Membrane topology of the ArsB protein, the membrane subunit of an anion-translocating ATPase

The ars operon of the conjugative R-factor R773 encodes an oxyanion pump that catalyzes extrusion of arsenicals from cells of Escherichia coli. The oxyanion translocation ATPase is composed of two polypeptides, the catalytic ArsA protein and the intrinsic membrane protein, ArsB. The topology of regions of the ArsB protein in the inner membrane was determined using a variety of gene fusions. Random gene fusions with lacZ and phoA were generated using transposon mutagenesis. A series of gene fusions with blaM were constructed in vitro using a beta-lactamase fusion vector. To localize individual segments of the ArsB protein, a ternary fusion method was developed, where portions of the arsB gene were inserted in-frame between the coding regions for two heterologous proteins, in this case a portion of a newly identified arsD gene and the blaM sequence encoding the mature beta-lactamase. The location of a periplasmic loop was determined from V8 protease digestion of an ArsA-ArsB chimera. From analysis of data from 26 fusions, a topological model of the ArsB protein with 12 membrane-spanning regions is proposed

tRNA Profiling of Mesenchymal Stem Cell Exosome

Background: Exosomes have great potential in regenerative medicine through the transfer of their bioactive cargos, such as RNA. tRF RNA and tiRNA are tRNAderived non-coding RNA. Here, we sought to identify the tRF/tiRNA profile in human mesenchymal stem cell (hMSC) exosomes. Methods: Bone marrow hMSCs were cultured with/without osteogenic differentiation medium and exosomes were harvested. RNA was extracted from: 1) control cells (Cell-NT); 2) control exosomes (EXO-NT); 3) differentiated cells (Cell-OM); 4) exosomes produced by differentiated cells (EXO-OM). RNA was sequenced to profile the small RNA with a focus on tRF/tiRNA. Results: tRF/tiRNA was highly enriched in hMSC exosomes. Less diversity was seen in the tRF/tiRNA profile in exosomes than that in parent cells. Selective tRF/tiRNA were packed into MSC exosomes and their profile is dependent on the cell maturation status. Conclusions: Our results suggest that tRF/tiRNA may play a role in mediating the function of exosomes in tissue regeneration

γ Heavy Chain Disease in Man: cDNA Sequence Supports Partial Gene Deletion Model

Human gamma heavy chain disease (HCD) is characterized by the presence in serum of a short monoclonal Ig gamma chain unattached to light chains. Although most HCD proteins have internal deletions, in some the defect is NH2-terminal. The OMM gamma 3 HCD serum protein is of the latter type, having undergone an extensive NH2-terminal deletion with a sequence starting within the hinge. A cell line synthesizing the OMM protein has enabled us to study the biogenesis of the abnormal molecule. In vitro translation of isolated mRNA yields a protein containing a hydrophobic NH2-terminal leader sequence. In the intact cell, the precursor molecule is processed normally to yield a protein with an NH2-terminal sequence homologous to the beginning of the variable (V) region. The nucleotide sequence of cDNA prepared from the OMM mRNA encodes a 19-amino acid leader followed by the first 15 residues of the V region. An extensive internal deletion encompasses the remainder of the V and the entire CHl domain. Immediately following the short V region, there is information in the cDNA for the entire normal hinge. The primary synthetic product is thus an internally deleted molecule that undergoes postsynthetic degradation to yield the NH2-terminally deleted serum protein. The structure of the OMM mRNA suggests that the protein abnormality results from a partial gene deletion rather than defective splicing

A Comparison of Nucleosome Organization in \u3cem\u3eDrosophila\u3c/em\u3e Cell Lines

