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Enzymatic conversion of arginine to citrulline by peptidyl arginine deiminase is associated with peptide presentation and development of autoimmunity in rheumatoid arthritis. In order to facilitate identification of the citrullination site, citrulline residue was modified using 4-bromophenyl glyoxal, and 194 Da mass increase and incorporation of the Br signature were confirmed by MALDI-TOF MS. Using this approach, we identified four and five citrullination sites of bovine serum albumin and bovine fibrinogen, respectively. MALDI-TOF/TOF MS was used to unambiguously identify two citrullination sites from bovine fibrinogen.

Controlled proteolytic activation of membrane-anchored transcription factors provides an adaptation strategy that guarantees rapid transcriptional responses to abrupt environmental stresses in both animals and plants. NTL6 is a plant-specific NAC [NAM/ATAF1/2/CUC2] transcription factor that is expressed as a dormant plasma membrane-associated form in Arabidopsis. Proteolytic processing of NTL6 is triggered by abiotic stresses and ABA (abscisic acid). In the present study, we show that NTL6 is linked directly with SnRK (Snf1-related protein kinase) 2.8-mediated signalling in inducing a drought-resistance response. SnRK2.8 phosphorylates NTL6 primarily at Thr142. NTL6 phosphorylation by SnRK2.8 is required for its nuclear import. Accordingly, a mutant NTL6 protein, in which Thr142 was mutated to an alanine, was poorly phosphorylated and failed to enter the nucleus. In accordance with the role of SnRK2.8 in drought-stress signalling, transgenic plants overproducing either NTL6 or its active form 6ΔC (35S:NTL6 and 35S:6ΔC) exhibited enhanced resistance to water-deficit conditions such as those overproducing SnRK2.8 (35S:SnRK2.8). In contrast, NTL6 RNAi (RNA interference) plants were susceptible to dehydration as observed in the SnRK2.8-deficient snrk2.8-1 mutant. Furthermore, the dehydration-resistant phenotype of 35S:NTL6 transgenic plants was compromised in 35S:NTL6 X snrk2.8-1 plants. These observations indicate that SnRK2.8-mediated protein phosphorylation, in addition to a proteolytic processing event, is important for NTL6 function in inducing a drought-resistance response.

Virus capsid structure is essential in virion maturation and durability, so disrupting capsid assembly could be an effective way to reduce virion count and cure viral diseases. However, currently there is no known antiviral which affects capsid inhibition, and only a small number of assembly inhibitors were experimentally successful. In this present study, we aimed to find hepatitis B virus (HBV) capsid assembly inhibitor which binds to the HBV core protein and changes protein conformation. Several candidate molecules were found to bind to certain structure in core protein with high specificity. Furthermore, these molecules significantly changed the protein conformation and reduced assembly affinity of core protein, leading to decrease of the number of assembled capsid or virion, both in vitro and in vivo. In addition, prediction also suggests that improvements in inhibition efficiency could be possible by changing functional groups and ring structures.

In tandem mass spectrometric peptide sequencing, simplifying the mass spectrum is often desirable. The b-series ions were distinguished from the y-series ions in the MALDI TOF-TOF spectra by incorporating a bromine-tag to the N-terminal amino group through rapid and selective acetylation using bromoacetic anhydride without blocking the lysine and tyrosine residues. The 51:49 ratio of Br-79 and Br-81 isotopes facilitated identification of ions carrying the tag. With the Br-tag in the b-series ions, N-terminal sequencing of tryptic peptides from hemoglobin as well as model peptides was straightforward. When the b-ions were low in intensity, ions without the Br-tag were identified as y-ions and used for sequencing.

Magnetic nanoparticles (MNPs) treated with phosphoric acid were used to improve sequence coverage in protein identification by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Sample solution of tryptic peptides from proteins was mixed with the MNPs, and the MNPs were separated from the supernatant using a magnet. MALDI mass spectra obtained separately from the supernatant and the MNPs were distinctly different and complementary to each other. Combination of the two spectra led to a significantly increased sequence coverage.

De novo analysis of protein N-terminal sequence is important for identification of N-terminal proteolytic processing such as N-terminal methionine or signal peptide removal, or for the genome annotation of uncharacterized proteins. We introduce a de novo sequencing method of protein N terminus utilizing matrix-assisted laser desorption/ionization (MALDI) signal enhancing picolinamidination with bromine isotopic tag incorporated to the N terminus. The doublet signature of bromine in the tandem mass (MS/MS) spectrum distinguished N-terminal ion series from C-terminal ion series, facilitating de novo N-terminal sequencing of protein. The dual advantage of MALDI signal enhancement by the basic picolinamidine and b-ion selection aided by Br signature is demonstrated using a variety of peptides. The N-terminal sequences of myoglobin and hemoglobin as model proteins were determined by incorporating the Br tag to the N terminus of the proteins and obtaining a series of b-ions with Br signature by MS/MS analysis after chymotryptic digestion of the tagged proteins. The N-terminal peptide was selected for MS/MS analysis from the chymotryptic digest based on the Br signature in the mass spectrum. Identification of phosphorylation site as well as N-terminal sequencing of a phosphopeptide was straightforward.

