Q-TOF LC-MS compounds evaluation of propolis extract derived from Malaysian stingless bees,Tetrigona apicalis,and their bioactivities in breast cancer cell,MCF7

Propolis is known to exhibit various phytochemical compounds that aid in several biological activities. The current study investigates the phytochemical compounds of ethanolic extract of propolis of Tetrigona apicalis (EEP) using Q-TOF LC-MS, its antioxidant properties using DPPH and ABTS⁺ radical scavenging assays, total phenolic (TPC) and flavonoid content (TFC), using Folin-Ciocalteu and Aluminium Chloride method, respectively, as well as proapoptotic effects, based on the selected IC₅₀ of the cytotoxic study conducted for EEP using annexin V-FITC assay. Terpene and polyphenol were among of 17 identified compounds. The EC₅₀ of EEP for DPPH and ABTS⁺ was 1.78 mg/mL and 1.68 mg/mL, while the EEP exhibited TPC and TFC values of 31.99 mgGAE/g and 66.4 mgQCE/g, respectively in which the parameters were strongly correlated. The IC₅₀ of EEP effectively induces apoptosis in MCF7 cells. In conclusion, EEP possessed important phytochemical compounds that work excellently as antioxidants and anticancer agents.     

Actin protein up-regulated upon infection and development of the filarial parasite, Wuchereria bancrofti (Spirurida: Onchocercidae), in the vector mosquito, Culex quinquefasciatus (Diptera: Culicidae)

Detection and identification of humoral proteins, which are up-regulated in Culex quinquefasciatus upon infection by Wuchereria bancrofti, is important in tracing out the biochemical consequences of the filarial parasite development in the vector mosquito. Analysis of the haemolymph of infected mosquitoes through SDS-PAGE and RP-HPLC showed up-regulation of five proteins of molecular weights 40, 66, 22, 14, and 7-kDa. Among these, only the 40-kDa was unknown and the others were comparable with those already reported as transferrin, attacin, lysozyme, and defensin, respectively. In the present study, the 40-kDa protein up-regulated upon infection was identified as actin through nano-LC-MS/MS analysis. Actin is known to be one of the cytoskeletal proteins up-regulated in the haemolymph, as part of the innate immune system, of Escherichia coli challenged Drosophila melanogaster larvae. For the first time, we have observed an increased level of actin in the haemolymph of W. bancrofti-infected Cx. quinquefasciatus. However, the exact mechanism of actin involvement in the immune system of this mosquito is yet to be studied.     

Type A-trichothecenes — Quantitative analysis using LC-MS and occurrence in Austrian maize and oats

During this study a method was developed for the quantitative determination of diacetoxyscirpenol (DAS), T-2 toxin and HT-2 toxin using deuterated T-2 toxin (T-2 d3 toxin) as an internal standard. The described method involves a clean up step of maize extract by the use of Mycosep® 227 columns a chromatographic separation on a Zorbax® bonus-RP-column (2,1×150mm) and the detection and quantification step on a mass spectrometer in SIM (Selected Ion Monitoring) mode.Data on the occurrence of three type A trichothecenes in Austrian maize, maize silage and oats were collected. It could be shown that maize and silage samples harvested in 2002 were only contaminated to a small extent with T-2 toxin (8% of the maize, 0% of the silage) and with HT-2 toxin (30% of the maize, 18% of the silage). But most of the analysed oats samples showed significant levels of T-2 toxin (64%) or HT-2 toxin (82%).     

Juvenile hormone titres and winged offspring production do not correlate in the pea aphid, Acyrthosiphon pisum

Pea aphids, Acyrthosiphon pisum, reproduce parthenogenetically and are wing-dimorphic such that offspring can develop into winged (alate) or unwinged (apterous) adults. Alate induction is maternal and offspring phenotype is entirely determined by changes in the physiology and environment of the mother. Juvenile hormones (JHs) have been implicated in playing a role in wing differentiation in aphids, however until recently, methods were not available to accurately quantify these insect hormones in small insects such as aphids. Using a novel LC-MS approach we were able to quantify JH III in pea aphids that were either producing a high proportion of winged morphs among their offspring or mainly unwinged offspring. We measured JH III titres by pooling the hemolymph of 12 or fewer individuals (1 μL hemolymph) treated identically. Levels of JH ranged from 30 to 163 pg/μL. While aphids in the two treatments strongly differed in the proportion of winged morphs among their offspring, their JH III titres did not differ significantly. There was also no correlation between JH III titre and the proportion of winged offspring in induced aphids. This supports earlier findings that wing dimorphism in aphids may be regulated by other physiological mechanisms.     

Structural basis for the substrate specificity of bone morphogenetic protein 1/tolloid-like metalloproteases

Procollagen C-peptidase, also known as bone morphogenetic protein 1 (BMP-1), is a multidomain, zinc endopeptidase of the astacin M12A family. BMP-1 is the prototype of a small group of proteases that have key roles in extracellular matrix formation and morphogenesis. BMP-1, its splice form mTLD, and the related proteases TLL-1 and TLL-2 are considered as promising drug targets for the treatment of excessive fibrosis and muscle wasting. We report here the crystal structures of the protease domains of human BMP-1 and the closely related Tolloid-like protease 1 (TLL-1). The crystal structures reveal an unexpected conformation of a cysteine-rich loop within the active site, and suggest that a flap movement is required in order to allow substrate binding. On the basis of these substantial differences between the BMP-1 and astacin active sites, a structural basis for their differing substrate specificities is proposed.     

Hydrogen-exchange mass spectrometry reveals activation-induced changes in the conformational mobility of p38alpha MAP kinase

Hydrogen-deuterium exchange measurements represent a powerful approach to investigating changes in conformation and conformational mobility in proteins. Here, we examine p38α MAP kinase (MAPK) by hydrogen-exchange (HX) mass spectrometry to determine whether changes in conformational mobility may be induced by kinase phosphorylation and activation. Factors influencing sequence coverage in the HX mass spectrometry experiment, which show that varying sampling depths, instruments, and peptide search strategies yield the highest coverage of exchangeable amides, are examined. Patterns of regional deuteration in p38α are consistent with tertiary structure and similar to deuteration patterns previously determined for extracellular-signal-regulated kinase (ERK) 2, indicating that MAPKs are conserved with respect to the extent of local amide HX. Activation of p38α alters HX in five regions, which are interpreted by comparing X-ray structures of unphosphorylated p38α and X-ray structures of phosphorylated p38γ. Conformational differences account for altered HX within the activation lip, the P + 1 site, and the active site. In contrast, HX alterations are ascribed to activation-induced effects on conformational mobility, within substrate-docking sites (αF-αG, β7-β8), the C-terminal core (αE), and the N-terminal core region (β4-β5, αL16, αC). Activation also decreases HX in a 3-10 helix at the C-terminal extension of p38α. Although this helix in ERK2 forms a dimerization interface that becomes protected from HX upon activation, analytical ultracentrifugation shows that this does not occur in p38α because both unphosphorylated and diphosphorylated forms are monomeric. Finally, HX patterns in monophosphorylated p38α are similar to those in unphosphorylated kinase, indicating that the major activation lip remodeling events occur only after diphosphorylation. Importantly, patterns of activation-induced HX show differences between p38α and ERK2 despite their similarities in overall deuteration, suggesting that although MAPKs are closely related with respect to primary sequence and tertiary structure, they have distinct mechanisms for dynamic control of enzyme function.     

Proteomic analysis of the response to high-salinity stress in <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Physcomitrella patens is well known because of its importance in the study of plant systematics and evolution. The tolerance of P. patens for high-salinity environments also makes it an ideal candidate for studying the molecular mechanisms by which plants respond to salinity stresses. We measured changes in the proteome of P. patens gametophores that were exposed to high-salinity (250, 300, and 350 mM NaCl) using two-dimensional gel electrophoresis (2-DE) via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sixty-five protein spots were significantly altered by exposure to the high-salinity environment. Among them, 16 protein spots were down-regulated and 49 protein spots were up-regulated. These proteins were associated with a variety of functions, including energy and material metabolism, protein synthesis and degradation, cell defense, cell growth/division, transport, signal transduction, and transposons. Specifically, the up-regulated proteins were primarily involved in defense, protein folding, and ionic homeostasis. In summary, we outline several novel insights into the response of P. patens to high-salinity; (1) HSP70 is likely to play a significant role in protecting proteins from denaturation and degradation during salinity stress, (2) signaling proteins, such as 14-3-3 and phototropin, may work cooperatively to regulate plasma membrane H(+)-ATPase and maintain ion homeostasis, (3) an increase in photosynthetic activity may contribute to salinity tolerance, and (4) ROS scavengers were up-regulated suggesting that the antioxidative system may play a crucial role in protecting cells from oxidative damage following exposure to salinity stress in P. patens.