Top-down proteomics reveals novel protein forms expressed in methanosarcina acetivorans

Combining our previous work with current platforms presented here that identified 99 new proteins (Supplemental Table 1), a total of 200 protein identities in M. acetivorans have been confirmed. Of these, five proteins were unannotated, 15 were mispredicted, two exhibited variable removal of the start methionine, and one was a proteolysis fragment from the identical C-terminus of two possible proteins. The incorporation of LC-MS/MS and offline automation speeds up the processing of samples through automation of the top-down process. For five data files that were automatically processed and iteratively searched, 2 h were required to complete analysis. In these, a total of 835 identifications were detected by the search algorithm in this time period. The expanded use of top-down will use both online and offline strategies, with increasingly sophisticated data acquisition strategies tailored for the challenges of top-down fragmentation [[16], [18], [19], [32] and [33]].     

Inverse 15N-metabolic labeling/mass spectrometry for comparative proteomics and rapid identification of protein markers/targets

The inverse labeling/mass spectrometry strategy has been applied to protein metabolic (15)N labeling for gel-free proteomics to achieve the rapid identification of protein markers/targets. Inverse labeling involves culturing both the perturbed (by disease or by a drug treatment) and control samples each in two separate pools of normal and (15)N-enriched culture media such that four pools are produced as opposed to two in a conventional labeling approach. The inverse labeling is then achieved by combining the normal (14)N-control with the (15)N-perturbed sample, and the (15)N-control with the (14)N-perturbed sample. Both mixtures are then proteolyzed and analyzed by mass spectrometry (coupled with on-line or off-line separation). Inverse labeling overcomes difficulties associated with protein metabolic labeling with regard to isotopic peak correlation and data interpretation in the single-experiment approach (due to the non-predictable/variable mass difference). When two data sets from inverse labeling are compared, proteins of differential expression are readily recognized by a characteristic inverse labeling pattern or apparent qualitative mass shifts between the two inverse labeling analyses. MS/MS fragmentation data provide further confirmation and are subsequently used to search protein databases for protein identification. The methodology has been applied successfully to two model systems in this study. Utilizing the inverse labeling strategy, one can use any mass spectrometer of standard unit resolution, and acquire only the minimum, essential data to achieve the rapid and unambiguous identification of differentially expressed protein markers/targets. The strategy permits quick focus on the signals of differentially expressed proteins. It eliminates the detection ambiguities caused by the dynamic range of detection. Finally, inverse labeling enables the detection of covalent changes of proteins responding to a perturbation that one might fail to distinguish with a conventional labeling experiment.     

Moringa oleifera leaves from Brazil: Influence of seasonality,regrowth age and,region in biochemical markers and antioxidant potential

Moringa oleifera leaves are cultivated in warm regions of Brazil for commercial ends due to their nutritional and biological properties. This study evaluated, by chemometric tools, the influence of seasonality (winter, spring, autumn, and summer), growing area in Brazil (South, Southeast, and Northeast), and regrowth age (40 and 80 days) in antioxidant potential, and biochemical markers determined by HPLC-DAD. The results obtained in the present study showed that all the parameters evaluated were affected by seasonality, regrowth age, and growing region. The content of phenolic compounds and antioxidant activity using the ABTS method was higher in samples cultivated in the southeast (59.4 mg GAE g^?1 and 346 µmol TEAC g^?1, respectively). The highest antioxidant activities evaluated by DPPH and FRAP methods were obtained from leaves of the southeast and northeast. In addition, young leaves presented higher antioxidant potential and total phenolic content. The cultivation region significantly influenced the content of chlorogenic acid, isoquercitrin, and astragalin, which ranged from 4.2 to 7.2 mg g^?1, 8.0 to 10.7 mg g^?1, and 2.2 to 3.8 mg g^?1, respectively. In addition, a positive correlation between solar radiation and temperature with caffeic acid, rutin, phenolic compounds, and antioxidant assay from the DPPH method was observed. Additionally, the RGB pattern of the images of these leaves was correlated with the levels of compounds with antioxidant activity. Models generated through machine learning showed good performances, and ABTS and rutin analyses developed the best models with a coefficient of determination above 75 %. Thus, color patterns can be used to measure the antioxidant activity by the ABTS method and to determine the rutin concentration in M. oleifera leaves.     

Variation in phytochemicals and bioactivity of the fruits of Eleutherococcus species cultivated in Poland

Neither the chemical profile and bioactivity of the fruits of Eleutherococcus species cultivated in Poland nor the influence of long-term domestic storage of the fruits are known. No changes in the phenols and flavonoids content in the freshly dried and 1-year storage fruits of Eleutherococcus senticosus and Eleutherococcus henryi were found. The 1-year storage fruits of both species exhibited a very high concentration of Mn, Zn and Cu (mg/kg) compared with the fresh-dried fruits. Spathulenol, β-longipinene and iso-longifolol were predominant essential oil compounds. It was observed that a high capacity of the freshly dried E. senticosus fruits were able to scavenge DPPH^* and inhibit Hyal (EC₅₀ 0.1 mg/mL; IC₅₀ 0.58 mg/mL). Due to their health and pharmacological benefits, a better understanding of the impact of the climate change is important to increase the pharmacological utility of the extracts.     

