Phytochemical Characterization, Antioxidant Activity, and Cytotoxicity of Methanolic Leaf Extract of Chlorophytum Comosum (Green Type) (Thunb.) Jacq.

Chlorophytum genus has been extensively studied due to its diverse biological activities. We evaluated the methanolic extract of leaves of Chlorophytum comosum (Green type) (Thunb.) Jacques, the species that is less studied compared to C. borivilianum. The aim was to identify phytoconstituents of the methanolic extract of leaves of C. comosum and biological properties of its different fractions. Water fraction was analyzed with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Nineteen compounds belonging to different chemical classes were identified in the methanolic extract of leaves of C. comosum (Green type) (Thunb.) Jacques. In addition to several fatty acids, isoprenoid and steroid compounds were found among the most abundant constituents. One of the identified compounds, 4'-methylphenyl-1C-sulfonyl-ß-d-galactoside, was not detected earlier in Chlorophytum extracts. The water fraction was toxic to HeLa cells but not to Vero cells. Our data demonstrate that methanolic extract of leaves of C. comosum can be a valuable source of bioactive constituents. The water fraction of the extract exhibited promising antitumor potential based on a high ratio of HeLa vs. Vero cytotoxicity.

Percutaneous closure of a secundum atrial septum defect performed under hypnosis: a case report.

Atrial septal defects (ASDs) are the third most common type of congenital heart diseases with ostium secundum defects (ASDsII) being responsible for approximately 75% of these defects. The treatment consists of either a transcatheter closure or a surgical intervention. The transcatheter approach offers a less invasive alternative for patients who fulfill anatomical and size criteria and is therefore increasingly used as the preferred primary intervention. As general anaesthesia is sometimes contra-indicated for some patients needing ASD or PFO closure and intracardiac echocardiography (ICE) is not always readily available, alternative "supportive" techniques as hypnosis could be of interest. In our case, a relative contra-indication for general anaesthesia was pronounced due to the severely impaired pulmonary function caused by severe deformative torsional scoliosis. Over the years several reviews have been published on hypnotic analgesia used together with usual care suggesting that hypnotic analgesia is a promising non-pharmacologic adjunct treatment for ameliorating pain and surgical distress. Also, various cardiological procedures under hypnosis have already been described in literature, but this is the first time that percutaneous ASD closure under virtual reality (VR) hypnosis is mentioned in literature.

The vasodilating effect of a Hintonia latiflora extract with antidiabetic action.

In the present study, it is shown for the first time that an extract of Hintonia latiflora (HLE) which is used as an antidiabetic herbal medicine, is not only able to decrease blood glucose concentration but additionally exerts a vasodilating effect. Accordingly, this extract might have a positive influence on diabetes-associated dysfunction of blood vessels. The vasodilating effect was demonstrated in vitro in aortic rings of guinea pigs as well as in vivo in rabbits. Aortic rings pre-contracted with noradrenaline (NA) could completely be relaxed by HLE (EC50: 51.98 mg/l). In contrast, potassium-induced contractions were not diminished by HLE. Therefore, it can be suggested that the vasodilating effect of HLE is primarily the result of an inhibition of G protein-induced increase in intracellular calcium and not of a blockade of voltage-operated L-type calcium channels. The neoflavonoid coutareagenin (COU), a constituent of HLE which in part is responsible for the blood glucose-lowering effect of HLE, also relaxed NA-induced contractions of aortic rings (EC50: 32.55 mg/l) and only weakly inhibited potassium-induced contractions. Experiments in aortic rat cells revealed that calcium transients evoked by vasopressin were suppressed by 60 mg/l COU supporting the idea of an inhibition of G protein-induced intracellular calcium release by a constituent of HLE. To study the effect of HLE on vascular tone under in vivo conditions, ultrasound measurements were carried out in conscious rabbits which received a single oral dose of HLE. Under the influence of HLE, a vasodilation combined with a lowering of blood flow velocity could be observed in the abdominal aorta and the common carotid artery. Additionally, a decrease in blood glucose concentration in the HLE group occurred. The combination of a blood glucose-lowering with a vasodilating effect may be helpful for reducing angiopathies, typical long-term complications in patients with diabetes mellitus.

