Cellulose TLC Research Articles

Rapid and sensitive naked eye detection of faecal pigments using their enhanced solid-state green fluorescence on a zinc acetate substrate.

The identification of trace faecal pigments in real-time and on-site detection remains a challenge for water quality monitoring. Herein, a simple, low-cost and rapid fluorescence-based analytical method has been developed in a solid matrix for faecal pigments like stercobilin and urobilin detection. This was made possible due to significant enhancement of green solid-state fluorescence (520 nm) by zinc(II) complexation with faecal pigments embedded in the surface of zinc acetate crystals. It enables naked-eye detection of these pigments even at a 10 μM level when excited with 365 nm blue-UV. It was demonstrated that easily available white cellulose paper strips or TLC silica plates coated with zinc acetate can be used as substrates. A photophysical study of solid-state faecal pigments-zinc(II) complexes suggests that green fluorescence enhancement results from the complexation, which can be attributed to the substantial decrease of the non-radiative decay rate (knr) as well as more efficient use of excitation light. The observation of reduced interference of humic acid fluorescence makes faecal pigment detection more efficient by this proposed method.

Functional definition of BirA suggests a biotin utilization pathway in the zoonotic pathogen Streptococcus suis.

Biotin protein ligase is universal in three domains of life. The paradigm version of BPL is the Escherichia coli BirA that is also a repressor for the biotin biosynthesis pathway. Streptococcus suis, a leading bacterial agent for swine diseases, seems to be an increasingly-important opportunistic human pathogen. Unlike the scenario in E. coli, S. suis lacks the de novo biotin biosynthesis pathway. In contrast, it retains a bioY, a biotin transporter-encoding gene, indicating an alternative survival strategy for S. suis to scavenge biotin from its inhabiting niche. Here we report functional definition of S. suis birA homologue. The in vivo functions of the birA paralogue with only 23.6% identity to the counterpart of E. coli, was judged by its ability to complement the conditional lethal mutants of E. coli birA. The recombinant BirA protein of S. suis was overexpressed in E. coli, purified to homogeneity and verified with MS. Both cellulose TLC and MALDI-TOFF-MS assays demonstrated that the S. suis BirA protein catalyzed the biotinylation reaction of its acceptor biotin carboxyl carrier protein. EMSA assays confirmed binding of the bioY gene to the S. suis BirA. The data defined the first example of the bifunctional BirA ligase/repressor in Streptococcus.

One-pot enzymatic reaction sequence for the syntheses of d-glyceraldehyde 3-phosphate and l-glycerol 3-phosphate

A one-pot enzymatic reaction sequence for the synthesis of optically pure d-glyceraldehyde 3-phosphate (d-GAP) and l-glycerol 3-phosphate (sn-G3P) was designed using fructose-bisphosphate aldolase from rabbit muscle (RAMA), sn-glycerol 3-phosphate dehydrogenase (sn-G3PDH) and formate dehydrogenase from Candida boidinii (FDH). The reaction sequence significantly improves the aldol cleavage of d-fructose 1,6-bisphosphate (d-F16BP) catalyzed by RAMA and yields 100% conversion of d-F16BP by overcoming thermodynamic limitation. The degradation kinetics of d-GAP under reaction conditions was investigated and a reaction kinetics model defining the entire cascade was developed. Validation of the model shows 98.5% correlation between experimental data and numerically simulated data matrices. The evaluation of different types of reactor was performed by combining the reaction kinetics model, mass balances and kinetics of the non-enzymatic degradation of d-GAP. Batch-wise operation in a stirred tank reactor (STR) is the most convenient procedure for the one-pot enzymatic syntheses of d-GAP and sn-G3P. The separation of the two products d-GAP and sn-G3P has been achieved using polyethylenimine (PEI)-cellulose TLC.

