Statistical discovery of site inter-dependencies in sub-molecular hierarchical protein structuring

Background

Much progress has been made in understanding the 3D structure of proteins using methods such as NMR and X-ray crystallography. The resulting 3D structures are extremely informative, but do not always reveal which sites and residues within the structure are of special importance. Recently, there are indications that multiple-residue, sub-domain structural relationships within the larger 3D consensus structure of a protein can be inferred from the analysis of the multiple sequence alignment data of a protein family. These intra-dependent clusters of associated sites are used to indicate hierarchical inter-residue relationships within the 3D structure. To reveal the patterns of associations among individual amino acids or sub-domain components within the structure, we apply a k-modes attribute (aligned site) clustering algorithm to the ubiquitin and transthyretin families in order to discover associations among groups of sites within the multiple sequence alignment. We then observe what these associations imply within the 3D structure of these two protein families.

Results

The k-modes site clustering algorithm we developed maximizes the intra-group interdependencies based on a normalized mutual information measure. The clusters formed correspond to sub-structural components or binding and interface locations. Applying this data-directed method to the ubiquitin and transthyretin protein family multiple sequence alignments as a test bed, we located numerous interesting associations of interdependent sites. These clusters were then arranged into cluster tree diagrams which revealed four structural sub-domains within the single domain structure of ubiquitin and a single large sub-domain within transthyretin associated with the interface among transthyretin monomers. In addition, several clusters of mutually interdependent sites were discovered for each protein family, each of which appear to play an important role in the molecular structure and/or function.

Conclusions

Our results demonstrate that the method we present here using a k- modes site clustering algorithm based on interdependency evaluation among sites obtained from a sequence alignment of homologous proteins can provide significant insights into the complex, hierarchical inter-residue structural relationships within the 3D structure of a protein family.

     

Prevalent role of TCR alpha-chain in the selection of the preimmune repertoire specific for a human tumor-associated self-antigen

The specificity of recognition of pMHC complexes by T lymphocytes is determined by the V regions of the TCR alpha- and beta-chains. Recent experimental evidence has suggested that Ag-specific TCR repertoires may exhibit a more V alpha- than V beta-restricted usage. Whether V alpha usage is narrowed during immune responses to Ag or if, on the contrary, restricted V alpha usage is already defined at the early stages of TCR repertoire selection, however, has remained unexplored. Here, we analyzed V and CDR3 TCR regions of single circulating naive T cells specifically detected ex vivo and isolated with HLA-A2/melan-A peptide multimers. Similarly to what was previously observed for melan-A-specific Ag-experienced T cells, we found a relatively wide V beta usage, but a preferential V alpha 2.1 usage. Restricted V alpha 2.1 usage was also found among single CD8(+) A2/melan-A multimer(+) thymocytes, indicating that V alpha-restricted selection takes place in the thymus. V alpha 2.1 usage, however, was independent from functional avidity of Ag recognition. Thus, interaction of the pMHC complex with selected V alpha-chains contributes to set the broad Ag specificity, as underlined by preferential binding of A2/melan-A multimers to V alpha 2.1-bearing TCRs, whereas functional outcomes result from the sum of these with other interactions between pMHC complex and TCR.     

Changing concepts in plant hormone action

Summary A plant hormone is not, in the classic animal sense, a chemical synthesized in one organ, transported to a second organ to exert a chemical action to control a physiological event. Any phytohormone can be synthesized everywhere and can influence different growth and development processes at different places. The concept of physiological activity under hormonal control cannot be dissociated from changes in concentrations at the site of action, from spatial differences and changes in the tissue's sensitivity to the compound, from its transport and its metabolism, from balances and interactions with the other phytohormones, or in their metabolic relationships, and in their signaling pathways as well. Secondary messengers are also involved. Hormonal involvement in physiological processes can appear through several distinct manifestations (as environmental sensors, homeostatic regulators and spatio-temporal synchronizers, resource allocators, biotime adjusters, etc.), dependent on or integrated with the primary biochemical pathways. The time has also passed for the hypothesized ‘specific’ developmental hormones, rhizocaline, canlocaline, and florigen: root, stem, and flower formation result from a sequential control of specific events at the right places through a coordinated control by electrical signals, the known phytohormones and nonspecific molecules of primary and secondary metabolism, and involve both cytoplasmic and apoplastic compartments. These contemporary views are examined in this review.     