Metal ion-mobilizing additives for comprehensive detection of femtomole amounts of phosphopeptides by reversed phase LC-MS.

It is hypothesized that metal ion-mediated adsorption of phosphorylated peptides on stationary phases of LC-columns is the major cause for their frequently observed poor detection efficiency in LC-MS. To study this phenomenon in more detail, sample solutions spiked with metal ion-mobilizing additives were analyzed by reversed phase µLC-ICP-MS or nanoLC-ESI-MS. Using µLC-ICP-MS, metal ions were analyzed directly as atomic ions. Using electrospray ionization, either metal ion chelates or phosphopeptide standard mixtures injected in subpicomole amounts were analyzed. Deferoxamine, imidazole, ascorbate, citrate, EDTA, and the tetrapeptide pSpSpSpS were tested as sample additives for the interlinked purposes of metal ion-mobilization and improvement of phosphopeptide recovery. Iron probably represents the major metal ion contamination of reversed phase columns. Based on the certified iron level in LC-grade solvents, a daily metal ion load of >10 pmol was estimated for typical nanoLC flow rates. In addition, phosphopeptide fractions from IMAC columns were identified as source for metal ion contamination of the LC column, as demonstrated for Ga(3+)-IMAC. The three metal ion-chelating additives, EDTA, citrate and pSpSpSpS, were found to perform best for improving the LC recovery of multiply phosphorylated peptides injected at subpicomole amounts. The benefits of metal ion-mobilizing LC (mimLC) characterized by metal ion complexing sample additives is demonstrated for three different instrumental setups comprising (a) a nanoUPLC-system with direct injection on the analytical column, (b) a nanoLC system with inclusion of a trapping column, and (c) the use of a HPLC-Chip system with integrated trapping and analytical column.

Structural and mechanistic Information on c(1) ion formation in collision-induced fragmentation of peptides.

The formation of c(1) ions during collision-induced fragmentation of peptides with asparagine, ornithine, or glutamine at the N-terminal position 2 has been studied. For this purpose, the corresponding fragment ion spectra of a large set of synthetic peptides were investigated. It is demonstrated that the c(1) ion intensity depends on the nature of the second residue in the N-terminal dipeptide motif as well as on the peptide length. It is shown that the formation of c(1) ions proceeds by two competing mechanisms. One mechanism is the secondary fragmentation of the b(2) ion, the efficiency of which shows only a minor dependency on the complete peptide sequence. The other mechanism is the direct formation from the molecular ion, which is identified to be connected with sequence-specific c(1) ion intensities. A model for this latter mechanism is proposed based on the analysis of the formation and secondary fragmentation of the z(max-1) ion, which is the complementary ion to the c(1) ion. Additional evidence is obtained by investigation of peptides with ornithine in N-terminal position 2, which in general exhibit c(1) ion intensities intermediate between the asparagine- and glutamine-containing species. The data presented support the reliable assignment of N-terminal dipeptide motifs using collision-induced dissociation.

Recombinant isotope labeled and selenium quantified proteins for absolute protein quantification.

A novel, widely applicable method for the production of absolutely quantified proteins is described, which can be used as internal standards for quantitative proteomic studies based on mass spectrometry. These standards are recombinant proteins containing an isotope label and selenomethionine. For recombinant protein expression, assembly of expression vectors fitted to cell-free protein synthesis was conducted using the gateway technology which offers fast access to a variety of genes via open reading frame libraries and an easy shuttling of genes between vectors. The proteins are generated by cell-free expression in a medium in which methionine is exchanged against selenomethionine and at least one amino acid is exchanged by a highly stable isotope labeled analogue. After protein synthesis and purification, selenium is used for absolute quantification by element mass spectrometry, while the heavy amino acids in the protein serve as reference in subsequent analyses by LC-ESI-MS or MALDI-MS. Accordingly, these standards are denominated RISQ (for recombinant isotope labeled and selenium quantified) proteins. In this study, a protein was generated containing Lys+6 ([(13)C(6)]-lysine) and Arg+10 ([(13)C(6),(15)N(4)]-arginine) so that each standard tryptic peptide contains a labeled amino acid. Apolipoprotein A1 was synthesized as RISQ protein, and its use as internal standard led to quantification of a reference material within the specified value. Owing to their cell-free expression, RISQ proteins do not contain posttranslational modifications. Thus, correct quantitative data by ESI- or MALDI-MS are restricted to quantifications based on peptides derived from unmodified regions of the analyte protein. Therefore, besides serving as internal standards, RISQ proteins stand out as new tools for quantitative analysis of covalent protein modifications.

