel Mazuraa, Lubomír Jandaa, et al.
Phytochemistry, Volume 74, February 2012, Pages 40–48

Abstract

The maize β-d-glucosidase Zm-p60.1 releases active cytokinins from their storage/transport forms, and its over-expression in tobacco disrupts zeatin metabolism. The role of the active-site microenvironment in fine-tuning Zm-p60.1 substrate specificity has been explored, particularly in the W373K mutant, using site-directed random mutagenesis to investigate the influence of amino acid changes around the 373 position. Two triple (P372T/W373K/M376L and P372S/W373K/M376L) and three double mutants (P372T/W373K, P372S/W373K and W373K/M376L) were prepared. Their catalytic parameters with two artificial substrates show tight interdependence between substrate catalysis and protein structure. P372T/W373K/M376L exhibited the most significant effect on natural substrate specificity: the ratio of hydrolysis of cis-zeatin-O-β-d-glucopyranoside versus the trans-zeatin-O-β-d-glucopyranoside shifted from 1.3 in wild-type to 9.4 in favor of the cis- isomer. The P372T and M376L mutations in P372T/W373K/M376L also significantly restored the hydrolytic velocity of the W373K mutant, up to 60% of wild-type velocity with cis-zeatin-O-β-d-glucopyranoside. These findings reveal complex relationships among amino acid residues that modulate substrate specificity and show the utility of site-directed random mutagenesis for changing and/or fine-tuning enzymes. Preferential cleavage of specific isomer-conjugates and the capacity to manipulate such preferences will allow the development of powerful tools for detailed probing and fine-tuning of cytokinin metabolism in planta.

Full article at: 10.1016/j.phytochem.2011.10.008

Rapid, simple, and highly sensitive analysis of drugs in biological samples using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry
Kenji Kuwayama, Kenji Tsujikawa, Hajime Miyaguchi, et al.
Analytical and Bioanalytical Chemistry, Volume 404, Issue 3, January 2012, Pages 1257-67

Abstract

Rapid and precise identification of toxic substances is necessary for urgent diagnosis and treatment of poisoning cases and for establishing the cause of death in postmortem examinations. However, identification of compounds in biological samples using gas chromatography and liquid chromatography coupled with mass spectrometry entails time-consuming and labor-intensive sample preparations. In this study, we examined a simple preparation and highly sensitive analysis of drugs in biological samples such as urine, plasma, and organs using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry (TLC/MALDI/MS). When the urine containing 3,4- methylenedioxymethamphetamine (MDMA) without sample dilution was spotted on a thin-layer chromatography (TLC) plate and was analyzed by TLC/MALDI/MS, the detection limit of the MDMA spot was 0.05 ng/spot. The value was the same as that in aqueous solution spotted on a stainless steel plate. All the 11 psychotropic compounds tested (MDMA, 4- hydroxy-3-methoxymethamphetamine, 3,4-methylenedioxyamphetamine, methamphetamine, p-hydroxymethamphetamine, amphetamine, ketamine, caffeine, chlorpromazine, triazolam, and morphine) on a TLC plate were detected at levels of 0.05−5 ng, and the type (layer thickness and fluorescence) of TLC plate did not affect detection sensitivity. In addition, when rat liver homogenate obtained after MDMA administration (10 mg/kg) was spotted on a TLC plate, MDMA and its main metabolites were identified using TLC/MALDI/MS, and the spots on a TLC plate were visualized by MALDI/imaging MS. The total analytical time from spotting of intact biological samples to the output of analytical results was within 30 min. TLC/MALDI/MS enabled rapid, simple, and highly sensitive analysis of drugs from intact biological samples and crude extracts. Accordingly, this method could be applied to rapid drug screening and precise identification of toxic substances in poisoning cases and postmortem examinations.

