RP 59500 prophylaxis of experimental endocarditis due to erythromycin-susceptible and -resistant isogenic pairs of viridans group streptococci.

RP 59500 is a new injectable streptogramin composed of two synergistic components (quinupristin and dalfopristin) which are active against a number of erythromycin-susceptible and -resistant gram-positive bacteria. The following experiments investigate the ability of RP 59500 to prevent experimental endocarditis due to either of two erythromycin-susceptible streptococcal isolates or their constitutively erythromycin-resistant Tn916 delta E transconjugants. RP 59500 had low MICs (0.125 to 0.5 mg/liter) for all four test organisms and was substantially bactericidal in vitro. Rats with catheter-induced aortic vegetations were given single-dose antibiotic prophylaxis 30 to 60 min before bacterial inoculation through a computerized pump system which permitted the simulation of drug kinetics for humans produced by either 7 mg of RP 59500 per kg of body weight or 1 g of vancomycin. Single-dose RP 59500 prophylaxis successfully prevented endocarditis due to both the erythromycin-susceptible parent strains and their erythromycin-resistant derivatives in rats challenged with the minimal inoculum infecting 90% of controls. In addition, RP 59500 also prevented infection in animals challenged with fivefold-larger inocula of the erythromycin-susceptible parent strains. Vancomycin successfully prevented endocarditis due to any of the four test organisms. These results underline the in vivo efficacy of RP 59500 against both erythromycin-susceptible and -resistant streptococci. Such good results against the resistant strains would not be expected with erythromycin or clindamycin, which are the standard macrolidelincosamide-streptogramin antibiotics used for endocarditis prophylaxis in humans. An oral form of RP 59500 which might advantageously replace some of the older prophylactic regimens is currently being developed.

Determinants for membrane association of the hepatitis C virus NS2 protease domain.

Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is required for HCV polyprotein processing and particle assembly. It comprises an N-terminal membrane domain and a C-terminal, cytosolically oriented protease domain. Here, we demonstrate that the NS2 protease domain itself associates with cellular membranes. A single charged residue in the second α-helix of the NS2 protease domain is required for proper membrane association, NS2 protein stability, and efficient HCV polyprotein processing.

Descemet membrane detachment after nonpenetrating filtering surgery.

PURPOSE: To make surgeons performing nonpenetrating filtering surgery aware of an unusual complication namely Descemet membrane detachment. METHODS: We retrospectively reviewed nine eyes of nine patients seen in our hospital with Descemet membrane detachment occurring after nonpenetrating filtering surgery from January 1994 to December 2000. RESULTS: Both planar and nonplanar detachments were reported. Neither scrolls nor tears in the Descemet membrane were observed in any patient. After viscocanalostomy (four patients), the detachment was generally noticed shortly after the procedure and the cornea maintained its clarity. After deep sclerectomy with a collagen implant (five patients), it developed weeks to months postoperatively with adjacent corneal edema. Four patients had descemetopexy. None required more than one procedure. However, at the last visit, two detachments persisted although they had diminished in size: one after viscocanalostomy and conservative treatment and one after descemetopexy after deep sclerectomy with a collagen implant. To date otherwise, no signs of significant corneal damage could be observed clinically nor by specular microscopy and pachymetry. CONCLUSIONS: The diagnosis of Descemet membrane detachment can be easily overlooked or misdiagnosed. The clinical presentation, clinical course, and pathogenesis depend on the type of nonpenetrating filtering surgery performed. Ophthalmologists should be aware of this unusual complication, which is likely to be more common after nonpenetrating filtering surgery than after trabeculectomy. A period of observation before attempting descemetopexy is recommended.

Structural determinants for membrane association and dynamic organization of the hepatitis C virus NS3-4A complex.

Hepatitis C virus (HCV) NS3-4A is a membrane-associated multifunctional protein harboring serine protease and RNA helicase activities. It is an essential component of the HCV replication complex and a prime target for antiviral intervention. Here, we show that membrane association and structural organization of HCV NS3-4A are ensured in a cooperative manner by two membrane-binding determinants. We demonstrate that the N-terminal 21 amino acids of NS4A form a transmembrane alpha-helix that may be involved in intramembrane protein-protein interactions important for the assembly of a functional replication complex. In addition, we demonstrate that amphipathic helix alpha(0), formed by NS3 residues 12-23, serves as a second essential determinant for membrane association of NS3-4A, allowing proper positioning of the serine protease active site on the membrane. These results allowed us to propose a dynamic model for the membrane association, processing, and structural organization of NS3-4A on the membrane. This model has implications for the functional architecture of the HCV replication complex, proteolytic targeting of host factors, and drug design.

Sequential analysis of trans-SNARE formation in intracellular membrane fusion.

SNARE complexes are required for membrane fusion in the endomembrane system. They contain coiled-coil bundles of four helices, three (Q(a), Q(b), and Q(c)) from target (t)-SNAREs and one (R) from the vesicular (v)-SNARE. NSF/Sec18 disrupts these cis-SNARE complexes, allowing reassembly of their subunits into trans-SNARE complexes and subsequent fusion. Studying these reactions in native yeast vacuoles, we found that NSF/Sec18 activates the vacuolar cis-SNARE complex by selectively displacing the vacuolar Q(a) SNARE, leaving behind a Q(bc)R subcomplex. This subcomplex serves as an acceptor for a Q(a) SNARE from the opposite membrane, leading to Q(a)-Q(bc)R trans-complexes. Activity tests of vacuoles with diagnostic distributions of inactivating mutations over the two fusion partners confirm that this distribution accounts for a major share of the fusion activity. The persistence of the Q(bc)R cis-complex and the formation of the Q(a)-Q(bc)R trans-complex are both sensitive to the Rab-GTPase inhibitor, GDI, and to mutations in the vacuolar tether complex, HOPS (HOmotypic fusion and vacuolar Protein Sorting complex). This suggests that the vacuolar Rab-GTPase, Ypt7, and HOPS restrict cis-SNARE disassembly and thereby bias trans-SNARE assembly into a preferred topology.

Vaccination of melanoma patients with Melan-A/Mart-1 peptide and Klebsiella outer membrane protein p40 as an adjuvant.

Toll-like receptor ligands are potentially useful adjuvants for the development of clinical T cell vaccination. Here we investigated the novel Toll-like receptor2 ligand P40, the outer membrane protein A derived from Klebsiella pneumoniae. Seventeen human leukocyte antigen-A*0201 positive stage III/IV melanoma patients were vaccinated with P40 and Melan-A/Mart-1 peptide subcutaneously in monthly intervals. Adverse reactions were mild-to-moderate. Fourteen patients received at least 8 vaccinations and were thus evaluable for clinical tumor and immune responses. Seven patients experienced progressive disease, whereas 2 patients had stable disease throughout the trial period, 1 of them with regression of multiple skin metastases. The remaining 5 patients had no measurable disease. Melan-A/Mart-1 specific CD8 T cells were analyzed ex vivo, with positive results in 6 of 14 evaluable patients. Increased percentages of T cells were found in three patients, memory/effector T cell differentiation in 4 patients, and a positive interferon-gamma Elispot assay in 1 patient. Antibody responses to P40 were observed in all patients. We conclude that vaccination with peptide and P40 was feasible and induced ex vivo detectable tumor antigen specific T cell responses in 6 of 14 patients with advanced melanoma.

Cytotoxic T cells deficient in both functional fas ligand and perforin show residual cytolytic activity yet lose their capacity to induce lethal acute graft-versus-host disease.

