Bacterial Ghosts of Escherichia coli Drive Efficient Maturation of Bovine Monocyte-Derived Dendritic Cells

Bacterial ghosts (BGs) are empty cell envelopes derived from Gram-negative bacteria. They not only represent a potential platform for development of novel vaccines but also provide a tool for efficient adjuvant and antigen delivery system. In the present study, we investigated the interaction between BGs of Escherichia coli (E. coli) and bovine monocyte-derived dendritic cells (MoDCs). MoDCs are highly potent antigen-presenting cells and have the potential to act as a powerful tool for manipulating the immune system. We generated bovine MoDCs in vitro from blood monocytes using E. coli expressed bovine GM-CSF and IL-4 cytokines. These MoDCs displayed typical morphology and functions similar to DCs. We further investigated the E. coli BGs to induce maturation of bovine MoDCs in comparison to E. coli lipopolysaccharide (LPS). We observed the maturation marker molecules such as MHC-II, CD80 and CD86 were induced early and at higher levels in BG stimulated MoDCs as compared to the LPS stimulated MoDCs. BG mediated stimulation induced significantly higher levels of cytokine expression in bovine MoDCs than LPS. Both pro-inflammatory (IL-12 and TNF-α) and anti-inflammatory (IL-10) cytokines were induced in MoDCs after BGs stimulation. We further analysed the effects of BGs on the bovine MoDCs in an allogenic mixed lymphocyte reaction (MLR). We found the BG-treated bovine MoDCs had significantly (p<0.05) higher capacity to stimulate allogenic T cell proliferation in MLR as compared to the LPS. Taken together, these findings demonstrate the E. coli BGs induce a strong activation and maturation of bovine MoDCs.     

Systematics of the Lutzomyia species of the verrucarum Theodor group, 1965 (Diptera: Psychodiadae)

The verrucarum group of phlebotomine sand flies includes vectors of Leishmania spp. and Bartonella bacilliformis, and from the perspective of public health is considered as one of the most important groups of neotropical phlebotomine sand flies. Due to marked morphological similarity among species constituting this group, the identification based on conventional taxonomic characters can be difficult. Consequently, the verrucarum group has been the focus of numerous taxonomic comparisons which have included the following methods: chaetotaxy, morphometry, larval spiracular system, chorionic structure, morphology of the genital atrium, cytogenetics, morphological phylogenetics, isoenzymes, random amplified polymorphic DNA, cuticular hydrocarbons, DNA probes, and nuclear and mitochondrial nucleotide sequences. Based on morphological characters of the male terminalia, the verrucarum group has been divided in four series, i.e., verrucarum, serrana, townsendi and pia. Since the revision of the group made by Young and Duncan in 1994, ten new species, principally of Andean origin, have been assigned to 3 of the series verrucarum (L. maranonensis, L. cajamarcensis, L. antioquiensis, L. falcaorum), serrana (L. robusta, L. guilvardae) and pia (L. suapiensis, L. tihuiliensis, L. tocaniensis, L. limafalcaoae). The total number of verrucarum group members is now 40. Explanations for this diversity of species include the isolation of ancestral populations in refugia of humid forest during the quaternary period, the Andean cordilleras as geographical barrier, and the appearance of the Isthmus of Panama. Biology systematics and evolution of the verrucarum group is reviewed with emphasis on the 19 species extant in Colombia.     

Ataxia-telangiectasia: Linkage analysis in highly inbred Arab and Druze families and differentiation from an ataxia-microcephaly-cataract syndrome

Summary Ataxia-telangiectasia (A-T) is a progressive autosomal recessive disease featuring neurodegeneration, immunodeficiency, chromosomal instability, radiation sensitivity and a highly increased proneness to cancer. A-T is ethnically widespread and genetically heterogeneous, as indicated by the existence of four complementation groups in this disease. Several A-T-like genetic diseases share various clinical and cellular characteristics with A-T. By using linkage analysis to study North American and Turkish A-O families, the ATA (A-T, complementation group A) gene has been mapped to chromosome 11q23. A number of Israeli Arab A-T patients coming from large, highly inbred families were assigned to group A In one of these families, an additional autosomal recessive disease was identified, characterized by ataxia, hypotonia, microcephaly and bilateral congenital cataracts. In two patients with this syndrome, normal levels of serum immunoglobulins and alpha-fetoprotein, chromosomal stability in peripheral blood lymphocytes and skin fibroblasts, and normal cellular response to treatments with X-rays and the radiomimetic drug neocarzinostatin indicated that this disease does not share, with A-T, any additional features other than ataxia. These tests also showed that another patient in this family, who is also mentally retarded, is affected with both disorders. This conclusion was further supported by linkage analysis with 11q23 markers. Lod scores between A-O and these markers, cumulated over three large Arab families, were significant and confirmed the localization of the ATA gene to aq23. However, another Druze family unassigned to a specific complementation group, showed several recombinants between A-T and the same markers, leaving the localization of the A-T gene in this family open.     

