Caffeoylquinic Acids,Cytotoxic, Antioxidant,Acetylcholinesterase and Tyrosinase Enzyme Inhibitory Activities of Six Inula Species from Bulgaria

Chlorogenic (5‐CQA), 1,5‐, 3,5‐, 4,5‐ and 3,4‐dicaffeoylquinic (DCQA) acids were identified and quantified in the methanol extracts of Inula oculus‐christi L., I. bifrons L., I. aschersoniana Janka var. aschersoniana, I. ensifolia L., I. conyza (Griess .) DC. and I. germanica L. by HPLC analysis. The amount of 5‐CQA varied from 5.48 to 28.44 mg/g DE and the highest content was detected in I. ensifolia. 1,5‐DCQA (4.05–55.25 mg/g DE) was the most abundant dicaffeoyl ester of quinic acid followed by 3,5‐DCQA, 4,5‐DCQA and 3,4‐DCQA. The extract of I. ensifolia showed the highest total phenolic content (119.92±0.95 mg GAE/g DE) and exhibited the strongest DPPH radical scavenging activity (69.41±0.55 %). I. bifrons extract was found to be the most active sample against ABTS^.+ (TEAC 0.257±0.012 mg/mL) and the best tyrosinase inhibitor. The studied extracts demonstrated a low inhibitory effect towards acetylcholinesterase and possessed low cytotoxicity in concentration range from 10 to 300 μg/mL toward non‐cancer (MDCK II) and cancer (A 549) cells.     

Functional analysis of PilT from the toxic cyanobacterium Microcystis aeruginosa PCC 7806

The evolution of the microcystin toxin gene cluster in phylogenetically distant cyanobacteria has been attributed to recombination, inactivation, and deletion events, although gene transfer may also be involved. Since the microcystin-producing Microcystis aeruginosa PCC 7806 is naturally transformable, we have initiated the characterization of its type IV pilus system, involved in DNA uptake in many bacteria, to provide a physiological focus for the influence of gene transfer in microcystin evolution. The type IV pilus genes pilA, pilB, pilC, and pilT were shown to be expressed in M. aeruginosa PCC 7806. The purified PilT protein yielded a maximal ATPase activity of 37.5 +/- 1.8 nmol P(i) min(-1) mg protein(-1), with a requirement for Mg(2+). Heterologous expression indicated that it could complement the pilT mutant of Pseudomonas aeruginosa, but not that of the cyanobacterium Synechocystis sp. strain PCC 6803, which was unexpected. Differences in two critical residues between the M. aeruginosa PCC 7806 PilT (7806 PilT) and the Synechocystis sp. strain PCC 6803 PilT proteins affected their theoretical structural models, which may explain the nonfunctionality of 7806 PilT in its cyanobacterial counterpart. Screening of the pilT gene in toxic and nontoxic strains of Microcystis was also performed.     

Biochemical and immunochemical characterization of boar sperm flagellar protein with role in hyperactivation/capacitation process

Investigations on specific and functionally active sperm antigens could bring about the elucidation of the mechanisms of gamete interaction and help the search for new approaches in prognosis and regulation of fertility. Previously, we reported that the monoclonal antibody (Mab) 3G4 against capacitated boar spermatozoa was capable of inhibiting boar sperm-porcine zona pellucida binding due to its inhibitory effect on sperm hyperactivation and capacitation. The cell and tissue specificity of Mab 3G4 was demonstrated in indirect immunofluorescence (IIF) and ELISA experiments against spermatozoa from different vertebrate species, as well as against extracts of boar reproductive and somatic organs. In the present IIF experiments, it was shown that Mab 3G4 recognized an antigen determinant on the flagellar midpiece region of ejaculated and capacitated boar spermatozoa. It was speculated that the Mab 3G4-corresponding antigen participates in pyruvate/lactate metabolism because of its specific localization in the sperm structure, which is responsible for producing forward motility and its involvement in processes that require the metabolism of pyruvate and lactate. As a possible approach toward investigating the participation of Ag 3G4 in pyruvate/lactate metabolism, Mab 3G4's effect on lactate dehydrogenase (LDH) was examined. Using an electrophoretic approach we provided evidence that Mab 3G4 stimulates LDH activity in the Triton X-100 and NP40 protein fractions of capacitated boar spermatozoa. In addition, we found that LDH isoenzymes stimulated by Mab 3G4 are of gametic C type. In Western blot, under nonreducing conditions, Mab 3G4 identified a single protein band with a molecular weight of 140 kDa. The biochemical and immunochemical experiments provided evidence supporting the involvement of 3G4 antigen in the sperm pyruvate/lactate metabolism.     

