Novel Ru(II) oximato complexes with silent oxygen atom: Synthesis,chemistry and biological activities

A series of octahedral complexes, [Ru(CO)(EPh₃)₂(bhmh)] (E = P or As; H₂bhmh = benzoic acid (2-hydroxyimino-1-methyl-propylidene)-hydrazide), [Ru(CO)(EPh₃)₂(ihmh)] (H₂ihmh = isonicotinic acid (2-hydroxyimino-1-methyl-propylidene)-hydrazide), [Ru(CO)(EPh₃)₂(hhmh)] (H₂hhmh = 2-hydroxy-benzoic acid (2-hydroxyimino-1-methyl-propylidene)-hydrazide) have been prepared by a facile procedure. X-ray structure determination of three of the complexes revealed that the hydrazone ligand coordinates through the imine and the oxime nitrogen and the amide oxygen atoms. In all the complexes, the N–OH moiety of the oxime is deprotonated to give an N–O^? species and this oxygen atom did not coordinate to the central metal atom. The oxidation–reduction processes for each of these complexes have been determined in CH₃CN by cyclic voltammetry. The complexes displayed two oxidation couples and one irreversible reduction response between +1.6 and ?1.6 V. The trend in the half wave potentials reflects the electronic nature of the hydrazone ligand. Antibacterial activity of the ligands and the complexes has been evaluated against five pathogenic bacteria. The binding of the complexes with herring sperm DNA has also been investigated by UV–Vis spectroscopy and cyclic voltammetry.     

Temporal Analyses of Barley Malting Stages Using Shotgun Proteomics

Malt derived from barley malting is an essential raw material for beer brewing. In this study, we performed the first dynamic proteome survey during barley malting using a gel‐free proteomics approach. This entailed in‐solution tryptic digestion of precipitated proteins and analysis of peptides by nanoliquid chromatography coupled with tandem mass spectrometry. A total of 1418 proteins were identified from the five malting stages: Steep, 1, 3, 5 days after germination, and end of Kiln. About 900 proteins identified in this analysis were uncharacterized or predicted proteins. Integrating information from Uniprot90, Uniprot50, Pfam, Interpro databases and gene ontologies from EnsemblPlants, 796 of the predicted and uncharacterized proteins were provided functional annotations. Nearly 63% of the identified proteins were present during all the five time points suggesting a coordinated activation of major metabolic pathways during malting. GO enrichment analysis showed over‐representation of proteins associated with translation, carbohydrate metabolism, and stress response. Analysis of variance of the spectral counts of proteins present in all the five malting stages identified 205 proteins with significant differences in their abundance. Proteins associated with carbohydrate metabolism especially enzyme activity regulation provide novel targets for malting barley breeding and for predicting malting quality.     

Naf1alpha is phosphorylated in mitotic phase and required to protect cells against apoptosis

Naf1α is an HIV Nef-associated factor expressed ubiquitously in human cells. Previously, we reported that Naf1α is phosphorylated with EGF through MEK/ERK2 pathway. In this study, we found an additional phosphorylation of Naf1α when cells are in mitotic phase (M phase) or arrested in M phase with anti-mitosis reagents, and disappeared when the cells exit from mitotic phase to G1 phase. Furthermore, we demonstrated that Naf1α plays an important role in preventing cells from apoptosis: over-expression of Naf1α in Saos-2 cells suppressed trichostatin A (TSA)-induced apoptosis either of random culture or of cell population synchronized in M phase. In addition, knock-down of Naf1α expression with small interfering RNA sensitized Saos-2 cells to TSA-induced apoptosis. Physiological significance of these findings is discussed in relation to protection of cells from the apoptosis induction.     

