Current-voltage studies on the thylakoid membrane in the presence of ionophores

The reversibility of the binding of ionophores to the thylakoid membrane is studied. While gramicidin binds practically irreversibly, valinomycin and nonactin bind reversibly, however, only a small fraction (about 1 %) of the membrane-bound valinomycin or nonactin is active in ion transport. The current-voltage relationship is evaluated under these circumstances. We have found that it is practically linear. This together with the relationship between current and ion concentration agrees qualitatively with the results reported for bimolecular lipid membranes, which contain a large fraction of negatively charged lipids. For the ionophores, valinomycin and nonactin, the binding equilibria (K ≈ 10⁴) and the turnover numbers (≈ 3 · 10⁴/s) are evaluated for their action on the thylakoid membrane. Possible reasons for the inactivity of the majority of membrane-bound ionophore molecules are discussed.     

Relationships between platelet and plasma monoamine oxidase,plasma dopamine-B-hydroxylase,and urinary 3-methoxy-4-hydroxy phenylglycol

The activity of dopamine-B-hydroxylase in blood has recently been demonstrated to be under genetic control and to correlate closely with urinary catecholamine excretion. The results of the present study did not demonstrate any relationship between a major catecholamine metabolite in urine, 3-methoxy-4-hydroxy phenylglycol, and dopamine-B-hydroxylase activity in plasma, monoamine oxidase activity in platelets, or monoamine oxidase activity in plasma. Differences in the origin of urinary catecholamines and urinary 3-methoxy-4-hydroxy phenyglycol may be responsible for these divergent results.     

Functional analysis of early secreted antigenic target-6, the dominant T-cell antigen of Mycobacterium tuberculosis, reveals key residues involved in secretion, complex formation, virulence, and immunogenicity

Proteins of the 6-kDa early secreted antigenic target (ESAT-6) secretion system-1 of Mycobacterium tuberculosis are not only strongly involved in the anti-mycobacterial Th1-host immune response but are also key players for virulence. In this study, protein engineering together with bioinformatic, immunological, and virulence analyses allowed us to pinpoint regions of the ESAT-6 molecule that are critical for its biological activity in M. tuberculosis. Mutation of the Trp-Xaa-Gly motif, conserved in a wide variety of ESAT-6-like proteins, abolished complex formation with the partner protein CFP-10, induction of specific T-cell responses, and virulence. Replacement of conserved Leu residues interfered with secretion, coiled-coil formation, and virulence, whereas certain mutations at the extreme C terminus did not affect secretion but caused attenuation, possibly because of altered ESAT-6 targeting or trafficking. In contrast, the mutation of several residues on the outer surface of the four-helical bundle structure of the ESAT-6.CFP-10 complex showed much less effect. Construction of recombinant BCG expressing ESAT-6 with a C-terminal hexahistidine tag allowed us to co-purify ESAT-6 and CFP-10, experimentally confirming their strong interaction both in and outside of the mycobacterial cell. The strain induced potent, antigen-specific T-cell responses and intermediate in vivo growth in mice, suggesting that it remained immunogenic and biologically active despite the tag. Together with previous NMR data, the results of this study have allowed a biologically relevant model of the ESAT-6.CFP-10 complex to be constructed that is critical for understanding the structure-function relationship in tuberculosis pathogenesis.     

Waterborne pathogen detection by use of oligonucleotide-based microarrays

A small-oligonucleotide microarray prototype was designed with probes specific for the universal 16S rRNA and cpn60 genes of several pathogens that are usually encountered in wastewaters. In addition to these two targets, wecE-specific oligonucleotide probes were included in the microarray to enhance its discriminating power within the Enterobacteriaceae family. Universal PCR primers were used to amplify variable regions of 16S rRNA, cpn60, and wecE genes directly in Escherichia coli and Salmonella enterica serovar Typhimurium genomic DNA mixtures (binary); E. coli, S. enterica serovar Typhimurium, and Yersinia enterocolitica genomic DNA mixtures (ternary); or wastewater total DNA. Amplified products were fluorescently labeled and hybridized on the prototype chip. The detection sensitivity for S. enterica serovar Typhimurium was estimated to be on the order of 0.1% (10(4) S. enterica genomes) of the total DNA for the combination of PCR followed by microarray hybridization. The sensitivity of the prototype could be increased by hybridizing amplicons generated by PCR targeting genes specific for a bacterial subgroup, such as wecE genes, instead of universal taxonomic amplicons. However, there was evidence of PCR bias affecting the detection limits of a given pathogen as increasing amounts of a different pathogen were spiked into the test samples. These results demonstrate the feasibility of using DNA microarrays in the detection of waterborne pathogens within mixed populations but also raise the problem of PCR bias in such experiments.     

