Folding and activity of the hammerhead ribozyme

The hammerhead is the smallest of the nucleolytic ribozymes, that undergo backbone cleavage by a transesterification reaction in the presence of magnesium ions. The RNA is induced to fold into its active conformation by the binding of metal ions in two stages. These generate domain 2, the scaffold on which the ribozyme is built, and domain 1, the active centre of the ribozyme. Further local structural rearrangement during the activation of the ribozyme is suggested by a number of crystal structures. The 10(5)-fold rate enhancement is probably brought about by a combination of metal-ion participation and stereochemical factors in the environment of the folded RNA structure.     

Selection of hammerhead ribozyme variants with low Mg2+ requirement: importance of stem-loop II

Variants of the hammerhead ribozyme with high in trans (intermolecular) cleavage activity at low Mg(2+) concentrations were in vitro selected from a library with 18 nucleotides randomised in the core and in helix II. The most active hammerhead ribozyme selected had the same sequence as the consensus ribozyme in the core but only two base pairs in stem II, G(10.1)-C(11.1) and U(10.2)-A(11.2), and a tetrauridine loop II. This ribozyme (clone 34) was found to be very active in single-turnover reactions at 1 mM Mg(2+) concentration in the context of several substrates with differences in the lengths of stem I and III, including the well-characterised HH16 substrate and a derivative thereof with a GUA triplet at the cleavage site, as well as a substrate used previously in a related study. For the HH16 substrate, a change of base pair 10.2-11.2 to C-G in stem II further improved activity by about 2.5-fold to 0.8 min(-1) (at 1 mM Mg(2+) concentration, 25 degrees C, pH 7.5). Interestingly, this very active variant was not identified by the selection procedure. Changing loop II from UUUU to GCAA or extension of stem II to three or four base pairs reduced the cleavage rate by 2.0-2.5-fold. Thus, small hammerhead ribozymes carrying a tetrauridine loop with two base pairs in stem II represent the most active versions known so far at low Mg(2+) concentrations; single-turnover rates of approximately 1 min(-1) are reached at 25 degrees C and pH 7.5 in monophasic reactions, with endpoints between 75 and 90 %. Such constructs promise to be advantageous for the inhibition of gene expression in vivo.     

Structural investigation of a high-affinity MnII binding site in the hammerhead ribozyme by EPR spectroscopy and DFT calculations. Effects of neomycin B on metal-ion binding

Electron paramagnetic resonance spectroscopy and density functional theory methods were used to study the structure of a single, high-affinity Mn(II) binding site in the hammerhead ribozyme. This binding site exhibits a dissociation constant Ke of 4.4 microM in buffer solutions containing 1 M NaCl, as shown by titrations monitored by continuous wave (cw) EPR. A combination of electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) experiments revealed that the paramagnetic manganese(II) ion in this binding site is coupled to a single nitrogen atom with a quadrupole coupling constant kappa of 0.7 MHz, an asymmetry parameter eta of 0.4, and an isotropic hyperfine coupling constant of Aiso(14N)=2.3 MHz. All three EPR parameters are sensitive to the arrangement of the Mn(II) ligand sphere and can therefore be used to determine the structure of the binding site. A possible location for this binding site may be at the G10.1, A9 site found to be occupied by Mn(II) in crystals (MacKay et al., Nature 1994, 372, 68 and Scott et al., Science 1996, 274, 2065). To determine whether the structure of the binding site is the same in frozen solution, we performed DFT calculations for the EPR parameters, based on the structure of the Mn(II) site in the crystal. Computations with the BHPW91 density function in combination with a 9s7p4d basis set for the manganese(II) center and the Iglo-II basis set for all other atoms yielded values of kappa(14N)=+0.80 MHz, eta=0.324, and Aiso(14N)=+2.7 MHz, in excellent agreement with the experimentally obtained EPR parameters, which suggests that the binding site found in the crystal and in frozen solution are the same. In addition, we demonstrated by EPR that Mn(II) is released from this site upon binding of the aminoglycoside antibiotic neomycin B (Kd=1.2 microM) to the hammerhead ribozyme. Neomycin B has previously been shown to inhibit the catalytic activity of this ribozyme (Uhlenbeck et al., Biochemistry 1995, 34, 11 186).     