주목열매 추출물 구조분석

주목열매에서 분리한 과육과 씨를 95% EtOH 용액을 사용하여 각각 1.15 kg과 0.94 kg을 유리 용기에 침지하여 추출하고, 농축된 추출용액을 용매의 극성에 따라 n-hexane, dichloromethane, ethyl acetate 및 수용성으로 순차 분획한 후, 동결건조하였다. 혼합물로부터 순수한 화합물을 분리하기 위하여 ethyl acetate 및 수용성 분획에 대하여 Sephadex-LH 20 칼럼크로마토그래피를 실시하였다. 단리된 화합물들의 구조는 flavan 화합물의 경우 <TEX>$^1H$</TEX>-NMR, <TEX>$^{13}C$</TEX>-NMR 및 EI-MS 스펙트럼을 측정하였으며, 탄수화물은 보다 정확한 구조를 결정하기 위해 COSY, HSQC와 같은 2-D NMR과 LC/MS 스펙트럼을 이용하여 구조를 결정하였다. 또한 구성당 분석을 위해 acid hydrolysis, permethylation을 실시하여 구성당의 성분과 결합위치를 확인하였다. 위의 실험을 통하여 (+)-catechin (1), (-)-epicatechin (2), (+)-gallocatechin (3), (-)-epigallocatechin(4) 및 <TEX>${\beta}$</TEX>-D-fructofuranose-(<TEX>$2{\rightarrow}4$</TEX>)-O-<TEX>${\beta}$</TEX>-D-glucopyranose(<TEX>$1{\rightarrow}4$</TEX>)-O-<TEX>${\alpha}$</TEX>-D-glucopyranose-(<TEX>$1{\rightarrow}2$</TEX>)-O-<TEX>${\beta}$</TEX>-D-fructofuranose (5)의 구조를 결정하였다. The fruits of Taxus cuspidata were collected, divided into seeds and fruits, and extracted with 95% EtOH. The extracts were evaporated under the reduced vacuum pressure, concentrated, then successively fractionated with a series of n-hexane, dichloromethane, ethyl acetate and water on a separatory funnel to get some freeze dried samples. A portion of the EtOAc (arils:1.65 g, seeds:1.04 g) and <TEX>$H_2O$</TEX> (arils:7 g, seeds:10 g) soluble samples were chromatographed on a Sephadex column using MeOH-<TEX>$H_2O$</TEX> (1:1, 1:3, 1:5, v/v), EtOH-hexane (3:1, v/v) mixture and 100% <TEX>$H_2O$</TEX> as eluting solvents to isolate pure compounds from the fractions. The isolates were developed by cellulose TLC using t-BuOH-HOAc-<TEX>$H_2O$</TEX> (TBA; 3:1:1, v/v/v) and 6% aqueous HOAc. Visualization was done under ultraviolet light and by spraying the vanillin-HCl-EtOH reagent (4.8:12:480, v/v/v). followed by heating. The structures of the isolates were characterized by <TEX>$^1H$</TEX>- and <TEX>$^{13}C$</TEX>-NMR, DEPT, 2D-NMR, LC/MS and EI-MS spectra. In addition to the NMR and MS spectra, acid hydrolysis and permethylation were used to determine the correct structure of the isolated sugar compound. Their structures were elucidated as (+)-catechin (1), (-)-epicatechin (2), (+)-gallocatechin (3), (-)-epigallocatechin (4) and <TEX>${\beta}$</TEX>-D-fructofuranose-(<TEX>$2{\rightarrow}4$</TEX>)-O-<TEX>${\beta}$</TEX>-D-glucopyranose(<TEX>$1{\rightarrow}4$</TEX>)-O-<TEX>${\alpha}$</TEX>-D-glucopyranose (<TEX>$1{\rightarrow}2$</TEX>)-O-<TEX>${\beta}$</TEX>-D-fructofuranose (5) on the basis of the above experimental evidences.

Saccharomyces cerevisiae Dmc1 and Rad51 Proteins Preferentially Function with Tid1 and Rad54 Proteins, Respectively, to Promote DNA Strand Invasion during Genetic Recombination

