Phosphorylation triggers domain separation in the DNA binding response regulator NarL

DNA binding proteins of two-component signal transduction systems in microorganisms are activated by phosphorylation through an unknown mechanism. NarL is an example from the nitrate/nitrite signal transduction system of Escherichia coli. NarL consists of N- and C-terminal domains, the latter of which contains the DNA binding elements. To explore the mechanism of activation, single nitroxide side chains were introduced, one at a time, at nine different sites throughout the C-terminal domain to monitor the tertiary structure and the status of the surface in contact with the N-terminal domain. In addition, three pairs of doubly labeled proteins were prepared to monitor the interdomain distance using the magnetic dipolar interaction. The results of these site-directed spin-labeling studies reveal that phosphorylation at a distant site in the N-terminal domain triggers domain separation, likely by a hinge-bending motion. This in turn presents key elements of the C-terminal domain for docking to the DNA target in the configuration described in the recent crystal structure. The data also imply that a single conformation of unphosphorylated NarL exists in solution, and there is no detectable equilibrium between the closed and open conformations.     

Gene structure-based splice variant deconvolution using a microarray platform

MOTIVATION: Alternative splicing allows a single gene to generate multiple mRNAs, which can be translated into functionally and structurally diverse proteins. One gene can have multiple variants coexisting at different concentrations. Estimating the relative abundance of each variant is important for the study of underlying biological function. Microarrays are standard tools that measure gene expression. But most design and analysis has not accounted for splice variants. Thus splice variant-specific chip designs and analysis algorithms are needed for accurate gene expression profiling. RESULTS: Inspired by Li and Wong (2001), we developed a gene structure-based algorithm to determine the relative abundance of known splice variants. Probe intensities are modeled across multiple experiments using gene structures as constraints. Model parameters are obtained through a maximum likelihood estimation (MLE) process/framework. The algorithm produces the relative concentration of each variant, as well as an affinity term associated with each probe. Validation of the algorithm is performed by a set of controlled spike experiments as well as endogenous tissue samples using a human splice variant array.     

Site-directed spin labeling of a bacterial chemoreceptor reveals a dynamic, loosely packed transmembrane domain

We used site-directed spin labeling and electron paramagnetic resonance spectroscopy to investigate dynamics and helical packing in the four-helix transmembrane domain of the homodimeric bacterial chemoreceptor Trg. We focused on the first transmembrane helix, TM1, particularly on the nine-residue sequence nearest the periplasm, because patterns of disulfide formation between introduced cysteines had identified that segment as the region of closest approach among neighboring transmembrane helices. Along this sequence, mobility and accessibility of the introduced spin label were characteristic of loosely packed or solvent-exposed side chains. This was also the case for eight additional positions around the circumference and along the length of TM1. For the continuous nine-residue sequence near the periplasm, mobility and accessibility varied only modestly as a function of position. We conclude that side chains of TM1 that face the interior of the four-helix domain interact with neighboring helices but dynamic movement results in loose packing. Compared to transmembrane segments of other membrane proteins reconstituted into lipid bilayers and characterized by site-directed spin labeling, TM1 of chemoreceptor Trg is the most dynamic and loosely packed. A dynamic, loosely packed chemoreceptor domain can account for many experimental observations about the transmembrane domains of chemoreceptors.     

Poly(ethylene glycol) block copolymers

The ubiquitous use of poly(ethylene glycol) in the biomaterials field has also boosted the research activity in the chemical derivatization of this polymer. We focused our interest on the preparation of tailor-made poly(ethylene glycol)-based structures and on the study of structure-activity relationships for its functionalization, as preliminary steps for the preparation of smart functional materials. More specifically, amphiphilic and cationic block copolymers were prepared for prospective use in the preparation of self-assembled carriers, and Michael-type addition of thiols onto acrylates was studied as a model for end-group reaction leading to hydrogel formation.     

Analysis of high density expression microarrays with signed-rank call algorithms

MOTIVATION: We consider the detection of expressed genes and the comparison of them in different experiments with the high-density oligonucleotide microarrays. The results are summarized as the detection calls and comparison calls, and they should be robust against data outliers over a wide target concentration range. It is also helpful to provide parameters that can be adjusted by the user to balance specificity and sensitivity under various experimental conditions. RESULTS: We present rank-based algorithms for making detection and comparison calls on expression microarrays. The detection call algorithm utilizes the discrimination scores. The comparison call algorithm utilizes intensity differences. Both algorithms are based on Wilcoxon's signed-rank test. Several parameters in the algorithms can be adjusted by the user to alter levels of specificity and sensitivity. The algorithms were developed and analyzed using spiked-in genes arrayed in a Latin square format. In the call process, p-values are calculated to give a confidence level for the pertinent hypotheses. For comparison calls made between two arrays, two primary normalization factors are defined. To overcome the difficulty that constant normalization factors do not fit all probe sets, we perturb these primary normalization factors and make increasing or decreasing calls only if all resulting p-values fall within a defined critical region. Our algorithms also automatically handle scanner saturation.     

