Origin and evolution of flavivirus 5'UTRs and panhandles: trans-terminal duplications?

Flavivirus replication is mediated by interactions between complementary ssRNA sequences of the 5'- and 3'-termini that form dsRNA cyclisation stems or panhandles, varying in length, sequence and specific location in the mosquito-borne, tick-borne, non-vectored and non-classified flaviviruses. In this manuscript we manually aligned the flavivirus 5'UTRs and adjacent capsid genes and revealed significantly more homology than has hitherto been identified. Analysis of the alignments revealed that the panhandles represent evolutionary remnants of a long cyclisation domain that probably emerged through duplication of one of the UTR termini.

Quantitative proteomics using uniform 15N-labeling, mascot and the trans-proteomic pipeline

Stable isotope labeling combined with MS is a powerful method for measuring relative protein abundances, for instance, by differential metabolic labeling of some or all amino acids with 14N and 15N in cell culture or hydroponic media. These and most other types of quantitative proteomics experiments using high-throughput technologies, such as LC-MS/MS, generate large amounts of raw MS data. This data needs to be processed efficiently and automatically, from the mass spectrometer to statistically evaluated protein identifications and abundance ratios. This paper describes in detail an approach to the automated analysis of uniformly 14N/15N-labeled proteins using MASCOT peptide identification in conjunction with the trans-proteomic pipeline (TPP) and a few scripts to integrate the analysis workflow. Two large proteomic datasets from uniformly labeled Arabidopsis thaliana were used to illustrate the analysis pipeline. The pipeline can be fully automated and uses only common or freely available software.

Analysing the ability to retain sidechain hydrogen-bonds in mutant proteins

Motivation: Hydrogen bonds are one of the most important inter-atomic interactions in biology. Previous experimental, theoretical and bioinformatics analyses have shown that the hydrogen bonding potential of amino acids is generally satisfied and that buried unsatisfied hydrogen-bond-capable residues are destabilizing. When studying mutant proteins, or introducing mutations to residues involved in hydrogen bonding, one needs to know whether a hydrogen bond can be maintained. Our aim, therefore, was to develop a rapid method to evaluate whether a sidechain can form a hydrogen-bond. Results: A novel knowledge-based approach was developed in which the conformations accessible to the residues involved are taken into account. Residues involved in hydrogen bonds in a set of high resolution crystal structures were analyzed and this analysis is then applied to a given protein. The program was applied to assess mutations in the tumour-suppressor protein, p53. This raised the number of distinct mutations identified as disrupting sidechain-sidechain hydrogen bonding from 181 in our previous analysis to 202 in this analysis.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine (DCVC) resulted in a >1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Role of polyhydroxybutyrate and glycogen as carbon storage compounds in pea and bean bacteroids

Rhizobium leguminosarum synthesizes polyhydroxybutyrate and glycogen as its main carbon storage compounds. To examine the role of these compounds in bacteroid development and in symbiotic efficiency, single and double mutants of R. legumosarum bv. viciae were made which lack polyhydroxybutyrate synthase (phaC), glycogen synthase (glgA), or both. For comparison, a single phaC mutant also was isolated in a bean-nodulating strain of R. leguminosarum bv. phaseoli. In one large glasshouse trial, the growth of pea plants inoculated with the R. leguminosarum bv. viciae phaC mutant were significantly reduced compared with wild-type-inoculated plants. However, in subsequent glasshouse and growth-room studies, the growth of pea plants inoculated with the mutant were similar to wildtype-inoculated plants. Bean plants were unaffected by the loss of polyhydroxybutyrate biosynthesis in bacteroids. Pea plants nodulated by a glycogen synthase mutants or the glgA/phaC double mutant, grew as well as the wild type in growth-room experiments. Light and electron micrographs revealed that pea nodules infected with the glgA mutant accumulated large amounts of starch in the II/III interzone. This suggests that glycogen may be the dominant carbon storage compound in pea bacteroids. Polyhydroxybutyrate was present in bacteria in the infection thread of pea plants but was broken down during bacteroid formation. In nodules infected with a phaC mutant of R. leguminosarum bv. viciae, there was a drop in the amount of starch in the II/III interzone, where bacteroids form. Therefore, we propose a carbon burst hypothesis for bacteroid formation, where polyhydroxybutyrate accumulated by bacteria is degraded to fuel bacteroid differentiation.