New insights on the sialidase protein family revealed by a phylogenetic analysis in metazoa

Sialidases are glycohydrolytic enzymes present from virus to mammals that remove sialic acid from oligosaccharide chains. Four different sialidase forms are known in vertebrates: the lysosomal NEU1, the cytosolic NEU2 and the membrane-associated NEU3 and NEU4. These enzymes modulate the cell sialic acid content and are involved in several cellular processes and pathological conditions. Molecular defects in NEU1 are responsible for sialidosis, an inherited disease characterized by lysosomal storage disorder and neurodegeneration. The studies on the biology of sialic acids and sialyltransferases, the anabolic counterparts of sialidases, have revealed a complex picture with more than 50 sialic acid variants selectively present in the different branches of the tree of life. The gain/loss of specific sialoconjugates have been proposed as key events in the evolution of deuterostomes and Homo sapiens, as well as in the host-pathogen interactions. To date, less attention has been paid to the evolution of sialidases. Thus we have conducted a survey on the state of the sialidase family in metazoan. Using an in silico approach, we identified and characterized sialidase orthologs from 21 different organisms distributed among the evolutionary tree: Metazoa relative (Monosiga brevicollis), early Deuterostomia, precursor of Chordata and Vertebrata (teleost fishes, amphibians, reptiles, avians and early and recent mammals). We were able to reconstruct the evolution of the sialidase protein family from the ancestral sialidase NEU1 and identify a new form of the enzyme, NEU5, representing an intermediate step in the evolution leading to the modern NEU3, NEU4 and NEU2. Our study provides new insights on the mechanisms that shaped the substrate specificity and other peculiar properties of the modern mammalian sialidases. Moreover, we further confirm findings on the catalytic residues and identified enzyme loop portions that behave as rapidly diverging regions and may be involved in the evolution of specific properties of sialidases.     

The contribution of TRPV4-mediated calcium signaling to calcium homeostasis in endothelial cells

A large variety of cation transport systems are involved in the regulation of calcium homeostasis in endothelial cells. The focus of the present study is to determine the contribution of nonselective cation channels from the TRP (transient receptor potential) family to cellular calcium homeostasis of porcine aortic endothelial cells (PAEC). One member of the TRPV (vanniloid) subfamily, TRPV4, has previously been shown to be involved in cation transport induced by a large variety of stimulations including osmolarity, temperature, mechanical stress, and phosphorylation. Here, we demonstrate the existence of several TRP proteins, including TRPV4, in PAEC using RT-PCR. To test whether this channel is functional, we performed FURA-2 calcium measurements and whole-cell patch-clamp experiments. We observed the induction of large calcium signals following mechanical stress, altered extracellular temperature, and the selective TRPV4 activator 4-alpha -PDD. These effects were diminished in the presence of the TRPV4 inhibitor miconazole, suggesting the involvement of this channel in mediating endothelial calcium signals. The large amounts of transported calcium and the short signaling ways suggest a potentially important role of this channel in many physiological processes.     

Changes in the ratio of free NEDD8 to ubiquitin triggers NEDDylation by ubiquitin enzymes

Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.     

Rapid parasite adaptation drives selection for high recombination rates

The Red Queen hypothesis proposes that sex is maintained through selection pressure imposed by coevolving parasites: susceptible hosts are able to escape parasite pressure by recombining their genome to create resistant offspring. However, previous theoretical studies have shown that the Red Queen typically selects against sex unless selection is strong, arguing that high rates of recombination cannot evolve when parasites are of low virulence. Here we show that under the biologically plausible assumption of a severe fitness cost for parasites that fail to infect, the Red Queen can cause selection for high recombination rates, and that the strength of virulence is largely irrelevant to the direction of selection for increased recombination rates. Strong selection on parasites and short generation times make parasites usually better adapted to their hosts than vice versa and can thus favor higher recombination rates in hosts. By demonstrating the importance of host-imposed selection on parasites, our findings resolve previously reported conflicting results.