Regulatory Mutations Impacting Antibiotic Susceptibility in an Established Staphylococcus aureus Biofilm

We previously determined the extent to which mutations of different Staphylococcus aureus regulatory loci impact biofilm formation as assessed under in vitro conditions. Here we extend these studies to determine the extent to which those regulatory loci that had the greatest effect on biofilm formation also impact antibiotic susceptibility. The experiments were done under in vitro and in vivo conditions using two clinical isolates of S. aureus (LAC and UAMS-1) and two functionally diverse antibiotics (daptomycin and ceftaroline). Mutation of the staphylococcal accessory regulator (sarA) or sigB was found to significantly increase susceptibilities to both antibiotics and in both strains in a manner that could not be explained by changes in the MICs. The impact of a mutation in sarA was comparable to that of a mutation in sigB and greater than the impact observed with any other mutant. These results suggest that therapeutic strategies targeting sarA and/or sigB have the greatest potential to facilitate the ability to overcome the intrinsic antibiotic resistance that defines S. aureus biofilm-associated infections.     

In vitro autoradiographic and in vivo scintigraphic localization of somatostatin receptors in human lymphatic tissue

Receptors for the neuropeptide somatostatin (SS) were evaluated in vitro and in vivo in various human lymphatic tissues, ie, thymus, spleen, and lymph nodes; thymic carcinoids and thymomas were also tested. The receptors were measured in vitro using receptor autoradiography on tissue sections incubated with the SS analog 125I- [Tyr3]-octreotide or 125I-[Leu8,D-Trp22,Tyr25]-SS-28. All tissues were SS-receptor positive for either radioligand, except the thymomas. In thymic tissue, the receptors were diffusely located in the medulla, presumably on epithelial cells. In the spleen, the red pulp was strongly labeled. In the lymph nodes, the germinal centers were preferentially labeled. In all tissues, the receptors were of high affinity (kd thymus, 0.84 nmol/L; kd spleen, 1.6 nmol/L; kd lymph node, 0.62 nmol/L) and specific for SS. Displacement by nanomolar concentrations of SS-14, SS-28, and octreotide was observed, as was guanosine triphosphate dependency. The in vivo visualization of somatostatin receptors was performed after injection of 111In-DTPA- octreotide and gamma-camera scintigraphy. The spleen, but not thymus or lymph nodes, were visualized. These data suggest an important role for SS in regulating immune functions through SS receptors in thymus, spleen, and lymph nodes. Furthermore, SS may regulate neuroendocrine functions in the thymus.     

Characterization of a factor IX variant with a glycine207 to glutamic acid mutation

Factor IXTaipei9 is a factor IX variant from a hemophilia B patient with reduced levels of circulating protein molecules (cross-reacting material reduced, CRM). This variant contained a glycine (Gly) to glutamic acid (Glu) substitution at the 207th codon of mature factor IX. The functional consequences of the Gly-->Glu mutation in factor IXTaipei9 (IXG207E) were characterized in this study. Plasma-derived IXG207E exhibited a mobility similar to that of normal factor IX on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its specific activity was estimated to be 3.5% that of the purified normal factor IX in a one-stage partial thromboplastin time assay (aPTT). Cleavage of factor IXG207E by factor XIa or factor VIIa-tissue factor complex appeared to be normal. When the calcium-dependent conformational change was examined by monitoring quenching of intrinsic fluorescence, both normal factor IX and IXG207E exhibited equivalent intrinsic fluorescence quenching. Activated factor IXG207E (IXaG207E) also binds antithrombin III equally as well as normal factor IXa. However, aberrant binding of the active site probe p-aminobenzamidine was observed for factor XIa-activated factor IXG207E, indicating that the active site pocket of the heavy chain of factor IXaG207E was abnormal. Moreover, the rate of activation of factor X by factor IXaG207E, as measured in a purified system using chromogenic substrates, was estimated to be 1/40 of that of normal factor IXa. A computer-modeled heavy-chain structure of factor IXa predicts a hydrophobic environment surrounding Gly-207 and this Gly forms a hydrogen bound to the active site serine-365. The molecular mechanism of the Gly-->Glu mutation in factor IXTaipei9 might result in the alteration of the microenvironment of the active site pocket which renders the active site serine-365 inaccessible to its substrate.     

Mapping of monoclonal antibodies to human factor IX

We used recombinant DNA techniques to map a panel of six monoclonal antibodies (MoAbs) to regions of the human factor IX molecule. A-2 maps to 17 amino acids at the amino terminus of the heavy chain of IXa; 2D5, an inhibitor of clotting, is defined to 36 amino acids of the first EGF- like domain of human factor IX. A-4, A-5, C10D, and FXC008 all map to a region of the heavy chain containing amino acids 180 through 310, suggesting an immunodominant site. FXC008 has been reported to interfere with binding of factor IXa to factor VIII:Ca.     

DIAPHRAGMATIC PARALYSIS IN MOTOR NEURONE DISEASE: USE OF NON-INVASIVE INVESTIGATIVE AND THERAPEUTIC TECHNIQUES

SUMMARY Two cases of bilateral diaphragmatic weakness are described in which the condition was the presenting feature of motor neurone disease. Inspiratory muscle strength was assessed by a non-invasive technique involving measurements of pressures generated within the mouth. One patient with severe inspiratory muscle weakness is being treated with domiciliary nasal ventilation and has returned to a good-quality life. The other patient with less severe weakness has thus far required no ventilatory support.