Synthesis and biological evaluation of novel 99mTcN-labeled bisnitroimidazole complexes containing monoamine-monoamide dithiol as potential tumor hypoxia markers

Tumor hypoxia can decrease the efficacy of clinical therapy due to resistance toward radiation damage and chemotherapy, thus detection of tumor hypoxia by radiolabeled hypoxia markers is important for the control of tumor. Radiopharmaceuticals with two bioreductive groups, such as propylene amine oxime-bisnitroimidazole or monoamine-monoamide dithiol (MAMA) -bisnitroimidazole, have potential to improve hypoxia selectivity. In order to obtain radiopharmaceuticals with better features, we synthesized two novel [^99mTcN]²⁺ complexes with bisnitroimidazole moieties and MAMA ligand for targeting tumor hypoxia. Their physicochemical characters and biodistribution were also investigated. Both the [^99mTcN]²⁺ complexes show good stability and hydrophilicity. They show faster clearance from blood and soft tissues, better tumor retention and favorable tumor-to-tissue ratios compared with a control complex without nitroimidazole group. In addition, both of them show more favorable biodistribution patterns than the corresponding [^99mTcO]³⁺ complexes. These results indicate that the ^99mTcN-labeled MAMA-bisnitroimidazole complexes would have potential to image tumor hypoxia in vivo.     

北京地区有机物种人为源排放量及O3生成潜势估算

在华北地区总VOCs排放清单的基础上,通过调研国、内外VOCs主要人为污染源排放源谱,计算了北京市各行业、各区县88个VOCs物种的排放量,发展了北京市2003年和2006年VOCs物种排放清单,并估算了北京地区VOCs排放的O3生成潜势.结果显示:北京市2003年和2006年人为源向大气排放VOCs总量分别为34.6×104t和55.0×104t,VOCs排放总OFP值分别为131.9×104t和209.5×104t;对北京地区O3生成潜势贡献最大的前10个VOCs物种依次为乙烯、间/对-二甲苯、甲苯、1-丁烯、异戊烷、反-2-丁烯、丙烯、顺-2-丁烯、邻-二甲苯和乙炔,这10个物种的累计排放量占VOCs排放总量的60%左右,但对总OFP值的贡献率接近80%;北京城区是VOCs排放的高值区,同时也是O3生成潜势最大的区域;降低交通排放、溶剂挥发及油品挥发排放是降低北京地区VOCs排放、控制O3生成的有效途径.     

Advances in the analysis of dynamic protein complexes by proteomics and data processing

Signal transduction governs virtually every cellular function of multicellular organisms, and its deregulation leads to a variety of diseases. This intricate network of molecular interactions is mediated by proteins that are assembled into complexes within individual signaling pathways, and their composition and function is often regulated by different post-translational modifications. Proteomic approaches are commonly used to analyze biological complexes and networks, but often lack the specificity to address the dynamic and hence transient nature of the interactions and the influence of the multiple post-translational modifications that govern these processes. Here we review recent developments in proteomic research to address these limitations, and discuss several technologies that have been developed for this purpose. The synergy between these proteomic and computational tools, when applied together with global methods to the analysis of individual proteins, complexes and pathways, may allow researchers to unravel the underlying mechanisms of signaling networks in greater detail than previously possible.     

Genetic aspects of variation of protein amounts in maize and pea

Using high-resolution two-dimensional polyacrylamide gel electrophoresis we studied the polymorphism of protein amounts in some genotypes of maize and pea. This type of variability seems to be rather common and insensitive to environmental conditions, as attested by the comparison of the patterns of two maize lines harvested in two different years. A large-scale experiment involving 5 lines, 7 of their hybrids, and 6 organs (or physiological stages) of maize allowed us to examine numerous polypeptides regarding their genetic variability, their amount differences between organs and the inheritance of their abundance. Genetic and organ variations are not independent: polypeptides whose amount varies from one organ to another are, for the most part, genetically variable (59%), while the stable polypeptides are not often genetically variable (18%). We found a striking organ specificity for (i) the extent of quantitative variability (from 2.3-15.4% of the polypeptides), (ii) the occurrence and the type of variation for a given polypeptide (an intensity difference seen in an organ can disappear or even be reversed in another one), (iii) the kind of inheritance (additive/non-additive): combining the 6 organs and the 7 hybrids we found 101 cases of non-additivity (4% of the total) which concern as many as 72 different spots, that is to say that in most cases a polypeptide displaying nonadditivity in an organ seems to display additivity in the other ones. Moreover, for most of the polypeptides with nonadditive inheritance the hybrid spot presents an intensity similar to that of the most intense parental spot.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of flavonoids in cultivated sugarcane leaves, bagasse, juice and in transgenic sugarcane by liquid chromatography-UV detection

