Fibrinogen induces alterations of endothelial cell tight junction proteins

We previously showed that an elevated content of fibrinogen (Fg) increased formation of filamentous actin and enhanced endothelial layer permeability. In the present work we tested the hypothesis that Fg binding to endothelial cells (ECs) alters expression of actin‐associated endothelial tight junction proteins (TJP). Rat cardiac microvascular ECs were grown in gold plated chambers of an electrical cell‐substrate impedance system, 8‐well chambered, or in 12‐well plates. Confluent ECs were treated with Fg (2 or 4 mg/ml), Fg (4 mg/ml) with mitogen‐activated protein kinase (MEK) kinase inhibitors (PD98059 or U0126), Fg (4 mg/ml) with anti‐ICAM‐1 antibody or BQ788 (endothelin type B receptor blocker), endothelin‐1, endothelin‐1 with BQ788, or medium alone for 24 h. Fg induced a dose‐dependent decrease in EC junction integrity as determined by transendothelial electrical resistance (TEER). Western blot analysis and RT‐PCR data showed that the higher dose of Fg decreased the contents of TJPs, occludin, zona occluden‐1 (ZO‐1), and zona occluden‐2 (ZO‐2) in ECs. Fg‐induced decreases in contents of the TJPs were blocked by PD98059, U0126, or anti‐ICAM‐1 antibody. While BQ788 inhibited endothelin‐1‐induced decrease in TEER, it did not affect Fg‐induced decrease in TEER. These data suggest that Fg increases EC layer permeability via the MEK kinase signaling pathway by affecting occludin, ZO‐1, and ZO‐2, TJPs, which are bound to actin filaments. Therefore, increased binding of Fg to its major EC receptor, ICAM‐1, during cardiovascular diseases may increase microvascular permeability by altering the content and possibly subcellular localization of endothelial TJPs. J. Cell. Physiol. 221: 195–203, 2009. © 2009 Wiley‐Liss, Inc     

Crystal Structure of Bfr A from Mycobacterium tuberculosis: Incorporation of Selenomethionine Results in Cleavage and Demetallation of Haem

Emergence of tuberculosis as a global health threat has necessitated an urgent search for new antitubercular drugs entailing determination of 3-dimensional structures of a large number of mycobacterial proteins for structure-based drug design. The essential requirement of ferritins/bacterioferritins (proteins involved in iron storage and homeostasis) for the survival of several prokaryotic pathogens makes these proteins very attractive targets for structure determination and inhibitor design. Bacterioferritins (Bfrs) differ from ferritins in that they have additional noncovalently bound haem groups. The physiological role of haem in Bfrs is not very clear but studies indicate that the haem group is involved in mediating release of iron from Bfr by facilitating reduction of the iron core. To further enhance our understanding, we have determined the crystal structure of the selenomethionyl analog of bacterioferritin A (SeMet-BfrA) from Mycobacterium tuberculosis (Mtb). Unexpectedly, electron density observed in the crystals of SeMet-BfrA analogous to haem location in bacterioferritins, shows a demetallated and degraded product of haem. This unanticipated observation is a consequence of the altered spatial electronic environment around the axial ligands of haem (in lieu of Met52 modification to SeMet52). Furthermore, the structure of Mtb SeMet-BfrA displays a possible lost protein interaction with haem propionates due to formation of a salt bridge between Arg53-Glu57, which appears to be unique to Mtb BfrA, resulting in slight modulation of haem binding pocket in this organism. The crystal structure of Mtb SeMet-BfrA provides novel leads to physiological function of haem in Bfrs. If validated as a drug target, it may also serve as a scaffold for designing specific inhibitors. In addition, this study provides evidence against the general belief that a selenium derivative of a protein represents its true physiological native structure.     

Characterization and phylogenetic analysis of environmental stress-responsive <Emphasis Type="Italic">SAP</Emphasis> gene family encoding A20/AN1 zinc finger proteins in tomato

Characterization of genes responsive to stress is important for efforts on improving stress tolerance of plants. To address components involved in stress tolerance of tomato (Solanum lycopersicum), a stress-responsive gene family encoding A20/AN1 zinc finger proteins was characterized. In the present study, 13 members of this gene family were cloned from tomato cultivar Pusa Ruby and named as Stress Associated Protein (SAP) genes. Out of 13 genes, 12 have been mapped on their respective chromosomes. Expression of these genes in response to cold, heat, salt, desiccation, wounding, abscisic acid, oxidative and submergence stresses was analysed. All tomato SAP genes were found to be responsive to one or other type of environmental stress. The phylogenetic analysis of these genes, along with their orthologs from Solanaceae species suggests the presence of a common set of SAP genes in the studied Solanaceae species. The present study characterizes a SAP gene family, which encodes A20/AN1 zinc finger containing proteins from tomato for the first time. Genes showing high expression in response to a particular stress can be exploited for improving stress tolerance of tomato and other Solanaceae members. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Amplification of Plant Beneficial Microbial Communities During Conversion of Coconut Leaf Substrate to Vermicompost by <Emphasis Type="Italic">Eudrilus</Emphasis> sp.