Sequencing of the thirteen structurally isomeric quartets of N-terminal dipeptide motifs in peptides by collision-induced dissociation.

N-terminal sequencing of protonated peptides is challenging, since each b(2) ion represents two sequence isomers, e.g., NE and EN. Additionally the occurrence of compositional isomers, such as NE and QD, further increases the number of isomers to four (NE, EN, QD, DQ). This leads to a subset of 13 b(2) ion masses where each value represents four individual species. The b(2) ions within such a quartet are characterized by the same elemental composition. To test the utility of CID for differentiation of isomeric b(2) ions, the CID spectra of 52 small synthetic peptides were recorded, representing the 13 isomeric b(2) ion quartets, which may be formed from unmodified amino acid residues. The CID spectra of protonated peptides containing these quartets were carefully inspected for N-terminal sequence information. Below the m/z value of the b(2) ion, individual differences were found in the b(2) fragment ion signatures (neutral loss of CO, H(2)O, NH(3), and other less common units). Recognition of N and Q in second position from the N-terminus is based on c(1) ion formation. Relative intensities of immonium ions were also used for differentiation between sequence isomers. In the complementary high-mass regions above the m/z value of the y(max-2) ion, individual differences were observed in the formation of y(max-1), x(max-1) and z(max-1) ions, which could be correlated to the complementary low-mass ions. In summary, de novo sequencing of the N-terminal dipeptide motif is feasible by considering all available sequence information present in CID spectra of protonated peptides.

Petroleum-degrading microbial numbers in rhizosphere and non-rhizosphere crude oil-contaminated soil.

Phytoremediation can be a cost-effective and environmentally acceptable method to clean up crude oil-contaminated soils in situ. Our research objective was to determine the effects of nitrogen (N) additions and plant growth on the number of total hydrocarbon (TH)-, alkane-, and polycyclic aromatic hydrocarbon (PAH)-degrading microorganisms in weathered crude oil-contaminated soil. A warm-season grass, sudangrass (Sorghum sudanense (Piper) Stapf), was grown for 7 wk in soil with a total petroleum hydrocarbon (TPH) level of 16.6 g TPH/kg soil. Nitrogen was added based upon TPH-C:added total N (TPH-C:TN) ratios ranging from 44:1 to 11:1. Unvegetated and unamended controls were also evaluated. The TH-, alkane-, and PAH-degrading microbial numbers per gram of dry soil were enumerated from rhizosphere and non-rhizosphere soil for vegetated pots and non-rhizosphere soil populations were enumerated from non-vegetated pots. Total petroleum-degrading microbial numbers were also calculated for each pot. The TH-, alkane-, and PAH-degrading microbial numbers per gram of dry soil in the sudangrass rhizosphere were 3.4, 2.6, and 4.8 times larger, respectively, than those in non-rhizosphere soil across all N rates. The presence of sudangrass resulted in significantly more TH-degrading microorganisms per pot when grown in soil with a TPH-C:TN ratio of 11:1 as compared to the control. Increased plant root growth in a crude oil-contaminated soil and a concomitant increase in petroleum-degrading microbial numbers in the rhizosphere have the potential to enhance phytoremediation.

Collision-induced reporter fragmentations for identification of covalently modified peptides.

