Improved PCR-based gene synthesis method and its application to the Citrobacter freundii phytase gene codon modification

Gene synthesis technologies provide a powerful tool for increasing protein expression through codon optimization and gene modification. Here we describe an improved PCR-based gene synthesis technology, which is accurate, simple and cheap. The improved PCR-based gene synthesis (IPS) method consists of two steps. The first one is the synthesis of 300-400 bp fragments by PCR reaction with Pfu DNA polymerase from 60-mer and 30-mer oligonucleotides with a 15 bp overlap. The second one is assembling of fragments from the first step into the full-length gene by PCR reaction. Using this approach, we have successfully synthesized a modified phytase gene with 1256 bp in length with optimal codons for expression in Pichia pastoris. P. pastoris strain that expressed the modified phytase gene (phyA-mod) showed a 50% increase in phytase activity level. In addition, we propose an inexpensive method for error correction, based on overlap-extension PCR (OE-PCR).     

Effects of GsMTx4 on bacterial mechanosensitive channels in inside-out patches from giant spheroplasts

GsMTx4 is a 34-residue peptide isolated from the tarantula Grammostola spatulata folded into an inhibitory cysteine knot and it selectively affects gating of some mechanosensitive channels. Here we report the effects of cytoplasmic GsMTx4 on the two bacterial channels, MscS and MscL, in giant Escherichia coli spheroplasts. In excised inside-out patches, GsMTx4 sensitized both channels to tension by increasing the opening rate and decreasing the closing rate. With ascending and descending pressure ramps, GsMTx4 increased the gating hysteresis for MscS, a consequence of slower gating kinetics. Quantitative kinetic analysis of the primary C↔O transition showed that the hysteresis is a result of the decreased closing rate. The gating barrier location relative to the open state energy well was unaffected by GsMTx4. A reconstructed energy profile suggests that the peptide prestresses the resting state of MscS, lowering the net barrier to opening and stabilizes the open conformation by ∼8 kT. In excised patches, both MscL and MscS exhibit reversible adaptation, a process separable from inactivation for MscS. GsMTx4 decreased the rate of reversible adaptation for both channels and the MscS recovery rate from the inactivation. These measurements support a mechanism where GsMTx4 binds to the lipid interface of the channel, increasing the local stress that is sensed by the channels and stabilizing the expanded conformations.     

Expression and distribution of cholinergic receptors in the human urothelium

The bladder urothelium not only provides a diffusion barrier but it also serves a sensor function and releases signalling molecules that are considered to act in a paracrine and autocrine fashion, e.g. by acetylcholine. Its actions are conferred by two classes of receptors, i.e. G-protein-coupled muscarinic receptors (MR) and ionotropic nicotinic receptors (nAChR). In this study we set out to determine the expression and distribution of all MR subtypes (M1R-M5R) and nAChR alpha-subunits 7, 9 and 10 in the human urothelium by means of RT-PCR and immunohistochemistry, respectively. Real-time RT-PCR revealed a rank order of MR subtype expression of M2R>M3R=M5R>M4R=M1R. Immunohistochemistry demonstrated differential distribution patterns with M1R being restricted to basal cells, M2R nearly exclusively found in umbrella cells, whereas M3R and M4R were homogenously distributed and M5R was seen in a decreasing gradient from luminal to basal. As for nAChR alpha-subunits, rank order of expression is alpha7>alpha10>alpha9, and they were observed throughout the urothelium with a gradient decreasing from luminal to basal in intensity. In conclusion, the human urothelium carries multiple cholinergic receptor subtypes, with predominant expression of M2R, M3R and alpha7-nAChR. Their distribution as well as that of the less expressed subtypes is layer-specific in the urothelium. In view of the multiplicity of pathways to which different cholinergic receptor subtypes are coupled, we propose that this layer-specific distribution serves to stratify cholinergic regulation of human urothelial function.     

Characterization of technetium(vII) reduction by cell suspensions of thermophilic bacteria and archaea

Washed cell suspensions of the anaerobic hyperthermophilic archaea Thermococcus pacificus and Thermoproteus uzoniensis and the anaerobic thermophilic gram-positive bacteria Thermoterrabacterium ferrireducens and Tepidibacter thalassicus reduced technetium [⁹⁹Tc(VII)], supplied as soluble pertechnetate with molecular hydrogen as an electron donor, forming highly insoluble Tc(IV)-containing grayish-black precipitate. Apart from molecular hydrogen, T. ferrireducens reduced Tc(VII) with lactate, glycerol, and yeast extract as electron donors, and T. thalassicus reduced it with peptone. Scanning electron microscopy and X-ray microanalysis of cell suspensions of T. ferrireducens showed the presence of Tc-containing particles attached to the surfaces of non-lysed cells. This is the first report on the reduction in Tc(VII) by thermophilic microorganisms of the domain Bacteria and by archaea of the phylum Euryarchaeota.     

Dynamics of electron transfer from high-potential cytochrome <Emphasis Type="Italic">c</Emphasis> to bacteriochlorophyll dimer in photosynthetic reaction centers as probed using laser-induced temperature jump

Laser-induced temperature jump experiments were used for testing the rates of thermoinduced conformational transitions of reaction center (RC) complexes in chromatophores of Chromatium minutissimum. The thermoinduced transition of the macromolecular RC complex to a state providing effective electron transport from the multiheme cytochrome c to the photoactive bacteriochlorophyll dimer within the temperature range 220–280 K accounts for tens of seconds with activation energy 0.166 eV/molecule. The rate of the thermoinduced transition in the cytochrome–RC complex was found to be three orders of magnitude slower than the rate of similar thermoinduced transition of the electron transfer reaction from the primary to secondary quinone acceptors studied in the preceding work (Chamorovsky et al. in Eur Biophys J 32:537–543, 2003). Parameters of thermoinduced activation of the electron transfer from the multiheme cytochrome c to the photoactive bacteriochlorophyll dimer are discussed in terms of cytochrome c docking onto the RC.     

Spontaneous class switch recombination in B cell lymphopoiesis generates aberrant switch junctions and is increased after VDJ rearrangement

Mature B cells replace the mu constant region of the H chain with a downstream isotype in a process of class switch recombination (CSR). Studies suggest that CSR induction is limited to activated mature B cells in the periphery. Recently, we have shown that CSR spontaneously occur in B lymphopoiesis. However, the mechanism and regulation of it have not been defined. In this study, we show that spontaneous CSR occurs at all stages of B cell development and generates aberrant joining of the switch junctions as revealed by: 1) increased load of somatic mutations around the CSR break points, 2) reduced sequence overlaps at the junctions, and 3) excessive switch region deletion. In addition, we found that incidence of spontaneous CSR is increased in cells carrying VDJ rearrangements. Our results reveal major differences between spontaneous CSR in developing B cells and CSR induced in mature B cells upon activation. These differences can be explained by deregulated expression or function of activation-induced cytidine deaminase early in B cell development.     

Conventional and PCR Detection of Aphelenchoides fragariae in Diverse Ornamental Host Plant Species

A PCR-based diagnostic assay was developed for early detection and identification of Aphelenchoides fragariae directly in host plant tissues using the species-specific primers AFragFl and AFragRl that amplify a 169-bp fragment in the internal transcribed spacer (ITS1) region of ribosomal DNA. These species-specific primers did not amplify DNA from Aphelenchoides besseyi or Aphelenchoides ritzemabosi. The PCR assay was sensitive, detecting a single nematode in a background of plant tissue extract. The assay accurately detected A. fragariae in more than 100 naturally infected, ornamental plant samples collected in North Carolina nurseries, garden centers and landscapes, including 50 plant species not previously reported as hosts of Aphelenchoides spp. The detection sensitivity of the PCR-based assay was higher for infected yet asymptomatic plants when compared to the traditional, water extraction method for Aphelenchoides spp. detection. The utility of using NaOH extraction for rapid preparation of total DNA from plant samples infected with A. fragariae was demonstrated.     

DNA accelerates the inhibition of human cathepsin V by serpins

A balance between proteolytic activity and protease inhibition is crucial to the appropriate function of many biological processes. There is mounting evidence for the presence of both papain-like cysteine proteases and serpins with a corresponding inhibitory activity in the nucleus. Well characterized examples of cofactors fine tuning serpin activity in the extracellular milieu are known, but such modulation has not been studied for protease-serpin interactions within the cell. Accordingly, we present an investigation into the effect of a DNA-rich environment on the interaction between model serpins (MENT and SCCA-1), cysteine proteases (human cathepsin V and human cathepsin L), and cystatin A. DNA was indeed found to accelerate the rate at which MENT inhibited cathepsin V, a human orthologue of mammalian cathepsin L, up to 50-fold, but unexpectedly this effect was primarily effected via the protease and secondarily by the recruitment of the DNA as a "template" onto which cathepsin V and MENT are bound. Notably, the protease-mediated effect was found to correspond both with an altered substrate turnover and a conformational change within the protease. Consistent with this, cystatin inhibition, which relies on occlusion of the active site rather than the substrate-like behavior of serpins, was unaltered by DNA. This represents the first example of modulation of serpin inhibition of cysteine proteases by a co-factor and reveals a mechanism for differential regulation of cathepsin proteolytic activity in a DNA-rich environment.