Informatics issues in large-scale sequence analysis: elucidating the protein kinases of C. elegans

With the availability of the nearly complete genomic sequence of C. elegans, the first multicellular organism to be sequenced, molecular biology has definitely entered the postgenomic era. Annotation of the genomic sequence, which refers to identifying the genes and other biologically relevant sections of the genome, is an important and nontrivial next step. A first-pass annotation will be necessarily incomplete but will drive further biological experiments, which in turn will help to annotate the genome better. Given the scale of the genome sequence analysis, it is clear that the annotation should be automated as much as possible without sacrificing the quality of analysis. In this work, we outline our approach to identifying the protein kinases of C. elegans from the genomic sequence. We describe new tools we have developed for analysis, management and visualization of genomic data. By developing modular and scalable solutions, this study has provided a framework for future analysis of the Drosophila and human genomes.     

The Streptococcal Binding Site in the Gelatin-binding Domain of Fibronectin Is Consistent with a Non-linear Arrangement of Modules

Fibronectin-binding proteins (FnBPs) of Staphylococcus aureus and Streptococcus pyogenes mediate invasion of human endothelial and epithelial cells in a process likely to aid the persistence and/or dissemination of infection. In addition to binding sites for the N-terminal domain (NTD) of fibronectin (Fn), a number of streptococcal FnBPs also contain an upstream region (UR) that is closely associated with an NTD-binding region; UR binds to the adjacent gelatin-binding domain (GBD) of Fn. Previously, UR was shown to be required for efficient streptococcal invasion of epithelial cells. Here we show, using a Streptococcus zooepidemicus FnBP, that the UR-binding site in GBD resides largely in the ⁸F1⁹F1 module pair. We also show that UR inhibits binding of a peptide from the α1 chain of type I collagen to ⁸F1⁹F1 and that UR binding to ⁸F1 is likely to occur through anti-parallel β-zipper formation. Thus, we propose that streptococcal proteins that contain adjacent NTD- and GBD-binding sites form a highly unusual extended tandem β-zipper that spans the two domains and mediates high affinity binding to Fn through a large intermolecular interface. The proximity of the UR- and NTD-binding sequences in streptococcal FnBPs is consistent with a non-linear arrangement of modules in the tertiary structure of the GBD of Fn.     

Spectroscopic studies of Rhodobacter capsulatus cytochrome c' in the isolated state and in intact cells.

Ferricytochrome c' from Rhodobacter capsulatus was investigated by 1H-NMR, EPR and optical spectroscopies. A haem-linked ionisation, occurring with a pKa of 8.4 at 25 degrees C, was observed and assigned to the ionisation of the axial histidine ligand by comparison with data for related proteins. At pH values below this pKa the spin-state of the haem Fe3+ is shown to be a quantum mechanically admixed S = 3/2, 5/2 state. Above the pKa the Fe3+ is high-spin. EPR studies of intact cells grown photoheterotrophically reveal that in situ cytochrome c' exists largely in the ferrous state. Upon the addition of [Fe(CN)6]3- the protein becomes oxidised and EPR spectra reveal that the Fe3+ spin-state is a quantum mechanically admixed S = 3/2, 5/2 state. These data indicate that the unusual spin-state of ferricytochrome c' is not a consequence of changes to the protein on its isolation, as had been suggested previously. They also indicate that in situ cytochrome c' is located in an environment with a pH less than 7.     

HIV antigens can induce TGF-beta(1)-producing immunoregulatory CD8+ T cells

HIV-infected individuals may progressively lose both HIV-specific and unrelated CTL responses despite the high number of circulating CD8+ T cells. In this study, we report that approximately 25% of HIV+ donors produced TGF-beta(1) in response to stimulation with HIV proteins or peptides. The production of TGF-beta(1) was sufficient to significantly reduce the IFN-gamma response of CD8+ cells to both HIV and vaccinia virus proteins. Ab to TGF-beta reversed the suppression. We found the source of the TGF-beta(1) to be predominantly CD8+ cells. Different peptide pools stimulated TGF-beta(1) and IFN-gamma in the same individual. The TGF-beta(1) secreting cells have distinct peptide specificity from the IFN-gamma producing cells. This represents an important mechanism by which an HIV-specific response can nonspecifically suppress both HIV-specific and unrelated immune responses.     

Interaction of microwave radiation undergoing stochastic phase jumps with plasmas or gases

New types of beam-plasma devices generating intense stochastic microwave radiation in the interaction of electron beams with hybrid plasma waveguides were developed and put into operation at the National Science Center Kharkov Institute of Physics and Technology (Ukraine). The objective of the paper is to discuss the results of theoretical and experimental studies and numerical simulations of the normal and oblique incidence of linearly polarized electromagnetic waves on an interface between a vacuum and an overcritical plasma. The main results of the reported investigations are as follows: (i) for the parameter values under analysis, the transmission coefficient for microwaves with a stochastically jumping phase is one order of magnitude greater than that for a broadband regular electromagnetic wave with the same spectral density; (ii) the electrons are heated most efficiently by obliquely incident waves with a stochastically jumping phase and, in addition, the electron distribution function has a high-energy tail; and (iii) necessary conditions for gas breakdown and for the initiation of a microwave discharge in stochastic fields in a light source are determined. The anomalously large transmission coefficient for microwaves, the anomalous character of the breakdown conditions, the anomalous behavior of microwave gas discharges, and the anomalous nature of collisionless electron heating, are attributed to stochastic jumps in the phase of microwave radiation.