Many faces of the unfolded state: conformational heterogeneity in denatured yeast cytochrome C

We have measured fluorescence energy-transfer (FET) kinetics from a dansyl fluorophore (Dns) introduced by derivatization of a Cys side-chain to the Fe(III) heme covalently attached to unfolded yeast iso-1 cytochrome c (cyt). To gain a global picture of the unfolded state, we examined variants with the fluorophore attached on three different helices (K4C, E66C, K99C) and in three different loops (H39C, D50C, L85C). Analysis of the FET kinetics data gave distributions of distances between the fluorescent donor and acceptor; these distributions demonstrate that the guanidine hydrochloride (GuHCl)-denatured polypeptide ensemble is not a simple random coil. Although misligation imposes some constraints, it is not the only source of structural complexity in the unfolded protein. Our FET kinetics data reveal a high degree of heterogeneity in the unfolded ensemble of cytochrome c. We detect relatively large populations of compact structures in unfolded Dns(C50)cyt, Dns(C39)cyt, and Dns(C66)cyt. These structures likely play a role in forming a hydrophobic core during the folding process.     

Nature of functional motor asymmetry in animals: state of the problem

Handedness in skilled movements of animals is a result of interaction of innate motor preference and learning. The nature of the innate preference is not clear. Breeding of right-handed and left-handed mice revealed that the degree rather than direction of motor preference is an inherited feature. There is, however, a correlation between the direction of preference and a number of morphological, functional, and neurochemical characters. Shifts of a preference direction were found in some strains of mice. Differences between right-handed and left-handed rats were revealed in social behavior, learning, and resistance to forced retraining. Strains of rats with different forms of genetic epilepsy were characterized by the predominance of animals with a certain direction of the motor preference. This evidence suggests some genetic influence on a direction of the motor preference. Perhaps, genetic and environmental factors closely interact in determining motor preference in animals.     

Comparison of the genome for phylogenetically related bacteriophages phiKZ and EL of Pseudomonas aeruginosa: evolutionary aspects and minimal genome size

Bacteriophages of the family Myoviridae represent one of the most widespread domains of the biosphere substantially affecting the ecological balance of microorganisms. Interestingly, sequence analysis of genomic DNAs of large bacteriophages revealed many genes coding for proteins with unknown functions. A new approach is proposed to improve the functional identification of genes. This approach is based on comparing the genome sequence for phylogenetically and morphologically related phages showing no considerable homology at the level of genomic DNA. It is assumed that gene functions essential for the development of phages of a given family are conserved and that the corresponding genes code for similar orthologous proteins even when lacking sequence homology. The genome was sequenced and compared for two Pseudomonas aeruginosa giant bacteriophages, phiKZ and EL, which belong to a group of (phiKZ-related phages. A substantial difference in genome organization was observed, suggesting specific features of phage evolution. In addition, the problem of the minimal genome of the superfamily is discussed on the basis of the difference in size and structure between the phiKZ and EL genomes.     

Cyclophosphamide resets dendritic cell homeostasis and enhances antitumor immunity through effects that extend beyond regulatory T cell elimination

Using a model of established malignancy, we found that cyclophosphamide (Cy), administered at a dose not requiring hematopoietic stem cell support, is superior to low-dose total body irradiation in augmenting antitumor immunity. We observed that Cy administration resulted in expansion of tumor antigen-specific T cells and transient depletion of CD4⁺Foxp3⁺ regulatory T cells (Tregs). The antitumor efficacy of Cy was not improved by administration of anti-CD25 monoclonal antibody given to induce more profound Treg depletion. We found that Cy, through its myelosuppressive action, induced rebound myelopoiesis and perturbed dendritic cell (DC) homeostasis. The resulting DC turnover led to the emergence of tumor-infiltrating DCs that secreted more IL-12 and less IL-10 compared to those from untreated tumor-bearing animals. These newly recruited DCs, originating from proliferating early DC progenitors, were fully capable of priming T cell responses and ineffective in inducing expansion of Tregs. Together, our results show that Cy-mediated antitumor effects extend beyond the well-documented cytotoxicity and lymphodepletion and include resetting the DC homeostasis, thus providing an excellent platform for integration with other immunotherapeutic strategies.     

Effect of perfluorodecalin, carbogal, and perfluoromethyldecalin on growth and ice-forming activity of bacteria

The isolates of pseudomonads (56) and of Plesiomonas shigelloides (7) exhibiting ice-forming activity were obtained from plant leaves and rhizosphere. The theoretical possibility of the application of perfluorocarbons (PFC) with a gas-transporting function (perfluorodecalin, carbogal, and perfluoromethyldecalin) for the cultivation of bacteria with different levels of ice-forming activity (IFA) in order to enhance their growth rates, biomass yields, and IFA was demonstrated. Introduction of 5% perfluorodecalin, carbogal, or perfluoromethyldecalin resulted for two strains in a 1.7–3.1-fold increase in biomass and a 3.2–24.5-fold increase in ice-forming activity compared to the control (without PFC).     

Three-dimensional structure of a pH-dependent fluorescent protein WasCFP with a tryptophan based deprotonated chromophore

WasCFP, a pH-dependent green fluorescent protein with a tryptophan-based chromophore (Thr65-Trp66-Gly67) in anionic state, was designed from a cyan precursor mCerulean. In this study, the three-dimensional structure of WasCFP has been determined by an X-ray method at pH 5.5, pH 8.0 and pH 10.0, with a resolution of 1.14, 1.25 and 1.5 Å, respectively. We show that changes in the acidity of the media are accompanied by a synchronous change of the side chain conformations of the residues in the near-chromophore environment. Subsequent changes in the local H-bond network interacting with the chromophore lead to considerable alterations in the protein spectral properties as a consequence of reversible processes of ionization-protonation of the Trp chromophore. These experimental results have been supported by quantum chemistry calculations.     

Blocking action of the soluble H-2 antigens and H-2 antibody isolated by various methods and mouse immune lymphocytes]

The treatment of tumour and lymphoid cells of mice with 3M KC1 solution having high ionic strength, nonionic detergent and by subsequent freeze-thawing resulted in obtaining serologically active H-2 antigen preparation capable of specifically blocking the cytotoxicity of H-2 antisera. The antigenic activity of the preparations thus obtained depended on the source from which they were isolated (spleen cells and their membrane fragments proved to be the best source), on the degree of maturity of tumor cells and the degree of purification of the preparation, as well as on the methods of solubilization. The blocking action of soluble H-2 antigens on the cytotoxicity of immune lymphocytes depended on the method used for isslating these antigens. The interaction of immune lymphocytes and H-2 antisera with soluble antigens was probably effected by different mechanisms.