Collision-induced reporter fragmentations of the currently most important covalent peptide modifications as detected by tandem mass spectrometry are summarized. These fragmentations comprise the formation of reporter ions, which are preferentially immonium ions, immonium ion-derived fragments or side chain fragments. In addition, the reporter neutral loss reactions for covalently modified amino acid residues are summarized. For each individual covalent modification which can be recognized by a reporter fragmentation, the accurate mass shift and the gross formula shift of the modified amino acid residue are given. The same set of data is provided for the reporter fragmentations. Finally, an extensive accurate mass and gross formula list is presented as supplementary material, describing mostly regular and modified y(1) and dipeptide a and b ions, which are helpful for identification of the peptide ends of covalently modified peptides.

Plant protein phosphorylation monitored by capillary liquid chromatography--element mass spectrometry.

Many essential cellular functions such as growth rate, motility, and metabolic activity are linked to reversible protein phosphorylation, since they are controlled by signaling cascades based mainly on phosphorylation/dephosphorylation events. Quantification of global or site-specific protein phosphorylation is not straightforward with standard proteomic techniques. The coupling of capillary liquid chromatography (microLC) with ICP-MS (inductively coupled plasma-mass spectrometry) is a method which allows a quantitative screening of protein extracts for their phosphorus and sulfur content, and thus provides access to the protein phosphorylation degree. In extension of a recent pilot study, we analyzed protein extracts from the model organisms Arabidopsis thaliana and Chlamydomonas reinhardtii as representatives for multicellular and unicellular green photosynthetically active organisms. The results indicate that the average protein phosphorylation level of the algae C. reinhardtii is higher than that of A. thaliana. Both the average phosphorylation levels were found to be between the extreme values determined so far for prokaryotes (C. glutamicum, lowest levels) and eukaryotes (Mus musculus, highest levels). Tissue samples of A. thaliana representing different stages of plant development showed varying levels of protein phosphorylation indicating a different adjustment of the kinase/phosphatase system. We also utilized the microLC-ICP-MS technology to estimate the efficiency of a novel phosphoprotein enrichment method based on aluminum hydroxide, since the enrichment of phosphorylated species is often an essential step for their molecular characterization.

Selecting plants and nitrogen rates to vegetate crude-oil-contaminated soil.

Phytoremediation can be effective for remediating contaminated soils in situ and generally requires the addition of nitrogen (N) to increase plant growth. Our research objectives were to evaluate seedling emergence and survival of plant species and to determine the effects of N additions on plant growth in crude-oil-contaminated soil. From a preliminary survival study, three warm-season grasses--pearlmillet (Pennisetum glaucum [L.] R. Br.), sudangrass (Sorghum sudanense [Piper] Stapf [Piper]), and browntop millet (Brachiaria ramosa L.)--and one warm-season legume--jointvetch (Aeschynomene americana L.)--were chosen to determine the influence of the N application rate on plant growth in soil contaminated with weathered crude oil. Nitrogen was added based on total petroleum hydrocarbon-C:added N ratios (TPH-C:TN) ranging from 44:1 to 11:1. Plant species were grown for 7 wk. Root and shoot biomass were determined and root length and surface area were analyzed. Pearlmillet and sudangrass had higher shoot and root biomass when grown at a TPH-C:TN (inorganic) ratio of 11:1 and pearlmillet had higher root length and surface area when grown at 11:1 compared with the other species. By selecting appropriate plant species and determining optimum N application rates, increased plant root growth and an extended rhizosphere influence should lead to enhanced phytoremediation of crude-oil-contaminated soil.

Protein tyrosine-O-sulfation analysis by exhaustive product ion scanning with minimum collision offset in a NanoESI Q-TOF tandem mass spectrometer.