The Saccharomyces cerevisiae Dmc1 and Tid1 proteins are required for the pairing of homologous chromosomes during meiotic recombination. This pairing is the precursor to the formation of crossovers between homologs, an event that is necessary for the accurate segregation of chromosomes. Failure to form crossovers can have serious consequences and may lead to chromosomal imbalance. Dmc1, a meiosis-specific paralog of Rad51, mediates the pairing of homologous chromosomes. Tid1, a Rad54 paralog, although not meiosis-specific, interacts with Dmc1 and promotes crossover formation between homologs. In this study, we show that purified Dmc1 and Tid1 interact physically and functionally. Dmc1 forms stable nucleoprotein filaments that can mediate DNA strand invasion. Tid1 stimulates Dmc1-mediated formation of joint molecules. Under conditions optimal for Dmc1 reactions, Rad51 is specifically stimulated by Rad54, establishing that Dmc1-Tid1 and Rad51-Rad54 function as specific pairs. Physical interaction studies show that specificity in function is not dictated by direct interactions between the proteins. Our data are consistent with the hypothesis that Rad51-Rad54 function together to promote intersister DNA strand exchange, whereas Dmc1-Tid1 tilt the bias toward interhomolog DNA strand exchange.

A Conserved Serine of Heterogeneous Nuclear Ribonucleoprotein L (hnRNP L) Mediates Depolarization-regulated Alternative Splicing of Potassium Channels

Molecular mechanisms of gene regulation underlying the activity-dependent long term changes of cellular electrical properties, such as those during memory, are largely unknown. We have shown that alternative splicing can be dynamically regulated in response to membrane depolarization and Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) activation, through special CaM kinase responsive RNA elements. However, proteins that mediate this regulation and how they are affected by CaMKIV are not known. Here we show that the regulation of the stress axis-regulated exon of the Slo1 potassium channel transcripts by membrane depolarization requires a highly conserved CaMKIV target serine (Ser-513) of the heterogeneous ribonucleoprotein L. Ser-513 phosphorylation within the RNA recognition motif 4 enhanced heterogeneous ribonucleoprotein L interaction with the CaMKIV-responsive RNA element 1 of stress axis-regulated exon and inhibited binding of the large subunit of the U2 auxiliary factor U2AF65. Both of these activities were abolished by a S513A mutation. Thus, through Ser-513, membrane depolarization/calcium signaling controls a critical spliceosomal assembly step to regulate the variant subunit composition of potassium channels.

Activity-guided supercritical CO2 isolation of antioxidative constituents from Eucommia ulmoides

Increasing evidence exhibited that antioxidants played very important role in protecting against various diseases like cancer, atherosclerosis, diabetes, cataracts, neurodegenerative disorders [1]. Eucommia ulmoides Oliv., the sole species in Eucommia genus and Eucommiaceae family, is a large medicinal hardwood native to China and widely cultured in Eastern Asia and has widely been used as a tonic to strengthen the kidney and liver, against diabetics, strong bones, ache knees, treat lower back pain, prevent fatigue and miscarriage [2, 3]. In present study, crude extracts of E. ulmoides wood were initially obtained by supercritical CO2 isolation. And DPPH free radical scavenging assay, a standard in vitro model, was employed for the activity-guided purification to identify the antioxidative constituents of E. ulmoides. The crude extracts were suspended in water, and then successively partitioned to a serious of polar solvents to yield fractions soluble in n-hexane, dichloromethane, ethyl acetate, n-buthanol and water. Ethyl acetate fraction exhibited most significant capacity to scavenge DPPH radical, and thus was further subjected to repeated sephadex LH-20 open column to separate the individual antioxidative compounds. Guided by DPPH assay, the fraction having superior activity was identified as containing five major yellow amorphous compounds, including gallic acid, ellagic acid, caffeic acid, (+)-catechin and kaempferol and were obtained. The structures of the isolated antioxidants were mainly elucidated and established by spectroscopic analysis, such as NMR and MS, as well as cellulose TLC. To the best of our knowledge, this was the first time of phytochemical investigation of E. ulmoides wood.

Pyrovanadolysis, a Pyrophosphorolysis-like Reaction Mediated by Pyrovanadate, Mn2+, and DNA Polymerase of Bacteriophage T7

DNA polymerases catalyze the 3'-5'-pyrophosphorolysis of a DNA primer annealed to a DNA template in the presence of pyrophosphate (PP(i)). In this reversal of the polymerization reaction, deoxynucleotides in DNA are converted to deoxynucleoside 5'-triphosphates. Based on the charge, size, and geometry of the oxygen connecting the two phosphorus atoms of PP(i), a variety of compounds was examined for their ability to carry out a reaction similar to pyrophosphorolysis. We describe a manganese-mediated pyrophosphorolysis-like activity using pyrovanadate (VV) catalyzed by the DNA polymerase of bacteriophage T7. We designate this reaction pyrovanadolysis. X-ray absorption spectroscopy reveals a shorter Mn-V distance of the polymerase-VV complex than the Mn-P distance of the polymerase-PP(i) complex. This structural arrangement at the active site accounts for the enzymatic activation by Mn-VV. We propose that the Mn(2+), larger than Mg(2+), fits the polymerase active site to mediate binding of VV into the active site of the polymerase. Our results may be the first documentation that vanadium can substitute for phosphorus in biological processes.