Nuclear export of simian immunodeficiency virus Vpx protein

Lentiviruses, human immunodeficiency viruses (HIVs), and simian immunodeficiency viruses (SIVs) are distinguished from oncoretroviruses by their ability to infect nondividing cells such as macrophages. Retroviruses must gain access to the host cell nucleus for replication and propagation. HIV and SIV preintegration complexes (PIC) enter nuclei after traversing the central aqueous channel of the limiting nuclear pore complex without membrane breakdown. Among the nucleophilic proteins, namely, matrix, integrase, Vpx, and Vpr, present in HIV type 2/SIV PIC, Vpx is implicated in nuclear targeting and is also available for incorporation into budding virions at the plasma membrane. The mechanisms of these two opposite functions are not known. We demonstrate that Vpx is a nucleocytoplasmic shuttling protein and contains two novel noncanonical nuclear import signals and a leptomycin B-sensitive nuclear export signal. In addition, Vpx interacts with the cellular tyrosine kinase Fyn through its C-terminal proline-rich motif. Furthermore, our data indicate that Fyn kinase phosphorylates Vpx and regulates its export from nucleus. Replacement of conserved tryptophan residues within domain 41 to 63 and tyrosine residues at positions 66, 69, and 71 in Vpx impairs its nuclear export, virion incorporation, and SIV replication in macrophages. Nuclear export is essential to ensure the availability of Vpx in the cytoplasm for incorporation into virions, leading to efficient viral replication within nondividing cells.     

Functional role of the "ionic lock"--an interhelical hydrogen-bond network in family A heptahelical receptors

Activation of family A G-protein-coupled receptors involves a rearrangement of a conserved interhelical cytoplasmic hydrogen bond network between the E(D)RY motif on transmembrane helix 3 (H3) and residues on H6, which is commonly termed the cytoplasmic “ionic lock.” Glu134^3.49 of the E(D)RY motif also forms an intrahelical salt bridge with neighboring Arg135^3.50 in the dark-state crystal structure of rhodopsin. We examined the roles of Glu134^3.49 and Arg135^3.50 on H3 and Glu247^6.30 and Glu249^6.32 on H6 on the activation of rhodopsin using Fourier transform infrared spectroscopy of wild-type and mutant pigments reconstituted into lipid membranes. Activation of rhodopsin is pH-dependent with proton uptake during the transition from the inactive Meta I to the active Meta II state. Glu134^3.49 of the ERY motif is identified as the proton-accepting group, using the Fourier transform infrared protonation signature and the absence of a pH dependence of activation in the E134Q mutant. Neutralization of Arg135^3.50 similarly leads to pH-independent receptor activation, but with structural alterations in the Meta II state. Neutralization of Glu247^6.30 and Glu249^6.32 on H6, which are involved in interhelical interactions with H3 and H7, respectively, led to a shift toward Meta II in the E247Q and E249Q mutants while retaining the pH sensitivity of the equilibrium. Disruption of the interhelical interaction of Glu247^6.30 and Glu249^6.32 on H6 with H3 and H7 plays its role during receptor activation, but neutralization of the intrahelical salt bridge between Glu134^3.49 and Arg135^3.50 is considerably more critical for shifting the photoproduct equilibrium to the active conformation. These conclusions are discussed in the context of recent structural data of the β₂-adrenergic receptor.     

Variation in melanin content and composition in type V and VI photoexposed and photoprotected human skin: the dominant role of DHI

A combination of techniques, including high-performance liquid chromatography (HPLC), spectrophotometric measurements, and a novel method for quantifying melanosome morphology, were applied to the analysis of melanin content and composition in highly pigmented (Fitzpatrick type V and VI) human skin. We found that total epidermal melanin content is significantly elevated in photoexposed type V and VI skin (approximately 1.6 x), while analysis of individual melanin components suggests that pheomelanin content increases only slightly, whereas 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-eumelanin and to a greater extent 5,6-dihydroxyindole (DHI)-eumelanin content are both markedly elevated. Analysis of the relative composition of epidermal melanin in these subjects revealed that DHI-eumelanin is the largest single component (approximately 60-70%), followed by DHICA-eumelanin (25-35%), with pheomelanin being a relatively minor component (2-8%). Moreover, there was a comparative enrichment of DHI-eumelanin at photoexposed sites, with a corresponding decline in the relative contributions from DHICA-eumelanin and pheomelanin. There was also a good correlation and close agreement between the concentration of spheroidal melanosomes determined by morphological image analysis and the concentration of pheomelanin determined by a combination of HPLC and spectrophotometric analysis (r = 0.89, P < 0.02). This study demonstrates the usefulness of melanosome morphology analysis as a sensitive new method for the quantification of melanin composition in human skin. The data also suggest that DHI-eumelanin formation is the dominant pathway for melanin synthesis in heavily pigmented (Fitzpatrick V and VI) skin types in vivo, and is the favoured pathway when melanin production is increased in chronically photoexposed skin.     

A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana

Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation (Ds) transposon lines with insertions on all 5 Arabidopsis chromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsis genome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsis lines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis.