Subject(s)
Lung Diseases/genetics , Lymphopenia/genetics , Primary Immunodeficiency Diseases/genetics , rac GTP-Binding Proteins/genetics , Adult , B-Lymphocytes/immunology , Disease Progression , GTPase-Activating Proteins/metabolism , Gain of Function Mutation , Graft vs Host Disease/drug therapy , Guanosine Triphosphate/metabolism , Hematopoietic Stem Cell Transplantation , Heterozygote , Humans , Immunologic Memory/immunology , Immunosuppressive Agents/therapeutic use , Infant , Lung Diseases/immunology , Lung Diseases/physiopathology , Lung Diseases/surgery , Lung Transplantation , Lymphopenia/immunology , Male , Molecular Docking Simulation , Neutrophils , Primary Immunodeficiency Diseases/immunology , Primary Immunodeficiency Diseases/therapy , Recurrence , Respiratory Tract Infections/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocytes/immunology , rac GTP-Binding Proteins/immunology , rac GTP-Binding Proteins/metabolism , rac GTP-Binding Proteins/ultrastructure , RAC2 GTP-Binding ProteinABSTRACT
Mevalonate kinase deficiency (MKD) is an autoinflammatory disorder caused by mutations in the MVK gene resulting in decreased activity of the enzyme mevalonate kinase (MK). Although MK is required for biosynthesis of all isoprenoids, in MKD, in particular, the timely synthesis of geranylgeranyl pyrophosphate appears to be compromised. Because small guanosine triphosphatases (GTPases) depend on geranylgeranylation for their proper signaling function, we studied the effect of MK deficiency on geranylgeranylation and activation of the two small GTPases, RhoA and Rac1. We demonstrate that both geranylgeranylation and activation of the two GTPases are more easily disturbed in MKD cells than in control cells when the flux though the isoprenoid biosynthesis pathway is suppressed by low concentrations of simvastatin. The limited capacity of geranylgeranylation in MKD cells readily leads to markedly increased levels of nonisoprenylated and activated GTPases, which will affect proper signaling by these GTPases.
Subject(s)
Fibroblasts/enzymology , Mevalonate Kinase Deficiency/enzymology , Phosphotransferases (Alcohol Group Acceptor)/deficiency , Protein Prenylation , rac1 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/metabolism , Case-Control Studies , Cell Line , Cell Membrane/enzymology , Enzyme Activation , Fibroblasts/drug effects , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Mevalonate Kinase Deficiency/genetics , Phosphotransferases (Alcohol Group Acceptor)/genetics , Protein Transport , Signal Transduction , Simvastatin/pharmacologyABSTRACT
The Structural Proteomics In Europe (SPINE) consortium contained a workpackage to address the automated X-ray analysis of macromolecules. The aim of this workpackage was to increase the throughput of three-dimensional structures while maintaining the high quality of conventional analyses. SPINE was able to bring together developers of software with users from the partner laboratories. Here, the results of a workshop organized by the consortium to evaluate software developed in the member laboratories against a set of bacterial targets are described. The major emphasis was on molecular-replacement suites, where automation was most advanced. Data processing and analysis, use of experimental phases and model construction were also addressed, albeit at a lower level.
Subject(s)
Crystallography, X-Ray/methods , Proteomics/methods , Algorithms , Automation , Data Interpretation, Statistical , Databases, Factual , Models, Chemical , Models, Molecular , Protein Conformation , Quality Control , SoftwareABSTRACT
In this report, we describe the validation of a rapid, single-step, microtiter plate method for quantifying bacterial adherence, based on fluorescent labeling of microorganisms with cell-permeable fluorescent DNA-binding probes. We have tested the binding to saliva-coated microtiter plates of bacteria, including Helicobacter pylori and viridans streptococci (S. mitis, S. gordonii, S. sanguis), known to interact with salivary components. Furthermore, we tested the short-term and longer-term temporal stability of a saliva-mediated adherence of these bacteria in a healthy population (N=30). The assay exhibited excellent reliability statistics, yielding within-assay variability coefficients ranging from 4.9% to 11%. A range of approximately 5 x 10(4)-1 x 10(7) cells could be detected. This method may be generally applicable to study surface binding of virtually any microbial species, while obviating the need of radioactive materials or specific antibodies for quantification, thus providing a procedure that is useful to both basic and clinical research.