An in Trans Interaction at the Interface of the Helicase and Primase Domains of the Hexameric Gene 4 Protein of Bacteriophage T7 Modulates Their Activities

DNA helicase and primase are essential for DNA replication. The helicase unwinds the DNA to provide single-stranded templates for DNA polymerase. The primase catalyzes the synthesis of oligoribonucleotides for the initiation of lagging strand synthesis. The two activities reside in a single polypeptide encoded by gene 4 of bacteriophage T7. Their coexistence within the same polypeptide facilitates their coordination during DNA replication. One surface of helix E within the helicase domain is positioned to interact with the primase domain and the linker connecting the two domains within the functional hexamer. The interaction occurs in trans such that helix E interacts with the primase domain and the linker of the adjacent subunit. Most alterations of residues on the surface of helix E (Arg(404), Lys(408), Tyr(411), and Gly(415)) eliminate the ability of the altered proteins to complement growth of T7 phage lacking gene 4. Both Tyr(411) and Gly(415) are important in oligomerization of the protein. Alterations G415V and K408A simultaneously influence helicase and primase activities in opposite manners that mimic events observed during coordinated DNA synthesis. The results suggest that Asp(263) located in the linker of one subunit can interact with Tyr(411), Lys(408), or Arg(404) in helix E of the adjacent subunit depending on the oligomerization state. Thus the switch in contacts between Asp(263) and its three interacting residues in helix E of the adjacent subunit results in conformational changes that modulate helicase and primase activity.

Polyphosphatase Activity of CthTTM, a Bacterial Triphosphate Tunnel Metalloenzyme

Triphosphate tunnel metalloenzymes (TTMs) are a superfamily of phosphotransferases with a distinctive active site located within an eight-stranded beta barrel. The best understood family members are the eukaryal RNA triphosphatases, which catalyze the initial step in mRNA capping. The RNA triphosphatases characteristically hydrolyze nucleoside 5'-triphosphates in the presence of manganese and are inept at cleaving inorganic tripolyphosphate. We recently identified a TTM protein from the bacterium Clostridium thermocellum (CthTTM) with the opposite substrate preference. Here we report that CthTTM catalyzes hydrolysis of guanosine 5'-tetraphosphate to yield GTP and P(i) (K(m) = 70 microm, k(cat) = 170 s(-1)) much more effectively than it converts GTP to GDP and P(i) (K(m) = 70 microm, k(cat) = 0.3 s(-1)), implying that a nucleoside interferes when positioned too close to the tunnel entrance. CthTTM is capable of quantitatively cleaving diadenosine hexaphosphate but has feeble activity with shorter derivatives diadenosine tetraphosphate and diadenosine pentaphosphate. We propose that the tunnel opens to accommodate the dumbbell-shaped diadenosine hexaphosphate and then closes around it to perform catalysis. We find that CthTTM can exhaustively hydrolyze a long-chain inorganic polyphosphate, a molecule that plays important roles in bacterial physiology. CthTTM differs from other known polyphosphatases in that it yields a approximately 2:1 mixture of P(i) and PP(i) end products. Bacterial/archaeal TTMs have a C-terminal helix located near the tunnel entrance. Deletion of this helix from CthTTM exerts pleiotropic effects. (i) It suppresses hydrolysis of guanosine 5'-tetraphosphate and inorganic PPP(i); (ii) it stimulates NTP hydrolysis; and (iii) it biases the outcome of the long-chain polyphosphatase reaction more strongly in favor of P(i) production. We discuss models for substrate binding in the triphosphate tunnel.

