Conformational preferences of the amylin nucleation site in SDS micelles: an NMR study

Human islet amyloid polypeptide (hIAPP), or amylin, is a 37 amino acid hormone secreted by pancreatic beta-cells. hIAPP constitutes approximately 90% of the amyloid deposits found in type II diabetic patients. It has been shown that the central region of the peptide (hIAPP(20-29)) constitutes the nucleation site for the amyloidogenic process with F23 playing a key role in the formation of the beta-pleated structures. In addition, it has been proposed that an important stage in the cytotoxicity of hIAPP is its interaction with the beta-cell membranes. As a first step toward the characterization of the interaction of hIAPP with cell membranes, we determined conformational preferences of hIAPP(20-29) in membrane-mimicking environments. We found that upon interacting with negatively charged micelles, the dominant conformation of hIAPP(20-29) is a distorted type I beta-turn centered on residues F23 and G24, with F23, A25, and I26 forming a small hydrophobic cluster that may facilitate the interaction of this peptide with the membrane bilayer. Moreover, we were able to elucidate the topological orientation of the peptide that is absorbed on the micelle surface, with the hydrophobic cluster oriented toward the hydrocarbon region of the micelles and both N- and C-termini exposed to the solvent.     

Unfolding of trp repressor studied using fluorescence spectroscopic techniques.

The unfolding properties of the trp repressor of Escherichia coli have been studied using a number of different time-resolved and steady-state fluorescence approaches. Denaturation by urea was monitored by the average fluorescence emission energy of the intrinsic tryptophan residues of the repressor. These data were consistent with a two-state transition from dimer to unfolded monomer with a free energy of unfolding of 19.2 kcal/mol. The frequency response profiles of the fluorescence emission brought to light subtle urea-induced modifications of the intrinsic tryptophan decay parameters both preceding and following the main unfolding transition. The increase of lifetime induced by urea required higher concentrations of urea than the increase in the total intensity described by Gittelman and Matthews [(1990) Biochemistry 29, 7011]. This indicates that the intensity increase has both dynamic and static origins. To assess the effect of tryptophan binding upon repressor stability, and to determine whether repressor oligomerization would be detectable in an unfolding experiment, we examined denaturation profiles of repressor labeled with the long-lived fluorescence probe 5-(dimethylamino)naphthalene-1-sulfonyl (DNS), by monitoring the average rotational correlation time of the probe. These experiments revealed a protein concentration dependent transition at low urea concentrations. This transition was promoted by tryptophan binding. We ascribe this transition to urea-induced dissociation of repressor tetramers. The main unfolding transition of the dimer to unfolded monomer was also observable using this technique, and the free energies associated with this transition were 18.3 kcal/mol in the absence of tryptophan and 24.1 kcal/mol in its presence, demonstrating that co-repressor binding stabilizes the repressor dimer against denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyperbolic Modeling of Starburst Dendrimers

Starburst dendrimers are highly branched oligomers. A rigid dendritic hydrocarbon, C₁₁₃₄H₁₁₄₆, has recently been synthesized. It consists of 94 phenylacetylene units displayed in a self-similar two-dimensional skeleton isomorphous to the three-coordinated Bethe lattice. The three-dimensional representation of phenylacetylene dendrimer shows a globular architecture with large voids and niches in its interior, characteristic of hyperbolic surfaces. This work investigates the geometrical scaling behavior of this starburst dendrimer using the symmetry properties of a Bethe lattice embedded in the hyperbolic plane. The results for C₁₁₃₄H₁₁₄₆ provide its density profile and an upper bound for its macromolecular size. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cation Permeability and Selectivity of a Root Plasma Membrane Calcium Channel

Calcium channels in the plasma membrane of root cells fulfill both nutritional and signaling roles. The permeability of these channels to different cations determines the magnitude of their cation conductances, their effects on cell membrane potential and their contribution to cation toxicities. The selectivity of the rca channel, a Ca²⁺-permeable channel from the plasma membrane of wheat (Triticum aestivum L.) roots, was studied following its incorporation into planar lipid bilayers. The permeation of K⁺, Na⁺, Ca²⁺ and Mg²⁺ through the pore of the rca channel was modeled. It was assumed that cations permeated in single file through a pore with three energy barriers and two ion-binding sites. Differences in permeation between divalent and monovalent cations were attributed largely to the affinity of the ion binding sites. The model suggested that significant negative surface charge was present in the vestibules to the pore and that the pore could accommodate two cations simultaneously, which repelled each other strongly. The pore structure of the rca channel appeared to differ from that of L-type calcium channels from animal cell membranes since its ion binding sites had a lower affinity for divalent cations. The model adequately accounted for the diverse permeation phenomena observed for the rca channel. It described the apparent submillimolar K _m for the relationship between unitary conductance and Ca²⁺ activity, the differences in selectivity sequences obtained from measurements of conductance and permeability ratios, the changes in relative cation permeabilities with solution ionic composition, and the complex effects of Ca²⁺ on K⁺ and Na⁺ currents through the channel. Having established the adequacy of the model, it was used to predict the unitary currents that would be observed under the ionic conditions employed in patch-clamp experiments and to demonstrate the high selectivity of the rca channel for Ca²⁺ influx under physiological conditions. Received: 23 August 1999/Revised: 12 November 1999     

Sequential Statistical Improvement of the Liquid Cultivation of Helicobacter pylori

Background: Colonization of the gastric mucosa by Helicobacter pylori is one of the most important causes of acute and chronic gastric pathologies in humans. Achieving the growth of H. pylori in liquid media is of great importance in the development of clinical studies. In this study, we developed a sequential optimization strategy based on statistical models to improve the conditions of liquid culture of H. pylori. Materials and Methods: Four statistical models were sequentially used. First, a Box‐Behnken design was used to select the best process conditions (shaking speed, inoculum concentration, and final volume of culture). Secondly, a general factorial design was used to evaluate the influence of adding gel blocks or gel beads (shape and composition). Then a D‐optimal reduce design was carried out to allow the selection of the most influential factors in increasing the cell concentration (culture media components). Finally, another Box‐Behnken design was used to optimize the concentration of the culture media components previously selected. Results: After 12 hours of liquid culture a concentration of 25 × 10⁸ cells per mL (9.4 log₁₀ cells per mL) of H. pylori was obtained, compared with a predicted 32 × 10⁸ (9.5 log₁₀ cells per mL), which means between 1 and 5 log₁₀ units higher than some previous reports. Conclusions: The sequential statistical approach increased the planktonic H. pylori cell culture. The final culture media and conditions were: Brain Heart Infusion, blood agarose (1.5% w/v), lamb’s blood (3.18% v/v), DENT (0.11% v/v), and Vitox (0.52% v/v) at 60 rpm and 37 °C with filtered CO₂ (5% v/v) bubbled directly into the culture media in a final volume of 76.22 mL.     

Phylogenetic position of Salinibacter ruber based on concatenated protein alignments

A total of 22 genes from the genome of Salinibacter ruber strain M31 were selected in order to study the phylogenetic position of this species based on protein alignments. The selection of the genes was based on their essential function for the organism, dispersion within the genome, and sufficient informative length of the final alignment. For each gene, an individual phylogenetic analysis was performed and compared with the resulting tree based on the concatenation of the 22 genes, which rendered a single alignment of 10,757 homologous positions. In addition to the manually chosen genes, an automatically selected data set of 74 orthologous genes was used to reconstruct a tree based on 17,149 homologous positions. Although single genes supported different topologies, the tree topology of both concatenated data sets was shown to be identical to that previously observed based on small subunit (SSU) rRNA gene analysis, in which S. ruber was placed together with Bacteroidetes. In both concatenated data sets the bootstrap was very high, but an analysis with a gradually lower number of genes indicated that the bootstrap was greatly reduced with less than 12 genes. The results indicate that tree reconstructions based on concatenating large numbers of protein coding genes seem to produce tree topologies with similar resolution to that of the single 16S rRNA gene trees. For classification purposes, 16S rRNA gene analysis may remain as the most pragmatic approach to infer genealogic relationships.     

Efficient genome editing and gene knockout in Setaria viridis with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway

The CRISPR/Cas9 system has been used for genome editing in several organisms, including higher plants. This system induces site-specific mutations in the genome based on the nucleotide sequence of engineered guide RNAs. The complex genomes of C4 grasses makes genome editing a challenge in key grass crops like maize (Zea mays), sorghum (Sorghum bicolor), Brachiaria spp., switchgrass (Panicum virgatum), and sugarcane (Saccharum spp.). Setaria viridis is a diploid C4 grass widely used as a model for these C4 crop plants. Here, an optimized CRISPR/Cas9 binary vector that exploits the non-homologous end joining (NHEJ) system was used to knockout a green fluorescent protein (gfp) transgene in S. viridis accession A10.1. Transformation of embryogenic callus by A. tumefaciens generated ten glufosinate-ammonium resistant transgenic events. In the T0 generation, 60% of the events were biallelic mutants in the gfp transgene with no detectable accumulation of GFP protein and without insertions or deletions in predicted off-target sites. The gfp mutations generated by CRISPR/Cas9 were stable and displayed Mendelian segregation in the T1 generation. Altogether, the system described here is a highly efficient genome editing system for S. viridis, an important model plant for functional genomics studies in C4 grasses. Also, this system is a potential tool for improvement of agronomic traits in C4 crop plants with complex genomes.     

A new methodology combining PCR, cloning, and sequencing of clones discriminated by RFLP for the study of microbial populations: application to an UASB reactor sample

This work describes a methodology combining DNA extraction, polymerase chain reaction amplification with primers targeting 16S ribosomal RNA genes, cloning, and sequencing of clones previously analyzed by restriction fragment length polymorphism (RFLP), which can be applied to study the microbial diversity in a given habitat. The methodology allows the minimization of the sequencing effort, which is particularly relevant when analyzing large numbers of clones. The methodology does not require particularly skilled personnel and can easily be adaptable to the molecular characterization of virtually any particular microbial population, provided that both adequate primers and suitable restriction enzymes for RFLP analysis of the clone library have been chosen. An example of application is presented, in which a sample taken from a continuously operating upflow anaerobic sludge blanket reactor was analyzed. RFLP analysis of the initial 162 clones with HaeIII allowed the identification of only 28 distinct profiles. As expected, identical RFLP profiles corresponded to identical nucleotide sequences.     

Production of rhamnolipids in solid-state cultivation using a mixture of sugarcane bagasse and corn bran supplemented with glycerol and soybean oil

Rhamnolipid biosurfactants are attracting attention due to their low toxicity, high biodegradability, and good ecological acceptability. However, production in submerged culture is made difficult by severe foaming problems. Solid-state cultivation (SSC) is a promising alternative production method. In the current work, we report the optimization of rhamnolipid production by Pseudomonas aeruginosa UFPEDA 614 on a solid substrate containing sugarcane bagasse and corn bran. The best rhamnolipid production, 45 g/l of impregnating solution used, was obtained with a 50:50 (m/m) mixture of sugarcane bagasse and corn bran supplemented with an impregnating solution containing 6% (v/v) of each of glycerol and soybean oil. This level is comparable with those of previous studies undertaken in solid-state cultivation; the composition of the biosurfactant is similar, but our medium is cheaper. Our work therefore provides a suitable basis for future studies of the development of an SSC-based process for rhamnolipid production.     

Three-dimensional pore space quantification of apple tissue using X-ray computed microtomography

The microstructure and the connectivity of the pore space are important variables for better understanding of the complex gas transport phenomena that occur in plant tissues. In this study, we present an experimental procedure for image acquisition and image processing to quantitatively characterize in 3D the pore space of apple tissues (Malus domestica Borkh.) for two cultivars (Jonagold and Braeburn) taken from the fleshy part of the cortex using X-ray computer microtomography. Preliminary sensitivity analyses were performed to determine the effect of the resolution and the volume size (REV, representative elementary volume analysis) on the computed porosity of apple samples. For comparison among cultivars, geometrical properties such as porosity, specific surface area, number of disconnected pore volumes and their distribution parameters were extracted and analyzed in triplicate based on the 3D skeletonization of the pore space (medial axis analysis). The results showed that microtomography provides a resolution at the micrometer level to quantitatively analyze and characterize the 3D topology of the pore space in apple tissue. The computed porosity was confirmed to be highly dependent of the resolution used, and the minimum REV of the cortical flesh of apple fruit was estimated to be 1.3 mm³. Comparisons among the two cultivars using a resolution of 8.5 μm with a minimum REV cube showed that in spite of the complexity and variability of the pore space network observed in Jonagold and Braeburn apples, the extracted parameters from the medial axis were significantly different (P-value < 0.05). Medial axis parameters showed potential to differentiate the microstructure between the two evaluated apple cultivars.