No abstract

HspA, a member of the GroES chaperonin family, is a small protein found in Helicobacter pylori with a unique histidineand cysteine-rich domain at the C terminus. In this work, we overexpressed, purified, and characterized this protein both in vitro and in vivo.

A newly developed nongenetically modified purple tomato V118 was investigated for its phytochemical compositions and antioxidant activities. A highly efficient and sensitive UPLC method was developed for both the phenolics and carotenoids, which showed that in addition to the phytochemicals commonly known for tomatoes, V118 had a unique composition of anthocyanins. The total carotenoid content of V118 was 234.78 μg/g dry weight (DW), and the total phenolic content was 659.11 mg GAE/100 g DW. The antioxidant activities of the lipophilic extract as measured by the PCL and ORAC-L assays were 30.11 μmol TE/g DW and 11.97 μmol TE/g DW, respectively, while the hydrophilic extracts as determined by the ORAC-H and FRAP assays were 323.23 μmol TE/g DW and 54.95 μmol AAE/g DW, respectively. The LC-MS study showed three major anthocyanins, which were mainly acylglycosides of petunidin and malvidin. This study showed that purple tomatoes such as V118 possess additional phytochemicals like anthocyanins, which can potentially have added health benefits.

Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni2+-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni2+-NTA–based probes. Unfortunately, previous Ni-NTA–based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni2+ ions. The probe, driven by Ni2+-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.

SlyD belongs to the FK506-binding protein (FKBP) family with both peptidylprolyl isomerase (PPIase) and chaperone activities, and is considered to be a ubiquitous cytosolic protein-folding facilitator in bacteria. It possesses a histidine- and cysteine-rich C-terminus binding to selected divalent metal ions (e.g., Ni2+, Zn2+), which is important for its involvement in the maturation processes of metalloenzymes. We have determined the solution structure of C-terminus-truncated SlyD from Helicobacter pylori (HpSlyDΔC). HpSlyDΔC folds into two well-separated, orientation-independent domains: the PPIase-active FKBP domain and the chaperone-active insert-in-flap (IF) domain. The FKBP domain consists of a four-stranded antiparallel β-sheet with an α-helix on one side, whereas the IF domain folds into a four-stranded antiparallel β-sheet accompanied by a short α-helix. Intact H. pylori SlyD binds both Ni2+ and Zn2+, with dissociation constants of 2.74 and 3.79 μM respectively. Intriguingly, binding of Ni2+ instead of Zn2+ induces protein conformational changes around the active sites of the FKBP domain, implicating a regulatory role of nickel. The twin-arginine translocation (Tat) signal peptide from the small subunit of [NiFe] hydrogenase (HydA) binds the protein at the IF domain. Nickel binding and the recognition of the Tat signal peptide by the protein suggest that SlyD participates in [NiFe] hydrogenase maturation processes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00775-011-0855-y) contains supplementary material, which is available to authorized users.

Mulberry leaves had been used as material of medicine, drink, and functional foods in many countries, but the variety suitability for different applications is still not clear. In this study, the nutritional and phytochemical components of mulberry tender shoots from 19 varieties in China were investigated. Obvious genotypic diversity was observed in all the assessed components. The contents of crude protein (CP), total soluble sugars (TSS), crude fiber (CF), 1-deoxynojirimycin (DNJ), total phenolic (TP), and total flavonoid content (TF) were 27.63-37.36 g/100 g dry weight (DW), 58.71-150.31 mg/g DW, 9.90-13.85 g/100 g DW, 0.08-1.12 mg/g DW, 8.76-20.26 mg gallic acid equivalents (GAE)/g DW, and 21.36-56.41 mg rutin equivalents (RE)/g DW, respectively. Chlorogenic acid, rutin, isoquercitrin, and astragalin were considered to be mainly potential antioxidant compounds in mulberry leaves. According to the result of correlational analysis, principal component analysis (PCA), and hierarchical cluster analysis (HCA), the NS14 variety had high comprehensive healthy properties. GS8, D10, XS, TP2, and DHS could be appropriate materials for functional food or drink because of high content of phenolics or DNJ. Some varieties may have a potential application as protein-rich vegetables. The study suggests that some variety of mulberry could be selected and utilized rationally for their dietary properties. ARTICLE HISTORY

No abstract

Trihelix transcription factors are characterized by containing a conserved trihelix (helix-loop-helix-loop-helix) domain that binds to GT elements required for light response, and they play roles in light stress and in abiotic stress responses. However, only a few of them have been functionally characterized. In the present study, we characterized the function of AST1 (Arabidopsis SIP1 clade Trihelix1) in response to salt and osmotic stress. AST1 shows transcriptional activation activity, and its expression is induced by osmotic and salt stress. A conserved sequence highly present in the promoters of genes regulated by AST1 was identified, which was bound by AST1, and termed the AGAG-box with the sequence [A/G][G/A][A/T]GAGAG. Additionally, AST1 also binds to some GT motifs including the sequence of GGTAATT, TACAGT, GGTAAAT and GGTAAA, but failed in binding to the sequence of GTTAC and GGTTAA. Chromatin immunoprecipitation combined with quantitative real-time reverse transcription-PCR analysis suggested that AST1 binds to the AGAG-box and/or some GT motifs to regulate the expression of stress tolerance genes, resulting in reduced reactive oxygen species, Na+ accumulation, stomatal apertures, lipid peroxidation, cell death and water loss rate, and increased proline content and reactive oxygen species scavenging capability. These physiological changes affected by AST1 finally improve salt and osmotic tolerance.