Metabolic Plasticity in Resting and Thrombin Activated Platelets

Platelet thrombus formation includes several integrated processes involving aggregation, secretion of granules, release of arachidonic acid and clot retraction, but it is not clear which metabolic fuels are required to support these events. We hypothesized that there is flexibility in the fuels that can be utilized to serve the energetic and metabolic needs for resting and thrombin-dependent platelet aggregation. Using platelets from healthy human donors, we found that there was a rapid thrombin-dependent increase in oxidative phosphorylation which required both glutamine and fatty acids but not glucose. Inhibition of fatty acid oxidation or glutamine utilization could be compensated for by increased glycolytic flux. No evidence for significant mitochondrial dysfunction was found, and ATP/ADP ratios were maintained following the addition of thrombin, indicating the presence of functional and active mitochondrial oxidative phosphorylation during the early stages of aggregation. Interestingly, inhibition of fatty acid oxidation and glutaminolysis alone or in combination is not sufficient to prevent platelet aggregation, due to compensation from glycolysis, whereas inhibitors of glycolysis inhibited aggregation approximately 50%. The combined effects of inhibitors of glycolysis and oxidative phosphorylation were synergistic in the inhibition of platelet aggregation. In summary, both glycolysis and oxidative phosphorylation contribute to platelet metabolism in the resting and activated state, with fatty acid oxidation and to a smaller extent glutaminolysis contributing to the increased energy demand.     

The ER-localized TWD1 immunophilin is necessary for localization of multidrug resistance-like proteins required for polar auxin transport in Arabidopsis roots

Multidrug resistance ABC transporters in plants are required for polar transport of the hormone auxin (indole-3-acetic acid). They are studied in animals primarily because their overexpression confers resistance to anticancer agents. Immunophilins are studied in both plants and animals for their roles in folding and trafficking of proteins, particularly those with signal transducing functions and susceptibility to immunosuppressant drugs. Previous genetic and molecular studies in Arabidopsis thaliana established a physical and functional interaction between some ABCB transporters and the TWISTED DWARF1 (TWD1) immunophilin. In this work, confocal microscopy of fluorescently tagged TWD1 shows it to reside at the endoplasmic reticulum (ER). Mutations in TWD1 caused mislocalization of ABCB1, ABCB4, and ABCB19 to the ER instead of the plasma membrane as shown by confocal microscopy of fluorescently tagged fusion proteins and transmission electron microscopy of immunogold-labeled samples in the case of ABCB19. Localization of the unrelated PIN-FORMED2 auxin transporter or plasma membrane marker proteins was not affected by loss of TWD1. Abnormal spread of auxin signaling into the elongation zone of twd1 roots, attributable to mislocalized ABCB transporters and suppressed by an auxin transport inhibitor, appeared to cause the twisted cell files characteristic of twd1 roots.     

Quantitative HIV-1 proviral DNA detection: a multicentre analysis

Despite the widespread use of molecular biology techniques, standardized methods for the measurement of HIV-1 proviral DNA are currently lacking and several discordant results are still present in different studies. To assess the clinical meaning of the proviral DNA load, a study group comprising seven different laboratories was set up to standardize a HIV-1 proviral DNA quantification method able to assess the DNA proviral load of the most relevant circulating HIV-1 subtypes. Reference samples (24 cellular samples infected with HIV-1 clade B, and 40 samples of peripheral blood mononuclear cells containing different concentrations of plasmids expressing different HIV-1 clades) were distributed and tested blindly. All laboratories employed hTERT gene as housekeeping gene and primers within the gag gene to quantify different HIV-1 clades. Inter-laboratory results did not differ statistically but showed only minor variations concerning HIV-1 DNA amounts and different HIV clades, with a good agreement among the laboratories participating in the study. Since test standardization represents a key step for future application in clinical practice, further studies of the patients' samples are in progress to establish the real meaning and utility of the proviral DNA load for clinical management of HIV-1 infected patients.     

Unraveling ferulate role in suberin and periderm biology by reverse genetics

Plant cell walls are dramatically affected by suberin deposition, becoming an impermeable barrier to water and pathogens. Suberin is a complex layered heteropolymer that comprises both a poly(aliphatic) and a poly(aromatic) lignin-like domain. Current structural models for suberin attribute the crosslinking of aliphatic and aromatic domains within the typical lamellar ultrastructure of the polymer to esterified ferulate. BAHD feruloyl transferases involved in suberin biosynthesis have been recently characterized in Arabidopsis and potato (Solanum tuberosum). In defective mutants, suberin, even lacks most of the esterified ferulate, but maintains the typical lamellar ultrastructure. However, suberized tissues display increased water permeability, in spite of exhibiting a similar lipid load to wild type. Therefore, the role of ferulate in suberin needs to be reconsidered. Moreover, silencing the feruloyl transferase in potato turns the typical smooth skin of cv. Desirée into a rough scabbed skin distinctive of Russet varieties and impairs the normal skin maturation that confers resistance to skinning. Concomitantly to these changes, the skin of silenced potatoes shows an altered profile of soluble phenolics with the emergence of conjugated polyamines.Key words: BAHD feruloyl acyltransferases, ferulate, periderm, potato tuber skin, suberin, suberized tissues, waxRecently published reverse genetic studies in Arabidopsis and potato identified two orthologous genes that encode a BAHD feruloyl transferase acting on aliphatics and showed that deficiency in these enzymes produces a decrease in suberin-associated ferulic acid. These results, here discussed, signify an important advance in suberin biochemistry and ultrastructure, providing a valuable new insight into the organization of the suberized tissues and their role in the control of water vapour loss.     

Diversity and mobility of integrative and conjugative elements in bovine isolates of Streptococcus agalactiae, S. dysgalactiae subsp. dysgalactiae, and S. uberis

Bovine isolates of Streptococcus agalactiae (n = 76), Streptococcus dysgalactiae subsp. dysgalactiae (n = 32), and Streptococcus uberis (n = 101) were analyzed for the presence of different integrative and conjugative elements (ICEs) and their association with macrolide, lincosamide, and tetracycline resistance. The diversity of the isolates included in this study was demonstrated by multilocus sequence typing for S. agalactiae and pulsed-field gel electrophoresis for S. dysgalactiae and S. uberis. Most of the erythromycin-resistant strains carry an ermB gene. Five strains of S. uberis that are resistant to lincomycin but susceptible to erythromycin carry the lin(B) gene, and one has both linB and lnuD genes. In contrast to S. uberis, most of the S. agalactiae and S. dysgalactiae tetracycline-resistant isolates carry a tet(M) gene. A tet(S) gene was also detected in the three species. A Tn916-related element was detected in 30 to 50% of the tetracycline-resistant strains in the three species. Tetracycline resistance was successfully transferred by conjugation to an S. agalactiae strain. Most of the isolates carry an ICE integrated in the rplL gene. In addition, half of the S. agalactiae isolates have an ICE integrated in a tRNA lysine (tRNA(Lys)) gene. Such an element is also present in 20% of the isolates of S. dysgalactiae and S. uberis. A circular form of these ICEs was detected in all of the isolates tested, indicating that these genetic elements are mobile. These ICEs could thus also be a vehicle for horizontal gene transfer between streptococci of animal and/or human origin.     

Complexes of 2-thiophenecarbonyl and isonicotinoyl hydrazones of 3-(N-methyl)isatin. A study of their antimicrobial activity