Pseudomonas aeruginosa guanidinobutyrase (GbuA) and guanidinopropionase (GpuA) catalyze the hydrolysis of 4-guanidinobutyrate and 3-guanidinopropionate, respectively. They belong to the ureohydrolase superfamily, which includes arginase, agmatinase, proclavaminate amidinohydrolase, and formiminoglutamase. In this study, we have determined the crystal structures of GbuA and GpuA from P. aeruginosa to provide a structural insight into their substrate specificity. Although GbuA and GpuA share a common structural fold of the typical ureohydrolase superfamily, they exhibit significant variations in two active site loops. Mutagenesis of Met161 of GbuA and Tyr157 of GpuA, both of which are located in the active site loop 1 and predicted to be involved in substrate recognition, significantly affected their enzymatic properties, implying their important roles in catalysis.

The unique Br signature was utilized for C-terminal amino acid sequencing of model peptides. C-terminal carboxyl group was selectively derivatized in peptides, containing side chain carboxyl group, using 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and Br was introduced using 4-bromophenylhydrazine hydrochloride (BPH) in a one pot reaction. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) tandem mass spectra were obtained carrying the Br signature in the y-series ions. The Br signature facilitated C-terminal sequencing and discrimination of C-terminal carboxyl groups in the free acid and amide forms.

There has been considerable interest in virulence genes in the plasticity region of Helicobacter pylori , but little is known about many of these genes. JHP940, one of the virulence factors encoded by the plasticity region of H. pylori strain J99, is a proinflammatory protein that induces tumor necrosis factor-alpha and interleukin-8 secretion as well as enhanced translocation of NF-κB in cultured macrophages. Here we have characterized the structure and function of JHP940 to provide the framework for better understanding its role in inflammation by H. pylori . Our work demonstrates that JHP940 is the first example of a eukaryotic-type Ser/Thr kinase from H. pylori . We show that JHP940 is catalytically active as a protein kinase and translocates into cultured human cells. Furthermore, the kinase activity is indispensable for indirectly up-regulating phosphorylation of NF-κB p65 at Ser276. Our results, taken together, contribute significantly to understanding the molecular basis of the role of JHP940 in inflammation and subsequent pathogenesis caused by H. pylori . We propose to rename the jhp940 gene as ctkA ( c ell t ranslocating k inase A ).

Bacterial phosphopantetheine adenylyltransferase (PPAT) catalyzes the pen­ultimate step in the coenzyme A (CoA) biosynthetic pathway. It catalyzes the reversible transfer of an adenylyl group from ATP to 4′-phosphopantetheine (Ppant) to form dephospho-CoA (dPCoA) and pyrophosphate. Previous structural studies have revealed how several ligands are recognized by bacterial PPATs. ATP, ADP, Ppant and dPCoA bind to the same binding site in a highly similar manner, while CoA binds to a partially overlapping site in a different mode. To provide further structural insights into ligand binding, the crystal structure of Staphylococcus aureus PPAT was solved in a binary complex with 3′-­phosphoadenosine 5′-phosphosulfate (PAPS). This study unexpectedly revealed a new mode of ligand binding to PPAT, thus providing potentially useful information for structure-based discovery of inhibitors of bacterial PPATs.

Two orders of magnitude matrix-assisted laser desorption/ionization (MALDI) signal enhancement of phosphopeptides has been achieved by picolinamidination of N-terminal amine group and epsilon-amine group of lysine residues. Due to the presence of picolinamidination tag at the N-terminal amine of peptides, MS/MS spectra with a strong b-ion series was obtained, which greatly facilitated sequencing and identification of the phosphorylation site. Phosphorylation site of a phosphopeptide could be identified from MALDI TOF/TOF spectrum obtained from a tryptic or a chymotryptic phosphopeptide, which was not even detected in the positive ion mode, without signal enhancement by picolinamidination, due to the negative charge of the phosphate group in the presence of other peptides.

