A model for proto-tRNA loading

A kinetic model for loading of proto-tRNA is presented. The kinetic parameters were first estimated from the results of Francklyn & Schimmel (1989;Nature337, 478--481), who studied the aminoacylation of both tRNA and its minihelix. Then these parameters were reduced several-fold, as is more appropriate for the prebiotic world. The simulations revealed a very slow time course of the loading reaction. We also consider a possibility for the proto-tRNA loading without a catalyst and discuss the feasibility of such processes. Analytical approximations are presented for the kinetics of proto-tRNA loading with and without enzyme. An estimate is given for the time required for the development of template- and sequence-directed systems.     

Detection of protein-protein interactions in plants using bimolecular fluorescence complementation

Protein function is often mediated via formation of stable or transient complexes. Here we report the determination of protein-protein interactions in plants using bimolecular fluorescence complementation (BiFC). The yellow fluorescent protein (YFP) was split into two non-overlapping N-terminal (YN) and C-terminal (YC) fragments. Each fragment was cloned in-frame to a gene of interest, enabling expression of fusion proteins. To demonstrate the feasibility of BiFC in plants, two pairs of interacting proteins were utilized: (i) the alpha and beta subunits of the Arabidopsis protein farnesyltransferase (PFT), and (ii) the polycomb proteins, FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA). Members of each protein pair were transiently co-expressed in leaf epidermal cells of Nicotiana benthamiana or Arabidopsis. Reconstitution of a fluorescing YFP chromophore occurred only when the inquest proteins interacted. No fluorescence was detected following co-expression of free non-fused YN and YC or non-interacting protein pairs. Yellow fluorescence was detected in the cytoplasm of cells that expressed PFT alpha and beta subunits, or in nuclei and cytoplasm of cells that expressed FIE and MEA. In vivo measurements of fluorescence spectra emitted from reconstituted YFPs were identical to that of a non-split YFP, confirming reconstitution of the chromophore. Expression of the inquest proteins was verified by immunoblot analysis using monoclonal antibodies directed against tags within the hybrid proteins. In addition, protein interactions were confirmed by immunoprecipitations. These results demonstrate that plant BiFC is a simple, reliable and relatively fast method for determining protein-protein interactions in plants.     

In silico screening of a saturated mutation library of tomato

A comprehensive mutant population is a basic resource for exploring gene function. We developed an isogenic tomato 'mutation library' in the genetic background of the inbred variety M82. A total of 13 000 M(2) families, derived from EMS (ethyl methane sulfonate) and fast-neutron mutagenesis, were visually phenotyped in the field and categorized into a morphological catalog that includes 15 primary and 48 secondary categories. Currently, 3417 mutations have been cataloged; among them are most of the previously described phenotypes from the monogenic mutant collection of The Tomato Genetics Resource Center, and over a thousand new mutants, with multiple alleles per locus. The phenotypic database indicates that most mutations fall into more than a single category (pleiotropic), with some organs such as leaves more prone to alterations than others. All data and images can be searched and accessed in the Solanaceae Genome Network (SGN) on a site called 'The Genes That Make Tomatoes' (http://zamir.sgn.cornell.edu/mutants/).     

PIVOT: protein interacions visualizatiOn tool

Protein Interaction VisualizatiOn Tool (PIVOT) is a visualization tool for protein-protein interactions. It allows the user to create personal data sets of interactions by combining information from private and public data sources. The user can gradually access the interactions' data using a clear interactive map that is focused on the researcher's protein of interest, and is reshaped and expanded in response to his/her queries. It also offers several visual enhancements and intelligent queries that help the user efficiently study it. PIVOT allows the user to search the interactions data set for paths connecting proteins that are expected to co-operate. The user can also employ PIVOT to predict unknown interactions among proteins, based on interactions among their homologous proteins in other species.     

