Mapping nucleosome position at single base-pair resolution by using site-directed hydroxyl radicals.

A base-pair resolution method for determining nucleosome position in vitro has been developed to com- plement existing, less accurate methods. Cysteaminyl EDTA was tethered to a recombinant histone octamer via a mutant histone H4 with serine 47 replaced by cysteine. When assembled into nucleosome core particles, the DNA could be cut site specifically by hydroxyl radical-catalyzed chain scission by using the Fenton reaction. Strand cleavage occurs mainly at a single nucleotide close to the dyad axis of the core particle, and assignment of this location via the symmetry of the nucleosome allows base-pair resolution mapping of the histone octamer position on the DNA. The positions of the histone octamer and H3H4 tetramer were mapped on a 146-bp Lytechinus variegatus 5S rRNA sequence and a twofold-symmetric derivative. The weakness of translational determinants of nucleosome positioning relative to the overall affinity of the histone proteins for this DNA is clearly demonstrated. The predominant location of both histone octamer and H3H4 tetramer assembled on the 5S rDNA is off center. Shifting the nucleosome core particle position along DNA within a conserved rotational phase could be induced under physiologically relevant conditions. Since nucleosome shifting has important consequences for chromatin structure and gene regulation, an approach to the thermodynamic characterization of this movement is proposed. This mapping method is potentially adaptable for determining nucleosome position in chromatin in vivo.

Mutagenesis in the C-terminal region of human interleukin 5 reveals a central patch for receptor alpha chain recognition.

Cassette mutagenesis was used to identify side chains in human interleukin 5 (hIL-5) that mediate binding to hIL-5 receptor alpha chain (hIL-5R alpha). A series of single alanine substitutions was introduced into a stretch of residues in the C-terminal region, including helix D, which previously had been implicated in receptor alpha chain recognition and which is aligned on the IL-5 surface so as to allow the topography of receptor binding residues to be examined. hIL-5 and single site mutants were expressed in COS cells, their interactions with hIL-5R alpha were measured by a sandwich surface plasmon resonance biosensor method, and their biological activities were measured by an IL-5-dependent cell proliferation assay. A pattern of mutagenesis effects was observed, with greatest impact near the interface between the two four-helix bundles of IL-5, in particular at residues Glu-110 and Trp-111, and least at the distal ends of the D helices. This pattern suggests the possibility that residues near the interface of the two four-helix bundles in hIL-5 comprise a central patch or hot spot, which constitutes an energetically important alpha chain recognition site. This hypothesis suggests a structural explanation for the 1:1 stoichiometry observed for the complex of hIL-5 with hIL-5R alpha.

Molecular basis of human mitochondrial very-long-chain acyl-CoA dehydrogenase deficiency causing cardiomyopathy and sudden death in childhood.

beta-Oxidation of long-chain fatty acids provides the major source of energy in the heart. Defects in enzymes of the beta-oxidation pathway cause sudden, unexplained death in childhood, acute hepatic encephalopathy or liver failure, skeletal myopathy, and cardiomyopathy. Very-long-chain acyl-CoA dehydrogenase [VLCAD; very-long-chain-acyl-CoA:(acceptor) 2,3-oxidoreductase, EC 1.3.99.13] catalyzes the first step in beta-oxidation. We have isolated the human VLCAD cDNA and gene and determined the complete nucleotide sequences. Polymerase chain reaction amplification of VLCAD mRNA and genomic exons defined the molecular defects in two patients with VLCAD deficiency who presented with unexplained cardiac arrest and cardiomyopathy. In one, a homozygous mutation in the consensus dinucleotide of the donor splice site (g+1-->a) was associated with universal skipping of the prior exon (exon 11). The second patient was a compound heterozygote, with a missense mutation, C1837-->T, changing the arginine at residue 613 to tryptophan on one allele and a single base deletion at the intron-exon 6 boundary as the second mutation. This initial delineation of human mutations in VLCAD suggests that VLCAD deficiency reduces myocardial fatty acid beta-oxidation and energy production and is associated with cardiomyopathy and sudden death in childhood.

Single-headed myosin II acts as a dominant negative mutation in Dictyostelium.

Conventional myosin II is an essential protein for cytokinesis, capping of cell surface receptors, and development of Dictyostelium cells. Myosin II also plays an important role in the polarization and movement of cells. All conventional myosins are double-headed molecules but the significance of this structure is not understood since single-headed myosin II can produce movement and force in vitro. We found that expression of the tail portion of myosin II in Dictyostelium led to the formation of single-headed myosin II in vivo. The resultant cells contain an approximately equal ratio of double- and single-headed myosin II molecules. Surprisingly, these cells were completely blocked in cytokinesis and capping of concanavalin A receptors although development into fruiting bodies was not impaired. We found that this phenotype is not due to defects in myosin light chain phosphorylation. These results show that single-headed myosin II cannot function properly in vivo and that it acts as a dominant negative mutation for myosin II function. These results suggest the possibility that cooperativity of myosin II heads is critical for force production in vivo.

A general model of invariant chain association with class II major histocompatibility complex proteins.

The binding of invariant chain to major histocompatibility complex (MHC) proteins is an important step in processing of MHC class II proteins and in antigen presentation. The question of how invariant chain can bind to all MHC class II proteins is central to understanding these processes. We have employed molecular modeling to predict the structure of class II-associated invariant chain peptide (CLIP)-MHC protein complexes and to ask whether the predicted mode of association could be general across all MHC class II proteins. CLIP fits identically into the MHC class II alleles HLA-DR3, I-Ak, I-Au, and I-Ad, with a consistent pattern of hydrogen bonds, contacts, and hydrophobic burial and without bad contacts. Our model predicts the burial of CLIP residues Met-91 and Met-99 in the deep P1 and P9 anchor pockets and other detailed interactions, which we have compared with available data. The predicted pattern of I-A allele-specific effects on CLIP binding is very similar to that observed experimentally by alanine-scanning mutations of CLIP. Together, these results indicate that CLIP may bind in a single, general way across products of MHC class II alleles.

Significance of bound water to local chain conformations in protein crystals.

We examine how the polypeptide chain in protein crystal structures exploits the multivalent hydrogen-bonding potential of bound water molecules. This shows that multiple interactions with a single water molecule tend to occur locally along the chain. A distinctive internal-coordinate representation of the local water-binding segments reveals several consensus conformations. The fractional water occupancy of each was found by comparison of the total number of conformations in the database regardless of the presence or absence of bound water. The water molecule appears particularly frequently in type II beta-turn geometries and an N-terminal helix feature. This work constitutes a first step into assessing not only the generality but also the significance of specific water binding in globular proteins.

A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides.

Bacteriophage T7 DNA polymerase efficiently incorporates a chain-terminating dideoxynucleotide into DNA, in contrast to the DNA polymerases from Escherichia coli and Thermus aquaticus. The molecular basis for this difference has been determined by constructing active site hybrids of these polymerases. A single hydroxyl group on the polypeptide chain is critical for selectivity. Replacing tyrosine-526 of T7 DNA polymerase with phenylalanine increases discrimination against the four dideoxynucleotides by > 2000-fold, while replacing the phenylalanine at the homologous position in E. coli DNA polymerase I (position 762) or T. aquaticus DNA polymerase (position 667) with tyrosine decreases discrimination against the four dideoxynucleotides 250- to 8000-fold. These mutations allow the engineering of new DNA polymerases with enhanced properties for use in DNA sequence analysis.

Persistence of immunoglobulin heavy chain/c-myc recombination-positive lymphocyte clones in the blood of human immunodeficiency virus-infected homosexual men.

We studied blood lymphocytes of human immunodeficiency virus (HIV)-seropositive and -negative homosexual men for the presence of T(8;14) translocations that recombine c-myc and immunoglobulin heavy-chain (IgH) mu/IgH alpha switch regions. Clones with T(8;14) translocations were detected in 10.5% (12/114) of the HIV-positive and in 2.0% of the 99 uninfected patients. The majority of recombinations were found at a single time point only. Four patients, however, harbored multiple (up to four) and persistent (up to 9 years) translocation-positive cell clones. No correlation between the presence of these aberrant lymphocytes and a later lymphoma could be established. The exon 1/intron 1 region of the recombined c-myc was investigated for the presence of point mutations and these were found in the nonpersistent clones. Additional alterations detected in these clones included duplications and a deletion in the c-myc gene. The pattern of base substitution indicates that they were introduced after the translocation event.

T-cell receptor (TCR) usage in Lewis rat experimental autoimmune encephalomyelitis: TCR beta-chain-variable-region V beta 8.2-positive T cells are not essential for induction and course of disease.

Predominant usage of V beta 8.2 gene segments, encoding a T-cell receptor (TCR) beta chain variable region, has been reported for pathogenic Lewis rat T cells reactive to myelin basic protein (MBP). However, up to 75% of the alpha/beta T cells in a panel of MBP-specific T-cell lines did not display TCR V beta 8.2, V beta 8.5, V beta 10, or V beta 16 elements. To further investigate TCR usage, we sorted the T-cell lines for V beta 8.2- and V beta 10-positive T cells or depleted the lines of cells with these TCRs. V beta 8.2-positive T cells and one of the depleted T-cell lines strongly reacted against the MBP peptide MBP-(68-88). The depleted T-cell line caused marked experimental autoimmune encephalomyelitis (EAE) even in Lewis rats in which endogenous V beta 8.2-positive T cells had been eliminated by neonatal treatment with anti-V beta 8.2 monoclonal antibodies. T-cell hybridomas generated from this line predominantly used V beta 3 TCR genes coexpressed with TCR V alpha 2 transcripts, which were also used by V beta 8.2-positive T cells. Furthermore, V beta 10-positive T cells reactive to MBP-(44-67) were encephalitogenic when injected immediately after positive selection. After induction of EAE by sorted V beta 8.2- or V beta 10-positive T-cell lines, immunocytochemical analysis of the spinal cord tissue showed a predominance of the injected TCR or of nontypable alpha/beta T cells after injection of the depleted line. Our results demonstrate heterogeneity of TCR beta-chain usage even for a single autoantigen in an inbred strain. Moreover, V beta 8.2-positive T cells are not essential for the induction and progression of adoptive-transfer EAE.

Genetic variability of Tomato spotted wilt virus in Australia and validation of real time RT-PCR for its detection in single and bulked leaf samples

The potential for large-scale use of a sensitive real time reverse transcription polymerase chain reaction (RT-PCR) assay was evaluated for the detection of Tomato spotted wilt virus (TSWV) in single and bulked leaf samples by comparing its sensitivity with that of DAS-ELISA. Using total RNA extracted with RNeasy (R) or leaf soak methods, real time RT-PCR detected TSWV in all infected samples collected from 16 horticultural crop species (including flowers, herbs and vegetables), two arable crop species, and four weed species by both assays. In samples in which DAS-ELISA had previously detected TSWV, real time RT-PCR was effective at detecting it in leaf tissues of all 22 plant species tested at a wide range of concentrations. Bulk samples required more robust and extensive extraction methods with real time RT-PCR, but it generally detected one infected sample in 1000 uninfected ones. By contrast, ELISA was less sensitive when used to test bulked samples, once detecting up to I infected in 800 samples with pepper but never detecting more than I infected in 200 samples in tomato and lettuce. It was also less reliable than real time RT-PCR when used to test samples from parts of the leaf where the virus concentration was low. The genetic variability among Australian isolates of TSWV was small. Direct sequencing of a 587 bp region of the nucleoprotein gene (S RNA) of 29 isolates from diverse crops and geographical locations yielded a maximum of only 4.3% nucleotide sequence difference. Phylogenetic analysis revealed no obvious groupings of isolates according to geographic origin or host species. TSWV isolates, that break TSWV resistance genes in tomato or pepper did not differ significantly in the N gene region studied, indicating that a different region of the virus genome is responsible for this trait.

Motor unit synchronization of the vasti muscles in closed and open chain tasks

Objectives: To investigate motor unit synchronization between medial and lateral vasti and whether such synchronization differs in closed and open chain tasks. Design: Electromyographic recordings of single motor unit action potentials were made from the vastus medialis obliquus (VMO) and multiunit recordings from vastus lateralis during isometric contractions at 30 degrees of knee flexion in closed and open chain conditions. Setting: Laboratory. Participants: Five volunteers with no history of knee pain (age, 30 +/- 3.32y). Interventions: Not applicable. Main Outcome Measure: The degree of synchronization between motor unit firing was evaluated by identifying peaks in the electromyographic averages of the vastus lateralis, triggered from motor unit action potentials in the VMO, and the proportion of power in the power spectral density of the triggered average at the firing frequency of the reference motor unit. The proportion of cases in which there was significant power and peaks in the triggered averages was calculated. Results: The proportion of trials with peaks in the triggered averages of the vastus lateralis electromyographic activity was greater than 61.5% in all tasks, and there was a significantly greater proportion of cases where power in the spectrum was greater than 7.5% (P = .01) for the closed chain condition. Conclusions: There was a high proportion of synchronized motor units between the 2 muscles during isometric contractions, with evidence for greater common drive between the VMO and vastus lateralis in closed chain tasks. This has implications for rehabilitation because it suggests that closed chain tasks may generate better coordination between the vasti muscles.

Single turn peptide alpha helices with exceptional stability in water

Cyclic pentapepticles are not known to exist in a-helical conformations. CD and NMR spectra show that specific 20-membered cyclic pentapepticles, Ac-(cyclo-1,5) [KxxxD]-NH2 and Ac-(cyclo-2,6)R[KxxxD]-NH2, are highly a-helical structures in water and independent of concentration, TFE, denaturants, and proteases. These are the smallest a-helical peptides in water.

Mechanistic aspects of proton chain transfer: A computational study for the green fluorescent protein chromophore

We explore several models for the ground-state proton chain transfer pathway between the green fluorescent protein chromophore and its surrounding protein matrix, with a view to elucidating mechanistic aspects of this process. We have computed quantum chemically the minimum energy pathways (MEPs) in the ground electronic state for one-, two-, and three-proton models of the chain transfer. There are no stable intermediates for our models, indicating that the proton chain transfer is likely to be a single, concerted kinetic step. However, despite the concerted nature of the overall energy profile, a more detailed analysis of the MEPs reveals clear evidence of sequential movement of protons in the chain. The ground-state proton chain transfer does not appear to be driven by the movement of the phenolic proton off the chromophore onto the neutral water bridge. Rather, this proton is the last of the three protons in the chain to move. We find that the first proton movement is from the bridging Ser205 moiety to the accepting Glu222 group. This is followed by the second proton moving from the bridging water to the Ser205for our model this is where the barrier occurs. The phenolic proton on the chromophore is hence the last in the chain to move, transferring to a bridging “water” that already has substantial negative charge.

Searching for convergence in phylogenetic Markov chain Monte Carlo

Markov chain Monte Carlo (MCMC) is a methodology that is gaining widespread use in the phylogenetics community and is central to phylogenetic software packages such as MrBayes. An important issue for users of MCMC methods is how to select appropriate values for adjustable parameters such as the length of the Markov chain or chains, the sampling density, the proposal mechanism, and, if Metropolis-coupled MCMC is being used, the number of heated chains and their temperatures. Although some parameter settings have been examined in detail in the literature, others are frequently chosen with more regard to computational time or personal experience with other data sets. Such choices may lead to inadequate sampling of tree space or an inefficient use of computational resources. We performed a detailed study of convergence and mixing for 70 randomly selected, putatively orthologous protein sets with different sizes and taxonomic compositions. Replicated runs from multiple random starting points permit a more rigorous assessment of convergence, and we developed two novel statistics, delta and epsilon, for this purpose. Although likelihood values invariably stabilized quickly, adequate sampling of the posterior distribution of tree topologies took considerably longer. Our results suggest that multimodality is common for data sets with 30 or more taxa and that this results in slow convergence and mixing. However, we also found that the pragmatic approach of combining data from several short, replicated runs into a metachain to estimate bipartition posterior probabilities provided good approximations, and that such estimates were no worse in approximating a reference posterior distribution than those obtained using a single long run of the same length as the metachain. Precision appears to be best when heated Markov chains have low temperatures, whereas chains with high temperatures appear to sample trees with high posterior probabilities only rarely. [Bayesian phylogenetic inference; heating parameter; Markov chain Monte Carlo; replicated chains.]

The Function and Consequences of Alternative Fresh Produce Supply Chain Models

Around the world, consumers and retailers of fresh produce are becoming more and more discerning about factors such as food safety and traceability, health, convenience and the sustainability of production systems, and in doing so they are changing the way in which fresh produce supply chains are configured and managed. When consumers demand fresh, safe, convenient, value-for-money produce, retailers in an increasingly competitive environment are attracted to those business models most capable of meeting these demands profitably. Traditional models are proving less and less able to deliver competitive advantage in such an environment. As a result, opportunistic, adversarial, price-based approaches to doing business between chain members are being replaced by approaches that are more strategic, collaborative and value-based. The shaping force behind this change is the need for producers, wholesalers, category managers, retailers and consumers to have more certainty about the performance of the supply chains upon which they rely. Certainty is generated through the supply chain's ability to create, deliver and share value. How to build supply chains that create, deliver and share value is arguably the single biggest challenge to the competitiveness of fresh produce firms, and therefore to the industries to which they belong.