Biochemical characterization of a novel ArsA ATPase complex from Alkaliphilus metalliredigens QYMF

The two putative ars operons in Alkaliphilus metalliredigens QYMF are distinctive in that the arsA gene is split in halves, amarsA1 and amarsA2, and, acr3 but not an arsB gene coexists with arsA. Heterologous expression of one of the A. metalliredigensars operons (ars1) conferred arsenite but not antimonite resistance to ΔarsEscherichia coli. Only the co-expressed AmArsA1 and AmArsA2 displayed arsenite or antimonite stimulated ATPase activity. The results show that AmArsA1-AmArsA2 interaction is needed to form the functional ArsA ATPase. This novel AmArsA1-AmArsA2 complex may provide insight in how it participates with Acr3 in arsenite detoxification.     

Design and synthesis of novel tri-aryl CB2 selective cannabinoid ligands

A novel series of cannabinoid ligands with a structurally unique tri-aryl core has been designed, synthesized and assayed. Receptor binding assays show that these compounds possess CB2 receptor sub-type selectivity with binding affinities ranging from 1.07 (±0.05) for 7 to 4.77 (±0.57) nM for 6. The selectivity of the compounds was enhanced 9–600-fold for the CB2 receptor over the CB1 receptor. The results of our present study identify a novel, highly selective cannabinoid scaffold with a non-classical core.     

Properties of Arsenite Efflux Permeases (Acr3) from Alkaliphilus metalliredigens and Corynebacterium glutamicum