Tyrosine-O-sulfated peptides were studied by nanoESI Q-TOF mass spectrometry and were found to exhibit an abundant loss of SO3 in positive ion mode under the usually nonfragmenting conditions of survey spectrum acquisition. A new strategy for the detection of tyrosine-O-sulfated peptides in total protein digests was designed based on exhaustive product ion scanning at the collision offset conditions typical for the recording of survey spectra (minimum collision offset). From these data, Q-TOF neutral loss scans for loss of 80/z and Q-TOF precursor ions scans were extracted. The specificity of this approach for analysis of tyrosine-O-sulfation was tested using a tryptic digest of bovine serum albumin spiked with sulfated hirudin (1:1 and 1000:1 molar ratio of BSA to sulfated hirudin, respectively) and using an in-solution digest of the recombinant extracellular domain of thyroid stimulating hormone receptor (ECD-TSHr). For both examples, the combination of in silico neutral loss scans for 80/z and subsequent in silico precursor ion scans resulted in a specific identification of sulfated peptides. In the analysis of recombinant ECD-TSHr, a doubly sulfated peptide could be identified in this way. Surprisingly, approximately 1/4 of the product ion spectra acquired from the tryptic digest of ECD-TSHr at minimum collision offset exhibited sequence-specific ions suitable for peptide identification. Complementary ion pairs were frequently observed, which either were b2/y(max-2) pairs or were induced by cleavage N-terminal to proline. MS/MS analysis at minimum collision offset followed by extraction of neutral loss and precursor ion scans is ideally suited for highly sensitive detection of analyte ions which exhibit facile gas-phase decomposition reactions.

Design and synthesis of a new fluorescent probe for cytochrome P450 3A4 (CYP 3A4).

Inhibition of CYP 3A4 catalytic activity is a principal mechanism for in vivo drug-drug interactions, sometimes leading to severe toxic effects. Rapid in vitro testing for CYP 3A4 high affinity/high inhibition potential has become part of the standard investigations for new drug candidates. Unfortunately, the complexity of the kinetics associated with CYP 3A4 catalyzed reactions (multiple substrates binding, non Michaelis-Menten kinetics) make these tests either inaccurate or tedious. We have designed and synthesized a new fluorescent probe, a testosterone substituted at the 6beta- position with a fluorescent deazaflavine moiety which is able to inhibit to the same extent the hydroxylation of compounds known to bind to different sites in the CYP 3A4 active site. Furthermore, the binding of this compound and its displacement from the active site can be followed by fluorescence measurements, which allows a rapid evaluation of the CYP 3A4 affinity of any new drug candidate.

A mathematical model of phytoremediation for petroleum contaminated soil: sensitivity analysis.

Phytoremediation is an attractive treatment technology for many contaminated sites due to its cost effectiveness and public acceptance. We present a sensitivity analysis of important parameters from a screening level model for phytoremediation by grass species of weathered petroleum-contaminated sites. The conceptual framework is that root movement through contaminated soil will enhance contaminant biodegradation by providing a local environment more favorable for petroleum degrading microorganisms--the so-called rhizosphere effect. Common questions in phytoremediation are, "What species should be planted?" and "What management practices should be followed?" These choices may affect degradation kinetics, root biomass (and therefore rhizosphere volume), and the root turnover. Important model parameters are the rate constants, rhizosphere volume, and the rate of root turnover. We present a sensitivity analysis with the aim of identifying the most important factors for improving phytoremediation effectiveness. For simulations of the phytoremediation of weathered diesel range organics, our results indicate that annual species, with higher root turnover, are preferred over perennial species with the caveat of equal degradation rate constants, that is, no species-dependent effects. In addition, the results suggest that the management of nonrhizosphere soil could play an important role in the overall effectiveness of phytoremediation. Finally, the effect of increasing root biomass or increasing the rhizosphere thickness is approximately equivalent with respect to the ultimate removal of the contaminants.

Phytoremediation of pyrene in a Cecil soil under field conditions.

We evaluated the effects of annual ryegrass (Lolium multiflorum Lam.) and phosphorus (P) availability on the dissipation of pyrene added at a concentration of approximately 600 mg kg-1 dry soil in the top 7.5 cm of a Cecil loamy sand (fine, kaolinitic, thermic Typic Kanhapludults) in a 10-month experiment under field conditions in Clemson, South Carolina. Plastic canopies were installed to prevent flooding of plots and raindrop dispersion of pyrene. Treatment factors were pyrene, vegetation, and available P levels. Each of the eight treatments had four replicates. The soil was adjusted to low and high P concentrations (an average of 41 and 66 kg extractable P ha-1, respectively). After a 175-d lag period for all treatments, the rate of pyrene removal followed first-order kinetics. The first-order rate constant was significantly higher in nonvegetated (0.098 d-1) than vegetated treatments (0.034 d-1). These data suggest that the presence of easily biodegradable organic matter from plant roots slowed the removal rate of pyrene. The levels of available P did not affect the rate of pyrene dissipation. Pyrene decreased below the detection limit of 6.25 mg kg-1 dry soil in all treatments after 301 d.