Ginseng is a well known herbal medicine in Asia, and ginsenoside Rg3 has anti-cancer and various pharmacological effects. In particular, 20S-ginsenoside Rg3 may increase the anti-proliferative effects of chemotherapy. The authors investigated the mechanism of the anti-proliferative effect of 20S-Rg3 at the protein level in HT29 colon cancer cells. MTT, caspase-3 assays, and flow cytometry analysis were performed to determine cytotoxicity and apoptosis, and proteomic analysis was performed by two-dimensional gel electrophoresis and MALDI-TOF/TOF MS, and a database was used to identify protein changes in 20S-Rg3 treated HT29 cells. The proteins identified included down-regulated Rho GDP dissociation inhibitor, up-regulated tropomyosin1, and annexin5 and glutathione s-transferase p1, which are apoptosis associated proteins. The anti-proliferative mechanism of 20S-Rg3 was found to be involved in mitotic inhibition, DNA replication, and repair and growth factor signaling. The findings of this study suggest that the cytotoxicity of 20S-Rg3 in colon cancer is dependent on several mechanisms, including apoptosis.

In order to gain insight into the molecular changes at the protein level in plants exposed to low temperature for a long period of time (vernalization), proteome analyses of vernalization-treated Arabidopsis thaliana have been carried out by two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry. Fourteen proteins including ATP binding/GTP binding/translation elongation factor and glycine-rich RNA-binding protein 7 (GRP7) showed differential expression in vernalization-treated Arabidopsis thaliana. GRP7 showed the most dramatic increase in expression suggesting its involvement in response to vernalization treatment.

This official contribution of the National Institute of Standards and Technology is not subject to copyright in the United States of America. This work was supported in part by the System IC 2010 Project of Korea, the Chemistry and Molecular Engineering Program of the Brain Korea 21 Project, and the NIST Office of Microelectronics Programs. The authors thank Dr. Robert Cook and Jack Douglas for their careful critique of the manuscript. Supporting Information is available online from Wiley InterScience or from the author.

c-Myc is instrumental in the progression of Burkitt's lymphoma including HL-60 human leukemia cells. We tested fatty acids for their inhibitory effect on the DNA binding of c-Myc/Max dimeric proteins of human origin, prepared as recombinant proteins encompassing DNA binding (basic) and dimerization (HLHZip) domain, and found that those suppress proliferation and induce apoptosis of DMSO-differentiated HL-60 cells. The analyzed IC50 values of myristic acid, stearic acid, gamma-linolenic acid, linoleic acid, linolenic acid and arachidonic acid by EMSA were 97(+/-3), 2.2(+/-1.2), 55(+/-5), 32(+/-2), 62(+/-12), 22(+/-2)microM for DNA binding of recombinant c-Myc/Max, respectively. According to the results shown by XTT assay, their influence on proliferation was quite different from the rank order of IC50. Whereas the degree of influence of the unsaturated fatty acids on the proliferation of DMSO-differentiated HL-60 cells was similar, the influence of saturated fatty acids, stearic acid in particular, was very weak at same concentrations. In addition, we confirmed that these fatty acids have no influence on the expression of c-Myc in DMSO-differentiated HL-60 cells. Our experiments demonstrated that the inhibitors for the DNA binding of c-Myc/Max contribute to the downregulation of Myc-dependent proliferation and to the inducement of apoptosis, and serve as an exploration of potent new inhibitors.

Electrospray ionization mass spectrometry (ESI MS) was used, along with gas chromatography-massspectrometry (GC-MS), to monitor Paal-Knorr synthesis of 2,5-dimethyl-1-phenylpyrrole by condensation ofaniline with 2,5-hexanedione. In addition to 2,5-dimethyl-1-phenylpyrrole observed as a single spot by TLC,unexpected dimer size compounds were observed by GC-MS. Dimers and trimers were observed by ESI MS.ESI tandem mass spectrometry was used to select plausible structures for the dimer. ESI MS with or withoutliquid chromatographic separation is useful for observing oligomeric byproducts with low volatility producedin organic reactions.

A simple, fast and sensitive high-performance liquid chromatography (HPLC)-mass spectrometric (MS) method has been developed for simultaneous determination of amoxicillin and clavulanic acid in human plasma using terbutaline as internal standard. After precipitation of the plasma proteins with acetonitrile, the analytes were separated on a C(8) reversed-phase column with formic acid-water-acetonirile (2:1000:100) and detected using electrospray ionization (ESI) mass spectrometry in negative selected ion monitoring (SIM) mode. The method was validated and successfully applied to analysis of amoxicillin and clavulanic acid in clinical studies. The limit of quantitation, 0.12 microg/ml for amoxicillin and 0.062 microg/ml for clavulanic acid, was five times lower than that of the published HPLC-UV method.