Members of the Acr3 family of arsenite permeases confer resistance to trivalent arsenic by extrusion from cells, with members in every phylogenetic domain. In this study bacterial Acr3 homologues from Alkaliphilus metalliredigens and Corynebacterium glutamicum were cloned and expressed in Esch e richia coli. Modification of a single cysteine residue that is conserved in all analyzed Acr3 homologues resulted in loss of transport activity, indicating that it plays a role in Acr3 function. The results of treatment with thiol reagents suggested that the conserved cysteine is located in a hydrophobic region of the permease. A scanning cysteine accessibility method was used to show that Acr3 has 10 transmembrane segments, and the conserved cysteine would be predicted to be in the fourth transmembrane segment.Arsenic is a carcinogen that ranks first on the Superfund List of Hazardous Substances (www.atsdr.cdc.gov). As a consequence of its environmental ubiquity, nearly every organism, from bacteria to humans, has genes that confer resistance to arsenic (1). The most common mechanism of arsenite resistance is efflux from cells catalyzed by members of three unrelated families of transporters. Homologues of the Mrp members of the ATP-binding cassette superfamily catalyze ATP-dependent pumping of As(III)-thiol complexes out of the cytosol. These include Mrp1 and Mrp2 in mammals that extrude As(GS)₃ into blood or bile (2), Ycf1p in yeast that extrudes As(GS)₃ into the vacuole (3), and PgpA in Leishmania that extrudes the As(III)-trypanothione complex into intracellular compartments (4). These pumps are generalized resistance pumps and are not specific for arsenite. In contrast, ArsB, the first identified member of the second family of arsenite efflux proteins, has the physiological role of conferring resistance to inorganic As(III) and Sb(III) (5, 6). The best characterized member of the ArsB family is that encoded by the arsRDABC operon of the conjugative R-factor R773 of Escherichia coli. ArsB is widespread in bacteria and archaea. It has 12 membrane-spanning segments (7), which is similar to members of the Major Facilitator Superfamily (8). It transports As(III) but has higher affinity for Sb(III). ArsB is an antiporter that catalyzes the exchange of trivalent metalloid for protons, coupling arsenite efflux to the electrochemical proton gradient (9).The third arsenic resistance transporter is Acr3, which is a member of the BART (bile/arsenite/riboflavin transporter) superfamily and includes members found in bacteria, archaea, and fungi and is more widely distributed than members of the ArsB family (10) (supplemental Fig. 1). Homologues have recently been identified in plant (Pteris vittata, NCBI accession number {type:entrez-protein,attrs:{text:ACN65413,term_id:224814384,term_text:ACN65413}}ACN65413) and animal genomes (Danio rerio, NCBI accession number {type:entrez-protein,attrs:{text:XP_001921075,term_id:189546053,term_text:XP_001921075}}XP_001921075). Unfortunately, the literature is confused by the fact that many members of the Acr3 family are annotated as ArsB even though they exhibit no significant sequence similarity to ArsB. The first identified member of this family is encoded by the ars operon of the skin (sigK intervening) element in the chromosome of Bacillus subtilis (11). The membrane topology of the B. subtilis Acr3 was recently investigated using translational fusions, but the results could not distinguish between 8 and 10 transmembrane-spanning segments (TMs)² (12). Fungal members of this family include the Saccharomyces cerevisiae Acr3p metalloid efflux protein (3, 13). Interestingly, yeast Acr3p appears to be selective for As(III) over Sb(III), which is surprising considering the similarity in chemical properties between the two metalloids. The properties of a more distant homologue from Shewanella oneidensis was examined recently (14). The S. oneidensis homologue confers resistance to arsenate but not arsenite. Similarly, the purified protein binds arsenate, not arsenite, indicating that this protein is not an Acr3 orthologue.Here we examined the properties of Acr3 orthologues from Alkaliphilus metalliredigens and Corynebacterium glutamicum (supplemental Fig. 1). A. metalliredigens is a borate-tolerant Gram-positive alkaliphile and strict anaerobe that uses reduction of metals as electron acceptors (15). It is a novel metal-reducing bacterium that is distantly related to other commonly studied iron-reducing microorganisms. The genome of A. metalliredigens QYMF (NCBI accession number {type:entrez-nucleotide,attrs:{text:NC_009633,term_id:150387853,term_text:NC_009633}}NC_009633) contains two novel ars operons, arsR₁B_acr3–1D₁A_1–1A_1–2 and arsR₂CB_acr3–2D₂A_2–1A_2–2. The two genes for the AmAcr3s were designated ars_acr3 because they are both in ars operons and are controlled by ArsR repressors, even though they are not homologues of ArsB. Interestingly, both ars operons have genes for ArsD and two genes corresponding to the two homologous halves of ArsA, which we designate AmArsA1 and AmArsA2. ArsD is an arsenic chaperone that transfers As(III) to ArsA (16), which then interacts with ArsB to extrude As(III) from the cells in an ATP-dependent manner (6, 17, 18). Whether or how Acr3 can replace ArsB in this process is a question of considerable interest.C. glutamicum is a Gram-positive soil bacterium that is used for commercial production of glutamate, lysine, and other amino acids, nucleotides, and vitamins and from which the genome sequence has been described (NCBI accession number {type:entrez-nucleotide,attrs:{text:NC_006958,term_id:62388892,term_text:NC_006958}}NC_006958). It is highly arsenic-resistant and has three genes encoding Acr3 homologues (19). Two of the homologues are in ars operons regulated by ArsRs (arsR₁B_acr3–1C₁C_1′ and arsR₂B_acr3–2arsC₂) and a third orphan gene (arsB_acr3–3) that is not in an operon and may not be expressed to the same extent as the other two. (Again, the genes were misnamed arsB even though they encode Acr3 homologues.)The genes for AmAcr3 and CgAcr3 from the ars1 operons of the respective species were cloned and expressed in the arsenite-hypersensitive E. coli strain AW3110, in which the chromosomal arsRBC operon had been deleted (20). Both conferred resistance to arsenite but not arsenate or antimonite. Examination of the sequence of Acr3 homologues from many species indicates that there is conserved cysteine residues, Cys¹³⁸ in AmAcr3 and Cys¹²⁹ in CgAcr3 (supplemental Fig. 1). Those and other nonconserved cysteine residues were changed by mutagenesis, and substitution of only Cys¹³⁸ in AmAcr3 and Cys¹²⁹ in CgAcr3 led to loss of function, suggesting that the conserved cysteine residue participates in As(III) transport. A scanning cysteine accessibility method (SCAM) (21) was used to determine the transmembrane topology of AmAcr3. SCAM analysis is preferable to the use of gene fusions because there are minimal structural changes in the membrane protein, and the sidedness of inserted cysteines can be unambiguously determined with maleimide reagents of differing membrane permeability. A series of single cysteine mutants of AmAcr3 was constructed and the reactivity of each cysteine residue assayed. The results unambiguously demonstrate that Acr3 has 10 TMs.     

Structural assembly of two-domain proteins by rigid-body docking

Background  

Modelling proteins with multiple domains is one of the central challenges in Structural Biology. Although homology modelling has successfully been applied for prediction of protein structures, very often domain-domain interactions cannot be inferred from the structures of homologues and their prediction requiresab initiomethods. Here we present a new structural prediction approach for modelling two-domain proteins based on rigid-body domain-domain docking.     

