Analysis of minor components in olive oil

Virgin olive oil is well known for its high content of phenolic substances that are thought to have health-promoting properties. These substances also contribute to the distinctive taste of the oil. In this study, tyrosol, vanillic acid, luteolin, and apigenin were identified and quantified by liquid chromatography mass spectrometry (LC-MS). In the seven samples analysed, tyrosol, the most abundant, was in the range of 1.4-29 mg/kg, vanillic acid was in the range of 0.67-4.0 mg/kg, luteolin was in the range of 0.22-7.0 mg/kg, and apigenin was in the range of 0.68-1.6 mg/kg. It was also shown that in olive oil, squalene can be analysed by using a refractive index detector. In the samples analysed, squalene occurred in the range of 3.9-9.6 g/l.     

Gene Expression Analysis Suggests Temporal Differential Response to Aluminum in Coffea arabica Cultivars

Aluminum (Al) is a limiting factor of crop yields on acidic soils. Ion aluminum (Al³⁺) acts primarily in plant root system retarding its growth and development, leading to the reduction of lateral roots number, and consequently the decrease of vegetal production. Most of coffee producing areas are located in acidic soils, which have Al³⁺ contents enough to damage plant development. Despite the advances in the understanding of physiological and genetic mechanisms of Al tolerance/susceptibility, few are known about Al ion action in coffee plants. This report describes the expression analysis of genes related to aluminum stress in germinating seeds of two cultivars of C. arabica (Catuaí Amarelo IAC 62 and Icatu Vermelho IAC 4045) when challenged with Al³⁺. In silico analyses of Brazilian Coffee Genome Project (BCGP) database were used to select genes previously found to be related with Al-stress. The expression profile of these genes in Catuaí and Icatu was evaluated through Quantitative PCR (qPCR). Based on our data, we suggest that both analyzed cultivars displays mechanisms of resistance or exclusion, which occurs outside the cell excluding Al³⁺ assimilation, and mechanisms of tolerance that occurs inside the cell after Al³⁺ absorption. The major difference is the timing of activation of each mechanism. While Catuaí tends to use resistance mechanisms in early stages of stress, Icatu uses tolerance strategies. In late stages, both cultivars seem to display tolerance mechanisms, but Icatu also displays Al-exclusion strategy.     

Genomic,Proteomic, and Biochemical Analysis of the Organohalide Respiratory Pathway in Desulfitobacterium dehalogenans

Desulfitobacterium dehalogenans is able to grow by organohalide respiration using 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) as an electron acceptor. We used a combination of genome sequencing, biochemical analysis of redox active components, and shotgun proteomics to study elements of the organohalide respiratory electron transport chain. The genome of Desulfitobacterium dehalogenans JW/IU-DC1^T consists of a single circular chromosome of 4,321,753 bp with a GC content of 44.97%. The genome contains 4,252 genes, including six rRNA operons and six predicted reductive dehalogenases. One of the reductive dehalogenases, CprA, is encoded by a well-characterized cprTKZEBACD gene cluster. Redox active components were identified in concentrated suspensions of cells grown on formate and Cl-OHPA or formate and fumarate, using electron paramagnetic resonance (EPR), visible spectroscopy, and high-performance liquid chromatography (HPLC) analysis of membrane extracts. In cell suspensions, these components were reduced upon addition of formate and oxidized after addition of Cl-OHPA, indicating involvement in organohalide respiration. Genome analysis revealed genes that likely encode the identified components of the electron transport chain from formate to fumarate or Cl-OHPA. Data presented here suggest that the first part of the electron transport chain from formate to fumarate or Cl-OHPA is shared. Electrons are channeled from an outward-facing formate dehydrogenase via menaquinones to a fumarate reductase located at the cytoplasmic face of the membrane. When Cl-OHPA is the terminal electron acceptor, electrons are transferred from menaquinones to outward-facing CprA, via an as-yet-unidentified membrane complex, and potentially an extracellular flavoprotein acting as an electron shuttle between the quinol dehydrogenase membrane complex and CprA.     

Mutations in the Lipopolysaccharide Biosynthesis Pathway Interfere with Crescentin-Mediated Cell Curvature in Caulobacter crescentus

Bacterial cell morphogenesis requires coordination among multiple cellular systems, including the bacterial cytoskeleton and the cell wall. In the vibrioid bacterium Caulobacter crescentus, the intermediate filament-like protein crescentin forms a cell envelope-associated cytoskeletal structure that controls cell wall growth to generate cell curvature. We undertook a genetic screen to find other cellular components important for cell curvature. Here we report that deletion of a gene (wbqL) involved in the lipopolysaccharide (LPS) biosynthesis pathway abolishes cell curvature. Loss of WbqL function leads to the accumulation of an aberrant O-polysaccharide species and to the release of the S layer in the culture medium. Epistasis and microscopy experiments show that neither S-layer nor O-polysaccharide production is required for curved cell morphology per se but that production of the altered O-polysaccharide species abolishes cell curvature by apparently interfering with the ability of the crescentin structure to associate with the cell envelope. Our data suggest that perturbations in a cellular pathway that is itself fully dispensable for cell curvature can cause a disruption of cell morphogenesis, highlighting the delicate harmony among unrelated cellular systems. Using the wbqL mutant, we also show that the normal assembly and growth properties of the crescentin structure are independent of its association with the cell envelope. However, this envelope association is important for facilitating the local disruption of the stable crescentin structure at the division site during cytokinesis.Most bacterial species display a particular cellular morphology that is generally preserved across generations. The production and maintenance of shape require coordination among multiple cellular systems positioned at different places within the cell. The peptidoglycan cell wall, located external to the cytoplasmic membrane, is an important structural element that is required for shape maintenance. The processes governing the localization and timing of cell wall growth and turnover are likewise critical (8, 9, 23). The bacterial cytoskeleton is thought to play a central role in cell morphogenesis by exerting spatiotemporal control over peptidoglycan growth (8, 9, 23). In order for it to do so, there are numerous proteins that are required to connect cytoskeletal control mechanisms to the periplasmic enzymes that directly synthesize and modify the peptidoglycan cell wall. These proteins, such as MreC, MreD, RodA, and RodZ, are essential for maintenance of cell shape and are positioned in the cytoplasmic membrane to presumably link cytoskeletal elements in the cytoplasm to the activities of peptidoglycan-modifying enzymes in the periplasm (2, 5, 9, 23, 29). Some bacterial species also contain additional components that make important contributions to cell shape, such as cell wall teichoic acids in Gram-positive bacteria (9) and periplasmic flagella in spirochetes (41).In the vibrioid bacterium Caulobacter crescentus, an intermediate filament-like protein, crescentin, is required for cell curvature (3). Crescentin forms an intracellular filamentous structure that is associated with the cell wall and is thought to mechanically govern cell wall growth to produce cell curvature (7). The crescentin structure is localized along the inner curvature of the cell under the cytoplasmic membrane (3, 7) and is highly stable, with no detectable subunit exchange (10). The association between the crescentin structure and the cell envelope appears essential for its function, since an attachment-defective crescentin mutant is unable to support cell curvature (7). The function of the actin-like protein MreB is also critical for the envelope association of the crescentin structure (10), and MreB may provide one part of the connection between the crescentin structure and the peptidoglycan cell wall.Since bacterial morphogenesis requires multiple cellular components and systems, we used a genetic screen to find other factors important for cell curvature in C. crescentus. Surprisingly, we found that an alteration in the lipopolysaccharide (LPS) biosynthesis pathway can have a catastrophic effect on the ability of the crescentin structure to associate with the cell envelope and govern cell curvature.     

Polar Mutations in the Left Arm of Bacteriophage λi434

By studying complementation between frameshift and nonsense mutants located in the structural genes for the head of bacteriophage lambdai434, we found mutations in gene B which are polar on genes C and D and one mutation in gene E which is polar on gene F.     

Analysis of bacterial polyhydroxybutyrate production by multimodal nanoimaging

In this paper, we will employ two microscopy techniques, transmission electron microscopy and infrared nanospectromicroscopy, to study the production of polyhydroxybutyrate in Rhodobacter capsulatus and to evaluate the influence of glucose and acetone on the production yield. The results overlap which leads us to a consistent conclusion, highlighting that each technique brings specific and complementary information. By using electron microscopy and infrared nanospectromicroscopy we have proved that both glucose and acetone had a positive effect on the biopolymer production, although the first study done by Fourier transform infrared spectroscopy only identified the effect of acetone. In conclusion, we have now established a method to be able to perform fast diagnostic for PHB production.     

Characterization of the Chemokine CXCL11-Heparin Interaction Suggests Two Different Affinities for Glycosaminoglycans

Chemokines orchestrate the migration of leukocytes in the context of homeostasis and inflammation. In addition to interactions of chemokines with receptors on migrating cells, these processes require interactions of chemokines with glycosaminoglycans (GAGs) for cell surface localization. Most chemokines are basic proteins with Arg/Lys/His residue clusters functioning as recognition epitopes for GAGs. In this study we characterized the GAG-binding epitopes of the chemokine I-TAC/CXCL11. Four separate clusters of basic residues were mutated to alanine and tested for their ability to bind to GAGs in vitro and to activate the receptor, CXCR3. Mutation of a set of basic residues in the C-terminal helix (the 50s cluster, ⁵⁷KSKQAR⁶²) along with Lys¹⁷, significantly impaired heparin binding in vitro, identifying these residues as components of the dominant epitope. However, this GAG mutant retained nearly wild type receptor binding affinity, and its ability to induce cell migration in vitro was only mildly perturbed. Nevertheless, the mutant was unable to induce cell migration in vivo, establishing a requirement of CXCL11 for GAG binding for in vivo function. These studies also led to some interesting findings. First, CXCL11 exhibits conformational heterogeneity, as evidenced by the doubling of peaks in its HSQC spectra. Second, it exhibits more than one affinity state for both heparin and CXCR3, which may be related to its structural plasticity. Finally, although the binding affinities of chemokines for GAGs are typically weaker than interactions with receptors, the high affinity GAG binding state of CXCL11 is comparable with typical receptor binding affinities, suggesting some unique properties of this chemokine.     

Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells

Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end–directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.     

Occurrence of Tn4371-Related Mobile Elements and Sequences in (Chloro)biphenyl-Degrading Bacteria

Tn4371, a 55-kb transposable element involved in the degradation and biphenyl or 4-chlorobiphenyl identified in Ralstonia eutropha A5, displays a modular structure including a phage-like integrase gene (int), a Pseudomonas-like (chloro)biphenyl catabolic gene cluster (bph), and RP4- and Ti-plasmid-like transfer genes (trb) (C. Merlin, D. Springael, and A. Toussaint, Plasmid 41:40–54, 1999). Southern blot hybridization was used to examine the presence of different regions of Tn4371 in a collection of (chloro)biphenyl-degrading bacteria originating from different habitats and belonging to different bacterial genera. Tn4371-related sequences were never detected on endogenous plasmids. Although the gene probes containing only bph sequences hybridized to genomic DNA from most strains tested, a limited selection of strains, all β-proteobacteria, displayed hybridization patterns similar to the Tn4371 bph cluster. Homology between Tn4371 and DNA of two of those strains, originating from the same area as strain A5, extended outside the catabolic genes and covered the putative transfer region of Tn4371. On the other hand, none of the (chloro)biphenyl degraders hybridized with the outer left part of Tn4371 containing the int gene. The bph catabolic determinant of the two strains displaying homology to the Tn4371 transfer genes and a third strain isolated from the A5 area could be mobilized to a R. eutropha recipient, after insertion into an endogenous or introduced IncP1 plasmid. The mobilized DNA of those strains included all Tn4371 homologous sequences previously identified in their genome. Our observations show that the bph genes present on Tn4371 are highly conserved between different (chloro)biphenyl-degrading hosts, isolated globally but belonging mainly to the β-proteobacteria. On the other hand, Tn4371-related mobile elements carrying bph genes are apparently only found in isolates from the environment that provided the Tn4371-bearing isolate A5.