Cellulose Thin Layer Research Articles

Study of the lipophilicity and retention behavior of some s-triazine derivatives on aminoplast and cellulose

The chromatographic behavior of four groups of s -triazine derivatives (15 compounds) has been studied on aminoplast and cellulose thin layers with two mobile phases, methanol-water-acetonitrile, 30 + 20 + X , where X = 1–10 ( v/v ), and methanol-dilute acetic acid, 30 + 20 ( v/v ). The mechanism of retention was investigated by changing of volume fraction of acetonitrile in the first mobile phase. Reversed-phase chromatography occurs on both supports. Retention constants, R M o were determined by extrapolation and good correlation was obtained between retention constants, R M o and log P . These retention constants can be used as the measure of the lipophilicity of compounds. The effect of mobile phase pH on the chromatographic retention of s -triazine derivatives was examined and approximate protonation constants, pK a , were determined on the basis of the dependence of retention on pH. These constants correlated well with Hammett substituent constants σ .

Effects of FTDP-17 mutations on the in vitro phosphorylation of tau by glycogen synthase kinase 3β identified by mass spectrometry demonstrate certain mutations exert long-range conformational changes

In vitro phosphorylation of recombinant wild-type 2N4R tau and FTDP-17 exonic mutant forms P301L, V337M and R406W by glycogen synthase kinase 3β (GSK3β) was examined by two dimensional phosphopeptide mapping analysis on thin layer cellulose plates. Comparison of these peptide maps with those generated from wild-type 1N4R tau isoform from which the phosphopeptide constituents and sites of phosphorylation had been determined previously, enabled us to monitor directly changes in phosphorylation of the individual tau proteins. No differences were found in the phosphorylation of wild-type, P301L or V337M tau by GSK3β but the R406W mutant showed at least two clear differences from the other three tau proteins. The peptides, identified by mass spectrometry corresponding to phosphorylation at both threonine 231 and serine 235 (spot 3), serines 396, 400 and 404 (spot 6a) and serines 195 and 199 (spot 6b) were absent from the R406W peptide map. The findings imply that the R406W mutation in tau exerts long-range conformational effects on the structure of tau.

Preconcentration of arsenic(V) as molybdoarsenic heteropoly acid by dynamic sorption

The sorption of molybdoarsenic heteropoly acid (MAA) and its ion pair with tri-n-octylamine (TOA) on sorbents of different nature and hydrophobic filters has been studied. It is demonstrated that MAA is efficiently extracted on the polyacrylate sorbent Amberlyte XAD-8; the distribution coefficient of arsenic is 1 x 104 cm3/g for the sorption from 0.35-1.5 M H2SO4. Conditions of the quantitative extraction of MAA in the presence of TOA on thin-layer cellulose triacetate filters are found. A procedure is proposed for the sorption-X-ray fluorescence determination of arsenic in potable water. The detection limit of arsenic is 10 Μg/L for the sorption from 100 mL of a solution.

Cytochemistry of developing cotton fibers:: A hypothesized relationship between motes and non-dyeing fibers

Developing lint fibers from normally developing seeds and from motes of Upland cotton ( Gossypium hirsutum L. cv Acala SJ2 and cv Acala Maxxa) were compared with respect to cell wall cytochemistry. Fibers from motes (unfertilized ovules or aborted seeds) were used as a model for non-dyeing fibers, a contributor to poor quality textiles. Primary and secondary fiber walls were stained for pectin, cutin, suberin, callose, and cellulose. Up to 25 days post-anthesis (DPA), mote fibers had pectinaceous primary walls with a thin layer of cellulose, indistinguishable from primary walls of normally developing fibers. However, secondary walls in mote fibers had scanty, irregularly deposited cellulose, while secondary walls in normal fibers were well developed with thick cellulosic layers. At 29 DPA and at later stages of development, fibers on normal seeds had well-developed secondary walls, which were proportionally less developed on medium-sized and small motes. Nonetheless, fibers, which varied in diameter and cell wall thickness, were found on all normal seeds, on medium-sized and on small motes; most fiber cell walls were smaller in thickness at the chalazal than at the micropylar end. Regardless of their thickness, fibers on seeds and motes had cellulosic secondary cell walls. As most industrial dyes are specific for cellulose, the paucity of cellulose in mote fibers is consistent with the suggested causal relationship between lack of cellulose and the non-dyeing nature of some fibers. There were indications that immature fibers at the chalazal end of seeds may also contribute to dye imperfections in fabrics.