Iron-independent Phosphorylation of Iron Regulatory Protein 2 Regulates Ferritin during the Cell Cycle

Iron regulatory protein 2 (IRP2) is a key iron sensor that post-transcriptionally regulates mammalian iron homeostasis by binding to iron-responsive elements (IREs) in mRNAs that encode proteins involved in iron metabolism (e.g. ferritin and transferrin receptor 1). During iron deficiency, IRP2 binds IREs to regulate mRNA translation or stability, whereas during iron sufficiency IRP2 is degraded by the proteasome. Here, we identify an iron-independent IRP2 phosphorylation site that is regulated by the cell cycle. IRP2 Ser-157 is phosphorylated by Cdk1/cyclin B1 during G(2)/M and is dephosphorylated during mitotic exit by the phosphatase Cdc14A. Ser-157 phosphorylation during G(2)/M reduces IRP2 RNA-binding activity and increases ferritin synthesis, whereas Ser-157 dephosphorylation during mitotic exit restores IRP2 RNA-binding activity and represses ferritin synthesis. These data show that reversible phosphorylation of IRP2 during G(2)/M has a role in modulating the iron-independent expression of ferritin and other IRE-containing mRNAs during the cell cycle.

Regulation of Proline-rich Akt Substrate of 40 kDa (PRAS40) Function by Mammalian Target of Rapamycin Complex 1 (mTORC1)-mediated Phosphorylation

The rapamycin-sensitive mammalian target of rapamycin (mTOR) complex 1 (mTORC1) contains mTOR, raptor, mLST8, and PRAS40 (proline-rich Akt substrate of 40 kDa). PRAS40 functions as a negative regulator when bound to mTORC1, and it dissociates from mTORC1 in response to insulin. PRAS40 has been demonstrated to be a substrate of mTORC1, and one phosphorylation site, Ser-183, has been identified. In this study, we used two-dimensional phosphopeptide mapping in conjunction with mutational analysis to show that in addition to Ser-183, mTORC1 also phosphorylates Ser-212 and Ser-221 in PRAS40 when assayed in vitro. Mutation of all three residues to Ala markedly reduces mTORC1-mediated phosphorylation of PRAS40 in vitro. All three sites were confirmed to be phosphorylated in vivo by [(32)P]orthophosphate labeling and peptide mapping. Phosphorylation of Ser-221 and Ser-183 but not Ser-212 is sensitive to rapamycin treatment. Furthermore, we demonstrate that mutation of Ser-221 to Ala reduces the interaction with 14-3-3 to the same extent as mutation of Thr-246, the Akt/protein kinase B-phosphorylated site. We also find that mutation of Ser-221 to Ala increases the inhibitory activity of PRAS40 toward mTORC1. We propose that after mTORC1 kinase activation by upstream regulators, PRAS40 is phosphorylated directly by mTOR, thus contributing to the relief of PRAS40-mediated substrate competition.

Structure and Function of the c-myc DNA-unwinding Element-binding Protein DUE-B

Local zones of easily unwound DNA are characteristic of prokaryotic and eukaryotic replication origins. The DNA-unwinding element of the human c-myc replication origin is essential for replicator activity and is a target of the DNA-unwinding element-binding protein DUE-B in vivo. We present here the 2.0A crystal structure of DUE-B and complementary biochemical characterization of its biological activity. The structure corresponds to a dimer of the N-terminal domain of the full-length protein and contains many of the structural elements of the nucleotide binding fold. A single magnesium ion resides in the putative active site cavity, which could serve to facilitate ATP hydrolytic activity of this protein. The structure also demonstrates a notable similarity to those of tRNA-editing enzymes. Consistent with this structural homology, the N-terminal core of DUE-B is shown to display both D-aminoacyl-tRNA deacylase activity and ATPase activity. We further demonstrate that the C-terminal portion of the enzyme is disordered and not essential for dimerization. However, this region is essential for DNA binding in vitro and becomes ordered in the presence of DNA.

The retention behavior of selected porphyrins on silica gel, polyamide, and cellulose TLC plates

The chromatographic behavior of uroporphyrin I, uroporphyrin III, coproporphyrin I, coproporphyrin III, and protoporphyrin IX has been examined using silica gel 60F 254 , polyamide 11F 254 , and cellulose F TLC plates as stationary phases. Homologous series of n -alcohols, acetonitrile, and tetrahydrofuran were used as mobile phases. Chromatographic retention data and a possible retention mechanism are discussed. The electrostatic interactions operating in these chromatographic systems were analyzed by studying relationship between log (1/ R F ) values and the molecular polarizabilities of the solvents.