Pre-clinical development of cord blood-derived progenitor cell graft expanded ex vivo with cytokines and the polyamine copper chelator tetraethylenepentamine

BACKGROUND: We have previously demonstrated that the copper chelator tetraethylenepentamine (TEPA) enables preferential expansion of early hematopoietic progenitor cells (CD34+CD38-, CD34+CD38-Lin-) in human umbilical cord blood (CB)-derived CD34+ cell cultures. This study extends our previous findings that copper chelation can modulate the balance between self-renewal and differentiation of hematopoietic progenitor cells. METHODS: In the present study we established a clinically applicative protocol for large-scale ex vivo expansion of CB-derived progenitors. Briefly, CD133+ cells, purified from CB using Miltenyi Biotec's (Bergisch Gladbach, Germany) CliniMACS separation device and the anti-CD133 reagent, were cultured for 3 weeks in a clinical-grade closed culture bag system, using the chelator-based technology in combination with early-acting cytokines (SCF, thrombopoietin, IL-6 and FLT-3 ligand). This protocol was evaluated using frozen units derived from accredited cord blood banks. RESULTS: Following 3 weeks of expansion under large-scale culture conditions that were suitable for clinical manufacturing, the median output value of CD34+ cells increase by 89-fold, CD34+CD38- increase by 30-fold and CFU cells (CFUc) by 172-fold over the input value. Transplantation into sublethally irradiated non-obese diabetic (NOD/SCID) mice indicated that the engraftment potential of the ex vivo expanded CD133+ cells was significantly superior to that of unexpanded cells: 60+/-5.5% vs. 21+/-3.5% CD45+ cells, P=0.001, and 11+/-1.8% vs. 4+/-0.68% CD45+CD34+ cells, P=0.012, n=32, respectively. DISCUSSION: Based on these large-scale experiments, the chelator-based ex vivo expansion technology is currently being tested in a phase 1 clinical trial in patients undergoing CB transplantation for hematological malignancies.     

Evolutionary analysis reveals collective properties and specificity in the C-type lectin and lectin-like domain superfamily

Members of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily share a common fold and are involved in a variety of functions, such as generalized defense mechanisms against foreign agents, discrimination between healthy and pathogen-infected cells, and endocytosis and blood coagulation. In this work we used ConSurf, a computer program recently developed in our lab, to perform an evolutionary analysis of this superfamily in order to further identify characteristics of all or part of its members. Given a set of homologous proteins in the form of multiple sequence alignment (MSA) and an inferred phylogenetic tree, ConSurf calculates the conservation score in every alignment position, taking into account the relationships between the sequences and the physicochemical similarity between the amino acids. The scores are then color-coded onto the three-dimensional structure of one of the homologous proteins. We provide here and at http://ashtoret.tau.ac.il/ approximately sharon a detailed analysis of the conservation pattern obtained for the entire superfamily and for two subgroups of proteins: (a) 21 CTLs and (b) 11 heterodimeric CTLD toxins. We show that, in general, proteins of the superfamily have one face that is constructed mostly of conserved residues and another that is not, and we suggest that the former face is involved in binding to other proteins or domains. In the CTLs examined we detected a region of highly conserved residues, corresponding to the known calcium- and carbohydrate-binding site of the family, which is not conserved throughout the entire superfamily, and in the CTLD toxins we found a patch of highly conserved residues, corresponding to the known dimerization region of these proteins. Our analysis also detected patches of conserved residues with yet unknown function(s).     

Contribution of the Atm protein to maintaining cellular homeostasis evidenced by continuous activation of the AP-1 pathway in Atm-deficient brains

Maintenance of genome stability is essential for keeping cellular homeostasis. The DNA damage response is a central component in maintaining genome integrity. Among of the most cytotoxic DNA lesions are double strand breaks (DSBs) caused by ionizing radiation or radiomimetic chemicals. ATM is missing or inactivated in patients with ataxia-telangiectasia. Ataxia-telangiectasia patients display a pleiotropic phenotype and suffer primarily from progressive ataxia caused by degeneration of cerebellar Purkinje and granule neurons. Additional features are immunodeficiency, genomic instability, radiation sensitivity, and cancer predisposition. Disruption of the mouse Atm locus creates a murine model of ataxia-telangiectasia that exhibits most of the clinical features of the human disease but very mild neuronal abnormality. The ATM protein is a multifunctional protein kinase, which serves as a master regulator of cellular responses to DSBs. There is growing evidence that ATM may be involved in addition to the DSB response in other processes that maintain processes in cellular homeostasis. For example, mounting evidence points to increased oxidative stress in the absence of ATM. Here we report that the AP-1 pathway is constantly active in the brains of Atm-deficient mice not treated with DNA damaging agents. A canonical activation (increased phosphorylation of mitogen-activated protein kinase kinase-4, c-Jun N-terminal kinase, and c-Jun) of the AP-1 pathway was found in Atm-deficient cerebra, whereas induction of the AP-1 pathway in Atm-deficient cerebella is likely to mediate elevated expression of c-Fos and c-Jun. Although Atm(+/+) mice are capable of responding to ionizing radiation by activating stress responses such as the AP-1 pathway, Atm-deficient mice display higher basal AP-1 activity but gradually lose their ability to activate AP-1 DNA-binding activity in response to ionizing radiation. Our results further demonstrate that inactivation of the ATM gene results in a state of constant stress.     

Interactions of hydrophobic peptides with lipid bilayers: Monte Carlo simulations with M2delta

We introduce here a novel Monte Carlo simulation method for studying the interactions of hydrophobic peptides with lipid membranes. Each of the peptide's amino acids is represented as two interaction sites: one corresponding to the backbone alpha-carbon and the other to the side chain, with the membrane represented as a hydrophobic profile. Peptide conformations and locations in the membrane and changes in the membrane width are sampled using the Metropolis criterion, taking into account the underlying energetics. Using this method we investigate the interactions between the hydrophobic peptide M2delta and a model membrane. The simulations show that starting from an extended conformation in the aqueous phase, the peptide first adsorbs onto the membrane surface, while acquiring an ordered helical structure. This is followed by formation of a helical-hairpin and insertion into the membrane. The observed path is in agreement with contemporary understanding of peptide insertion into biological membranes. Two stable orientations of membrane-associated M2delta were obtained: transmembrane (TM) and surface, and the value of the water-to-membrane transfer free energy of each of them is in agreement with calculations and measurements on similar cases. M2delta is most stable in the TM orientation, where it assumes a helical conformation with a tilt of 14 degrees between the helix principal axis and the membrane normal. The peptide conformation agrees well with the experimental data; average root-mean-square deviations of 2.1 A compared to nuclear magnetic resonance structures obtained in detergent micelles and supported lipid bilayers. The average orientation of the peptide in the membrane in the most stable configurations reported here, and in particular the value of the tilt angle, are in excellent agreement with the ones calculated using the continuum-solvent model and the ones observed in the nuclear magnetic resonance studies. This suggests that the method may be used to predict the three-dimensional structure of TM peptides.     

Sphingomyelin and cholesterol promote HIV-1 gp41 pretransmembrane sequence surface aggregation and membrane restructuring

The interfacial sequence DKWASLWNWFNITNWLWYIK, preceding the transmembrane anchor of gp41 glycoprotein subunit, has been shown to be essential for fusion activity and incorporation into virions. HIV(c), a peptide representing this region, formed lytic pores in liposomes composed of the main lipids occurring in the human immunodeficiency virus, type 1 (HIV-1), envelope, i.e. 1-palmitoyl-2-oleoylphosphatidylcholine (POPC):sphingomyelin (SPM):cholesterol (Chol) (1:1:1 mole ratio), at low (>1:10,000) peptide-to-lipid mole ratio, and promoted the mixing of vesicular lipids at >1:1000 peptide-to-lipid mole ratios. Inclusion of SPM or Chol in POPC membranes had different effects. Whereas SPM sustained pore formation, Chol promoted fusion activity. Even if partitioning into membranes was not affected in the absence of both SPM and Chol, HIV(c) had virtually no effect on POPC vesicles. Conditions described to disturb occurrence of lateral separation of phases in these systems reproduced the high peptide-dose requirements for leakage as found in pure POPC vesicles and inhibited fusion. Surface aggregation assays using rhodamine-labeled peptides demonstrated that SPM and Chol promoted HIV(c) self-aggregation in membranes. Employing head-group fluorescent phospholipid analogs in planar supported lipid layers, we were able to discern HIV(c) clusters associated to ordered domains. Our results support the notion that the pretransmembrane sequence may participate in the clustering of gp41 monomers within the HIV-1 envelope, and in bilayer architecture destabilization at the loci of fusion.