Conserved in VivoPhosphorylation of Calnexin at Casein Kinase II Sites as Well as a Protein Kinase C/Proline-directed Kinase Site

Calnexin is a lectin-like chaperone of the endoplasmic reticulum (ER) that couples temporally and spatially N-linked oligosaccharide modifications with the productive folding of newly synthesized glycoproteins. Calnexin was originally identified as a major type I integral membrane protein substrate of kinase(s) associated with the ER. Casein kinase II (CK2) was subsequently identified as an ER-associated kinase responsible for the in vitro phosphorylation of calnexin in microsomes (Ou, W-J., Thomas, D. Y., Bell, A. W., and Bergeron, J. J. M. (1992) J. Biol. Chem. 267, 23789-23796). We now report on the in vivo sites of calnexin phosphorylation. After 32PO4 labeling of HepG2 and Madin-Darby canine kidney cells, immunoprecipitated calnexin was phosphorylated exclusively on serine residues. Using nonradiolabeled cells, we subjected calnexin immunoprecipitates to in gel tryptic digestion followed by nanoelectrospray mass spectrometry employing selective scans specific for detection of phosphorylated fragments. Mass analyses identified three phosphorylated sites in calnexin from either HepG2 or Madin-Darby canine kidney cells. The three sites were localized to the more carboxyl-terminal half of the cytosolic domain: S534DAE (CK2 motif), S544QEE (CK2 motif), and S563PR. We conclude that CK2 is a kinase that phosphorylates calnexin in vivo as well as in microsomes in vitro. Another yet to be identified kinase (protein kinase C and/or proline-directed kinase) is directed toward the most COOH-terminal serine residue. Elucidation of the signaling cascade responsible for calnexin phosphorylation at these sites in vivo may define a novel regulatory function for calnexin in cargo folding and transport to the ER exit sites.

The perchlorate effect with metallo-cyanide complexes on cellulose anion exchange thin layers

The effect of NaClO 4 concentration on the R f value for [Fe(CN) 6] 4−, [Fe(CN) 6] 3−, [FeNO(CN) 5] 2−, [Au(CN) 2] −, CrO 2− 4, [PtCl 4] 2−, CNS − and [AuCl 4] − on DEAE cellulose thin layers is studied. Perchlorate is shown to have strong desorbing effect on all the anions, known as the perchlorate effect. Nitrate behaves similarly but less effectively.

Characterization of Yeast Protein Deg1 as Pseudouridine Synthase (Pus3) Catalyzing the Formation of Ψ38 and Ψ39 in tRNA Anticodon Loop

The enzymatic activity of yeast gene product Deg1 was identified using both disrupted yeast strain and cloned recombinant protein expressed in yeast and in Escherichia coli. The results show that the DEG1-disrupted yeast strain lacks synthase activity for the formation of pseudouridines psi 38 and psi 39 in tRNA whereas the other activities, specific for psi formation at positions 13, 27, 28, 32, 34, 35, 36, and 55 in tRNA, remain unaffected. Also, the His6-tagged recombinant yeast Deg1p expressed in E. coli as well as a protein fusion with protein A in yeast display the enzymatic activity only toward psi 38 and psi 39 formation in different tRNA substrates. Therefore, Deg1p is the third tRNA:pseudouridine synthase (Pus3p) characterized so far in yeast. Disruption of the DEG1 gene is not lethal but reduces considerably the yeast growth rate, especially at an elevated temperature (37 degrees C). Deg1p localizes both in the nucleus and in the cytoplasm, as shown by immunofluorescence microscopy. Identification of the pseudouridine residues present (or absent) in selected naturally occurring cytoplasmic and mitochondrial tRNAs from DEG1-disrupted strain points out a common origin of psi 38- and psi 39-synthesizing activity in both of these two cellular compartments. The sensitivity of Pus3p (Deg1p) activity to overall three-dimensional tRNA architecture and to a few individual mutations in tRNA was also studied. The results indicate the existence of subtle differences in the tRNA recognition by yeast Pus3p and by its homologous tRNA:pseudouridine synthase truA from E. coli (initially called hisT or PSU-I gene product).

