Subcellular measurements of mechanical and chemical properties using dual Raman‐Brillouin microspectroscopy

Brillouin microspectroscopy is a powerful technique for noninvasive optical imaging. In particular, Brillouin microspectroscopy uniquely allows assessing a sample's mechanical properties with microscopic spatial resolution. Recent advances in background‐free Brillouin microspectroscopy make it possible to image scattering samples without substantial degradation of the data quality. However, measurements at the cellular‐ and subcellular‐level have never been performed to date due to the limited signal strength. In this report, by adopting our recently optimized VIPA‐based Brillouin spectrometer, we probed the microscopic viscoelasticity of individual red blood cells. These measurements were supplemented by chemically specific measurements using Raman microspectroscopy.

     

Creation and analysis of a bank of chromosomal genes of Streptococcus group A

The representative genomic library of chromosomal genes has been constructed for streptococcus group A serotype M48 strain 1/64 on the vector lambda L 47.1. Screening of the obtained genomic library by hybridization and immunological techniques revealed about 50 clones producing the streptococcal antigens (extracellular nonidentified products and non-type specific structural streptococcal proteins). Among the recombinant clones three were found to harbour the genetic determinants for M-protein. One the clones contains a determinant coding for epitopes crossreacting with antisera to M-proteins of other serotypes and a protective epitope. The presence of the latter was tested in an indirect bactericidal test.     

Na+,K+-ATPase activity in cultured C6 glioma cells

Rat C₆ glioma cells were cultured for 4 days in MEM medium supplemented with 10% bovine serum and Na⁺,K⁺-ATPase activity was determined in homogenates of harvested cells. Approximately 50% of enzyme activity was attained at 1.5 mM K⁺ and the maximum (2.76±0.13 mol Pi/h/mg protein) at 5 mM K⁺. The specific activity of Na⁺,K⁺-ATPase was not influenced by freezing the homogenates or cell suspensions before the enzyme assay. Ten minutes' exposure of glioma cells to 10^–4 or 10^–5 M noradrenaline (NA) remained without any effect on NA⁺,K⁺-ATPase activity. Neither did the presence of NA in the incubation medium, during the enzyme assay, influence the enzyme activity. The nonresponsiveness of Na⁺,K⁺-ATPase of C₆ glioma cells to NA is consistent with the assumption that (+) form of the enzyme may be preferentially sensitive to noradrenaline. Na⁺,K⁺-ATPase was inhibited in a dose-dependent manner by vanadate and 50% inhibition was achieved at 2×10^–7 M concentration. In spite of the fact that Na⁺,K⁺-ATPase of glioma cells was not responsive to NA, the latter could at least partially reverse vanadate-induced inhibition of the enzyme. Although the present results concern transformed glial cells, they suggest the possibility that inhibition of glial Na⁺,K⁺-ATPase may contribute to the previously reported inhibition by vanadate of Na⁺,K⁺-ATPase of the whole brain tissue.     

MASDET—A fast and user-friendly multiplatform software for mass determination by dark-field electron microscopy

Electron microscopy has been used to measure the mass of biological nanoparticles since the early 60s, and is the only way to obtain the mass of large structures or parameters such as the mass-per-length of filaments. The ability of this method to sort heterogeneous samples both in terms of mass and shape promises to make it a key tool for proteomics down to the single cell level. A new multiplatform software package, MASDET, that can be run under MATLAB or as a standalone program is described. Based on a user-friendly graphical interface MASDET streamlines mass evaluation and greatly increases the speed of required optimisation procedures. Importantly, the immediate application of Monte-Carlo simulations to describe multiple scattering is possible, allowing the mass analysis of thicker samples and the generation of mass thickness maps.     

Stabilization of secondary structure elements by specific combinations of hydrophilic and hydrophobic amino acid residues is more important for proteins encoded by GC-poor genes

Stabilization of secondary structure elements by specific combinations of hydrophobic and hydrophilic amino acids has been studied by the way of analysis of pentapeptide fragments from twelve partial bacterial proteomes. PDB files describing structures of proteins from species with extremely high and low genomic GC-content, as well as with average G + C were included in the study. Amino acid residues in 78,009 pentapeptides from alpha helices, beta strands and coil regions were classified into hydrophobic and hydrophilic ones. The common propensity scale for 32 possible combinations of hydrophobic and hydrophilic amino acid residues in pentapeptide has been created: specific pentapeptides for helix, sheet and coil were described. The usage of pentapeptides preferably forming alpha helices is decreasing in alpha helices of partial bacterial proteomes with the increase of the average genomic GC-content in first and second codon positions. The usage of pentapeptides preferably forming beta strands is increasing in coil regions and in helices of partial bacterial proteomes with the growth of the average genomic GC-content in first and second codon positions. Due to these circumstances the probability of coil-sheet and helix-sheet transitions should be increased in proteins encoded by GC-rich genes making them prone to form amyloid in certain conditions. Possible causes of the described fact that importance of alpha helix and coil stabilization by specific combinations of hydrophobic and hydrophilic amino acids is growing with the decrease of genomic GC-content have been discussed.     

Conformational changes in the photocycle of Anabaena sensory rhodopsin: absence of the Schiff base counterion protonation signal

Anabaena sensory rhodopsin (ASR) is a novel microbial rhodopsin recently discovered in the freshwater cyanobacterium Anabaena sp. PCC7120. This protein most likely functions as a photosensory receptor as do the related haloarchaeal sensory rhodopsins. However, unlike the archaeal pigments, which are tightly bound to their cognate membrane-embedded transducers, ASR interacts with a soluble cytoplasmic protein analogous to transducers of animal vertebrate rhodopsins. In this study, infrared spectroscopy was used to examine the molecular mechanism of photoactivation in ASR. Light adaptation of the pigment leads to a phototransformation of an all-trans/15-anti to 13-cis/15-syn retinylidene-containing species very similar in chromophore structural changes to those caused by dark adaptation in bacteriorhodopsin. Following 532 nm laser-pulsed excitation, the protein exhibits predominantly an all-trans retinylidene photocycle containing a deprotonated Schiff base species similar to those of other microbial rhodopsins such as bacteriorhodopsin, sensory rhodopsin II, and Neurospora rhodopsin. However, no changes are observed in the Schiff base counterion Asp-75, which remains unprotonated throughout the photocycle. This result along with other evidence indicates that the Schiff base proton release mechanism differs significantly from that of other known microbial rhodopsins, possibly because of the absence of a second carboxylate group at the ASR photoactive site. Several conformational changes are detected during the ASR photocycle including in the transmembrane helices E and G as indicated by hydrogen-bonding alterations of their native cysteine residues. In addition, similarly to animal vertebrate rhodopsin, perturbations of the polar head groups of lipid molecules are detected.