Roles of Conserved Proline and Glycosyltransferase Motifs of EmbC in Biosynthesis of Lipoarabinomannan

D-Arabinans, composed of D-arabinofuranose (D-Araf), dominate the structure of mycobacterial cell walls in two settings, as part of lipoarabinomannan (LAM) and arabinogalactan, each with markedly different structures and functions. Little is known of the complexity of their biosynthesis. beta-D-Arabinofuranosyl-1-monophosphoryldecaprenol is the only known sugar donor. EmbA, EmbB, and EmbC, products of the paralogous genes embA, embB, and embC, the sites of resistance to the anti-tuberculosis drug ethambutol (EMB), are the only known implicated enzymes. EmbA and -B apparently contribute to the synthesis of arabinogalactan, whereas EmbC is reserved for the synthesis of LAM. The Emb proteins show no overall similarity to any known proteins beyond Mycobacterium and related genera. However, functional motifs, equivalent to a proline-rich motif of several bacterial polysaccharide co-polymerases and a superfamily of glycosyltransferases, were found. Site-directed mutagenesis in glycosyltransferase superfamily C resulted in complete ablation of LAM synthesis. Point mutations in three amino acids of the proline motif of EmbC resulted in marked reduction of LAM-arabinan synthesis and accumulation of an unknown intermediate and of the known precursor lipomannan. Yet the pattern of the differently linked d-Araf units observed in wild type LAM-arabinan was largely retained in the proline motif mutants. The results allow for the presentation of a unique model of arabinan synthesis.

Daphnetin Methylation by a Novel O-Methyltransferase Is Associated with Cold Acclimation and Photosystem II Excitation Pressure in Rye

In plants, O-methylation of phenolic compounds plays an important role in such processes as lignin synthesis, flower pigmentation, chemical defense, and signaling. However, apart from phenylpropanoids and flavonoids, very few enzymes involved in coumarin biosynthesis have been identified. We report here the molecular and biochemical characterization of a gene encoding a novel O-methyltransferase that catalyzes the methylation of 7,8-dihydroxycoumarin, daphnetin. The recombinant protein displayed an exclusive methylation of position 8 of daphnetin. The identity of the methylated product was unambiguously identified as 7-hydroxy-8-methoxycoumarin by co-chromatography on cellulose TLC and coelution from high performance liquid chromatography, with authentic synthetic samples, as well as by UV, mass spectroscopy, (1)H NMR spectral analysis, and NOE correlation signals of the relevant protons. Northern blot analysis and enzyme activity assays revealed that the transcript and corresponding enzyme activity are up-regulated by both low temperature and photosystem II excitation pressure. Using various phenylpropanoid and flavonoid substrates, we demonstrate that cold acclimation of rye leaves increases O-methyltransferase activity not only for daphnetin but also for the lignin precursors, caffeic acid, and 5-hydroxyferulic acid. The significance of this novel enzyme and daphnetin O-methylation is discussed in relation to its putative role in modulating cold acclimation and photosystem II excitation pressure.

Optimization of separation of (+)-catechin and (−)-epicatechin on cellulose TLC plates

The separation of (+)-catechin and (−)-epicatechin on cellulose TLC plates with 1-butanol-water-acetic acid, 4 + 2 + 1 ( v/v ), has been studied under different development conditions on prewashed and untreated TLC plates. Plates were developed in horizontal chambers (tank and sandwich configuration) and in twin trough (unsaturated and saturated) developing chambers. Tank configurations were used with and without preconditioning.Prewashing of the TLC plates, preconditioning, and the type of development chamber used had a large effect on the chromatographic results. Without prewashing of the TLC plate unsatisfactory or misleading results were obtained.Anisaldehyde-sulfuric acid dipping reagent proved to be more selective than vanillin-phosphoric acid as detection reagent for the detection of flavan-3-ols in oak bark extract; the latter was, however, more sensitive.