Studies of platinum(II) compounds in aqueous solution. Part 1. K 2PtCl 4

The hydrolysis of K 2PtCl 4 in water, 0.17 M NaCl and 0.08 M KBr was studied at room temperature using adsorption chromatography on cellulose thin layers, ion exchange chromatography on DEAE cellulose thin layers and paper electrophoresis. Two hydrolysis species could be separated in solutions aged in water or dilute NaCl. In dilute KBr mixed bromochloroplatinates are formed.

FAP52, a Novel, SH3 Domain-containing Focal Adhesion Protein

Src-homology 3 (SH3) domain is a 60-70-amino acid motif present in a large variety of signal transduction and cytoskeletal proteins. We used reverse transcriptase-polymerase chain reaction with degenerate and specific primers and chicken brain mRNA to clone a cDNA that codes for a novel SH3 domain-containing protein. The sequence predicts a 448-amino acid polypeptide with a molecular mass of 51, 971 daltons. In the amino terminus, it shows a very high propensity for alpha-helicity, suggesting coiled-coil and possibly a higher order oligomeric arrangement. In the carboxyl terminus, there is a unique SH3 sequence. In Northern blotting, a major 3.7-kilobase and a minor 7.2-kilobase transcript was detected in most chicken tissues. In immunofluorescence microscopy and immunoelectron microscopy on cultured chicken fibroblasts, the protein was localized to focal adhesions in which it showed a distinct codistribution with the focal adhesion proteins vinculin, talin, and paxillin. Phosphoamino acid analysis showed that in cultured chicken heart fibroblasts, the protein contains phosphoserine, but no phosphothreonine or phosphotyrosine, and that the phosphorylation is not dependent on fibronectin. We propose this protein the name FAP52, for Focal Adhesion Protein of 52 kDa, and suggest that it forms part of the multimolecular complex constituting focal adhesion sites.

Thin-Layer Chromatography Experiments That Illustrate General Problems in Chromatography

A number of chromatography experiments using cellulose thin layers and aqueous eluents are proposed to illustrate:1. How chromatography can be used for "pattern recognition" of the inks of felt-tip pens.2. The principle of displacement chromatography of the constituents of the antiseptic Mercurochrome.3. Salting-out chromatography for the separation of halides using a nearly saturated ammonium sulfate solution as eluent.4. The impossibility to use chromatography alone as a method of identification: Methyl Orange travels differently in presence and absence of cyclodextrins or starches in the sample to be analysed.

Adsorption chromatography on cellulose XIV. Some results using aqueous solutions of soluble cyclodextrin polymers as eluents

Commercial α-, β- and γ-cyclodextrin polymers were examined for their eluent properties in adsorption chromatography on cellulose thin layers using aqueous solvents. Azo dyes were found to be eluted better by the polymers than by a solution of α-cyclodextrin. Chloroauric acid (HAuCl 4) was eluted on the liquid front by polymers but not by α-cyclodextrin. No improvement could be seen in the separation of tryptophan enantiomers compared with α-cyclodextrin. α-Cyclodextrin (monomer) yields much better separations than any of the polymers. Attempts to use the polymers as non-specific desorbing agents for coloured inks were unsuccessful.

Quantitative thin-layer chromatography of perbromate

A thin-layer densitometric micromethod for the determination of perbromate is described using Merck 5577 microcrystalline cellulose thin layers with butanol-10% ammonia as solvent. The spots are evaluated by a starch-iodide reagent in 6 M HCl using the Cybertech Gel documentation system WINCAM 2.1. The method was used to determine perbromate in an aged dilute solution and in an impure solid sample.