A high-performance liquid chromatography (HPLC) method with photo-diode array (DAD) detection was developed to separate and quantify flavonoids in sugarcane leaves and bagasse (= the crushed sugarcane refuse from juice extraction), and in sugarcane juice. Sugarcane flavonoids consist of a complex mixture of aglycones and glycosides (including flavonolignan glycosides), and the HPLC-UV method herein proposed is suitable for their quantification as total flavonoids. This method was applied to analyze samples of cultivated sugarcane, commercial juice and transgenic sugarcane leaves. Sugarcane leaves proved a promising source of flavonoids: an average of 1.10 mg of total flavonoids/g plant material was found in fresh leaves. Moreover, the flavonoid content of sugarcane juice (0.6 mg/mL) is comparable to other food sources of flavonoids previously reported. Transgenic sugarcane leaves ("Bowman-Birk" and "Kunitz") were compared with non-modified ("control") plant samples using the proposed HPLC-UV method, which indicated that the content of total flavonoids in transgenic plants is different from that in non-modified sugarcane.     

Alleviation of adverse effects of drought stress on growth and some potential physiological attributes in maize (Zea mays L.) by seed electromagnetic treatment

Effects of varying preseed magnetic treatments on growth, chlorophyll pigments, photosynthesis, water relation attributes, fluorescence and levels of osmoprotectants in maize plants were tested under normal and drought stress conditions. Seeds of two maize cultivars were treated with different (T0 [0 mT], T1 [100 mT for 5 min], T2 [100 mT for 10 min], T3 [150 mT for 5 min] and T4 [150 mT for 10 min]) electromagnetic treatments. Drought stress considerably suppressed growth, chlorophyll a and b pigments, leaf water potential, photosynthetic rate (A), stomatal conductance (g(s)) and substomatal CO(2) concentration (C(i)), while it increased leaf glycinebetaine and proline accumulation in both maize cultivars. However, pretreated seeds with different magnetic treatments significantly alleviated the drought-induced adverse effects on growth by improving chlorophyll a, A, E, g(s), C(i) and photochemical quenching and nonphotochemical quenching, while it had no significant effect on other attributes. However, different magnetic treatments negatively affected the g(s) and C(i) particularly in cv. Agaiti-2002 under drought stress conditions. Of all magnetic treatments, 100 and 150 mT for 10 min were most effective in alleviating the drought-induced adverse effects. Overall, preseed electromagnetic treatments could be used to minimize the drought-induced adverse effects on different crop plants.     

Biolabel-led research pattern reveals serum profile in rats after treatment with Herba Lysimachiae: Combined analysis of metabonomics and proteomics

In traditional Chinese medicine, Herba Lysimachiae (HL) is mainly used to treat rheumatic arthralgia. Current pharmacological studies also showed that HL has therapeutic potential for synovial diseases. HL is an oral drug, whose compounds need to enter the blood circulation before reaching the injured tissue, thus potentially causing activity or toxicity to the blood system. In this study, the biolabel-led research pattern was used to analyze the serum profile after HL intervention, based on which the safety and efficacy of HL were explored. Metabonomics and proteomics were combined to analyze the biolabels responsible for the interventions of HL in serum. Bioinformatics databases were used to screen for the material basis that may interfere with biolabels. Omics analysis showed that differentially expressed proteins (19) and metabolites (5) were identified and considered as the potential biolabels, which were involved in 8 biochemical processes (platelet activation and aggregation, blood glucose release, immune and inflammatory regulation, oxidative stress, endoplasmic reticulum stress, tumor progression, blood pressure regulation, and uric acid synthesis). Thirty-one compounds may be the material basis to interfere with 11 biolabels. The present research reveals that the potential activities and toxicities of HL can be explored based on the biolabel-led research pattern.     

LC-ESI-MS/MS profiling of phenolics in the leaves of Eleutherococcus senticosus cultivated in the West Europe and anti-hyaluronidase and anti-acetylcholinestarase activities

Neither secondary metabolites of the spring leaves nor the autumn leaves of Eleutherococcus senticosus species cultivated in Poland, or the bioactivity are known. The richest in polyphenols was the autumn leaves (171.1 mg/g DE), while in flavonoids the spring leaves (107.9 mg/g DE). Using LC-ESI-MS/MS, protocatechuic acid has been identified as the most abundant compound in the spring and autumn leaves (200 and 70 μg/g DE, respectively). Amongst flavonoids, naringenin 7-O-glucoside occurred in the largest amount (20 and 10 mg/g DE in the spring and autumn leaves, respectively). The autumn leaves inhibited Hyal the strongest (74.3%), comparing to the spring leaves (33%). A weak inhibition was found towards AChE (0.64 and 5.8% for the autumn and spring leaves, respectively). To our best knowledge, no information was available on the phytochemical composition and activity of the leaves of E. senticosus cultivated in Poland.     