Full article at: 10.1007/s00216-011-5576-0

A Comparative Study Concerning The Image Analysis in Thin Layer Chromatography of Fluorescent Compounds
Lucian Alexandru Fazakaşa, Rodica Domnica Naşcu-Briciua, Costel Sârbua
Journal of Liquid Chromatography & Related Technologies, Volume 34, Issue 19, 2011, Pages 2315-2325

Abstract

The use of image analysis software for the detection and quantitative evaluation of fluorescent compounds in thin layer chromatography is investigated. Four different image analysis software packages (Sorbfil TLC, Biodit, JustTLC, and Melanie) were applied and compared. The image analysis methods were developed and validated for the simultaneous determination of quercetin and kaempferol in food supplements. The chromatographic separation used Silica gel 60 TLC plates as the stationary phase and carbon tetrachloride:acetone:formic acid (35:11:3, v/v/v) as the mobile phase. The plate images were recorded by a digital camera and imported in the image analysis software. The final results were validated by evaluating the linearity, precision, and accuracy of the methods used to quantify synthetic and real samples. All the results suggest that the image analysis software packages are proper to investigate the fluorescent compounds in quantitative thin layer chromatography.

Full article at: 10.1080/10826076.2011.587226

Proteomic Analysis of Two Types of Exosomes in Human Whole Saliva
Yuko Ogawa, Yuri Miura, Akira Harazono, et al.
Biological and Pharmaceutical Bulletin, Volume 34, No 1, 2011, Pages 13-23

Abstract

Saliva contains a large number of proteins that participate in the protection of oral tissue. Exosomes are small vesicles (30—100 nm in diameter) with an endosome-derived limiting membrane that are secreted by a diverse range of cell types. We have recently demonstrated that exosomes are present in human whole saliva. In this study, we found that whole saliva contained at least two types of exosomes (exosome I and exosome II) that are different in size and protein composition. Proteomic analysis revealed that both types of exosomes contained Alix, Tsg101 and Hsp70, all exosomal markers, immunoglobulin A and polymeric immunoglobulin receptor, whereas they had different protein compositions. Most of dipeptidyl peptidase IV known as CD26 in whole saliva, was present on the exosome II and metabolically active in cleaving chemokines (CXCL11 and CXCL12). Human whole saliva exosomes might participate in the catabolism of bioactive peptides and play a regulatory role in local immune defense in the oral cavity.

Full article at: 10.1248/bpb.34.13

Thin-layer chromatographic analysis of carotenoids in plant and animal samples
Alam Zeb1, Michael Murkovic
JPC - Journal of Planar Chromatography - Modern TLC, Volume 23, Number 2/April 2010, Pages 94-103

Abstract

Carotenoids are among the most widespread and important pigments in living organisms. They are found in common foods and vegetables. The characteristic pattern of alternating single and double bonds in the polyene backbone enables them to absorb excess energy from other molecules. The nature of the specific end groups on carotenoids may effect their polarity, thus solubility ranges from acetone to hexane. Because of this wide range of polarity, specific extraction and separation procedures are required. In these procedures use of planar chromatography in food analysis might seem a minor aspect of carotenoid analysis. This review describes available data on analysis of carotenoids by thin-layer chromatography (TLC). It has been found that petroleum ether, acetone, and hexane are the major mobile phases used for TLC. Thin-layer chromatography was found to have the potential to be the first choice for analysis of carotenoids in biological samples. The uses of other, orthogonal chromatographic methods, for example HPLC, spectroscopy (mass spectroscopy), scanning densitometry, and image analysis with TLC can enable precise analysis of carotenoids.

Full article at: 10.1556/JPC.23.2010.2.1

Quantitative Thin Layer Chromatography

Gustav Träff and Ulf Ellervik

Poster at 21st Organic Days 2008 conference 9-12 June Åhus, Sweden

Abstract

Thin layer chromatography (TLC) has been a very popular qualitative analysis for over 50 years. However, many have not considered it as a quantitative alternative. We have revealed the true power of quantitative TLC by developing a simple yet powerful solution based on modern image analysis. The solution only adds two extra steps compared to traditional TLC, but gives measuring accuracy suitable for high-throughput screening or physical organic investigations in return.

