Differential inhibition of 30S and 70S translation initiation complexes on leaderless mRNA by kasugamycin

In contrast to canonical mRNAs, translation of leaderless mRNA has been previously reported to continue in the presence of the antibiotic kasugamycin. Here, we have studied the effect of the antibiotic on determinants known to affect translation of leadered and leaderless mRNAs. Kasugamycin did not affect the Shine-Dalgarno (SD)-anti-SD (aSD) interaction or the function of translation initiation factor 3 (IF3). Thus, the preferential translation of leaderless mRNA in the presence of kasugamycin can neither be attributed to an expanding pool of 30S subunits with a "blocked" aSD nor to a lack of action of IF3, which has been shown to discriminate against translation initiation at 5'-terminal start codons. Using toeprinting, we observed that on leaderless mRNA 70S in contrast to 30S translation initiation complexes are comparatively resistant to the antibiotic. These results taken together with the known preference of 70S ribosomes for 5'-terminal AUGs lend support to the hypothesis that translation of leaderless mRNAs may as well proceed via an alternative initiation pathway accomplished by intact 70S ribosomes.     

Drebrin particles: components in the ensemble of proteins regulating actin dynamics of lamellipodia and filopodia

Drebrin, an actin-binding 70-kDa protein with an unusually slow SDS-PAGE mobility corresponding to approximately 120 kDa, containing a proline-rich, profilin-binding motif, had originally been reported from neuronal cells, but recently has also been found in diverse other kinds of tissues and cell lines. In biochemical analyses of various cells and tissues, employing gel filtration, sucrose gradient centrifugation, immunoprecipitation and -blotting, we have identified distinct states of soluble drebrin: a approximately 4S monomer, an 8S, ca. 217-kDa putative trimer, a 13S and a > 20S oligomer. In the 8S particles only [35S]methionine-labelled drebrin but no other actin-binding protein has been detected in stoichiometric amounts. By immunofluorescence and immunoelectron microscopy, drebrin-positive material often appeared as "granules" up to 400 nm in diameter, in some cell types clustered near the Golgi apparatus or in lamellipodia, particularly at leading edges, or in dense-packed submembranous masses at tips (acropodia) or ruffles of leading edges, in filopodia and at plaques of adhering junctions. We conclude that these drebrin complexes and drebrin-rich structures allow the build-up and maintenance of high local drebrin concentrations in strategic positions for the regulation of actin filament assembly, thereby contributing to cell motility and morphology, in particular local changes of plasticity and the formation of protrusions.     

Attractive interhelical electrostatic interactions in the proline- and acidic-rich region (PAR) leucine zipper subfamily preclude heterodimerization with other basic leucine zipper subfamilies

Basic region-leucine zipper (B-ZIP) proteins homo- or heterodimerize to bind sequence-specific double-stranded DNA. We present circular dichroism (CD) thermal denaturation data on vitellogenin promoter-binding protein (VBP), a member of the PAR subfamily of B-ZIP proteins that also includes thyroid embryonic factor, hepatocyte leukemia factor, and albumin site D-binding protein. VBP does not heterodimerize with B-ZIP domains from C/EBP alpha, JUND, or FOS. We describe a dominant negative protein, A-VBP, that contains the VBP leucine zipper and an acidic amphipathic protein sequence that replaces the basic region critical for DNA binding. The acidic extension forms a coiled coil structure with the VBP basic region in the VBP.A-VBP heterodimer. This new alpha-helical structure extends the leucine zipper N-terminally, stabilizing the complex by 2.0 kcal/mol. A-VBP abolishes DNA binding of VBP in an equimolar competition assay, but does not affect DNA binding even at 100-fold excess of CREB, C/EBP alpha, or FOS/JUND. Likewise, proteins containing the acidic extension appended to seven other leucine zippers do not inhibit VBP DNA binding. We show that conserved g <--> e' or i, i' +5 salt bridges are sufficient to confer specificity to VBP by mutating the C/EBPalpha leucine zipper to contain the g <--> e' salt bridges that characterize VBP. A-VBP heterodimerizes with this mutant C/EBP, preventing it from binding to DNA. These conserved g <--> e' electrostatic interactions define the specificity of the PAR subfamily of B-ZIP proteins and preclude interaction with other B-ZIP subfamilies.     