Ex vivo IFN-gamma secretion by circulating CD8 T lymphocytes: implications of a novel approach for T cell monitoring in infectious and malignant diseases

To elucidate the functional heterogeneity of Ag-specific T lymphocyte populations, we combined labeling of lymphocytes with MHC/peptide tetramers and a cell surface affinity matrix for IFN-gamma. Magnetic cell sorting of IFN-gamma-positive lymphocytes allowed the selective enrichment and identification of live Ag-specific cytokine-secreting cells by flow cytometry. Naive, memory, and effector Ag-specific populations were evaluated in healthy HLA-A2 individuals. Significant fractions of influenza- and CMV-specific cells secreted IFN-gamma upon challenge with cognate peptide, consistent with an effector/memory status. The sensitivity of the approach allowed the detection of significant numbers of CMV-specific IFN-gamma-secreting cells ex vivo (i.e., without Ag stimulation). This was not apparent when using previously described assays, namely, ELISPOT or intracellular IFN-gamma staining (cytospot). CD8+ T cells specific for the melamoma-associated Ag Melan-A/MART-1 did not produce IFN-gamma upon challenge with cognate peptide, reminiscent with their naive functional state in healthy individuals. In contrast, CD45RA(low) Melan-A/MART-1 tumor-specific cells from three of three melanoma patients presented levels of activity similar to those found for influenza- or CMV virus-specific lymphocytes, compatible with a functional differentiation into competent effector/memory T lymphocytes in vivo. Notably, a sizable fraction of Melan-A/MART-1-specific cells from a patient secreted IFN-gamma ex vivo following peptide-based vaccination. Thus, the high sensitivity of the assay provides a valuable tool to monitor effector T cell responses in different clinical situations.     

An easy and reliable method for establishment and maintenance of leaf and root cell cultures ofArabidopsis thaliana

Cell suspension cultures are useful for a wide range of biochemical and physiological studies, yet their production can be technically demanding and often unreliable. Here we describe a protocol for producing Arabidopsis cell suspension cultures that is reliable and easy to use.     

Comparative analysis and validation of different assays for glycopeptide susceptibility among methicillin-resistant Staphylococcus aureus strains

Detection of methicillin-resistant Staphylococcus aureus (MRSA) isolates exhibiting intermediate susceptibility to glycopeptides (GISA) is challenging for clinical microbiology laboratories. We compared three different screening assays for evaluating trends in decreased glycopeptide susceptibility during two periods. Ninety four and ninety five consecutive MRSA blood isolates from 189 bacteremic patients collected during periods A (1989-1994) and B (1999-2001), respectively, were screened in parallel for vancomycin or teicoplanin susceptibility by glycopeptide-containing brain-heart infusion agar (BHIA) tests, Etest MICs performed at a standard (0.5 McFarland) or high (2.0 McFarland) inoculum on Mueller-Hinton (MHA) or BHIA, respectively. Any MRSA isolate yielding <50 CFU (representing <10(-6) of the plated inoculum) on either BHIA containing 2 mg/l of vancomycin (V2-BHIA) or 5 mg/l of teicoplanin (T5-BHIA) was considered as fully susceptible to vancomycin or teicoplanin, respectively. The proportion of MRSA isolates yielding>50 CFU on either V2-BHIA or T5-BHIA significantly (P<0.01) increased from 7/94 (7.4 %) or 8/94 (8.5%) in period A to 16/95 (16.8 %) or 14/95 (14.7%) in period B, respectively. Vancomycin Etests MICs on MHA were of lower sensitivity (<30% and<65%), but high specificity (100% and 99%) on periods A and B isolates, respectively, compared to those on V2-BHIA. Vancomycin Etests MICs on BHIA were of a higher sensitivity (57% and 81%) but lower specificity (91% and 65%) compared to those on V2-BHIA, on periods A and B isolates, respectively, reflecting an unexpectedly high number of false positive isolates on period B isolates (28/95). Screening of decreased susceptibility to vancomycin or teicoplanin on V2-BHIA or T5-BHIA, respectively, may represent simple low-cost alternatives to Etest MICs, minimising the risk of missing potential GISA isolates.     