Phosphorylation and Activation of the DNA Binding Activity of Purified Stat1 by the Janus Protein-tyrosine Kinases and the Epidermal Growth Factor Receptor

The activation of Janus protein-tyrosine kinases (Jaks) and the subsequent phosphorylation and activation of latent signal transducers and activators of transcription (Stats) are common elements in signal transduction through the cytokine receptor superfamily. To assess the role and specificity of Jaks in Stat activation, we have utilized baculovirus expression systems to produce Stat1 and the Jaks. Co-expression of Stat1 with Tyk2, Jak1, or Jak2 resulted in the specific tyrosine phosphorylation of Stat1 at Tyr701, the residue phosphorylated in mammalian cells stimulated with interferon gamma. Alternatively, Stat1, purified to apparent homogeneity from insect cell extracts, was phosphorylated at Tyr701 in Jak immune complex kinase reactions. Phosphorylation of purified Stat1 was necessary and sufficient for the acquisition of DNA binding activity. The specificity in both systems was indicated by the inability of a Jak2 catalytically inactive mutant (Jak2-Glu882) or the Tec protein-tyrosine kinase to phosphorylate Stat1. However, immune complex-purified epidermal growth factor receptor was capable of phosphorylating purified Stat1 at Tyr701 and activating its DNA binding activity in in vitro reactions.

A new method to detect homocysteine in dried blood spots using thin layer chromatography

Introduction: Homocystine had been difficult to detect by conventional methods in the newborn period even in patients with homocystinuria because homocystine easily conjugates with plasma proteins. However, the method of Kang et al. [1] enabled us to measure the total amount of homocysteine (free and protein-bound) in blood and urine by using 2-mercaptoethanol. We applied their method to thin layer chromatography (TLC) on the specimens from patients with homocystinuria due to cystathionine β-synthase deficiency, including two newborn patients. Methods: Extract from dried blood discs was applied to a cellulose thin layer plate after treatment with 2-mercaptoethanol (2-ME). Homocysteine-2-ME compound was identified by liquid chromatograph atmospheric pressure chemical ionization mass spectrography. Results: Using this technique, we identified total homocysteine in the blood not only of 10- and 12-year-old children with homocystinuria, but also of two newborn homocystinurics on the 11th and 20th day after birth, respectively. However, both newborns on day 5 showed only a trace amount of total homocysteine. Homocysteine in the dried blood discs of the two newborns was detectable even after 4 and 6.5 years storage at −15 °C. Conclusions: (1) Homocystinuria can be diagnosed by the TLC described here in the newborn period, possibly later than 2 weeks after birth; (2) a newly identified homocysteine-2-ME compound is S-(2-hydroxyethylthio)homocysteine; and (3) the methionine test had priority over the homocysteine test as a primary screening in our survey.

Overview of Protein Phosphorylation

This overview provides a history of protein phosphorylation research and provides the reader with an understanding of how and why labeling studies are performed. The various sites of protein phosphorylation are described along with the roles of the many kinases and phosphatases that regulate phosphorylation. Methods for detecting unlabeled phosphoamino acids, including high-voltage electrophoresis on thin-layer cellulose acetate plates, gel-shift assays, and the use of anti-phosphopeptide antibodies.

Direct analysis of thin-layer chromatographic spots of narcotics by means of diffuse reflectance Fourier transform infrared spectroscopy

The technique of diffuse reflectance Fourier transform infrared spectrometry (DRIFT) as an in situ detection method was used for the qualitative and quantitative analysis of drugs (heroin, cocaine and codeine) separated by thin layer chromatography. It was found that at a given interferometer throughput and detector sensitivity the quality of the spectrum depends strongly in the type of the chromatographic thin layer used. A detection limit of approx. 2 μg was attained on a microcrystalline cellulose thin layer with a dynamically aligned Bio-Rad Digilab FTS 60A/896 type interferometer and room temperature DTGS detector. A reliable qualitative analysis can be made with as little as 10 to 15 μg drug per spot.

Utilization of Micelle-Mimetic Intramolecular Ionene Aggregates as the Mobile Phase in Pseudophase Thin-Layer Liquid Chromatography.

The use of aqueous or aqueous methanolic ionene (cationic polyelectrolytes consisting of dimethylammonium charge centers interconnected by alternating alkyl chain segments containing x and y methylene groups) solutions as mobile phases in thin layer chromatography (TLC) is described. The ortho, meta and para isomers of chlorobenzoic acid and phenylphenol as well as dinitroanilines and 1- and 2-naphthol were successfully chromatographed using aqueous solutions of [3, 16]-ionene with polyamide thin layer sheets. Some quinones were successfully resolved using this same aqueous ionene mobile phase but with cellulose thin layer plates. For some analytes, the TLC retention results obtained with the [3, 16]-ionene were compared to that obtained with [3, 6]-ionene, which does not form intramolecular aggregated species. The relevant properties of micelle-mimetic intramolecular ionene aggregates are described as are their possible advantages over conventional normal surfactant micelle systems. A brief prospectus on the future use of such micelle-mimetic ionene aggregates in separation science is presented.