C, 4 h),aminopeptidase, and carboxypeptidase Y. A single peptide mass map obtained from truncated peptides in thepartial acid hydrolysate of angiotensin and its glycosylation product mixture by matrix-assisted laserdesorption/ionization time-of-flight (MALDI-TOF) mass spectrometry enabled sequencing of angiotensin bya combinatorial procedure. MALDI-TOF and electrospra y ionization (ESI) tandem mass spectrometric resultsindicate that both the N-terminal amino group of aspartic acid and the guanidinium group of the second residuearginine are glycosylated.

Posttranscriptional RNA metabolism plays versatile roles in the regulation of gene expression during eukaryotic growth and development. It is mediated by a group of RNA binding proteins with distinct conserved motifs. In this study, an Arabidopsis (Arabidopsis thaliana) gene, designated FLK, was identified and shown to encode a putative RNA binding protein with K homology motifs. A mutant in which FLK was inactivated by T-DNA insertion exhibited a severe late flowering phenotype both in long and short days. The late flowering phenotype was reversed by gibberellin and vernalization treatments. The FLOWERING LOCUS C (FLC) transcription was greatly upregulated, whereas those of FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 decreased in the mutant. These observations demonstrate that FLK regulates the autonomous flowering pathway via FLC. It is now evident that a battery of different RNA binding proteins are involved in the posttranscriptional regulation of flowering time in Arabidopsis.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to observe folic acid and its derivatives such as tetrahydrofolate and 5-methyltetrahydrofolate in a vitamin tablet and in foods. Folic acid in a vitamin tablet was determined using angiotensin I as an internal reference. Tetrahydrofolic acid, 5-methyltetrahydrofolic acid, and an oxygenated folate were observed from a human blood sample using graphite plate. The results show that these mass spectrometric methods are useful for quickly obtaining a profile of folates.

Determination of the phosphorylation site in peptides by conventional tandem mass spectrometry is subject to ambiguity due to the neutral loss of the phosphate groups, especially in multiphosphorylated peptides. To prevent the neutral loss, the phosphate groups in phosphoserine or phosphothreonine peptides were replaced by p-bromobenzyl mercaptan via β-elimination and Michael addition. The unique isotopic signature of the Br introduced facilitated definitive localization of phosphorylation sites in multiphosphorylated peptides with highly adjacent serine or threonine residues. This method could be used to confirm phosphorylation sites determined by conventional tandem mass spectrometry after phosphopeptide enrichment.

When acetate buffer was used in chiral separation of d , l -serine derivatives using a γ-cyclodextrin (CD) column, both retention factor and resolution were high below the pKa of acetic acid and decreased sharply as the pH approached the pKa. A similar result was obtained by increasing the buffer concentration at a fixed pH. These observations suggest that hydrogen bonding interaction between the carboxylate group of the amino acid and the secondary hydroxyl groups at the CD rim plays an important role in chiral separation and is disrupted by the buffer anion.

Heat-induced (90 degrees C, 30 min) beta-elimination of a cystine residue leads to cleavage of a disulfide bond and produces a set of three peptides with a cysteine residue, a thiocysteine residue (+32Da), and a dehydroalanine residue (-34Da). This characteristic feature was observed from somatostatin and insulin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Mass spectrometric observation of this triplet is useful in identifying the presence of a cystine residue in a peptide, and could assist mass spectrometric identification of the peptide from a database.

All eight cysteine residues in 92 kDa cabbage phospholipase D (PLD), deduced from the cDNA sequence, were shown to have free sulfhydryl groups by analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of tryptic peptides of PLD derivatized with p-chloromercurybenzoate, iodoacetic acid, and N-ethylmaleimide, as well as of underivatized PLD. Assignment of sulfhydryl groups by any one method was not conclusive. However, complementary information derived from tryptic peptides derivatized with different reagents made full assignment of sulfhydryl groups possible.

Sulfite was detected in 7 varieties of Oriental herbal medicines (Pueraria radix, Zingiberis rhizoma, Platycodon radix, Adenophora radix, Pinellia tuber, Astragalus radix, and Paeonia radix) on the Korean market. Sulfiting of commercial Oriental herbal medicines by fumigation with burning bituminous coal was simulated, and the accumulation of sulfite was investigated by using fresh Platycodon radix roots obtained from a growing field. The sulfite level reached a plateau in 9 h, and the maximum sulfite level found by the Monier-Williams (MW) method (AOAC 990.28) was 1020 ppm. The sulfite content in the simulated Platycodon radix sample determined by alkali extraction followed by ion-exclusion chromatography with electrochemical detection (AOAC 990.31) was approximately 17% lower on average than the MW results. Free-sulfite levels determined by acid extraction and ion-exclusion chromatography with electrochemical detection were between 19 and 49% of the MW results. The advantages of different methods for sulfite determination and the significance of the results are discussed.

Tyrosinase-induced oxidation of tyrosine is known to lead to melanin by cross-linking of 5,6-dihydroxyindole (DHI) and indole-5,6-quinone intermediates. However, tyrosinase-induced cross-linking of tyrosine-containing peptides has not been reported. We observed tyrosinase-induced adducts of tyrosine-containing peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). MALDI-TOFMS was also used to observe tyrosine adducts at various levels of oxidation derived from acid hydrolysis of the peptide adducts. The rate of tyrosinase-induced browning of lys-tyr-lys was about half of that of tyrosine. These results indicate that tyrosinase-induced browning of tyrosine-containing peptides via direct oxidation and cross-linking of the benzene ring of the tyrosine residue occurs at a significant rate and needs to be considered in melanogenesis. Copyright © 2000 John Wiley & Sons, Ltd.

Nonenzymatic glycosylation products of several peptides and lysozyme were observed directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) operating in a linear mode, and the number of glucose units added was determined from the observed mass. Oxidation products of the peptides and the glycosylated peptides were also observed. Lysine residues were readily glycosylated; however, a nonapeptide without lysine residue also showed addition of up to four glucose units, suggesting that other amino acid residues can also be glycosylated. The MALDI-TOF-MS technique was useful for comparing reaction products from the glycosylation of different peptides as well as for demonstrating inhibition of glycosylation by N-α-acetyl-l-lysine. Keywords: MALDI-TOF-MS; nonenzymatic glycosylation; Maillard reaction; glucagon; lysozyme

Revered-phase LC-electrospray ionization mass spectrometry was used to selectively determine enalapril from plasma with minimal sample preparation. Detection limit of the method was 1 ng/mL. Precision (within day and between days) and accuracy of the method at various concentrations were acceptable. The analytical technique was used for pharmacokinetic studies after administration of enalapril to human test subjects.

The intracellular pathogen Mycobacterium tuberculosis ( Mtb ) causes tuberculosis. Enhanced intracellular survival (Eis) protein, secreted by Mtb , enhances survival of Mycobacterium smegmatis ( Msm ) in macrophages. Mtb Eis was shown to suppress host immune defenses by negatively modulating autophagy, inflammation, and cell death through JNK-dependent inhibition of reactive oxygen species (ROS) generation. Mtb Eis was recently demonstrated to contribute to drug resistance by acetylating multiple amines of aminoglycosides. However, the mechanism of enhanced intracellular survival by Mtb Eis remains unanswered. Therefore, we have characterized both Mtb and Msm Eis proteins biochemically and structurally. We have discovered that Mtb Eis is an efficient N ɛ -acetyltransferase, rapidly acetylating Lys55 of dual-specificity protein phosphatase 16 (DUSP16)/mitogen-activated protein kinase phosphatase-7 (MKP-7), a JNK-specific phosphatase. In contrast, Msm Eis is more efficient as an N α -acetyltransferase. We also show that Msm Eis acetylates aminoglycosides as readily as Mtb Eis. Furthermore, Mtb Eis, but not Msm Eis, inhibits LPS-induced JNK phosphorylation. This functional difference against DUSP16/MKP-7 can be understood by comparing the structures of two Eis proteins. The active site of Mtb Eis with a narrow channel seems more suitable for sequence-specific recognition of the protein substrate than the pocket-shaped active site of Msm Eis. We propose that Mtb Eis initiates the inhibition of JNK-dependent autophagy, phagosome maturation, and ROS generation by acetylating DUSP16/MKP-7. Our work thus provides insight into the mechanism of suppressing host immune responses and enhancing mycobacterial survival within macrophages by Mtb Eis.

The feasibility of using thermospray liquid chromatography-mass spectrometry using water as chemical ionization (CI) reagent in discharge mode for molecular weight determination was demonstrated with two known thermally produced compounds. Subsequently, the method was used to determine the molecular weight of a third compound thermally produced in meats. The same molecular weight was obtained using pure water as CI reagent with discharge and a 0.1 M ammonium acetate solution without discharge. The molecular weight of 114 was confirmed by gas chromatography-mass spectrometry (GC-MS). An electron impact mass spectrum obtained by GC-MS matched a previously published spectrum for 4-hydroxy-5-methyl-3(2H)-furanone