Calciosome, a sarcoplasmic reticulum-like organelle involved in intracellular Ca2+-handling by non-muscle cells: studies in human neutrophils and HL-60 cells

Calciosomes are intracellular organelles in HL-60 cells, neutrophils and various other cell types, characterized by their content of a Ca2+-binding protein that is biochemically and immunologically similar to calsequestrin (CS) from muscle cells. In subcellular fractionation studies the CS-like protein copurifies with functional markers of the inositol 1,4,5-trisphosphate (IP3) releasable Ca2+-store. These markers (ATP-dependent Ca2+-uptake and IP3-induced Ca2+-release) show a subcellular distribution which is clearly distinct from the endoplasmic reticulum and other organelles. In morphological studies, antibodies against rabbit skeletal muscle CS protein specifically stained hitherto unrecognized vesicles with a diameter between 50 and 250 nm. Thus both, biochemical and morphological studies indicate that the calsequestrin containing intracellular Ca2+-store, now referred to as the calciosome, is distinct from other known organelles such as endoplasmic reticulum. Calciosomes are likely to play an important role in intracellular Ca2+-homeostasis. They are possibly the intracellular target of inositol 1,4,5-trisphosphate and thus the source of Ca2+ that is redistributed into the cytosol following surface receptor activation in non-muscle cells.     

Mass changes in inositol tetrakis- and pentakisphosphate isomers induced by chemotactic peptide stimulation in HL-60 cells

Absolute concentrations of inositol phosphate isomers (InsP(s] were quantified in the myeloid cell line HL-60 using the metal-dye detection technique. Stimulation with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) led to distinct alterations in at least seven different inositol phosphate species. Whereas the intracellular concentrations of the tetrakisphosphate isomers (InsP4(s] were found below the micromolar range, inositol 1,3,4,5,6-pentakis- and hexakisphosphate levels were about two orders of magnitude higher (36 and 54 +/- 2 microM (mean +/- S.D.), respectively). The three InsP4(s) showed distinct kinetic pattern upon receptor activation, the transient elevation of inositol 1,3,4,5-tetrakisphosphate being faster both in onset and in redecrease than inositol 1,3,4,6-tetrakisphosphate. Whereas the two latter isomers reached maximally 2.75 and 2.9 +/- 0.2 microM, respectively, 1 min after stimulation, inositol 3,4,5,6-tetrakisphosphate remained elevated (3.5 +/- 0.4 microM) up to 5 min after fMLP. Unexpected changes in highly phosphorylated InsP(s) were observed, notably a rise in inositol 1,3,4,5,6-pentakisphosphate and in inositol hexakisphosphate to 52 +/- 3 and 60 +/- 1 microM, respectively. In terms of mass, the increases in highly phosphorylated inositols are by far highest among all InsP(s). Combining radiotracer method with mass determination it was observed that the specific radioactivity of various InsP(s) was different and changed markedly upon fMLP stimulation, in spite of a prolonged labeling period leading to apparent isotopic steady state. The data presented demonstrate agonist-induced elevations of highly phosphorylated InsP(s) and suggest that inositol 1,4,5-trisphosphate, product of receptor-activated phospholipase C, is metabolized rather via phosphorylation than only by dephosphorylation pathways.     

The variability of the hepatitis B virus genome: statistical analysis and biological implications

A statistical analysis of the nucleotide sequence variability in 14 published hepatitis B virus (HBV) genomes was carried out using parametric and nonparametric methods. A parametric statistical model revealed that the different regions of the genome differed significantly in their variability. The conclusion was supported by a nonparametric kernel-density model of the HBV genome. Genes S, C, and P, region X, the precore region, and the pre-S2/pre-S1 regions were ranked in order of increasing variability. In many instances, conserved regions of the genome identified with sequences of known function in HBV biology. However, other characterized regions (such as pre-S) showed much variability despite the involvement of their encoded peptides in specific functions. Point mutations that may result in the formation of stop codons and amino acid changes may affect the clinical picture of HBV infection and may be reflected in atypical serological patterns.