Qualitative identification of metal ions in honey by two-dimensional thin-layer chromatography

Two-dimensional thin-layer chromatography (2D TLC) has been used as a screening method for detection of the metal ions Al 3+ , Cr 3+ , Cu 2+ , Ni 2+ , Mg 2+ , Zn 2+ , Mn 2+ , Pb 2+ , Sn(IV), and Fe 3+ in honey. Different mobile phases and visualization reagents were tested. The best separation was achieved on precoated microcrystalline cellulose TLC plates with acetonitrile-HCl-water, 73 + 15 + 12 ( v/v ), as mobile phase in the direction x , and 2-pentanol-acetonitrile-HCl, 30 + 20 + 50 ( v/v ), in the direction y . A mixture of quercetin, dimethylglyoxime, and NH 3 was used for visualization of the chromatographic spots. This reagent mixture produced intensely colored spots for all the ions tested and enabled their identification at concentrations ranging from 50 to 1000 ng per spot.The method was used to identify metal ions in honey samples, dissolved in 2 mol L −1 hydrochloric acid, after microwave digestion. Seven metal ions, Al 3+ , Cr 3+ , Fe 3+ , Mn 2+ , Pb 2+ , Ni 2+ and Sn(IV), were identified.

A Single Amidotransferase Forms Asparaginyl-tRNA and Glutaminyl-tRNA in Chlamydia trachomatis

Aminoacyl-tRNA is generally formed by aminoacyl-tRNA synthetases, a family of 20 enzymes essential for accurate protein synthesis. However, most bacteria generate one of the two amide aminoacyl-tRNAs, Asn-tRNA or Gln-tRNA, by transamidation of mischarged Asp-tRNA(Asn) or Glu-tRNA(Gln) catalyzed by a heterotrimeric amidotransferase (encoded by the gatA, gatB, and gatC genes). The Chlamydia trachomatis genome sequence reveals genes for 18 synthetases, whereas those for asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase are absent. Yet the genome harbors three gat genes in an operon-like arrangement (gatCAB). We reasoned that Chlamydia uses the gatCAB-encoded amidotransferase to generate both Asn-tRNA and Gln-tRNA. C. trachomatis aspartyl-tRNA synthetase and glutamyl-tRNA synthetase were shown to be non-discriminating synthetases that form the misacylated tRNA(Asn) and tRNA(Gln) species. A preparation of pure heterotrimeric recombinant C. trachomatis amidotransferase converted Asp-tRNA(Asn) and Glu-tRNA(Gln) into Asn-tRNA and Gln-tRNA, respectively. The enzyme used glutamine, asparagine, or ammonia as amide donors in the presence of either ATP or GTP. These results suggest that C. trachomatis employs the dual specificity gatCAB-encoded amidotransferase and 18 aminoacyl-tRNA synthetases to create the complete set of 20 aminoacyl-tRNAs.

In vitro analysis of cis-elements and trans-factors for the RNA editing of tobacco chloroplast psbE and petB mRNAs

RNA editing is one of the posttranscriptional processes in higher plant chloroplasts. Editing is found in many cases in internal protein-coding regions, resulting in amino acid substitutions. Therefore, it is important to understand editing events to use the chloroplast as a factory for protein production. The number of editing sites so far observed is 31 in tobacco and these editing events are C- to -U conversions. To analyze the molecular mechanism, we have developed an in vitro RNA editing system from tobacco green chloroplasts (1). Extracts were prepared from intact chloroplasts isolated from 5-10 cm tobacco leaves. Pre-mRNA portions in which the C residue to be edited was specifically labelled with 32P were used as in vitro substrates. Editing activity was assayed by detecting 32P-labelled U mononucleotides on cellulose TLC after digestion of substrates with nuclease P1. Using the in vitro system, cis-acting elements for editing were defined in tobacco chloroplast mRNAs from psbE and petB. In vitro competition assays indicated that trans-acting factors interact with these cis-elements and that the trans-factor is site-specific. UV cross-linking assays showed the existence of site-specific trans-factors for these mRNA editing. (1) T. Hirose and M. Sugiura, Involvement of a site-specific trans-acting factor and a common RNA-binding protein in the editing of chloroplast mRNAs: development of a chloroplast in vitro RNA editing system. EMBO J. 20: 1144-1152 (2001)