A bioanalytical method for evaluating the extent of protein cross-linking was developed. The method is based on reversed-phase HPLC determination of tryptophan released from the cross-linked proteins by Pronase, a nonspecific proteolytic enzyme. The amounts of tryptophan released from un-cross-linked bovine serum albumin (BSA), a nanopeptide, and Trp-Phe in control experiments were consistent with the tryptophan content of the protein and peptides. However, the amount of tryptophan released decreased significantly when BSA was cross-linked extensively by heating at 120 o C in the presence of glucose

Tyrosine phosphorylation and sulfation play many key roles in the cell. Isobaric phosphotyrosine and sulfotyrosine residues in peptides were determined by mass spectrometry using phosphatase or sulfatase to remove the phosphate or the sulfate group. Unique Br signature was introduced to the resulting tyrosine residues by incubation with 32% HBr at -20 °C for 20 min. MS/MS analysis of the brominated peptide enabled unambiguous determination of the phosphotyrosine and the sulfotyrosine sites. When phosphotyrosine and sulfotyrosine as well as free tyrosine were present in the same peptide, they could be determined simultaneously using either phosphatase or sulfatase following acetylation of the free tyrosine.

Due to almost identical chemical properties of C-terminal and side-chain carboxylic groups, selective C-terminal derivatization has been difficult. Although oxazolone-based C-terminal derivatization is the only selective C-terminal modification available, it has not been used widely because of its low derivatization efficiency. In this paper, an improved oxazolone chemistry for incorporation of Br signature to C-terminus is reported. MS/MS analysis of the brominated peptides led to a series of y ions with Br signature, facilitating de novo C-terminal sequencing.

Thiol-specific antioxidant protein (Protector Protein; PRP) from Saccharomyces cerevisiae was found to remove hydrogen peroxide and hydroxyl radical in the presence of dithiothreitol (DTT). Without DTT as a reducing equivalent, the antioxidant protein did not show the activities for destroying hydrogen peroxide and hydroxyl radical. N-ethylmaleimide (NEM) was observed to prevent the PRP from both removing hydrogen peroxide and protecting the cleavage of DNA. These observations suggest that the sulfhydryl of cysteine in PRP could function as a strong nucleophile to attack and destroy H2O2 and .OH.

Changes in membrane fluidity are the earliest cellular events that occur in plant cells upon exposure to cold. This subsequently triggers physiological processes, such as calcium influx and reorganization of actin cytoskeletons, and induces expression of cold-responsive genes. The plasma-membrane-anchored NAC (NAM/ATAF/CUC) transcription factor NTL6 is of particular interest. Cold triggers proteolytic activation of the dormant NTL6 protein, which in turn elicits pathogen-resistance responses by inducing a small group of cold-inducible PR (pathogenesis-related) genes in Arabidopsis. In the present study, we show that proteolytic processing of NTL6 is regulated by cold-induced remodelling of membrane fluidity. NTL6 processing was stimulated rapidly by cold. The protein stability of NTL6 was also enhanced by cold. The effects of cold on NTL6 processing and protein stability were significantly reduced in cold-acclimatized plants, supporting the regulation of NTL6 processing by membrane fluidity. Consistent with this, although NTL6 processing was stimulated by pharmacological agents that reduce membrane fluidity and thus mimic cold, it was inhibited when plants were treated with a 18:3 unsaturated fatty acid, linolenic acid. In addition, the pattern of NTL6 processing was changed in Arabidopsis mutants with altered membrane lipid compositions. Assays employing chemicals that inhibit activities of the proteasome and proteases showed that NTL6 processing occurs via the regulated intramembrane proteolysis mechanism. Interestingly, a metalloprotease inhibitor blocked the NTL6 processing. These observations indicate that a metalloprotease activity is responsible for NTL6 processing in response to cold-induced changes in membrane fluidity.

5-Hydroxymethylfurfural (HMF) was determined without interferences in juices, honey, syrup, tomato paste, grape juice concentrate and dehydrated pear by anion-exclusion chromatographic separation and UV detection at 285 nm. The samples were mixed with water, filtered and injected without extensive sample treatment, which is normally required in conventional spectrophotometric or reversed-phase high-performance liquid chromatographic methods, HMF at the 50 ppb level was determined with a signal-to-noise ratio of 6. The recovery of HMF added to honey was 98% at the 10 ppm level and 91% at 30 ppm.