Domain swapping in the human histamine H-1 receptor

G-protein-coupled receptors (GPCRs) represent the largest family of receptors involved in transmembrane signaling. Although these receptors were generally believed to be monomeric entities, accumulating evidence supports the presence of GPCRs in multimeric forms. Here, using immunoprecipitation as well as time-resolved fluorescence resonance energy transfer to assess protein-protein interactions in living cells, we unambiguously demonstrate the occurrence of dimerization of the human histamine H-1 receptor. We also show the presence of domain-swapped H-1 receptor dimers in which there is the reciprocal exchange of transmembrane domain TM domains 6 and 7 between the receptors present in the dimer. Mutation of aspartate(107) in transmembrane (TM) 3 or phenylalanine(432) in TM6 to alanine results in two radioligand-binding-deficient mutant H-1 receptors. Coexpression of H-1 D(107)A and H-1 F(432)A, however, results in a reconstituted radioligand binding site that exhibits a pharmacological profile that corresponds to the wildtype H-1 receptor. Interestingly, the H-1 receptor radioligands [H-3] mepyramine and [H-3]-(-)- trans-1-phenyl-3-N, N-dimethylamino-1,2,3,4-tetrahydronaphthalene show differential saturation binding values (B-max) for wild-type H-1 receptors but not for the radioligand binding site that is formed upon coexpression of H-1 D(107)A and H-1 F(432)A receptors, suggesting the presence of different H-1 receptor populations.

Characterization of an escherichia coli elaC deletion mutant

The elaC gene of Escherichia coli encodes a binuclear zinc phosphodiesterase (ZiPD). ZiPD homologs from various species act as 3' tRNA processing endoribonucleases, and although the homologous gene in Bacillus subtilis is essential for viability [EMBO J. 22 (2003) 4534], the physiological function of E. coli ZiPD has remained enigmatic. In order to investigate the function of E. coli ZiPD we generated and characterized an E. coli elaC deletion mutant. Surprisingly, the E. coli elaC deletion mutant was viable and had wild-type like growth properties. Micro array-based transcriptional analysis indicated expression of the E. coli elaC gene at basal levels during aerobic growth. The elaC gene deletion had no effect on the expression of genes coding for RNases or amino-acyl tRNA synthetases or any other gene among a total of > 1300 genes probed. 2D-PAGE analysis showed that the elaC mutation, likewise, had no effect on the proteome. These results strengthen doubts about the involvement of E. coli ZiPD in tRNA maturation and suggest functional diversity within the ZiPD/ElaCl protein family. In addition to these unexpected features of the E. coli elaC deletion mutant, a sequence comparison of ZiPD (ElaCl) proteins revealed specific regions for either enterobacterial or mammalian ZiPD (ElaCl) proteins. (C) 2004 Elsevier Inc. All rights reserved.

Mutant alleles at the rugosus loci in pea affect seed moisture sorption isotherms and the relations between seed longevity and moisture content

Pea (Pisum sativum L.) mutant near-isogenic lines (RRrbrb, rrRbRb, rrrbrb) with lower starch but higher lipid contents, brought about by lesions in the starch biosynthetic pathway, had seed moisture sorption isotherms displaced below that of the wild type (RRRbRb). The negative logarithmic relationship between seed longevity and seed storage moisture content (%, f.wt basis), determined in hermetic storage at 65 °C, also differed: longevity in the mutant near-isogenic lines was poorer and less sensitive to moisture content than in the wild type (i.e. CW was lower). The low-moisture-content limit (mc) to this relation also differed, being lower in the mutant near-isogenic lines (5.4–5.9%) than in the wild type (6.1%). In contrast, all four near-isogenic lines showed no difference (P >0.25) in the negative semi-logarithmic relationship between equilibrium relative humidity (ERH) and seed longevity. It is concluded that the effect of these alleles at the r and rb loci on seed longevity was largely indirect; a consequence of their effect on seed composition and hence on moisture sorption isotherms. However, this explanation could not be invoked at moisture contents below mc where differences in longevity remained substantial (RRRbRb double that of rrrbrb). Hence, these mutant alleles affected seed longevity directly at very low moisture contents.