Comparison of the activity levels and localization of dipeptidyl peptidase IV in normal and tumor human lung cells

Dipeptidyl peptidase IV (DPPIV) was studied in three human lung cells - P (fetal lung-derived cells), A549 (lung adenocarcinoma) and SK-MES-1 (squamous cell carcinoma) using a fluorescent cytochemical procedure developed on the basis of the substrate 4-(glycyl-l-prolyl hydrazido)-N-hexyl-1,8-naphthalimide. The observed differences in the enzyme expression were confirmed by measuring the enzyme hydrolysis of glycyl-l-prolyl-para-nitroanilide. The surface and total dipeptidyl peptidase activities of P cells were correspondingly 7-8 and 3-10 times higher than those of SK-MES-1 and A549 cells. The ratio surface per total activity showed that in P (95%) and A549 (93%) cells the enzyme is associated with the plasmalemma while in SK-MES-1 cells (35%) it is bound to intracellular membranes. In order to compare the results from cell cultures with those in human tumor, the enzyme activity was investigated in cryo-sections of three cases of diagnosed squamous lung carcinoma. DPPIV activity was restricted to the connective tissue stroma surrounding the DPPIV-negative tumor foci.     

Comments about Joint Modeling of Cluster Size and Binary and Continuous Subunit-Specific Outcomes

In longitudinal studies and in clustered situations often binary and continuous response variables are observed and need to be modeled together. In a recent publication Dunson, Chen, and Harry (2003, Biometrics 59, 521-530) (DCH) propose a Bayesian approach for joint modeling of cluster size and binary and continuous subunit-specific outcomes and illustrate this approach with a developmental toxicity data example. In this note we demonstrate how standard software (PROC NLMIXED in SAS) can be used to obtain maximum likelihood estimates in an alternative parameterization of the model with a single cluster-level factor considered by DCH for that example. We also suggest that a more general model with additional cluster-level random effects provides a better fit to the data set. An apparent discrepancy between the estimates obtained by DCH and the estimates obtained earlier by Catalano and Ryan (1992, Journal of the American Statistical Association 87, 651-658) is also resolved. The issue of bias in inferences concerning the dose effect when cluster size is ignored is discussed. The maximum-likelihood approach considered herein is applicable to general situations with multiple clustered or longitudinally measured outcomes of different type and does not require prior specification and extensive programming.     

Effect of halothane on lung carcinoma cells A 549

The halogenated hydrocarbons, such as halothane, are widely used as anesthetics in clinical practice; however their application is often accompanied with metabolic, cardiovascular and respiratory complications. One of the possible factors for this negative outcome might be the severe toxicity of these agents. In this paper, we investigate in vitro effects of halothane on human lung carcinoma A 549 cells, namely on their cytotoxicity, adhesive properties and metabolic activity. The cytotoxicity response of lung carcinoma A 549 cells to halothane was determined by lactate dehydrogenase (LDH) assay (for cytotoxicity), by detachment assay after adhesion to type IV collagen (for cell adhesive properties) and by surface tension measurements of culture medium (for cell metabolic activity). Regarding the cytotoxicity, the determined maximal non-toxic concentration of halothane on A 549 cells, given here as volume percentages (vol.%) was 0.7 vol.% expressed as aqueous concentration in the culture medium. Direct measurement of the actual halothane concentration in the culture medium showed that 0.7 vol.% corresponds to 1.05 mM and 5.25 aqueous-phase minimum alveolar concentration (MAC). Concentrations equal or higher than 1.4 vol.% (2.1 mM; 10.5 MAC) of halothane provoked complete detachment (cell death), or reduction of initial adhesion to collagen IV in half of the cell population. Surfactant production of A 549 cells, registered up to 48 h after halothane treatment, was inhibited by halothane concentrations as low as 0.6 vol.% (0.9 mM; 4.5 MAC). Our results demonstrate that sub toxic halothane concentrations of 0.6 vol.% inhibits surfactant production; concentrations in the range 0.8-1.4 vol.% induce membrane damages and concentrations equal and higher than 1.4 vol.%--cell death of approximately 50% of the cells.     