In search of the ideal protein sequence

The inverse of a folding problem is to find the ideal sequencethat folds into a particular protein structure. This problemhas been addressed using the topology fingerprintbased threadingalgorithm, capable of calculating a score (energy) of an arbitrarysequence-structure pair. At first, the search is conducted byunconstrained minimization of the energy in sequence space.It is shown that using energy as the only design criterion leadsto spurious solutions with incorrect amino acid composition.The problem lies in the general features of the protein energysurface as a function of both structure and sequence. The proposedsolution is to design the sequence by maximizing the differencebetween its energy in the desired structure and in other knownprotein structures. Depending on the size of the database ofstructures ‘to avoid’, sequences bearing significantsimilarity to the native sequence of the target protein areobtained using this procedure.     

In vitro DNA recombination by L-Shuffling during ribosome display affinity maturation of an anti-Fas antibody increases the population of improved variants

The use of random mutagenesis in concert with protein display technologies to rapidly select high affinity antibody variants is an established methodology. In some cases, DNA recombination has been included in the strategy to enable selection of mutations which act cooperatively to improve antibody function. In this study, the impact of L-Shuffling DNA recombination on the eventual outcome of an in vitro affinity maturation has been experimentally determined. Parallel evolution strategies, with and without a recombination step, were carried out and both methods improved the affinity of an anti-Fas single chain variable fragment (scFv). The recombination step resulted in an increased population of affinity-improved variants. Moreover, the most improved variant, with a 22-fold affinity gain, emerged only from the recombination-based approach. An analysis of mutations preferentially selected in the recombined population demonstrated strong cooperative effects when tested in combination with other mutations but small, or even negative, effects on affinity when tested in isolation. These results underline the ability of combinatorial library approaches to explore very large regions of sequence space to find optimal solutions in antibody evolution studies.     

In vitro selection of proteins that undergo covalent labeling with small molecules by thiol-disulfide exchange by using ribosome display

There is a great deal of interest in proteins that can bind covalently to target molecules, as they allow unambiguous experiments by tight binding to molecules of interest. Here, we report the generation of proteins that undergo covalent labeling with small molecules through in vitro selection by using ribosome display. Selection was performed from a mutant library of the WW domain with a biotinylated peptide as its binding target, in which the biotin and the peptide are connected by a disulfide bond. After five rounds of selection, we identified mutants carrying a particular cysteine mutation. The binding target reacted specifically with the selected mutant, even in the presence of other proteins, and resulted in the generation of biotin- or peptide-labeled WW domains by thiol-disulfide exchange. When the mutant was fused to a protein of interest, the fusion protein was also labeled with biotin. Thus, the characteristics of the selected mutant should be suitable as a tag sequence that can be covalently labeled with small synthetic molecules. These results indicate that the rapid and efficient generation of such proteins is possible by ribosome display.     

In vitro degradation of willow salicylates

Salicylates are defensive compounds against a great variety of generalist herbivores. Salicortin and its derivatives are labile compounds that degrade immediately when cell compartmentalization is ruptured, producing a 6-hydroxy-2-cyclohexenone (6-HCH) moiety that is a strong antifeeding cue. We studied the In Vitro degradation of willow salicylates in the presence and absence of foliar enzymes at acidic, neutral, and alkaline pHs. Higher substituted salicylates were degraded in the absence of foliar enzymes at alkaline pH and in the presence of foliar enzymes at all three pHs. Salicin and its diglucoside, on the other hand, were degraded only in the presence of foliar enzymes at acidic pH, probably by -glucosidase activity. The main degradation products of higher substituted salicylates were salicin, 6-HCH, and catechol in both the absence and presence of enzymes, suggesting that the production of 6-HCH and catechol do not necessarily demand enzymatic activity. We propose that the degradation of salicylates begins with the cleavage of a 1-hydroxy-6-oxo-2-cyclohexen-1-carbonyl moiety by foliar esterases and/or alkaline condition. This moiety is decarboxylated in nonenzymatic reaction to an anion of 2-hydroxy-3-cyclohexenone, which is tautomerized to the enol form and further to the keto form, 6-HCH. Hydroxyketone can be also oxidized to catechol, a substrate of polyphenol oxidases.     

In vitro studies of carbon nanotubes biocompatibility

Cellular tests have been applied to study the biocompatibility of high purity multiwalled carbon nanotubes (MWNTs). The viability of fibroblasts, osteoblasts and osteocalcin concentrations in osteoblasts cultures in the presence of nanotubes has been examined, as well as the degree of cells stimulation, based on the amount of released collagen type I, IL-6 and oxygen free radicals. The high level of viability of the examined cells in contact with the nanotubes, the slight increase of collagen formation, the lack of pro-inflammatory IL-6 cytokine as well as the induction of free radicals, confirm a good biocompatibility of nanotubes, which is similar to that of polysulfone currently used in medicine. The collagen synthesis induced on nanotubes by both fibroblasts and osteoblasts may be significant for future medical applications of nanotubes, in particular as substrates for the regeneration of tissues.     

Estimates of energy consumption and selection of optimal distillation sequence for multicomponent distillation

An estimate has been obtained of the energy consumption on 1 mol of separated mixture based on thermodynamic balances for processes that use mechanical and heat energy. Only irreversible losses depend on the order of separation for mixtures in mechanical systems, while for the systems using the heat, the order of separation also affects the reversible energy consumption. In the latter case, in order to obtain a reversible estimate of the heat consumption, one should solve the problem of selecting the order of separation. An algorithm has been obtained for its solution that enables one to approximately choose the order of separation at the stage of preliminary calculations knowing only the properties of the feed.     

In vitro effects of nanoparticles on renal cells

Background  

The ability of nanoparticles to cross the lung-blood barrier suggests that they may translocate to blood and to targets distant from their portal of entry. Nevertheless, nanotoxicity in organs has received little attention. The purpose of this study was to evaluate nanotoxicity in renal cells using in vitro models. Various carbon black (CB) (FW2–13 nm, Printex60-21 nm and LB101-95 nm) and titanium dioxide (TiO₂-15 and TiO₂-50 nm) nanoparticles were characterized on size by electron microscopy. We evaluated theirs effects on glomerular mesangial (IP15) and epithelial proximal tubular (LLC-PK₁) renal cells, using light microscopy, WST-1 assay, immunofluorescence labeling and DCFH-DA for reactive oxygen species (ROS) assay.     

In vitro bioactivity evaluation of magnesium-substituted fluorapatite nanopowders

Properties of apatites, such as bioactivity, biocompatibility, solubility, and adsorption properties can be tailored over a wide range by modifying the composition via ionic substitutions. The aim of this work was preparation, characterization and in vitro bioactivity evaluation of Mg-doped fluorapatite (Mg-FA) nanopowders. Mg-FA nanopowders with different Mg contents were prepared via sol-gel method. In vitro bioactivity evaluation of powders was performed by soaking the powders in simulated body fluid (SBF). Results indicated that Mg ions entered into the fluorapatite lattice and occupied Ca²⁺ sites, and the obtained powders had crystallite size about 30-100 nm. With increasing the Mg-substitution, the solubility of powders and the adsorption of Ca²⁺ ions onto the powders surfaces increased simultaneously. It was concluded that Mg-substitution improves the bioactivity of FA.     

Utilisation of key descriptors from protein sequence data to aid bioprocess route selection

The large-scale manufacture of biological products results in the generation of significant quantities of process information that can be used to inform future design decisions. Currently this information is not exploited to its full potential. The challenge is thus to identify and/or develop tools that allow the utilisation of this valuable resource. The main objective of the research reported in this paper was to investigate whether it was possible to utilise information, in particular that extracted from protein sequence data, from previous processes, with the goal of informing process route selection early in development. The approach adopted draws on tools in the areas of data mining and pattern recognition including the techniques of Fisher correlation score and self-organising maps. The methodology developed was applied to two case studies utilising data from the amino acid sequences of 41 proteins previously developed at Avecia Biologics, along with associated information relating to the downstream processing steps used during their large scale manufacture. The results demonstrate that information from previous processes can be used to inform process route selection.     

In vitro drug release from chitosan membranes

Summary In vitro studies of controlled release from chitosan membranes were carried out using drugs of different molecular weight. The release rate was measured in water at 37°±0.5°C by determining the amount of drug released by spectrophotometric analysis. It was found that release of acetamide, nicotinamide, sodium benzoate, sodium salicylate, phenobarbital sodium and sodium cefazoline followed zero-order kinetics. The relation between molecular weight and diffusion coefficient was established.     

In vitro apatite-forming ability of calcium aluminate blends

Calcium aluminate cement (CAC) blends show great potential as biomaterial when compared to commercial products used in odontology and orthopedics. Mixtures of CAC +4 wt% of different additives (alumina, zirconia, zinc oxide, tricalcium phosphate or hydroxyapatite) containing compositions, resulted in samples with low porosity levels and smaller pore sizes after their contact with simulated body fluid (SBF) solution, which was associated with apatite precipitation on the materials’ surface. In order to certify these aspects, the in vitro apatite-formation ability (bioactivity) of CAC blends was evaluated by pH and calcium concentration measurements in SBF for samples previously treated (or not) with sodium silicate (SS) solution. The surface of the samples after immersion in SBF or SBF/SS was analyzed by scanning electron microscopy (SEM), energy dispersive X-ray (EDX) analysis and confocal Raman spectroscopy. In addition, the in vitro apatite deposition and the osteoblastic cell viability were also evaluated. SEM results showed that the precipitation of phases was detected on the CAC blend samples’ surfaces. The presence of calcium and mainly phosphorus by EDX indicated the formation of calcium phosphate phases. Moreover, the presence of a more homogeneous apatite-like layer on the samples’ surface was observed after treatment with sodium silicate solution. The detection of the Raman signature at 960 cm⁻¹, confirmed the presence of an apatite-like layer on the surface of the compositions after immersion in SBF or SBF/SS. Regarding the osteoblastic cell viability results, blends with collagen, zinc oxide and zirconia presented better results when compared to commercial products.     

In vitro biosynthesis of fatty acids inDrosophila melanogaster

A new in vitro technique, utilizing ruptured larvae ofDrosophila melanogaster, was employed to study the incorporation of³H-acetate into long-chain fatty acids. Preparative gas-liquid chromatography and scintillation spectroscopy were used to determine the relative activity of each fatty acid from total lipid extracts. Quantitative changes were observed in the distribution of label during the course of the incubation times, which ranged from five minutes to nine hours. All fatty acids which were observed to incorporate acetate in previous in vivo studies also showed incorporation of label under these in vitro conditions. It is concluded that this system may be useful for studying aspects on insect metabolism for short intervals of time.     

In vitro properties of nano-hydroxyapatite/chitosan biocomposites

In vitro behavior of the composites was performed in simulated body fluid (SBF) to induce the formation of bone-like apatite layer onto their surfaces and its enhancement in the presence of citric acid (CA). The results proved the mineralization of calcium (Ca²⁺) and phosphorus (P) ions onto the composites which contain high chitosan concentration especially after longer time of immersion. The degradation data decreased with increase chitosan content especially C2 composites (containing 30% chitosan) and highly decreased in the presence of CA which increased binding strength through the composite. The swelling % increased with increase of chitosan content in HA composite but it decreased with CA addition as increase of interaction between three matrices. The Fourier Transformed Infrared Spectroscopy (FT-IR) and Scanning Electron Microscope (SEM-EDAX) confirmed the formation of bone-like apatite layer on the surface of the composites especially these containing CA. These biocomposites have unique in vitro properties for bone substitute's applications in the future.     

In vitro skin permeation of cubosomes containing triclosan

Monoolein (MO) cubosomes containing triclosan (TCA) were prepared by micronizing MO cubic phases containing TCA in a bath-type sonicator. Pluronic F127 was used as a dispersant and no cubosomes could be produced from monoolein alone in the absence of the dispersant. The maximum tolerable ratio of TCA to MO for the formation of cubic phase was 16:84 (w/w). According to TEM photos and size distributions, the sizes of cubosomes were hundreds of nanometers. Using hydroxypropyl-β-cyclodextrin as a solubilizer for TCA, the in vitro skin permeation of TCA loaded in cubosomes was investigated on a diffusion cell. Even though the concentrations of TCA were the same in the suspensions of cubosomes, the skin permeations were increased more when the ratios of TCA to MO were higher in the cubosomes. Moreover, the permeations were higher than those of TCA suspension in aqueous solution of ethanol or propylene glycol.     

防晒原料的体外生物学评测方法

总结了不同波长紫外线损伤皮肤的生物学效应,分析了防晒剂体外生物学评测的步骤、原则和影响因素,重点介绍了角质细胞、成纤维细胞、黑色素细胞、固有免疫细胞等细胞水平的防晒剂测试方法,以及离体皮肤、3D皮肤等三维组织的评测方法,提出利用多重功能防晒原料优化配方的思路,最后总结了不同体外方法的权重及选择原则。