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Reductive Openings of Benzylidene Acetals. Kinetic Studies of Borane and Alane Activation by Lewis Acids
Richard Johnsson, Risto Cukalevski, Fanny Dragén, Damir Ivanisevic, Ida Johansson, Linn Petersson, Erika Elgstrand Wettergren, Kabo Yam, Beatrice Yang and Ulf Ellervik
Carbohydrate Research, Volume 343, Issue 17, 24 November 2008, Pages 2997-3000

Abstract

The reaction kinetics for a number of reductive openings of methyl 2,3-di- O-benzyl-4,6-O-benzylidene-α-D-glucopyranoside have been investigated. Openings to give free HO-6 (using BH3·THF–AlCl3–THF or LiAlH4–AlCl3–Et2O) follow first order kinetics, while reactions yielding free HO-4 (using BH3·NMe3–AlCl3–THF or BH3·NMe3–BF3·OEt2–THF) follow higher order kinetics. The addition of water to the BH3·NMe3–AlCl3–THF results in faster reactions. The BH3·SMe2–AlCl3–THF system constitutes a borderline case, yielding both free HO-6 (by a first order reaction) and free HO-4 (by a higher order reaction). These results correlate well with the concept of regioselectivity by activation of borane complexes.

Full article at: 10.1016/j.carres.2008.08.022

Planar Chromatography
Joseph Sherma
Analytical Chemistry, 80 (12), 2008, Pages 4253–4267

Abstract

This review covers the literature of thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) found by computer-assisted searching in Chemical Abstracts and the ISI Web of Science from November 1, 2005 to November 1, 2007. The literature search was augmented by consulting Analytical Abstracts, and the following journals that regularly publish papers on TLC were searched directly: Journal of Chromatography (parts A and B and the bibliography issues), Journal of Chromatographic Science, Chromatographia, Analyti- cal Chemistry , Journal of Liquid Chromatography & Related Technologies, Journal of AOAC International, Journal of Planar Chromatography-Modern TLC, Acta Chromatographica, and Acta Universitatis Cibiniensis, Seria F, Chemia.

Full article at: 10.1021/ac7023415

Reductive Openings of Acetals: Explanation of Regioselectivity in Borane Reductions by Mechanistic Studies
Richard Johnsson, Dan Olsson and Ulf Ellervik
Journal of Organic Chemistry, 73 (14), 2008, Pages 5226–5232

Abstract

The mechanisms of regioselective reductive openings of acetals were investigated in several model systems by a combination of Hammett plots, kinetic experiments, density functional calculations, and 11B NMR. The regioselectivity of borane reductions of cyclic acetals can be controlled by the choice of borane. Lewis acid activation of BH3·NMe3 increases the reaction rate and renders the borane the most electrophilic species, which associates to the more electron-rich oxygen of the acetal. In contrary, without activation, the regioselectivity is instead directed by the Lewis acid, as exemplified by the reaction with BH3·THF.

Full article at: 10.1021/jo800396g

Evaluation of quantitative thin layer chromatography using staining reagents
Richard Johnsson, Gustav Träff, Martin Sundén and Ulf Ellervik
Journal of Chromatography A, Volume 1164, Issues 1-2, 14 September 2007, Pages 298-305

Abstract

Thin layer chromatography (TLC) using staining reagents is a superior method for analyzing organic compounds without chromophores. It is fast, versatile and sometimes the only viable method. We have investigated quantitative TLC using staining reagents, in combination with modern image analysis software. Our results show that it is possible to get reliable measurements, suitable for high-throughput screening or physical organic investigations. The range of detection and the errors for the different parts of the process are illustrated. We show that the errors are largely due to the staining process and can be diminished by measuring ratios of compounds.

Full article at: 10.1016/j.chroma.2007.07.029

The proper citation for JustTLC is as follows: JustTLC Version 4.0.3, Sweday, Lund, Sweden