A mathematical model of phytoremediation for petroleum-contaminated soil: model development.

We present a simple model for root length density that combines the generally accepted spatial (exponential decrease with depth) and temporal (sinusoidal) variability of root length. Parameters in this model for root length density can be determined from assumed or measured information regarding the annual biomass turnover, maximum standing biomass, and maximum depth of root penetration. The root length density model, coupled with information regarding the average root lifespan, gives specific root growth and senescence functions that are the forcing functions for the phytoremediation model. We present a screening level mathematical model for phytoremediation that accounts for the growth and senescence of roots in the system. This is an important factor for recalcitrant, immobile compounds found in weathered crude oil contaminated soils. The phytoremediation model is based on variable volume compartments that have individual first-order degradation rate constants; as the roots move through the soil, the soil cycles through the rhizosphere zone, decaying root zone and bulk soil zone. Thus, although the oil is immobile, as the roots penetrate through the soil the oil is brought into contact with the rhizosphere.

Influence of organic and inorganic soil amendments on plant growth in crude oil-contaminated soil.

Phytoremediation can be a viable alternative to traditional, more costly remediation techniques. Three greenhouse studies were conducted to evaluate plant growth with different soil amendments in crude oil-contaminated soil. Growth of alfalfa (Medicago sativa L., cultivar: Riley), bermudagrass (Cynodon dactylon L., cultivar: Common), crabgrass (Digitaria sanguinalis, cultivar: Large), fescue (Lolium arundinaceum Schreb., cultivar: Kentucky 31), and ryegrass (Lolium multiflorum Lam., cultivar: Marshall) was determined in crude oil-contaminated soil amended with either inorganic fertilizer, hardwood sawdust, papermill sludge, broiler litter or unamended (control). In the first study, the addition of broiler litter reduced seed germination for ryegrass, fescue, and alfalfa. In the second study, bermudagrass grown in broiler litter-amended soil produced the most shoot biomass, bermudagrass produced the most root biomass, and crabgrass and bermudagrass produced the most root length. In the third study, soil amended with broiler litter resulted in the greatest reduction in gravimetric total petroleum hydrocarbon (TPH) levels across the six plant treatments following the 14-wk study. Ryegrass produced more root biomass than any other species when grown in inorganic fertilizer- or hardwood sawdust + inorganic fertilizer-amended soil. The studies demonstrated that soil amendments and plant species selection were important considerations for phytoremediation of crude oil-contaminated soil.

Protective effect of XY99-5038 on hydrogen peroxide induced cell death in cultured retinal neurons.

XY99-5038 is a preparation from a specific formula provided by Dr. Yan Xin, a renowned Traditional Chinese Medicine specialist. This formulation has proven to be effective without side effects but has not been studied under rigorous laboratory conditions. In order to investigate the role of XY99-5038 in protecting neurons from degeneration, we used retinal neuronal culture as a model system to study the protective effects of XY99-5038 against hydrogen peroxide (H2O2) induced cytotoxicity. Basic fibroblast growth factor (bFGF), a potent neurotrophic factor, was employed as comparable agent. Retinas of 0-2 days old Sprague-Dawley rats were isolated, dissociated, and the neurons maintained for 2 weeks in a synthetic serum-free media. Cytotoxicity was determined by MTT (3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide) and LDH (Lactate dehydrogenase) assays, whereas apoptotic cell death was evaluated by the TUNEL (TdT-mediated digoxigenin-dUTP nick-end labeling) assay. Treatment with H2O2 significantly induced death of retinal neurons. Pretreatment with XY99-5038 prior to insult greatly inhibited or attenuated H2O2 induced cytotoxicity and apoptosis. These effects were shown to be dose-dependent. Our data also show that the protective effect of XY99-5038 is more potent than that of bFGF. Our data suggest that XY99-5038 could be a potential agent in the treatment of disorders associated with oxidative damage.