Characterization of model bile using fluorescence energy transfer from dehydroergosterol to dansylated lecithin

Fluorescence energy transfer from dehydroergosterol (DHE) to dansylated lecithin (DL) was used to characterize lecithin-cholesterol vesicles in the presence of the bile salt, sodium taurocholate. At lipid concentrations approximating physiological levels, exposure of fluorescently labeled vesicles to the bile salt led to a dose-dependent increase in the DHE-to-DL fluorescence ratio during the first 24 h after mixing. The initial changes in the fluorescence ratio correlated well with conventional turbidity measurements that quantify partial micellization of vesicles as a function of bile salt loading. In addition, fluorescence energy transfer from DHE to DL revealed cholesterol enrichment of vesicles and re-vesiculation of micelles at bile salt loadings for which vesicles and micelles coexisted. Samples containing the cholesterol-enriched vesicle fraction exhibited further increases in the DHE-to-DL fluorescence ratio during a 4-week observation period but only after a significant lag period of several days. The lag period decreased with cholesterol loading, and the increase in the fluorescence ratio always preceded the appearance of microscopic, birefringent, either needlelike or platelike, cholesterol crystals, in samples that were initially supersaturated with cholesterol. Cholesterol crystals were not observed, and the fluorescence ratio did not increase, for any sample that was undersaturated with cholesterol.Taken together, these results suggest that the latter changes in fluorescence are the result of cholesterol nucleation. Fluorescence energy transfer from DHE to DL is therefore a promising technique for the characterization of model bile and, possibly, provides a direct measurement of cholesterol nucleation.     

Proteins with High Turnover Rate in Barley Leaves Estimated by Proteome Analysis Combined with in Planta Isotope Labeling