Ginseng is a representative herbal medicine in Asia and various pharmacological activities of ginsenoside Rd isolated from ginseng have been reported. However, anti-cancer activity and mechanism of ginsenoside Rd in HT29 colon cancer cell lines were not studied yet. We performed proteomic analysis through two-dimensional gel electrophoresis, MALDI-TOF/TOF-MS and database to identify altered protein induced by ginsenoside Rd treatment in HT29. We can identify fourteen proteins contributed to cell growth inhibition induced by Rd. Proteins involved in the inhibition of mitosis (Stathmin1, Microtubule-associated protein RP/EB family and Stratifin) were significantly up- and down-regulated. And proteins associated with apoptosis (Rho GDP dissociation inhibitor, Tropomyosin1 and Annexin5) were significantly changed. Furthermore, ginsenoside Rd in HT29 was involved in cytoprotection, DNA replication and repair, protein synthesis and degradation, metastasis and mutagenesis. It was supposed that ginsenoside Rd contributed to induce anti-cancer activity by complementary functions of these proteins in colon cancer cells.

The specificity of bromelain and papain for degradation of actin and myosin in meat was compared. Meat was treated with 0·1% enzyme for 0–60 min at 24°C and proteins, extracted with 6 m urea containing 2% sodium dodecylsulfate (SDS) solution, were separated by SDS-polyacrylamide gel electrophoresis (PAGE). The profile of the extracted proteins clearly indicated that papain degrades myosin and actin at similar rates, whereas bromelain degrades myosin preferentially. This distinction could be useful in the development of freeze-dried meat products in which toughening of the meat due to the formation of high molecular weight aggregates from myosin cross-linking is minimized.

Proteome analysis was performed to identify proteins differentially expressed in an Arabidopsis mutant, ntm1-D. In this mutant the NAC transcription factor NTM1 is constitutively expressed and the resultant phenotypic changes include dwarfism, serrated leaves, and altered floral structures, probably due to reduced cell division. Marked elevation of proteins mediating environmental stress responses, including annexin, vegetative storage proteins, beta-glucosidase homolog 1, and glutathione transferases was observed. Overexpression of annexin was confirmed by RT-PCR and Western blotting. These observations suggest that the reduced growth observed in the ntm1-D mutant is caused by enhancement of its stress responses, possibly resulting in a cost in fitness.

Results for determination of sulfur dioxide In grapes were compared by 3 methods: the modified Monier-Wllllams method, acid dlstlllatlon/lon exclusion chromatography with electrochemical detection (AD/IEC-EC), and alkali extraction/ ion exclusion chromatography with electrochemical detection (AE/IEC-EC). An unusual positive response was observed during the later stage of the Monier-Wllllams distillation of both control grapes and sulfited grapes. Development of volatile acidic compounds in parallel with this Monier- Wllllams response and darkening of sample was also observed by collection In an alkali trap and analysis using anion exclusion chromatography and photodlode array detection. No parallel Increase In sulfite was observed by the more selective AD/IEC-EC method, which clearly demonstrated that the response observed during the later stage of the Monier-Wllllams method Is a false positive, probably due to caramellzatlon reaction products. Monier-Williams results for grapes containing ca 10 ppm sulfite were In reasonably good agreement with those by either the AD/IEC-EC or AE/ IEC-EC methods, presumably because the false positive response In the Monier-Wllllams analysis compensated for the somewhat Incomplete recovery of sulfite. The AE/IECEC method Is recommended because it is rapid, sensitive, straightforward, and free from interference. Accurate results by Monier-Wllllams analysis could be obtained by limiting distillation to 60 min and correcting for recovery.

A rapid liquid chromatographic (LC) method was developed for a sensitive determination of nitrite In cured meats, using ion-exclusion chromatographic separation and electrochemical detection (IEC-EC). The current AOAC colorimetric method requires 2 h shaking In a steam bath to eliminate Interference from reducing compounds such as ascorbic acid. In the present method, nitrite was analyzed In the presence of ascorbic acid without interference, and the extraction time was reduced to 1 mln. The extracted nitrite was determined by ion chromatography using anion-excluslon/ HS column and amperometric detector equipped with platinum or glassy carbon electrode operating at +1.0 V vs Ag/ AgCI reference electrode. The detection limit was 1 ppb as NO-2. The recoveries of 50 ppm nitrite added to frankfurter and meat stick were 103 and 99.6%, respectively, with relative standard deviations less than 4%. The high speed, sensitivity, and selectivity make the new method a useful alternative to the AOAC colorimetric method.

A liquid chromatographic (LC) method for determination of total sulfite in foods and beverages by alkali extraction followed by ion exclusion chromatographic separation and electrochemical detection (IEC-EC) was collaboratively studied by 9 laboratories. Blind duplicate samples of starch, diluted lemon juice, wine cooler, dehydrated seafood, and instant mashed potatoes were analyzed without spiking and with added sulfite at 2 levels. The initial sulfite levels varied from 0 to 384 ppm S02, and the levels added varied from 10 to 400 ppm. The initial sulfite levels determined by the IECEC method and the Monler-Williams method were in good agreement. Recovery of added sulfite by the IEC-EC method was generally higher than that by the Monler-Williams method. Within-laboratory repeatability (RSDr) for the IEC-EC method varied from 4.4 to 26.0%, and overall reproducibility (RSDR) varied from 8.5 to 39.3 %. The collaborators found the method to be fast, sensitive, and easy to use, which makes it a useful alternative to the Monler-Williams method. The method has been adopted official first action.