Coronamic acid, an intermediate in coronatine biosynthesis by Pseudomonas syringae

Experiments were undertaken on the biosynthetic pathway to coronatine in Pseudomonas syringae pv. glycinea, using mutants blocked in coronatine synthesis, wild type strains and radioactive labelling with l-[U- 14C] isoleucine and 14C-coronamic acid. Evaluation of the kinetics of coronatine appearance in the medium revealed an initial exponential phase with a maximum rate of 0.86 μmol hr − 1 culture − 1 after 3.1 days growth. Beyond this the rate declined to a steady rate of 0.20 μmol hr − 1 culture t $ ̄ 1 . With P. syringae 4185, 8.6% Of l-[U- 14C] isoleucine added at three days growth was incorporated into coronatine during a 90 min exposure period. At the same time there was negligible incorporation (0.04%) to coronamic acid. Two mutants blocked in coronatine synthesis were shown to produce coronamic acid by isolation and purification of this product from culture supernatants. l-[U- 14C]Isoleucine was added to one of these mutants at four days growth and 1.9% of the radioactivity was incorporated into coronamic acid, these data showed that coronamic acid is a distinct biosynthetic entity. However, the coronatine-producing strain from which these mutants were derived, P. syringae 4180, showed only 0.08% incorporation into a coronamic acid fraction, although culture supernatants after five days growth did reveal a trace of coronamic acid as detected by ninhydrin after electrophoresis and chromatography on thin layer cellulose plates. When a three-day culture of P. syringae 4185 was fed coronamic acid prior to 14C-isoleucine, a 3.7-fold reduction in the incorporation of label into coronatine occurred; instead the radioactivity was diverted to extracelluar coronamic acid which showed 7% incorporation. This result demonstrates that coronamic acid is a biosynthetic intermediate of coronatine. Pseudomonas syringae 4185 very efficiently utilized 14C-coronamic acid, giving 94% incorporation into coronatine after 90 min exposure in a four-day culture, showing the specific utilization of this compound. Other experiments were undertaken to evaluate the possible involvement of coronafacoylisoleucines in the biosynthesis of coronatine, by use of a cormutant blocked in coronafacic acid synthesis, with the conclusion that this was not an operative pathway to coronatine.

Phosphopeptide mapping and phosphoamino acid analysis by electrophoresis and chromatography on thin-layer cellulose plates.

Identification of protein phosphorylation sites is essential in order to evaluate the contribution of individual sites to the regulation of a particular protein by phosphorylation. Here we review a method we have developed for the identification of phosphorylation sites based on digestion of 32P-labeled proteins with site-specific proteases and separation of the digestion products in two dimensions on thin-layer cellulose plates using electrophoresis in the first dimension followed by chromatography. This method is very sensitive, requiring only a few hundred 32P-disintegrations per minute to obtain reproducible phosphopeptide maps. We also report methods for the analysis of the phosphoamino acid content of both intact phosphoproteins and individual phosphopeptides recovered from two-dimensional separations, in which the material is subjected to partial acid hydrolysis, and the hydrolysis products are separated on thin-layer cellulose plates by electrophoresis in one or two dimensions. Finally, we describe methods for analyzing the structure of isolated phosphopeptides by secondary digestion with site-specific proteases, by manual Edman degradation, and by immunoprecipitation, and indicate how this information can be used in conjunction with the two-dimensional mobility of the peptide to deduce the identity of a phosphopeptide from the known sequence of a protein.

Adsorption chromatography on cellulose: X. Adsorption of tryptophan and derivatives from CuSO 4-containing eluents

The adsorption of tryptophan and substituted tryptophans on cellulose thin layers developed with CuSO 4 solutions is reported. The presence of CuSO 4 decreases the chiral discrimination of cellulose for tryptophan and substituted tryptophans. The results are compared with those from previously reported work where chiral separations were obtained.