An in situ time-dependent study of the photodimerisation of chloro-derivatives of trans-cinnamic acid using infrared microspectroscopy with a synchrotron radiation source

The photodimerisation of single crystals of substituted cinnamic acid has been monitored continuously by infrared microscopy using a synchrotron source. The beta-form of 2,4-dichloro-trans-cinnamic acid dimerises under ultraviolet irradiation to form the corresponding beta-truxinic acid derivative in a reaction which follows strictly first order kinetics. By contrast the corresponding reactions in single crystals of beta-2-chloro-trans-cinnamic acid and beta-4-chloro-trans-cinnamic acid deviate somewhat from first order kinetics as a result of solid-state effects. In all three cases the reactions proceed smoothly from monomer to dimer with no hint of any reaction intermediate.

The photodimerisation of the alpha- and beta-forms of trans-cinnamic acid: a study of single crystals by vibrational microspectroscopy

Infrared and Raman microspectroscopy have been used to follow the photodimerisation reactions of single crystals, the alpha- and beta-forms of trans-cinnamic acid. This approach allows the starting materials and products -alpha-truxillic acid that has C-i symmetry and beta-truxinic acid, which has C-s symmetry-to be identified. It also allows the topotactic nature of the reaction to be confirmed. Attempts to produce the poorly-defined unreactive gamma-form of trans-cinnamic acid resulted only in a mixture of the alpha- and beta-forms. The findings suggest a wide role for these spectroscopic methods in monitoring solid-state organic reactions. (C) 2002 Elsevier Science B.V. All rights reserved.

Ultrafast excited state dynamics controlling photochemical isomerization of N-methyl-4-[trans-2-(4-pyridyl)ethenyl]pyridinium coordinated to a {Re-1(CO)(3)(2,2 '-bipyridine)} chromophore

Two Multifunctional photoactive complexes [Re(Cl)(CO)(3)-(MeDpe(+))(2)](2+) and [Re(MeDpe(+))(CO)(3)(bpy)](2+) (MeDpe(+) = N-methyl-4-[trans-2-(4-pyridyl)ethenyl]pyridinium, bpy = 2,2'-bipyridine) were synthesized. characterized. and their redox and photonic properties were investigated by cyclic voltammetry: ultraviolet-visible-infrared (UV/Vis/IR) spectroelectrochemistry, stationary UV/Vis and resonance Raman spectroscopy; photolysis; picosecond time-resolved absorption spectroscopy in the visible and infrared regions: and time-resolved resonance Raman spectroscopy. The first reduction step of either complex Occurs at about -1.1 V versus Fc/Fc(+) and is localized at MeDpe(+). Reduction alone does not induce a trans -> cis isomerization of MeDpe(+). [Re(Cl)(CO)(3)(MeDPe(+))(2)](2+) is photostable, while [Re(MeDpe(+))(CO)(3)(bpy)](2+) and free MeDpe(+) isomerize under near-UV irradiation. The lowest excited state of [Re(Cl)(CO)(3)(MeDPe(+))(2)](2+) has been identified as the Re(Cl)(CO)(3) -> MeDpe(+) (MLCT)-M-3 (MLCT = metal-to-ligand charge transfer), decaying directly to the ground state with lifetimes of approximate to 42 (73%) and approximate to 430ps (27%). Optical excitation of [Re(MeDpe(+))(CO)(3)(bpy)](2+) leads to population of Re(CO)(3) -> MeDpe(+) and Re(CO)(3) -> bpy (MLCT)-M-3 states, from which a MeDpe(+) localized intraligand 3 pi pi* excited state ((IL)-I-3) is populated with lifetimes of approximate to 0.6 and approximate to 10 ps, respectively. The 3IL state undergoes a approximate to 21 ps internal rotation, which eventually produces the cis isomer on a much longer timescale. The different excited-state behavior of the two complexes and the absence of thermodynamically favorable interligand electron transfer in excited [Re(MeDpe(+))(CO)(3)(bpy)](2+) reflect the fine energetic balance between excited states of different orbital origin, which can be tuned by subtle Structural variations. The complex [Re(MeDpe+)(CO)(3)(bpy)](2+) emerges as a prototypical, multifunctional species with complementary redox and photonic behavior.