Protein turnover is a key component in cellular homeostasis; however, there is little quantitative information on degradation kinetics for individual plant proteins. We have used ¹⁵N labeling of barley (Hordeum vulgare) plants and gas chromatography-mass spectrometry analysis of free amino acids and liquid chromatography-mass spectrometry analysis of proteins to track the enrichment of ¹⁵N into the amino acid pools in barley leaves and then into tryptic peptides derived from newly synthesized proteins. Using information on the rate of growth of barley leaves combined with the rate of degradation of ¹⁴N-labeled proteins, we calculate the turnover rates of 508 different proteins in barley and show that they vary by more than 100-fold. There was approximately a 9-h lag from label application until ¹⁵N incorporation could be reliably quantified in extracted peptides. Using this information and assuming constant translation rates for proteins during the time course, we were able to quantify degradation rates for several proteins that exhibit half-lives on the order of hours. Our workflow, involving a stringent series of mass spectrometry filtering steps, demonstrates that ¹⁵N labeling can be used for large-scale liquid chromatography-mass spectrometry studies of protein turnover in plants. We identify a series of abundant proteins in photosynthesis, photorespiration, and specific subunits of chlorophyll biosynthesis that turn over significantly more rapidly than the average protein involved in these processes. We also highlight a series of proteins that turn over as rapidly as the well-known D1 subunit of photosystem II. While these proteins need further verification for rapid degradation in vivo, they cluster in chlorophyll and thiamine biosynthesis.New proteins need to be synthesized while others must be degraded so that plants can respond to the daily requirements of cellular maintenance while also allowing progression through different developmental stages. Ubiquitinated proteins are targeted to the proteasome, which is responsible for a large part of the protein degradation process in both plants and animals (Vierstra, 2009). Its action is supplemented by vacuolar protein degradation during autophagy (Araújo et al., 2011) as well as compartment-specific ATP-dependent metalloproteases (Janska et al., 2013). The plant cell proteome differs most markedly from other eukaryotic cells by the abundance of chloroplasts, which can account for up to three-fourths of soluble protein in green plant tissues (Huber et al., 1976). Besides capturing energy from sunlight into fixed carbon, chloroplasts are also responsible for vitamin and cofactor generation as well as other anabolic processes needed to synthesize amino acids, lipids, and pigments (Rolland et al., 2012). Determining the turnover characteristics of chloroplast proteins relative to those in other parts of the plant cell will be important for understanding how metabolic functions are coordinated across plant organelles.Over the life cycle of plants, the turnover of the chloroplast proteome is critical on several occasions. First, plastid protein degradation and the cytosolic ubiquitination system are known to be critical factors in deetiolation, the process of converting etioplasts to chloroplasts (Ling et al., 2012). Second, under carbon starvation induced by leaf shading or darkness, the catabolism of plastid proteins via autophagy is important in selectively degrading some proteins and tissues in order to sustain others (Araújo et al., 2011). Third, amino acids derived from degradation of the photosynthetic machinery are the primary source of nitrogen during the seed-filling process (Masclaux et al., 2000); therefore, plastid degeneration during natural senescence is central to the yield potential of crops. The protein content of chloroplasts is also modified on a shorter time scale in order to respond to environmental perturbations, to account for changing metabolic processes in diurnal cycles, and to renew the machinery required for the steady-state operations of the organelle. As a result, turnover of plastid proteins contributes to the significant energetic cost of protein synthesis required for cell maintenance (Penning De Vries, 1975). The most studied protein that exhibits fast turnover in plants is the D1 subunit protein of PSII in chloroplasts. The half-life of D1 is inversely correlated with light intensity, and environmental stress conditions are also associated with faster degradation of D1 (Aro et al., 1993; Marutani et al., 2012; Mittal et al., 2012). Plants generate their circadian rhythms by regulating gene expression and protein abundance over the diurnal cycle (McWatters and Devlin, 2011), and recently, these pathways have been shown to be interconnected with plastid proteins involved in thiamine metabolism (Bocobza et al., 2013). A deeper understanding of protein turnover dynamics within the plastid will enhance our understanding of plastid maintenance (van Wijk and Baginsky, 2011; Nevo et al., 2012) and also allow researchers to make more informed decisions regarding the energetic costs of genetic engineering strategies in the plastid compartment (Maliga and Bock, 2011).The simultaneous measurement of degradation rate (K_d) and synthesis rate (K_s) for a range of proteins in parallel is a new proteomic tool available to biologists, resulting from improved mass spectrometry (MS) equipment, computational power, and algorithm development. Large data sets have been generated in yeast (Saccharomyces cerevisiae), mammalian cell cultures, as well as intact animals (Price et al., 2010; Cambridge et al., 2011; Schwanhäusser et al., 2011). Proteomic analyses of protein turnover are just beginning to gather momentum in plants. Several studies have assessed the utility of various metabolic labels (²H, ¹³C, and ¹⁵N) for this purpose (Yang et al., 2010; Chen et al., 2011; Li et al., 2012b). More focused reports have refined our understanding of the dynamics in the assembly of mitochondrial electron transport complexes using these tools (Li et al., 2012a, 2013). Most recently, we performed a shotgun study of mitochondrial proteins from Arabidopsis (Arabidopsis thaliana) cell culture, measured K_d values of 224 proteins, and assessed the turnover of several protein complexes by adaptation of this approach to assess larger scale liquid chromatography-tandem mass spectrometry (LC-MS/MS) data sets (Nelson et al., 2013). The important next step for the utility of this approach for plant biologists is to conduct and analyze whole-plant isotope labeling experiments in order to define the turnover of proteins in planta. Application of these techniques to a model species such as Arabidopsis will build upon the large body of information already available for this species (Lamesch et al., 2012). However, because there are significant differences between Arabidopsis and agriculturally relevant crop species, it is important to conduct studies in crops that can generate targets for geneticists and breeders.Here, we assess the utility of ¹⁵N labeling to better understand protein turnover during the steady-state operation of plant leaves, with an emphasis on chloroplast proteins. We have analyzed leaves from barley (Hordeum vulgare), which has been a long-standing model for photosynthesis research, chloroplast biology, and genetic dissection of photosynthesis and photorespiration (Christopher and Mullet, 1994; Leegood et al., 1996; Dal Bosco et al., 2003; Rollins et al., 2013). Barley is also an important cereal crop for a variety of food and feed products (Schulte et al., 2009), and recent genome sequencing has scaffolded most of its expressed genes to a genetic map, opening new opportunities for in-depth proteome analysis of this species and linkage to quantitative trait loci of agronomic importance (Mayer et al., 2012). We have examined ¹⁵N labeling at the amino acid and protein levels and discuss the advantages and limitations of this method. We report K_d values for more than 500 protein groups, with most of these being localized to the chloroplast, and compare turnover rates for proteins across different organelles and functional categories. We observed a series of abundant protein subunits in photosynthesis, photorespiration, and cofactor synthesis pathways that are degraded much more rapidly than the average barley or plastid protein involved in these processes. We also highlight a series of proteins that turn over rapidly that have not previously been described in terms of their degradation kinetics, and we show that they cluster into certain metabolic pathways, specifically branches of chlorophyll and thiamine biosynthesis.