Changes in the distribution of nucleosomes along the genome influence chromatin structure and impact gene expression by modulating the accessibility of DNA to transcriptional machinery. However, the role of genome-wide nucleosome positioning in gene expression and in maintaining differentiated cell states remains poorly understood. Drosophila melanogastercell lines represent distinct tissue types and exhibit cell-type specific gene expression profiles. They thus could provide a useful tool for investigating cell-type specific nucleosome organization of an organism’s genome. To evaluate this possibility, we compared genome-wide nucleosome positioning and occupancy in five different Drosophila tissue-specific cell lines, and in reconstituted chromatin, and then tested for correlations between nucleosome positioning, transcription factor binding motifs, and gene expression. Nucleosomes in all cell lines were positioned in accordance with previously known DNA-nucleosome interactions, with helically repeating A/T di-nucleotide pairs arranged within nucleosomal DNAs and AT-rich pentamers generally excluded from nucleosomal DNA. Nucleosome organization in all cell lines differed markedly from in vitro reconstituted chromatin, with highly expressed genes showing strong nucleosome organization around transcriptional start sites. Importantly, comparative analysis identified genomic regions that exhibited cell line-specific nucleosome enrichment or depletion. Further analysis of these regions identified 91 out of 16,384 possible heptamer sequences that showed differential nucleosomal occupation between cell lines, and 49 of the heptamers matched one or more known transcription factor binding sites. These results demonstrate that there is differential nucleosome positioning between these Drosophila cell lines and therefore identify a system that could be used to investigate the functional significance of differential nucleosomal positioning in cell type specification

The Search for Antibiotic Leads: Targeting Pantothenate Kinase in Pseudomonas aeruginosa

Master's Thesis in Biomedical SciencesBMED395MAMD-MEDB

On the Specificity of Heparin/Heparan Sulfate Binding to Proteins. Anion-Binding Sites on Antithrombin and Thrombin Are Fundamentally Different

Background The antithrombin–heparin/heparan sulfate (H/HS) and thrombin–H/HS interactions are recognized as prototypic specific and non-specific glycosaminoglycan (GAG)–protein interactions, respectively. The fundamental structural basis for the origin of specificity, or lack thereof, in these interactions remains unclear. The availability of multiple co-crystal structures facilitates a structural analysis that challenges the long-held belief that the GAG binding sites in antithrombin and thrombin are essentially similar with high solvent exposure and shallow surface characteristics. Methodology Analyses of solvent accessibility and exposed surface areas, gyrational mobility, symmetry, cavity shape/size, conserved water molecules and crystallographic parameters were performed for 12 X-ray structures, which include 12 thrombin and 16 antithrombin chains. Novel calculations are described for gyrational mobility and prediction of water loci and conservation. Results The solvent accessibilities and gyrational mobilities of arginines and lysines in the binding sites of the two proteins reveal sharp contrasts. The distribution of positive charges shows considerable asymmetry in antithrombin, but substantial symmetry for thrombin. Cavity analyses suggest the presence of a reasonably sized bifurcated cavity in antithrombin that facilitates a firm ‘hand-shake’ with H/HS, but with thrombin, a weaker ‘high-five’. Tightly bound water molecules were predicted to be localized in the pentasaccharide binding pocket of antithrombin, but absent in thrombin. Together, these differences in the binding sites explain the major H/HS recognition characteristics of the two prototypic proteins, thus affording an explanation of the specificity of binding. This provides a foundation for understanding specificity of interaction at an atomic level, which will greatly aid the design of natural or synthetic H/HS sequences that target proteins in a specific manner

Streptomyces sporulation - Genes and regulators involved in bacterial cell differentiation

Streptomycetes are Gram-positive bacteria with a complex developmental life cycle. They form spores on specialized cells called aerial hyphae, and this sporulation involves alterations in growth, morphogenesis and cell cycle processes like cell division and chromosome segregation. Understanding the developmental mechanisms that streptomycetes have evolved for regulating for example cell division is of general interest in bacterial cell biology. It can also be valuable in the design of new drugs against bacterial pathogens. Very few sporulation genes have been found with an impact on cell cycle-related processes. Finding of such genes is important for a clarification of the molecular mechanisms that are underlying the developmental control of fundamental cellular processes in Streptomyces. The work of this thesis has led to the identification of genes previously unknown to be developmentally regulated in Streptomyces. By comparing the transcriptome of the wildtype S. coelicolor strain M145 to two developmental mutants, whiA and whiH, which specifically affect sporulation processes, it was possible to identify differentially expressed genes. Genes that so far have been characterized proved to have important roles during sporulation, affecting spore maturation, chromosome condensation and cell division. WhiH is a central regulator in the early sporulation process and required for the developmentally controlled form of cell division in S. coelicolor. In this thesis the role of WhiH as a transcription factor has been established and WhiH was found to bind to a specific site in its own promoter and function as an autoregulator. A whiHp-mCherry reporter fusion was used to monitor cell type-specific activity of the whiH promoter in aerial hyphae, and showed that it is active before delimitation of the sporogenic cell in which multiple developmentally controlled cell divisions will be triggered. A new Streptomyces model organism, S. venezuelae, was finally exploited to identify target genes for control by WhiH. This organism sporulates efficiently in liquid culture and is well suited for global transcriptomic approaches. In this study, microarray analysis of whiH-dependent gene expression was used to find putative targets for WhiH. Combined with chromatin-immunoprecipitation (ChIP-chip) and protein-DNA binding assays this identified a group of genes that are directly repressed by WhiH during a late stage of sporulation, and also some candidate genes that could be activated by WhiH at an earlier stage. Future analyses should shed light on the functions of these genes and their potential roles in developmental and cell cycle-related processes