The population densities of 15 microbial communities in the coconut leaves + cow manure mixture (10:1 ratio, w/w) and pure cow manure, gut contents of the earthworm, Eudrilus sp., reared on the above substrates and vermicompost produced by the worm were studied. The enumeration was done by dilution plate and most probable number method using several selective and semi-selective microbial media. In the vermicompost produced from coconut leaves + cow manure (CLV) mixture, 9 out of 15 microbial communities, particularly the plant beneficial ones, were amplified whereas five communities were amplified in case of pure cow manure (CMV). The CLV contained significantly high population of fungi, free-living nitrogen fixers, phosphate solubilizers, fluorescent pseudomonads, and silicate solubilizers. The CMV was preponderant with aerobic heterotrophic bacteria, actinomycetes, and Trichoderma spp. Spore formers were present in similar numbers in both the vermicomposts. Presence of Azotobacter was detected only in CMV. The results obtained in this study suggest coconut leaf litter to be a good alternative for cow manure for the production of vermicompost, especially in the areas where coconut is grown in plenty.     

Bimodal Analysis Reveals a General Scaling Law Governing Nondirected and Chemotactic Cell Motility

Cell motility is a fundamental process with relevance to embryonic development, immune response, and metastasis. Cells move either spontaneously, in a nondirected fashion, or in response to chemotactic signals, in a directed fashion. Even though they are often studied separately, both forms of motility share many complex processes at the molecular and subcellular scale, e.g., orchestrated cytoskeletal rearrangements and polarization. In addition, at the cellular level both types of motility include persistent runs interspersed with reorientation pauses. Because there is a great range of variability in motility among different cell types, a key challenge in the field is to integrate these multiscale processes into a coherent framework. We analyzed the motility of Dictyostelium cells with bimodal analysis, a method that compares time spent in persistent versus reorientation mode. Unexpectedly, we found that reorientation time is coupled with persistent time in an inverse correlation and, surprisingly, the inverse correlation holds for both nondirected and chemotactic motility, so that the full range of Dictyostelium motility can be described by a single scaling relationship. Additionally, we found an identical scaling relationship for three human cell lines, indicating that the coupling of reorientation and persistence holds across species and making it possible to describe the complexity of cell motility in a surprisingly general and simple manner. With this new perspective, we analyzed the motility of Dictyostelium mutants, and found four in which the coupling between two modes was altered. Our results point to a fundamental underlying principle, described by a simple scaling law, unifying mechanisms of eukaryotic cell motility at several scales.     

webFOG: A web tool to map genomic features onto genes

A large number of new genomic features are being discovered using high throughput techniques. The next challenge is to automatically map them to the reference genome for further analysis and functional annotation. We have developed a tool that can be used to map important genomic features to the latest version of the human genome and also to annotate new features. These genomic features could be of many different source types, including miRNAs, microarray primers or probes, Chip-on-Chip data, CpG islands and SNPs to name a few. A standalone version and web interface for the tool can be accessed through: http://populationhealth.qimr.edu.au/cgi-bin/webFOG/index.cgi. The project details and source code is also available at http://www.bioinformatics.org/webfog.     

Isolation of C-phycocyanin from <Emphasis Type="Italic">Synechococcus</Emphasis> sp., (<Emphasis Type="Italic">Anacystis nidulans</Emphasis> BD1)

We report a procedure for obtaining fairly pure phycocyanin from a local isolate of the cyanobacterium Synechococcus sp (Anacystis nidulans BD1). Cells were incubated with 1 mg∙mL⁻¹ of lysozyme at 37°C for 16 h with shaking. The cell-free extract was treated with activated charcoal and chitosan. The purity (A _620/280) of phycocyanin obtained after lysozyme treatment was up to 2.18, which could be improved to 4.72 after incubation with activated charcoal and chitosan. The yield of phycocyanin was 80–100 mg∙g⁻¹ dry weight of cells. The method reported here is a single-step and efficient procedure and has the potential to be adopted for large-scale production of phycocyanin.     