Synthesis and in vitro antimycobacterial potential of novel hydrazones of eugenol

Fifty one hydrazone derivatives of eugenol were designed and docked with 2NSD and 2X22 (enzymes of H37Rv strain) using Schrodinger v7.4. The selective ten hydrazone derivatives (4, 5, 11, 18, 30, 34, 35, 37, 42, and 45) of eugenol were synthesized via esterification, hydrazination and treatment with different aldehydes. Synthesized compounds were characterized by IR, ¹H NMR, and LCMS data. The compounds were evaluated for their antitubercular potential against H37Rv using microplate alamar blue assay (MABA). The study revealed that all synthesized compounds were significantly active at concentration 50 and 100 μg/ml, whereas compound 11 exhibited activity at 25 μg/ml. Present study showed that antitubercular activity of novel hydrazone derivatives of eugenol is strongly connected with the position of the substituent on aromatic aldehyde or ketones.     

Chromatographic investigations of flavonoid compounds in the leaves and flowers of some species of the genusAlthaea

Summary The flavonoids present in the leaves and flowers ofAlthaea armeniaca Ten., A. cannabina L., A. narbonesis Pourr. andA. broussonetiifolia Iljin were investigated and compared to the flavonoids present in the leaves and flowers ofA. officinalis L. The inliquid chromatography and two-dimensional paper chromatography. The same flavonoids were found in the flowers of all the investigated species while differences could be noted in the flavonoid composition of the leaves. Both the qualitative and quantitative difference was the greatest in the flavonoids of the leaves ofA. cannabina L.     

Influence of mass resolution on species matching in accurate mass and retention time (AMT) tag proteomics experiments

Diverse mass spectrometric instruments have been used to provide data for accurate mass and retention time (AMT) tag proteomics analyses, including ion trap, quadrupole time-of-flight, and Fourier transform mass spectrometry (FTMS). An important attribute of these instruments, beside mass accuracy, is their spectral resolution. In fact, the ability to separate peaks with close m/z values is likely to play a major role in enabling species identification and matching in analyses of very complex proteomics samples. In FTMS, resolution is directly proportional to the detection period and can therefore be easily tuned. We took advantage of this feature to investigate the effect of resolution on species identification and matching in an AMT tag experiment. Using an Arabidopsis thaliana chloroplast protein extract as prototypical 'real-life' sample, we have compared the number of detected features, the optimal mass tolerance for species matching, the number of matched species and the false discovery rate obtained at various resolution settings. It appears that while the total number of matches is not significantly affected by a reduction of resolution in the range investigated, the confidence level of identifications significantly drops as evidenced by the estimated false discovery rate.     

Thin-layer chromatography-densitometry and liquid chromatography analysis of alkaloids in leaves of Papaver somniferum under stress conditions

The effect of stress conditions on the concentrations of secondary metabolites were examined during various developmental stages of Papaver somniferum plants. P. somniferum plants were grown in laboratory conditions (Budakalász). The experiment consisted of 22 treatments. Significantly different alkaloid contents can be observed under different stress conditions. In general, the alkaloid contents of plants are very low; therefore, a highly sensitive and reliable method has to be developed for analysis. The amount of alkaloids was measured by 2 separation and detection techniques. Accuracy of the thin-layer chromatography method for quantitative analysis is limited. Without purification of samples the background is too noisy. Column liquid chromatography is a sensitive and relatively inexpensive method that allows precise quantitative determination of the alkaloid content.     

Evaluation of chlorophyll fluorescence and membrane injury in the leaves of barley cultivars under osmotic stress

Two physiological tests for screening drought tolerance of barley (Hordeum vulgare, L.) plants are compared in this work. Water deficit is induced by treating the plants' roots with polyethylene glycol (PEG 8000). The relative water content (RWC) of the plants is used as a measure of the water status. Conductometrically determined electrolyte leakage from the leaf tissue demonstrates the membrane injury caused by dehydration. It is shown that the injury index increases with the decrease of the RWC of the leaves. The F(v)/F(m) ratio is employed to assess changes in the primary photochemical reactions of the photosynthetic apparatus after dehydration. The results suggest that PSII is weakly affected by the imposed osmotic stress. The fluorescence behaviour of the examined cultivars is related to their RWC.