Proteome analysis by 2-DE and PMF by MALDI-TOF MS was performed on human amnion and amniotic fluid at term. Ninety-two soluble and nineteen membrane proteins were identified from amnion. Thirty-five proteins were identified from amniotic fluid. Calgranulin A and B were found in all patients infected with Ureaplasma urealyticum, but not in any of the patients without infection, indicating that they are potential markers of intrauterine infection. Identity of calgranulin A and B was confirmed by MALDI-TOF/TOF MS. This study represents the first extensive analysis of the human amnion and amniotic fluid proteome at term and demonstrates that 2-DE and MALDI-TOF MS is a useful tool for identifying clinically significant biomarkers of problematic pregnancies.

A database search using peptide mass fingerprints obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry leads to protein identification with incomplete sequence coverage, because certain peptides are preferentially desorbed/ionized and some are not detected at all. We show that certain tryptic peptides mainly with C-terminal arginine not detected before derivatization become detectable upon dansylation. Others, mainly with C-terminal lysine, are suppressed. An increase in protein sequence coverage and protein identification score by combined data from underivatized and dansylated peptides in database search is demonstrated using human amnion proteins (human serum albumin precursor, calmodulin, collagen alpha 2(VI) chain precursor, galectin-3) separated by two-dimensional gel electrophoresis as well as femtomole amounts of BSA in solution.

AIM: Expression of heat shock proteins (HSPs) is frequently up-regulated in hepatocellular carcinoma (HCC), which evolves from dysplastic nodule (DN) and early HCC to advanced HCC. However, little is known about the differential expression of HSPs in multistep hepatocarcinogenesis. It was the purpose of this study to monitor the expression of HSPs in multistep hepatocarcinogenesis and to evaluate their prognostic significance in hepatitis B virus (HBV)-related HCC. METHODS: Thirty-eight HCC and 19 DN samples were obtained from 52 hepatitis B surface antigen-positive Korean patients. Immunohistochemical and dot immunoblot analyses of HSP27, HSP60, HSP70, HSP90, glucose regulated protein (GRP)78, and GRP94 were performed and their expression at different stages of HCC development was statistically analyzed. RESULTS: Expression of HSP27, HSP70, HSP90, GRP78, and GRP94 increased along with the stepwise progression of hepatocarcinogenesis. Strong correlation was found only in GRP78 (Spearman’s r = 0.802). There was a positive correlation between the expressions of GRP78, GRP94, HSP90, or HSP70 and prognostic factors of HCC. Specifically, the expression of GRP78, GRP94, or HSP90 was associated significantly with vascular invasion and intrahepatic metastasis. CONCLUSION: The expressions of HSPs are commonly up-regulated in HBV-related HCCs and GRP78 might play an important role in the stepwise progression of HBV-related hepatocarcinogenesis. GRP78, GRP94, and HSP90 may be important prognostic markers of HBV-related HCC, strongly suggesting vascular invasion and intrahepatic metastasis.

A simple, fast and sensitive high-performance liquid chromatography (HPLC)–electrospray ionization (ESI) tandem mass spectrometric method (LC–MS/MS) has been developed for determination of propiverine and propiverine N-oxide metabolite in human plasma using oxybutynin as internal standard. Instead of extracting propiverine from plasma using organic solvents, which should be separated from the aqueous phase and evaporated before injecting the sample into the chromatograph, plasma sample containing propiverine and N-oxide was directly injected after precipitating proteins with acetonitrile. Numerous compounds in the plasma did not interfere with the highly specific multiple reaction monitoring in tandem mass spectrometric detection following C 8 reversed-phase chromatographic separation under conditions that eluted propiverine, N-oxide and oxybutynin within 2 min (0.1% formic acid in water/acetonitrile, 25:75, v/v). The LC–MS/MS method and an alternative LC–MS method, using methyl- t -butyl ether extraction and selected ion monitoring, were validated over 1–250 ng ml −1 of propiverine and 2 to 500 ng ml −1 of N-oxide, and successfully applied in a pharmacokinetic study. The lower limit of quantitation was 1 ng ml −1 for propiverine and 2 ng ml −1 for N-oxide in both methods.