Graft-versus-host disease (GVHD) is the main complication after allogeneic bone marrow transplantation. Although the tissue damage and subsequent patient mortality are clearly dependent on T lymphocytes present in the grafted inoculum, the lethal effector molecules are unknown. Here, we show that acute lethal GVHD, induced by the transfer of splenocytes from C57BL/6 mice into sensitive BALB/c recipients, is dependent on both perforin and Fas ligand (FasL)-mediated lytic pathways. When spleen cells from mutant mice lacking both effector molecules were transferred to sublethally irradiated allogeneic recipients, mice survived. Delayed mortality was observed with grafted cells deficient in only one lytic mediator. In contrast, protection from lethal acute GVHD in resistant mice was exclusively perforin dependent. Perforin-FasL-deficient T cells failed to lyse most target cells in vitro. However, they still efficiently killed tumor necrosis factor alpha-sensitive fibroblasts, demonstrating that cytotoxic T cells possess a third lytic pathway.

Prévention des adhérences péricardiques avec une membrane biorésorbable [Prevention of pericardial adhesions with a bioresorbable membrane]

A bioresorbable membrane made of sodium hyaluronate and carboxymethycellulose, has been reported to prevent peritoneal adhesion. This study was designed to test its efficiency in the prevention of pericardial adhesions. Two groups of six pigs (mean weight 72 +/- 8 kg) were chosen for the experiment. The heart was exposed through a left thoracotomy and a wide patch of pericardium was excised. In the test group (n = 6), the left ventricular area without pericardium was divided into two areas: area A where six stitches of Prolene were performed, and area B which was left intact. The membrane was applied on the both areas as well as on the adjacent area covered with pericardium (area C). In the control group (n = 6), the same protocol was performed except for the membrane application. The animals were sacrificed one month later. The adhesion status as well as the visibility of the coronary anatomy was assessed according to severity scores ranging from 0 to 3 for the adhesions and from 0 to 2 for the visibility. The difference between groups was considered significant when p < 0.05. The adhesion score of the area A was 1.7 +/- 0.5 in the test group versus 2.5 +/- 0.5 in the control group (p = 0.02) and the visibility score was 1.3 +/- 0.8 and 2 +/- 0 respectively (p = 0.07). In the area B, the adhesion score was 1 +/- 0 in the test group versus 2 +/- 0.6 in the control group (p = 0.03) and the visibility score was 0.7 +/- 0.5 and 2 +/- 0 respectively (p = 0.001). Lastly, in the area C, the adhesion score was 1 +/- 0 in both groups (n.s.) and the visibility score was 0.7 +/- 0.4 in the test group versus 0.5 +/- 0.5 in the control group (n.s.). In this animal model, the role of the bioresorbable membrane in the prevention of pericardial adhesions is limited to the areas without pericardial cover and without foreign material. The presence of foreign material neutralizes its effect.

Compartmentalization in membrane rafts defines a pool of N-cadherin associated with catenins and not engaged in cell-cell junctions in melanoma cells.

Melanoma progression is associated with changes in adhesion receptor expression, in particular upregulation of N-cadherin which promotes melanoma cell survival and invasion. Plasma membrane lipid rafts contribute to the compartmentalization of signaling complexes thereby regulating their function, but how they may affect the properties of adhesion molecules remains elusive. In this study, we addressed the question whether lipid rafts in melanoma cells may contribute to the compartmentalization of N-cadherin. We show that a fraction of N-cadherin in a complex with catenins is associated with cholesterol/sphingolipid-rich membrane microdomains in aggressive melanoma cells in vitro and experimental melanomas in vivo. Partitioning of N-cadherin in membrane rafts is not modulated by growth factors and signaling pathways relevant to melanoma progression, is not necessary for cell-cell junctions' establishment or maintenance, and is not affected by cell-cell junctions' and actin cytoskeleton disruption. These results reveal that two independent pools of N-cadherin exist on melanoma cell surface: one pool is independent of lipid rafts and is engaged in cell-cell junctions, while a second pool is localized in membrane rafts and does not participate in cell-cell adhesions. Targeting to membrane rafts may represent a previously unrecognized mechanism regulating N-cadherin function in melanoma cells.

A phosphorylation cycle shapes gradients of the DYRK family kinase Pom1 at the plasma membrane.