Cobalt(II), nickel(II), copper(II) and zinc(II) complexes of 2-thiophenecarbonyl and isonicotinoyl hydrazones of 3-(N-methyl)isatin (HL(1) and HL(2), respectively) were synthesized and characterized, being the crystal structures of HL(1), HL(2) and [Ni(L(1))(2)].2CHCl(3) elucidated by X-ray diffraction techniques. The in vitro antimicrobial activity of all these compounds was tested against several bacteria and fungi. HL(1)and its complexes exhibited a strong inhibition of the growth of Haemophilus influenzae (MIC 0.15-1.50microg/mL) and good antibacterial properties towards Bacillus subtilis (MIC 3-25microg/mL). The minimal inhibitory concentration (MIC) was defined as the lowest concentration of compound inhibiting the growth of each strain. The antibacterial effectiveness was confirmed against a number of Gram positive bacteria, including methicillin-resistant Staphylococcus aureus. Yeasts and moulds showed a low susceptibility, except the dermatophyte mould Epidermophyton floccosum that is inhibited at concentrations ranging from 6 to 50microg/mL. In general, the antimicrobial activity of the thiophene derivatives was greater than that of the isonicotinic analogues.     

The contribution of heme propionate groups to the conformational dynamics associated with CO photodissociation from horse heart myoglobin

Photoacoustic calorimetry and transient absorption spectroscopy were used to study conformational dynamics associated with CO photodissociation from horse heart myoglobin (Mb) reconstituted with either Fe protoporphyrin IX dimethylester (FePPDME), Fe octaethylporphyrin (FeOEP), or with native Fe protoporphyrin IX (FePPIX). The volume and enthalpy changes associated with the Fe-CO bond dissociation and formation of a transient deoxyMb intermediate for the reconstituted Mbs were found to be similar to those determined for native Mb (DeltaV1 = -2.5+/-0.6 ml mol(-1) and DeltaH1 = 8.1+/-3.0 kcal mol(-1)). The replacement of FePPIX by FeOEP significantly alters the conformational dynamics associated with CO release from protein. Ligand escape from FeOEP reconstituted Mb was determined to be roughly a factor of two faster (tau=330 ns) relative to native protein (tau=700 ns) and accompanying reaction volume and enthalpy changes were also found to be smaller (DeltaV2 = 5.4+/-2.5 ml mol(-1) and DeltaH2 = 0.7+/-2.2 kcal mol(-1)) than those for native Mb (DeltaV2 = 14.3+/-0.8 ml mol(-1) and DeltaH2 = 7.8+/-3.5 kcal mol(-1)). On the other hand, volume and enthalpy changes for CO release from FePPIX or FePPDME reconstituted Mb were nearly identical to those of the native protein. These results suggest that the hydrogen bonding network between heme propionate groups and nearby amino acid residues likely play an important role in regulating ligand diffusion through protein matrix. Disruption of this network leads to a partially open conformation of protein with less restricted ligand access to the heme binding pocket.     

Genome sequence, structural proteins, and capsid organization of the cyanophage Syn5: a "horned" bacteriophage of marine synechococcus

Marine Synechococcus spp and marine Prochlorococcus spp are numerically dominant photoautotrophs in the open oceans and contributors to the global carbon cycle. Syn5 is a short-tailed cyanophage isolated from the Sargasso Sea on Synechococcus strain WH8109. Syn5 has been grown in WH8109 to high titer in the laboratory and purified and concentrated retaining infectivity. Genome sequencing and annotation of Syn5 revealed that the linear genome is 46,214 bp with a 237 bp terminal direct repeat. Sixty-one open reading frames (ORFs) were identified. Based on genomic organization and sequence similarity to known protein sequences within GenBank, Syn5 shares features with T7-like phages. The presence of a putative integrase suggests access to a temperate life cycle. Assignment of 11 ORFs to structural proteins found within the phage virion was confirmed by mass-spectrometry and N-terminal sequencing. Eight of these identified structural proteins exhibited amino acid sequence similarity to enteric phage proteins. The remaining three virion proteins did not resemble any known phage sequences in GenBank as of August 2006. Cryo-electron micrographs of purified Syn5 virions revealed that the capsid has a single “horn”, a novel fibrous structure protruding from the opposing end of the capsid from the tail of the virion. The tail appendage displayed an apparent 3-fold rather than 6-fold symmetry. An 18 Å resolution icosahedral reconstruction of the capsid revealed a T = 7 lattice, but with an unusual pattern of surface knobs. This phage/host system should allow detailed investigation of the physiology and biochemistry of phage propagation in marine photosynthetic bacteria.