Efficient micropropagation and chlorocholine chloride induced stevioside production of Stevia rebaudiana Bertoni

A promising method of micropropagation of Stevia rebaudiana Bertoni has been developed with an aim to increase the biomass, survivability of the plantlets and stevioside production, using chlorocholine chloride (CCC). Microshoots transferred to the MS medium containing different combinations CCC and IBA were found to be most effective in terms of growth pattern, hardening ability of the plantlets and stevioside content, compared to MS medium containing either IBA or CCC. Among other combinations tested, MS medium supplemented with 3 mg/l CCC and 3 mg/l IBA was found most effective in inducing significant changes like reduced shoot length, increased number of roots, higher leaf size, increased biomass and chlorophyll retaining capacity, higher survival percentage and most importantly the elevated stevioside content. Collectively, the major observations of this research indicate that application of CCC in micropropagation of S. rebaudiana Bertoni is a promising approach and has commercial prospects.     

Crosstalk between medulloblastoma cells and endothelium triggers a strong chemotactic signal recruiting T lymphocytes to the tumor microenvironment

Cancer cells can live and grow if they succeed in creating a favorable niche that often includes elements from the immune system. While T lymphocytes play an important role in the host response to tumor growth, the mechanism of their trafficking to the tumor remains poorly understood. We show here that T lymphocytes consistently infiltrate the primary brain cancer, medulloblastoma. We demonstrate, both in vitro and in vivo, that these T lymphocytes are attracted to tumor deposits only after the tumor cells have interacted with tumor vascular endothelium. Macrophage Migration Inhibitory Factor (MIF)" is the key chemokine molecule secreted by tumor cells which induces the tumor vascular endothelial cells to secrete the potent T lymphocyte attractant "Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES)." This in turn creates a chemotactic gradient for RANTES-receptor bearing T lymphocytes. Manipulation of this pathway could have important therapeutic implications.     

Regulation of acetyl-CoA synthetase of Saccharomyces cerevisiae.

Acetyl-coenzyme A synthetase (EC 6.2.1.1) activity of Saccharomyces cerevisiae was determined by a radioactive assay procedure. The activity in vitro was inhibited significantly by NADPH, NADH, or AMP and to a lesser extent by NADP, NAD, or ADP. Glutamic acid and alpha-ketoglutaric acid were not inhibitory. The enzyme level was repressed when the cells were grown in a complex nutrient medium as opposed to the minimal medium. However, a glutamic acid auxotroph glul, when grown in excess glutamic acid, demonstrated a fivefold increase of acetyl-CoA synthetase.     

High-Glucose-Induced Endothelial Cell Injury Is Inhibited by a Peptide Derived from Human Apolipoprotein E

Although the importance of human apolipoprotein E (apoE) in vascular diseases has clearly been established, most of the research on apoE has focused on its role in cholesterol metabolism. In view of the observation that apoE and its functional domains impact extracellular matrix (ECM) remodeling, we hypothesized that apoE could also confer protection against ECM degradation by mechanisms independent of its role in cholesterol and lipoprotein transport. The ECM degrading enzyme, heparanase, is secreted by cells as pro-heparanase that is internalized through low-density lipoprotein (LDL) receptor-related protein-1 (LRP-1) to become enzymatically active. Both apoE and pro-heparanase bind the LRP-1. We further hypothesized that an apoE mimetic peptide (apoEdp) would inhibit the production of active heparanase by blocking LRP-1-mediated uptake of pro-heparanase and thereby decrease degradation of the ECM. To test this hypothesis, we induced the expression of heparanase by incubating human retinal endothelial cells (hRECs) with high glucose (30 mM) for 72 hours. We found that elevated expression of heparanase by high glucose was associated with increased shedding of heparan sulfate (ΔHS) and the tight junction protein occludin. Treatment of hRECs with 100 µM apoEdp in the presence of high glucose significantly reduced the expression of heparanase, shedding of ΔHS, and loss of occludin as detected by Western blot analysis. Either eye drop treatment of 1% apoEdp topically 4 times a day for 14 consecutive days or intraperitoneal injection (40 mg/kg) of apoEdp daily for 14 consecutive days in an in vivo mouse model of streptozotocin-induced diabetes inhibited the loss of tight junction proteins occludin and zona occludin- 1 (ZO-1). These findings imply a functional relationship between apoE and endothelial cell matrix because the deregulation of these molecules can be inhibited by a short peptide derived from the receptor-binding region of apoE. Thus, strategies targeting ECM-degrading enzymes could be therapeutically beneficial for treating diabetic retinopathy.