Analysis of Two Polyhydroxyalkanoate Synthases in Bradyrhizobium japonicum USDA 110

Bradyrhizobium japonicum USDA 110 has five polyhydroxyalkanoate (PHA) synthases (PhaC) annotated in its genome: bll4360 (phaC1), bll6073 (phaC2), blr3732 (phaC3), blr2885 (phaC4), and bll4548 (phaC5). All these proteins possess the catalytic triad and conserved amino acid residues of polyester synthases and are distributed into four different PhaC classes. We obtained mutants in each of these paralogs and analyzed phaC gene expression and PHA production in liquid cultures. Despite the genetic redundancy, only phaC1 and phaC2 were expressed at significant rates, while PHA accumulation in stationary-phase cultures was impaired only in the phaC1 mutant. Meanwhile, the phaC2 mutant produced more PHA than the wild type under this condition, and surprisingly, the phaC3 transcript increased in the phaC2 background. A double mutant, the phaC2 phaC3 mutant, consistently accumulated less PHA than the phaC2 mutant. PHA accumulation in nodule bacteroids followed a pattern similar to that seen in liquid cultures, being prevented in the phaC1 mutant and increased in the phaC2 mutant in relation to the level in the wild type. Therefore, we used these mutants, together with a phaC1 phaC2 double mutant, to study the B. japonicum PHA requirements for survival, competition for nodulation, and plant growth promotion. All mutants, as well as the wild type, survived for 60 days in a carbon-free medium, regardless of their initial PHA contents. When competing for nodulation against the wild type in a 1:1 proportion, the phaC1 and phaC1 phaC2 mutants occupied only 13 to 15% of the nodules, while the phaC2 mutant occupied 81%, suggesting that the PHA polymer is required for successful competitiveness. However, the bacteroid content of PHA did not affect the shoot dry weight accumulation.Fil: Quelas, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Mongiardini, Elias Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Pérez Giménez, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Parisi, Gustavo Daniel. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lodeiro, Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

Identification of hydroxyapatite spherules provides new insight into subretinal pigment epithelial deposit formation in the aging eye.

Accumulation of protein- and lipid-containing deposits external to the retinal pigment epithelium (RPE) is common in the aging eye, and has long been viewed as the hallmark of age-related macular degeneration (AMD). The cause for the accumulation and retention of molecules in the sub-RPE space, however, remains an enigma. Here, we present fluorescence microscopy and X-ray diffraction evidence for the formation of small (0.5-20 μm in diameter), hollow, hydroxyapatite (HAP) spherules in Bruch's membrane in human eyes. These spherules are distinct in form, placement, and staining from the well-known calcification of the elastin layer of the aging Bruch's membrane. Secondary ion mass spectrometry (SIMS) imaging confirmed the presence of calcium phosphate in the spherules and identified cholesterol enrichment in their core. Using HAP-selective fluorescent dyes, we show that all types of sub-RPE deposits in the macula, as well as in the periphery, contain numerous HAP spherules. Immunohistochemical labeling for proteins characteristic of sub-RPE deposits, such as complement factor H, vitronectin, and amyloid beta, revealed that HAP spherules were coated with these proteins. HAP spherules were also found outside the sub-RPE deposits, ready to bind proteins at the RPE/choroid interface. Based on these results, we propose a novel mechanism for the growth, and possibly even the formation, of sub-RPE deposits, namely, that the deposit growth and formation begin with the deposition of insoluble HAP shells around naturally occurring, cholesterol-containing extracellular lipid droplets at the RPE/choroid interface; proteins and lipids then attach to these shells, initiating or supporting the growth of sub-RPE deposits