Localization and organization of phenol degradation genes ofPseudomonas putida strain H

The genetic organization of the DNA region encoding the phenol degradation pathway ofPseudomonas putida H has been investigated. This strain can utilize phenol or some of its methylated derivatives as its sole source of carbon and energy. The first step in this process is the conversion of phenol into catechol. Catechol is then further metabolized via themeta-cleavage pathway into TCA cycle intermediates. Genes encoding these enzymes are clustered on the plasmid pPGH1. A region of contiguous DNA spanning about 16 kb contains all of the genetic information necessary for inducible phenol degradation. The analysis of mutants generated by insertion of transposons and cassettes indicates that all of the catabolic genes are contained in a single operon. This codes for a multicomponent phenol hydroxylase andmeta-cleavage pathway enzymes. Catabolic genes are subject to positive control by the gene product(s) of a second locus.     

Regulatory conversation between antigen presenting cells and regulatory T cells enhance immune suppression

Regulatory T cells (Treg) were originally described by their suppressive function exerted on effector T cells, but recent evidence also reveals interactions with antigen presenting cells (APCs). In general, all major subpopulations of APCs, i.e., dendritic cells (DC), B cells and monocytes/macrophages (Mvarphi), respond to exposure to Treg by down regulation of their antigen presenting function, upregulation of immunosuppressive molecules and secretion of immunosuppressive cytokines. Thus, Treg gain influence on the innate immune system and are able to augment their immunosuppressive capacities by blocking the effective priming of T effector cells by APCs. Conversely, APCs have an important role in nurturing peripheral Treg populations, since it has been shown that immature DC, as well as alternatively activated Mvarphi, are able to induce Treg de novo. These properties are dependent on the expression of surface molecules (CTLA-4, F4/80) and the production of soluble factors such as IL-10 and Indoleamine 2,3-dioxygenase by the APC subpopulations. On the whole, the mutual interaction of Treg and APCs enables Treg to sustain their immunosuppressive functions which, in healthy individuals, may be crucial for the maintenance of peripheral tolerance.     

Tn5: A molecular window on transposition

DNA transposition is an underlying process involved in the remodeling of genomes in all types of organisms. We analyze the multiple steps in cut-and-paste transposition using the bacterial transposon Tn5 as a model. This system is particularly illuminating because of the existence of structural, genetic, and biochemical information regarding the two participating specific macromolecules: the transposase and the 19-bp sequences that define the ends of the transposon. However, most of the insights should be of general interest because of similarities to other transposition-like systems such as HIV-1 DNA integration into the host genome.     

Rapid in vitro biocompatibility assay of endovascular stents by flow cytometry using platelet activation and platelet-leukocyte aggregation

Clinical studies suggest that stent design and surface texture are responsible for differences in biocompatibility of metallic endovascular stents. A simple in vitro experimental setup was established to test stent-induced degree of platelet and leukocyte activation and platelet-leukocyte aggregation by flow cytometry. Heparin-coated tantalum stents and gold-coated and uncoated stainless steel stents were tested. Stents were implanted into silicone tubes and exposed to blood from healthy volunteers. Platelet and leukocyte activation and percentage of leukocyte-platelet aggregates were determined in a whole-blood assay by subsequent staining for activation-associated antigens (CD41a, CD42b, CD62p, and fibrinogen binding) and leukocyte antigens (CD14 and CD45) and flow cytometric analysis. Blood taken directly after venous puncture or exposed to the silicone tube alone was used as negative controls. Positive control was in vitro stimulation with thrombin receptor activating peptide (TRAP-6). Low degree of platelet activation and significant increase in monocyte- and neutrophil-platelet aggregation were observed in blood exposed to stents (P < 0.05). In addition, leukocyte activation was induced as measured by increased CD45 and CD14 expression. Heparin coated stents continuously induced less platelet activation and leukocyte-platelet aggregation than uncoated stainless steel stents of the same length and shorter stents of the same structure. Stent surface coating and texture plays a role in platelet and leukocyte activation and leukocyte-platelet aggregation. Using this simple in vitro assay and whole blood and flow cytometry, it seems possible to differentiate stents by their potency to activate platelets and/or leukocytes. This assay could be applied for improving the biocompatibility of coronary stents.     