Concentration gradients regulate many cell biological and developmental processes. In rod-shaped fission yeast cells, polar cortical gradients of the DYRK family kinase Pom1 couple cell length with mitotic commitment by inhibiting a mitotic inducer positioned at midcell. However, how Pom1 gradients are established is unknown. Here, we show that Tea4, which is normally deposited at cell tips by microtubules, is both necessary and, upon ectopic cortical localization, sufficient to recruit Pom1 to the cell cortex. Pom1 then moves laterally at the plasma membrane, which it binds through a basic region exhibiting direct lipid interaction. Pom1 autophosphorylates in this region to lower lipid affinity and promote membrane release. Tea4 triggers Pom1 plasma membrane association by promoting its dephosphorylation through the protein phosphatase 1 Dis2. We propose that local dephosphorylation induces Pom1 membrane association and nucleates a gradient shaped by the opposing actions of lateral diffusion and autophosphorylation-dependent membrane detachment.

Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response.

Mice with homologous disruption of the gene coding for either the p35 subunit or the p40 subunit of interleukin-12 (IL-12) and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in resistance to infection and the differentiation of functional CD4+ T cell subsets in vivo. Wild-type 129/Sv/Ev mice are resistant to infection with L. major showing only small lesions which resolve spontaneously within a few weeks and develop a type 1 CD4+ T cell response. In contrast, mice lacking bioactive IL-12 (IL-12p35-/- and IL-12p40-/-) developed large, progressing lesions. Whereas resistant mice were able to mount a delayed-type hypersensitivity (DTH) response to Leishmania antigen, susceptible BALB/c mice as well as IL-12-deficient 129/Sv/Ev mice did not show any DTH reaction. To characterize the functional phenotype of CD4+ T cells triggered in infected wild-type mice and IL-12-deficient mice, the expression of mRNA for interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in purified CD4+ lymph node cells was analyzed. Wild-type 129/Sv/Ev mice showed high levels of mRNA for IFN-gamma and low levels of mRNA for IL-4 which is indicative of a Th1 response. In contrast, IL-12- deficient mice and susceptible BALB/c mice developed a strong Th2 response with high levels of IL-4 mRNA and low levels of IFN-gamma mRNA in CD4+ T cells. Similarly, lymph node cells from infected wild-type 129 mice produced predominantly IFN-gamma in response to stimulation with Leishmania antigen in vitro whereas lymph node cells from IL-12-deficient mice and susceptible BALB/c mice produced preferentially IL-4. Taken together, these results confirm in vivo the importance of IL-12 in induction of Th1 responses and protective immunity against L. major.

Overcoming the heterologous bias: an in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata.

We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplished by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the artefactual concerns encountered in using heterologous systems are totally excluded.

Usefulness of double locus sequence typing (DLST) for regional and international epidemiological surveillance of methicilin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial infections worldwide. To differentiate reliably among S. aureus isolates, we recently developed double locus sequence typing (DLST) based on the analysis of partial sequences of clfB and spa genes. In the present study, we evaluated the usefulness of DLST for epidemiological investigations of MRSA by routinely typing 1242 strains isolated in Western Switzerland. Additionally, particular local and international collections were typed by pulsed field gel electrophoresis (PFGE) and DLST to check the compatibility of DLST with the results obtained by PFGE, and for international comparisons. Using DLST, we identified the major MRSA clones of Western Switzerland, and demonstrated the close relationship between local and international clones. The congruence of 88% between the major PFGE and DLST clones indicated that our results obtained by DLST were compatible with earlier results obtained by PFGE. DLST could thus easily be incorporated in a routine surveillance procedure. In addition, the unambiguous definition of DLST types makes this method more suitable than PFGE for long-term epidemiological surveillance. Finally, the comparison of the results obtained by DLST, multilocus sequence typing, PFGE, Staphylococcal cassette chromosome mec typing and the detection of Panton-Valentine leukocidin genes indicated that no typing scheme should be used on its own. It is only the combination of data from different methods that gives the best chance of describing precisely the epidemiology and phylogeny of MRSA.