Murine macrophage behavior on peptide-grafted polyethyleneglycol-containing networks

Polyethyleneglycol-based networks were employed as substrates to graft bioactive peptides to study macrophage interactions with materials. Our overall objective was to utilize biologically active factors to stimulate certain macrophage function on materials suitable for implantation in connective tissues. In this study, we sought to explore the bioactivity of several peptides derived from extracellular matrix adhesion proteins and macrophage-active proteins that are normally soluble. The candidate peptides examined corresponded to residues 63 to 77 of complement component C3a (C3a(63-77)), residues 178 to 207 of interleukin-1 beta (IL1beta(178-207)), residues 1615 to 1624 of fibronectin (FN(1615-1624)), endothelial-macrophage activating polypeptide II, complement component C5a inhibitory sequence, macrophage inhibitory peptide, and YRGDG; materials lacking peptides were used as negative controls. An established murine cell-line IC-21 was employed as a macrophage model, and human dermal fibroblasts were used for comparison. Our results showed that the substrates without grafted peptides were free from artifactual cell adhesion associated with the adsorption of serum or cellularly secreted proteins for long duration of culture. Of all grafted samples, IL1beta(178-207)- and C3a(63-77)-grafted surfaces supported higher adherent macrophage densities. C3a(63-77)- and FN(1615-1624)-grafted surfaces supported higher adherent fibroblast densities. From competitive inhibition studies, cell adhesion was determined to occur in a receptor-peptide specific manner. The presence of grafted YRGDG in addition to IL1beta(178-207), C3a(63-77), or FN(1615-1624) synergistically increased macrophage and fibroblast adhesion. Materials grafted with IL1beta(178-207) or C3a(63-77) co-grafted with or without YRGDG did not support the formation of multinucleated giant cells from the fusion of adherent macrophages in vitro.     

A Role for the MEK/MAPK Pathway in PMA-Induced Cell Cycle Arrest: Modulation of Megakaryocytic Differentiation of K562 Cells

In vitromegakaryocytic differentiation of the pluripotent K562 human leukemia cell line is induced by PMA. Treatment of K562 cells with PMA results in growth arrest, polyploidy, morphological changes, and increased cell–cell and cell–substrate adhesion. These PMA-induced changes in K562 cells are preceded by a rapid rise in the activity of MEK (MAP kinase/extracellular regulated kinases) that leads to a sustained activation of ERK2 (extracellular regulated kinase; MAPK). Blockade of MEK1 activation by PD098059, a recently described specific MEK inhibitor [D. T. Dudleyet al.(1995).Proc. Natl. Acad. Sci. USA92, 7686–7689], reverses both the growth arrest and the morphological changes of K562 cells induced by PMA treatment. These changes are not associated with a disruption of PMA-induced down-regulation of BCR-ABL kinase or early integrin signaling events but are associated with a block of the cell-surface expression of the gpIIb/IIIa (CD41) integrin, a cell marker of megakaryocytic differentiation. These results demonstrate that the PMA-induced signaling cascade initiated by protein kinase C activation requires the activity of the MEK/ERK signaling complex to regulate cell cycle arrest, thus regulating the program that leads to the cell-surface expression of markers associated with megakaryocytic differentiation.     

Density and distance-to-adult effects of a canker disease of trees in a moist tropical forest

We compared the spatial distribution of stem cankers on the canopy tree Ocotea whitei (Lauraceae) in a 20-ha plot on Barro Colorado Island, Panama, with spatial and temporal patterns of mortality in this host over the previous decade. The cankers occur both on adult and juvenile individuals, aothough juveniles are much more likely the adults to show symptoms. Disease incidence is host-density dependent, and both the presence of the disease and host mortality are more likely close to than far from a conspecific adult, which resulted in a net spatial shift of the juvenile population away from conspecific adults through time. Disease incidence is lower than expected among juveniles of O. whitei growing near to adults of the non-susceptible canopy tree Beilschmiedia pendula. The coincidence of spatial patterns of canker incidence and host mortality suggest a role for the disease in regulating host spatial distribution, in agreement with predictions of the Janzen-Connell hypothesis.     

Stability of rhodopsin in detergent solutions.

The thermal stability of lipid-free rhodopsin in solutions of a homologous series of alkyltrimethylammonium bromide detergents and one nonionic detergent, dodecyl-beta-maltoside, has been studied as a function of detergent concentration. Rhodopsin thermal stability increases with increasing chain length within the homologous series of ionic detergents, and for chain lengths greater than 10 carbon atoms increases with increasing detergent concentration up to a "critical" concentration that depends on the chain length. Stability also increases with increasing detergent concentration for rhodopsin in solutions of the nonionic detergent. These results may be rationalized in terms of the dependence of micelle packing density on the detergent chain length, head group, and concentration.     

Structural features of the C-terminal domain of bovine rhodopsin: a site-directed spin-labeling study.

Eleven single-cysteine substitution mutants have been prepared in the sequence 325-340 of rhodopsin, corresponding to the C-terminal domain. Each of the cysteine mutants was modified with a selective nitroxide reagent to introduce a spin-labeled side chain. The electron paramagnetic resonance spectra of the labeled proteins were analyzed in terms of side chain dynamics. At all sites, the spectra reflected the presence of two populations of different mobility, although one was always dominant. The mobility of the dominant population increased in a regular fashion from the palmitoylation sites at 322C and 323C to the C-terminus, where the spectra resembled those of an unfolded protein. This apparent mobility gradient is only slightly affected in mutants lacking the palmitoyl groups, suggesting that they are not responsible for physically anchoring the C-terminal peptide at one end. Binding of a monoclonal antibody to its epitope at the C-terminus dramatically reduces the mobility of nearby residues, creating a local mobility gradient opposite that in the absence of the antibody. These results indicate that the C-terminal domain of rhodopsin, beyond the palmitoylation sites, is highly disordered and dynamic, resembling an unfolded peptide tethered at one end.