The dendritic cell receptor for endocytosis, DEC-205, can recycle and enhance antigen presentation via major histocompatibility complex class II-positive lysosomal compartments

Many receptors for endocytosis recycle into and out of cells through early endosomes. We now find in dendritic cells that the DEC-205 multilectin receptor targets late endosomes or lysosomes rich in major histocompatibility complex class II (MHC II) products, whereas the homologous macrophage mannose receptor (MMR), as expected, is found in more peripheral endosomes. To analyze this finding, the cytosolic tails of DEC-205 and MMR were fused to the external domain of the CD16 Fcgamma receptor and studied in stable L cell transfectants. The two cytosolic domains each mediated rapid uptake of human immunoglobulin (Ig)G followed by recycling of intact CD16 to the cell surface. However, the DEC-205 tail recycled the CD16 through MHC II-positive late endosomal/lysosomal vacuoles and also mediated a 100-fold increase in antigen presentation. The mechanism of late endosomal targeting, which occurred in the absence of human IgG, involved two functional regions: a membrane-proximal region with a coated pit sequence for uptake, and a distal region with an EDE triad for the unusual deeper targeting. Therefore, the DEC-205 cytosolic domain mediates a new pathway of receptor-mediated endocytosis that entails efficient recycling through late endosomes and a greatly enhanced efficiency of antigen presentation to CD4(+) T cells.     

Direct Fluorescent-Antibody Technique for the Microbiological Examination of Food and Environmental Swab Samples for Salmonellae

Comparative studies of a modified fluorescent-antibody procedure and the 5 to 7 day method used by the Association of Official Analytical Chemists for the detection of Salmonella were made on 151 samples of wheat products and 183 swab samples. The agreement between the two methods for the 334 samples tested was 92.5%. Food samples yielded 94.7% agreement, whereas the swab samples yielded 90.7% agreement. There were 7.5% false positives for the total number of samples tested. No false negatives were obtained by using the fluorescent-antibody method.     

Transfection of immature murine bone marrow-derived dendritic cells with the granulocyte-macrophage colony-stimulating factor gene potently enhances their in vivo antigen-presenting capacity.

Ag presentation by dendritic cells (DC) is crucial for induction of primary T cell-mediated immune responses in vivo. Because DC culture from blood or bone marrow-derived progenitors is now clinically applicable, this study investigated the effectiveness of in vitro-generated murine bone marrow-derived DC (Bm-DC) for in vivo immunization protocols. Previous studies demonstrated that GM-CSF is an essential growth and differentiation factor for DC in culture and that in vivo administration of GM-CSF augments primary immune responses, which renders GM-CSF an attractive candidate to further enhance the effectiveness of DC-based immunotherapy protocols. Therefore, immature Bm-DC were transiently transfected with the GM-CSF gene and tested for differentiation, migration, and Ag-presenting capacity in vitro and in vivo. In vitro, GM-CSF gene-transfected Bm-DC were largely unaltered with regard to MHC and costimulatory molecule expression as well as alloantigen or peptide Ag-presenting capacity. When used for in vivo immunizations, however, the Ag-presenting capacity of GM-CSF gene-transfected Bm-DC was greatly enhanced compared with mock-transfected or untransfected cells, as determined by their effectiveness to induce primary immune reactions against hapten, protein Ag, and tumor Ag, respectively. Increased effectiveness in vivo correlated with the better migratory capacity of GM-CSF gene-transfected Bm-DC. These results show that GM-CSF gene transfection significantly enhances the capacity of DC to induce primary immune responses in vivo, which might also improve DC-based vaccines currently under clinical investigation.     

Rapid, Direct Fluorescent-Antibody Method for the Detection of Salmonellae in Food and Feeds

An improved immunofluorescent-antibody (FA) method for the detection of salmonellae in foods and feeds was developed. This FA method combines a rapid cultural phase and a serological phase that allow for propagation of salmonellae in a minimum time, employing the industrial 8-hr work day as a guide. Two hundred fifty naturally contaminated human food and animal feed samples, representing 647 trials, were tested by the FA method. A total of 18 different food and feed samples was used. The method used by the Association of Official Analytical Chemists (AOAC) for the detection of salmonellae was the control method. The percent agreement when comparing the FA slide method to the AOAC method ranged from 87.1 to 95.3%, depending upon the conjugated antisera used in comparative studies.     

Membrane association, mechanism of action, and structure of Arabidopsis embryonic factor 1 (FAC1)

Embryonic factor 1 (FAC1) is one of the earliest expressed plant genes and encodes an AMP deaminase (AMPD), which is also an identified herbicide target. This report identifies an N-terminal transmembrane domain in Arabidopsis FAC1, explores subcellular fractionation, and presents a 3.3-A globular catalytic domain x-ray crystal structure with a bound herbicide-based transition state inhibitor that provides the first glimpse of a complete AMPD active site. FAC1 contains an (alpha/beta)(8)-barrel characterized by loops in place of strands 5 and 6 that places it in a small subset of the amidohydrolase superfamily with imperfect folds. Unlike tetrameric animal orthologs, FAC1 is a dimer and each subunit contains an exposed Walker A motif that may be involved in the dramatic combined K(m) (25-80-fold lower) and V(max) (5-6-fold higher) activation by ATP. Normal mode analysis predicts a hinge motion that flattens basic surfaces on each monomer that flank the dimer interface, which suggests a reversible association between the FAC1 globular catalytic domain and intracellular membranes, with N-terminal transmembrane and disordered linker regions serving as the anchor and attachment to the globular catalytic domain, respectively.