Gastric H-K-ATPase and acid-resistant surface proteins

Despite the fact that mucus and bicarbonate are important macroscopic components of the gastric mucosal barrier, severe acidic and peptic conditions surely exist at the apical membrane of gastric glandular cells, and these membranes must have highly specialized adaptations to oppose external insults. Parietal cells abundantly express the heterodimeric, acid-pumping H-K-ATPase in their apical membranes. Its beta-subunit (HKbeta), a glycoprotein with >70% of its mass and all its oligosaccharides on the extracellular side, may play a protective role. Here, we show that the extracellular domain of HKbeta is highly resistant to trypsin in the native state (much more than that of the structurally related Na-K-ATPase beta-subunit) and requires denaturation to expose tryptic sites. Native HKbeta also resists other proteases, such as chymotrypsin and V8 protease, which hydrolyze at hydrophobic and anionic amino acids, respectively. Removal of terminal alpha-anomeric-linked galactose does not appreciably alter tryptic sensitivity of HKbeta. However, full deglycosylation makes HKbeta much more susceptible to all proteases tested, including pepsin at pH <2.0. We propose that 1) intrinsic folding of HKbeta, 2) bonding forces between subunits, and 3) oligosaccharides on HKbeta provide a luminal protein domain that resists gastric lytic conditions. Protein folding that protects susceptible charged amino acids and is maintained by disulfide bonding and hydrophilic oligosaccharides would provide a stable structure in the face of large pH changes. The H-K-ATPase is an obvious model, but other gastric luminally exposed proteins are likely to possess analogous protective specializations.     

In vitro propagation of three <Emphasis Type="Italic">Agave</Emphasis> species used for liquor distillation and three for landscape

In vitro bud clusters of Calathea orbifolia (Linden) Kennedy were obtained and subcultured in semi-solid (agar) medium and temporary immersion system (TIS) for 12 weeks. Uniform young plants were selected and transferred to soilless mix in a growth chamber for ex vitro acclimatization during 35 days, followed by growing in a shaded greenhouse for 65 days. Comparison of in vitro leaf anatomy, ex vitro photosynthetic behaviors and growth was made between two cultural systems. Plants in TIS produced thicker leaf chlorenchyma and aquiferous parenchyma, lower stomatal frequency and more epicuticular wax than did those in semi-solid medium. Plants from semi-solid medium had consistently lower leaf Fv/Fm values than plants from TIS. Leaf Fv/Fm value in plants from TIS decreased to 0.65 at day 7 after transfer and increased soon up to 0.76 thereafter. In contrast, leaf Fv/Fm value in plants from semi-solid medium reduced to 0.27 at day 7 after transfer and increased slowly up to 0.68 at day 35. During ex vitro acclimatization, plants in TIS had substantial higher photosynthetic rates than plants in semi-solid medium. Plants from TIS had subsequent higher leaf area, fresh and dry weights than plants from semi-solid medium.     

Proximal effects in the modulation of nitric oxide synthase reactivity: a QM-MM study

Nitric oxide synthases (NOS) are heme proteins that have a cysteine residue as axial ligand, which generates nitric oxide (NO). The proximal environment, specifically H-bonding between tryptophan (Trp) 178 and thiolate, has been proposed to play a fundamental role in the modulation of NOS activity. We analyzed the molecular basis of this modulation by performing electronic structure calculations on isolated model systems and hybrid quantum-classical computations of the active sites in the protein environment for wild-type and mutant (Trp 178 × Gly) proteins. Our results show that in the ferrous proteins NO exhibits a considerable trans effect. We also showed that in the ferrous (Fe⁺²) mutant NOS the absence of Trp, experimentally associated to a protonated cysteine, weakens the Fe–S bond and yields five coordinate complexes. In the ferric (Fe⁺³) state, the NO dissociation energy is shown to be slightly smaller in the mutant NOS, implying that the Fe⁺³–NO complex has a shorter half-life. We found computational evidence suggesting that ferrous NOS is favored in wild-type NOS when compared to the Trp mutant, consistently with the fact that Trp mutants have been shown to accumulate less Fe⁺²–NO dead end species. We also found that the heme macrocycle showed a significant distortion in the wild-type protein, due to the presence of the nearby Trp 178. This may also play a role in the subtle tuning of the electronic structure of the heme moiety.     

Laser-based single-axon transection for high-content axon injury and regeneration studies

The investigation of the regenerative response of the neurons to axonal injury is essential to the development of new axoprotective therapies. Here we study the retinal neuronal RGC-5 cell line after laser transection, demonstrating that the ability of these cells to initiate a regenerative response correlates with axon length and cell motility after injury. We show that low energy picosecond laser pulses can achieve transection of unlabeled single axons in vitro and precisely induce damage with micron precision. We established the conditions to achieve axon transection, and characterized RGC-5 axon regeneration and cell body response using time-lapse microscopy. We developed an algorithm to analyze cell trajectories and established correlations between cell motility after injury, axon length, and the initiation of the regeneration response. The characterization of the motile response of axotomized RGC-5 cells showed that cells that were capable of repair or regrowth of damaged axons migrated more slowly than cells that could not. Moreover, we established that RGC-5 cells with long axons could not recover their injured axons, and such cells were much more motile. The platform we describe allows highly controlled axonal damage with subcellular resolution and the performance of high-content screening in cell cultures.     

Eggplant latent viroid,the candidate type species for a new genus within the family Avsunviroidae (hammerhead viroids)

Viroids, small circular RNAs that replicate independently and in most cases incite diseases in plants, are classified into the families Pospiviroidae, composed of species with a central conserved region (CCR) and without hammerhead ribozymes, and Avsunviroidae, composed of three members lacking CCR but able to self-cleave in both polarity strands through hammerhead ribozymes. Here we report the biological and molecular properties of Eggplant latent viroid (ELVd). Purified circular ELVd induces symptomless infections when inoculated into eggplant seedlings. ELVd can be transmitted horizontally and through seed. Sequencing 10 complete cDNA clones showed that ELVd is a circular RNA of 332 to 335 nucleotides with high variability. This RNA can adopt a quasi-rod-like secondary structure of minimal free energy and alternative foldings that permit formation of stable hammerhead structures in plus and minus strands. The ribozymes are active in vitro and, most likely, in vivo. Considering the ELVd properties to be intermediate between those of the two genera of family Avsunviroidae, we propose ELVd as the type species of a third genus with the name ELAVIROID: