tRNA residues evolved to promote translational accuracy

The decoding properties of 22 structurally conservative base-pair and base-triple mutations in the anticodon hairpin and tertiary core of Escherichia coli tRNA^Ala_GGC were determined under single turnover conditions using E. coli ribosomes. While all of the mutations were able to efficiently decode the cognate GCC codon, many showed substantial misreading of near-cognate GUC or ACC codons. Although all the misreading mutations were present in the sequences of other E. coli tRNAs, they were never found among bacterial tRNA^Ala_GGC sequences. This suggests that the sequences of bacterial tRNA^Ala_GGC have evolved to avoid reading incorrect codons.     

Use of a gel-forming dipeptide derivative as a carrier for antigen presentation

A dipeptide of the formula Fmoc-Leu-Asp and some other related dipeptides were synthesized in solution by standard methods. When such peptides are dissolved in water at concentrations below 1% at 100 °C and cooled below 60 °C they form turbid solutions and eventaully visocelastic gels at lower temperatures. Such gels are thermoreversible and can also be disrupted by mechanical agitation. At a concentration of 2 mg/ml the peptide Fmoc-Leu-Asp forms an aqueous gel at 60 °C with a shear modulus of 80 Pa measured at a frequency of 1 rad/s. Peptide solutions undergo an abrupt increase in light scattering between 1 and 1.5 mg/ml at both 23 and 60 °C. By analogy with previous observations of other systems, this increse appears to be due to the formation of filamentous micelles and the aggregation of filamaents into a three-dimensional network. When low molecular weight adamantanamine derivatives, which are inherently non-antigenci antiviral drugs, were incorporated into the Fmoc-Leu-Asp gel and injected into rabbits, high titre specific antibodies were efficiently produced without the need for additional adjuvant. Both the physical properties of the gel and its effect on the antigenicity of low molecular weight compounds suggest a number of practical applications.     

Formation of potential titanium antigens based on protein binding to titanium dioxide nanoparticles

Degradation products of titanium implants include free ions, organo-metallic complexes, and particles, ranging from nano to macro sizes. The biological effects, especially of nanoparticles, is yet unknown. The main objective of this study was to develop Ti-protein antigens in physiological solutions that can be used in testing of cellular responses. For this purpose, 0.1% TiO2 nanoparticles less than 100 nm were mixed with human serum albumin (HSA), 0.1% and 1%, in cell culture medium (DMEM, pH 7.2). The Ti concentrations in the resulting solutions were analyzed by inductively coupled plasma mass spectrometry. The stability of the nanoparticles in suspension was analyzed by UV-vis spectrophotometer and Dynamic Light Scattering. The concentration of Ti in suspension was dependent on the presence and concentration of HSA. Albumin prevented high aggregation rate of TiO2 nanoparticles in cell culture medium. It is shown that nano TiO2-protein stable aggregates can be produced under physiological conditions at high concentrations, and are candidates for use in cellular tests.     

An RNA microchip containing immobilized oligoribonucleotides with protective groups at 2'-O-positions

To analyze RNA interactions with RNA binding molecules an RNA microchip containing immobilized oligoribonucleotides with protective groups [t-butyldimethylsilyl (tBDMS)] at 2'-O- positions was developed. The oligonucleotides were immobilized within three-dimensional (3-D) hydrogel pads fixed on a glass support. The protective groups preserved the oligoribonucleodes from degradation and were suitable to be removed directly on the microchip when needed, right before its use. These immobilized, deprotected oligoribonucleotides were tested for their interaction with afluorescently labeled oligodeoxyribonucleotide and analyzed for their availability to be cleaved enzymatically by the RNase binase. Stability of tBDMS-protected immobilized oligoribonucleotides after 2.5 years of storage as well as after direct RNase action was also tested. Melting curves of short RNA/DNA hybrids that had formed into gel pads of the microchip were found to exhibit clearly defined S-like shapes, with the melting temperatures in full accordance with those theoretically predicted for the same ionic strength. This approach, based on keeping the protective groups attached to oligoribonucleotides, can be applied for manufacturing any RNA microchips containing immobilized oligoribonucleotides, including microchips with two-dimensional (2-D) features. These RNA microchips can be used to measure thermodynamic parameters of RNA/RNA or RNA/DNA duplexes as well as to study ligand- or protein-RNA interactions.     

Simultaneous Surface Display and Secretion of Proteins from Mammalian Cells Facilitate Efficient in Vitro Selection and Maturation of Antibodies

A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein also being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cell-based functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Presentation and secretion of monomeric Fab can also be achieved to minimize avidity effects. Manipulation of the splice donor site sequence enables control of the relative amounts of cell surface and secreted antibody. Multi-domain proteins may be presented and secreted in different formats to enable flexibility in experimental design, and secreted proteins may be produced with epitope tags to facilitate high-throughput testing. This system is particularly useful in the context of in situ mutagenesis, as in the case of in vitro somatic hypermutation.     

Potassium chloride released from contracting skeletal muscle may stimulate development of its hypertrophy

The effects of potassium chloride on the expression of IGF-1 splice forms and myoblast proliferation were investigated. KCl at the concentrations of 7–12 mM stimulated the synthesis of IGF-1 and mechano growth factor (MGF) in murine myoblasts as well as in myotubes both at the mRNA and protein levels. Pan-calcium channel blocker CdCl₂ completely abolished stimulation of growth factor expression, whereas blocker of HCN and Na_v1.4 channels ZD7288 drastically reduced it. In addition, potassium chloride stimulated myoblast proliferation, while IGF-1 autocrine signaling inhibition partially suppressed these mitogenic effects.     

Petrosal and inner ear anatomy and allometry amongst specimens referred to Litopterna (Placentalia)

New isolated petrosals from the Itaboraí beds of Brazil (late Palaeocene or early Eocene) are here described and referred to the early diverging litoptern Miguelsoria parayirunhor, based on phylogenetic, size, and abundance arguments. Both the external and internal anatomy of these specimens were investigated, which for the first time document many details of the auditory region of a Palaeogene litoptern. Our cladistic analysis, which included our new observations, failed to recover a monophyletic Litopterna but did not exclude it. A constrained analysis for the monophyly of this order showed that several features such as a (sub)quadrangular and anteroposteriorly elongated tensor tympani fossa and a large notch in the vicinity of the external opening of the cochlear canaliculus may constitute synapomorphies for Litopterna. The evolution of several other auditory characters amongst Litopterna is discussed and the relative dimensions of the inner ear and surrounding petrosal in the group were also investigated. This allowed detection of negative allometry of the bony labyrinth within the petrosal, which was confirmed by measurements and regression analysis across a larger sample of placental mammals. This scaling effect probably has an important influence on several characters of the bony labyrinth and petrosal, amongst which are the length of the vestibular aqueduct and cochlear canaliculus. It demonstrates that many aspects of the morphological variation of the bony labyrinth need to be thoroughly investigated before being incorporated into phylogenetic analyses. © 2015 The Linnean Society of London     

Sensitivity of Superfolder GFP to Ionic Agents

Superfolder variant of the green fluorescent protein (sfGFP) became a favorite probe for examination of the unfolding–refolding processes of fluorescent proteins with beta-barrel structure owing to its reversible unfolding in comparison with other fluorescent proteins. Its benefit is the proper folding even in fusion constructions with poorly folded polypeptides. We noticed that guanidine thiocyanate affects not only the structure of protein but its chromophore directly. Therefore we studied the influence of ionic denaturants and salts including guanidine thiocyanate, guanidine hydrochloride, sodium chloride and sodium thiocyanate on spectral features of sfGFP. It was shown that moderate amounts of the studied agents do not disrupt sfGFP structure but provoke pronounced alteration of its spectral characteristics. Changes in absorption and CD spectra in visible spectral range indicate the specific binding of SCN⁻ and Cl⁻ anions in the sfGFP chromophore vicinity. The anion binding results in the redistribution of sfGFP molecules with neutral and anionic chromophores. This also hinders the proton transfer in the chromophore excited state, considerably decreasing the fluorescence intensity of sfGFP. Our results indicate that when ionic denaturants are used in the studies of fluorescent protein folding their effect on fluorophore charge state should be taken into account.     

Functional properties of proteins from the coelomic fluid of the wounded sea star Asterias rubens (L)

Impact on viability and adhesion of three protein fractions, separated by size, from the coelomic fluid of wounded Asterias rubens′, was tested on autologous coelomocytes. In addition antimicrobial property of the protein fractions was tested on the Gram-negative bacterium Vibrio parahaemolyticus. All fractions promoted viability and the larger proteins facilitated adhesion of the coelomocytes. The strongest antimicrobial effect was caused by the fraction with the smallest proteins.     

Muscodor fengyangensis sp. nov. from southeast China: morphology, physiology and production of volatile compounds

The fungal genus Muscodor was erected on the basis of Muscodor albus, an endophytic fungus originally isolated from Cinnamomum zeylanicum. It produces a mixture of volatile organic compounds (VOCs) with antimicrobial activity that can be used as mycofumigants. The genus currently comprises five species. Here we describe the isolation and characterization of a new species of Muscodor on the basis of five endophytic fungal strains from leaves of Actinidia chinensis, Pseudotaxus chienii and an unidentified broad leaf tree in the Fengyangshan Nature Reserve, Zhejiang Province, Southeast of China. They exhibit white colonies on potato dextrose agar (PDA) media, rope-like mycelial strands, but did not sporulate. The optimum growth temperature is 25°C. The results of a phylogenetic analysis based on four loci (ITS1-5.8S-ITS2, 28S rRNA, rpb2 and tub1) are consistent with the hypothesis that these five strains belong to a single taxon. All five strains also produce volatile chemical components with antimicrobial activity in vitro, which were different from those previously described for other Muscodor species.