Hydrogen sulfide protects against vascular remodeling from endothelial damage

Remodeling by its very nature implied synthesis and degradation of extracellular matrix (ECM) proteins. Although oxidative stress, matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) have been implicated in vascular remodeling, the differential role of MMPs versus TIMPs and oxidative stress in vascular remodeling was unclear. TIMP-3 induced vascular cell apoptosis, therefore, we hypothesized that during vascular injury TIMP-3, MMP-9 and -12 (elastin-degrading MMP) were increased, whereas MMP-2 (constitutive MMP) and TIMP-4 (cardioprotective TIMP) decreased. Because of the potent anti-oxidant, vasorelaxing, anti-hypertensive agent, hydrogen sulfide (H₂S) was used to mitigate the vascular remodeling due to the differential expression of MMP and TIMP. Carotid artery injury was created by inserting a PE-10 catheter and rotating several times before pulling out. The insertion hole was sealed. Mice were grouped: wild type (WT), wild-type damaged artery (WTD), WT + NaHS (sodium hydrogen sulfide, precursor of H₂S) treatment (30 μmol/L in drinking water/6 weeks) and WTD + NaHS treatment. Carotid arteries were analyzed for oxidative stress and remodeling, by measuring super oxide dismutase-1 (SOD1), p47 (NADPH oxidase subunit), nitrotyrosine, MMPs and TIMPs by in situ immunolabeling and by Western blot analyses. The results suggested robust increase in p47, nitrotyrosine, MMP-9, MMP-12, TIMP-3 and decrease in SOD1 and MMP-2 levels in the injured arteries. The treatment with H₂S ameliorated these effects. We concluded that p47, TIMP-3, MMP-9 and -12 were increased where as SOD-1, MMP-2 and TIMP-4 were decreased in the injured arteries. The treatment with H₂S mitigated the vascular remodeling by normalizing the levels of redox stress, MMPs and TIMPs.     

Genome-Wide Survey and Expression Analysis Suggest Diverse Roles of Glutaredoxin Gene Family Members During Development and Response to Various Stimuli in Rice

Glutaredoxins (GRXs) are glutathione-dependent oxidoreductase enzymes involved in a variety of cellular processes. In this study, our analysis revealed the presence of 48 genes encoding GRX proteins in the rice genome. GRX proteins could be classified into four classes, namely CC-, CGFS-, CPYC- and GRL-type, based on phylogenetic analysis. The classification was supported with organization of predicted conserved putative motifs in GRX proteins. We found that expansion of this gene family has occurred largely via whole genome duplication events in a species-specific manner. We explored rice oligonucleotide array data to gain insights into the function of GRX gene family members during various stages of development and in response to environmental stimuli. The comprehensive expression analysis suggested diverse roles of GRX genes during growth and development in rice. Some of the GRX genes were expressed in specific organs/developmental stages only. The expression of many of rice GRX genes was influenced by various phytohormones, abiotic and biotic stress conditions, suggesting an important role of GRX proteins in response to these stimuli. The identification of GRX genes showing differential expression in specific tissues or in response to environmental stimuli provide a new avenue for in-depth characterization of selected genes of importance.     

Structural Ordering of Disordered Ligand-Binding Loops of Biotin Protein Ligase into Active Conformations as a Consequence of Dehydration

Mycobacterium tuberculosis (Mtb), a dreaded pathogen, has a unique cell envelope composed of high fatty acid content that plays a crucial role in its pathogenesis. Acetyl Coenzyme A Carboxylase (ACC), an important enzyme that catalyzes the first reaction of fatty acid biosynthesis, is biotinylated by biotin acetyl-CoA carboxylase ligase (BirA). The ligand-binding loops in all known apo BirAs to date are disordered and attain an ordered structure only after undergoing a conformational change upon ligand-binding. Here, we report that dehydration of Mtb-BirA crystals traps both the apo and active conformations in its asymmetric unit, and for the first time provides structural evidence of such transformation. Recombinant Mtb-BirA was crystallized at room temperature, and diffraction data was collected at 295 K as well as at 120 K. Transfer of crystals to paraffin and paratone-N oil (cryoprotectants) prior to flash-freezing induced lattice shrinkage and enhancement in the resolution of the X-ray diffraction data. Intriguingly, the crystal lattice rearrangement due to shrinkage in the dehydrated Mtb-BirA crystals ensued structural order of otherwise flexible ligand-binding loops L4 and L8 in apo BirA. In addition, crystal dehydration resulted in a shift of ∼3.5 Å in the flexible loop L6, a proline-rich loop unique to Mtb complex as well as around the L11 region. The shift in loop L11 in the C-terminal domain on dehydration emulates the action responsible for the complex formation with its protein ligand biotin carboxyl carrier protein (BCCP) domain of ACCA3. This is contrary to the involvement of loop L14 observed in Pyrococcus horikoshii BirA-BCCP complex. Another interesting feature that emerges from this dehydrated structure is that the two subunits A and B, though related by a noncrystallographic twofold symmetry, assemble into an asymmetric dimer representing the ligand-bound and ligand-free states of the protein, respectively. In-depth analyses of the sequence and the structure also provide answers to the reported lower affinities of Mtb-BirA toward ATP and biotin substrates. This dehydrated crystal structure not only provides key leads to the understanding of the structure/function relationships in the protein in the absence of any ligand-bound structure, but also demonstrates the merit of dehydration of crystals as an inimitable technique to have a glance at proteins in action.