Microbial Characterization of Biological Filters Used for Drinking Water Treatment

The impact of preozonation and filter contact time (depth) on microbial communities was examined in drinking water biofilters treating Ohio River water which had undergone conventional treatment (coagulation, flocculation, sedimentation) or solutions of natural organic matter isolated from groundwater (both ozonated and nonozonated). With respect to filter depth, compared to filters treating nonozonated waters, preozonation of treated water led to greater differences in community phospholipid fatty acid (PLFA) profiles, utilization of sole carbon sources (Biolog), and arbitrarily primed PCR fingerprints. PLFA profiles indicated that there was a shift toward anaerobic bacteria in the communities found in the filter treating ozonated water compared to the communities found in the filter treating nonozonated settled water, which had a greater abundance of eukaryotic markers.     

Mechanistic Properties of the Two-Component Bacteriocin Lactococcin G

Lactococcin G is a bacteriocin whose activity depends on the complementary action of two peptides, termed α and β. Biologically active, synthetic lactococcin G was used to study the mode of action on sensitive cells of Lactococcus lactis. The α and β peptides can bind independently to the target cell surface, but activity requires the complementary peptide. Once bound to the cell surface, the peptides cannot be displaced to the surfaces of other cells. A complex of α and β peptides forms a transmembrane pore that conducts monovalent cations but not protons. Efflux of potassium ions is observed only above pH 5.0, and the rate of efflux increases steeply with the pH. The consequences of cation fluxes for the viability of the target cells are discussed.     

Integrating Community-Based Interventions to Reverse the Convergent TB/HIV Epidemics in Rural South Africa

The WHO recommends integrating interventions to address the devastating TB/HIV co-epidemics in South Africa, yet integration has been poorly implemented and TB/HIV control efforts need strengthening. Identifying infected individuals is particularly difficult in rural settings. We used mathematical modeling to predict the impact of community-based, integrated TB/HIV case finding and additional control strategies on South Africa’s TB/HIV epidemics. We developed a model incorporating TB and HIV transmission to evaluate the effectiveness of integrating TB and HIV interventions in rural South Africa over 10 years. We modeled the impact of a novel screening program that integrates case finding for TB and HIV in the community, comparing it to status quo and recommended TB/HIV control strategies, including GeneXpert, MDR-TB treatment decentralization, improved first-line TB treatment cure rate, isoniazid preventive therapy, and expanded ART. Combining recommended interventions averted 27% of expected TB cases (95% CI 18–40%) 18% HIV (95% CI 13–24%), 60% MDR-TB (95% CI 34–83%), 69% XDR-TB (95% CI 34–90%), and 16% TB/HIV deaths (95% CI 12–29). Supplementing these interventions with annual community-based TB/HIV case finding averted a further 17% of TB cases (44% total; 95% CI 31–56%), 5% HIV (23% total; 95% CI 17–29%), 8% MDR-TB (68% total; 95% CI 40–88%), 4% XDR-TB (73% total; 95% CI 38–91%), and 8% TB/HIV deaths (24% total; 95% CI 16–39%). In addition to increasing screening frequency, we found that improving TB symptom questionnaire sensitivity, second-line TB treatment delays, default before initiating TB treatment or ART, and second-line TB drug efficacy were significantly associated with even greater reductions in TB and HIV cases. TB/HIV epidemics in South Africa were most effectively curtailed by simultaneously implementing interventions that integrated community-based TB/HIV control strategies and targeted drug-resistant TB. Strengthening existing TB and HIV treatment programs is needed to further reduce disease incidence.     

Evasion of Immunity to Plasmodium falciparum: Rosettes of Blood Group A Impair Recognition of PfEMP1

The ABO blood group antigens are expressed on erythrocytes but also on endothelial cells, platelets and serum proteins. Notably, the ABO blood group of a malaria patient determines the development of the disease given that blood group O reduces the probability to succumb in severe malaria, compared to individuals of groups A, B or AB. P. falciparum rosetting and sequestration are mediated by PfEMP1, RIFIN and STEVOR, expressed at the surface of the parasitized red blood cell (pRBC). Antibodies to these antigens consequently modify the course of a malaria infection by preventing sequestration and promoting phagocytosis of pRBC. Here we have studied rosetting P. falciparum and present evidence of an immune evasion mechanism not previously recognized. We find the accessibility of antibodies to PfEMP1 at the surface of the pRBC to be reduced when P. falciparum forms rosettes in blood group A RBC, as compared to group O RBC. The pRBC surrounds itself with tightly bound normal RBC that makes PfEMP1 inaccessible to antibodies and clearance by the immune system. Accordingly, pRBC of in vitro cloned P. falciparum devoid of ABO blood group dependent rosetting were equally well detected by anti-PfEMP1 antibodies, independent of the blood group utilized for their propagation. The pathogenic mechanisms underlying the severe forms of malaria may in patients of blood group A depend on the ability of the parasite to mask PfEMP1 from antibody recognition, in so doing evading immune clearance.