Exploration of the Arrest Peptide Sequence Space Reveals Arrest-enhanced Variants

Translational arrest peptides (APs) are short stretches of polypeptides that induce translational stalling when synthesized on a ribosome. Mechanical pulling forces acting on the nascent chain can weaken or even abolish stalling. APs can therefore be used as in vivo force sensors, making it possible to measure the forces that act on a nascent chain during translation with single-residue resolution. It is also possible to score the relative strengths of APs by subjecting them to a given pulling force and ranking them according to stalling efficiency. Using the latter approach, we now report an extensive mutagenesis scan of a strong mutant variant of the Mannheimia succiniciproducens SecM AP and identify mutations that further increase the stalling efficiency. Combining three such mutations, we designed an AP that withstands the strongest pulling force we are able to generate at present. We further show that diproline stretches in a nascent protein act as very strong APs when translation is carried out in the absence of elongation factor P. Our findings highlight critical residues in APs, show that certain amino acid sequences induce very strong translational arrest and provide a toolbox of APs of varying strengths that can be used for in vivo force measurements.     

Effects of simultaneous changes in light, nutrients, and herbivory levels, on the structure and function of a subtropical turtlegrass meadow

The interactive effects of light, nutrients, and simulated herbivory on the structure and functioning of a subtropical turtlegrass bed were analyzed monthly from May to October 2001 in Perdido Bay, FL. For each of the three factors, two levels were evaluated in a factorial design with four replicates per treatment. The variables included: light, at ambient and 40% reduction; nutrients, at ambient and 2× ambient concentrations; and herbivory, with no herbivory and simulated effects of a density of 15 sea urchins/m². In practice, light levels turned out to be 40% of surface PAR for ambient conditions, and 16% for shaded plots. Biomass removed as herbivory represented, on average, slightly less than 20% of the above-ground biomass. Separate three-way ANOVAs found no significant three-way interactions for any of the response variables, and few two-way interactions. There were no significant nutrient effects on turtlegrass above-ground biomass, although nutrient additions produced significant decreases in epibiont biomass, and net above-ground primary production (NAPP); significant increases in below-ground biomass during the peak of the growing season. Shoot density and average number of leaves per shoot increased significantly, while the C/N ratio of the oldest leaf in the enriched plots decreased significantly. Light reduction significantly negatively affected all response variables, except below-ground biomass, shoot density and leaf length. Herbivory had isolated and inconsistent significant effects on below-ground biomass, shoot density, average number of leaves per shoot, and leaf length and width. Overall, our results indicate that nutrients are not limiting in Perdido Bay, and that nutrient additions had mostly detrimental effects. Light appeared to be the most important variable limiting seagrasses growth and abundance, and as with terrestrial plants, seagrasses seemed to respond more to light and nutrients than to herbivory. However, it is essential that additional tests of the single and interactive effects of the three key factors of light, nutrients and herbivory be done to evaluate the generality of our work, since our study is the first of its kind in seagrass meadows.     

Functional assessment of cryopreserved pig aortas for pharmaceutical research

Several in vitro studies have demonstrated diminished post-thaw functional activity. Therefore, the aim of this study was to investigate the consequences of thawing and storage method used on the post-thaw functional activity of cryopreserved pig aortas with the aim of adjusting the freezing and thawing protocol so that the vascular segments are preserved in the best possible state, maintaining structure and functionality so that they can later be transplanted with success. In vitro responses of frozen, thawed pig aortas were used to investigate the functional activity after thawing at 15 degrees C and 100 degrees C/min and after storage in gas or liquid phase of liquid nitrogen. Cryopreservation was performed in RPMI 1640 medium + 10% dimethylsulfoxide and the rate of cooling was -1 degrees C/min, until -150 degrees C was reached.After thawing the maximal contractile responses to all the contracting agonists tested (KCl, noradrenaline) were in the ranges of 13-27% compared with the responses in unfrozen pig aortas. Contractile responses were slightly better when thawing was performed at 15 degrees C/min compared with 100 degrees C/min. The endothelium independent relaxant responses to sodium nitroprusside were reduced ( P < 0.05). Cryostorage of pig arteries also resulted in a loss of the endothelium-dependent relaxant response to acetylcholine. The cryopreservation method used provided a limited preservation of pig aorta contractibility, a reduction of the endothelium independent relaxant responses, and no apparent preservation of the endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol might allow better post-thaw functional recovery of pig aortas.     

A Retained Secretory Signal Peptide Mediates High Density Lipoprotein (HDL) Assembly and Function of Haptoglobin-related Protein

Haptoglobin-related protein (Hpr) is a component of a minor subspecies of high density lipoproteins (HDL) that function in innate immunity. Here we show that assembly of Hpr into HDL is mediated by its retained N-terminal signal peptide, an unusual feature for a secreted protein and the major difference between Hpr and the soluble acute phase protein haptoglobin (Hp). The 18-amino acid signal peptide is necessary for binding to HDL and interacts directly with the hydrocarbon region of lipids. Utilizing model liposomes, we show that the rate of assembly and steady-state distribution of Hpr in lipid particles is mediated by the physical property of lipid fluidity. Dye release assays reveal that Hpr interacts more rapidly with fluid liposomes. Conversely, steady-state binding assays indicate that more rigid lipid compositions stabilize Hpr association. Lipid association also plays a role in facilitating hemoglobin binding by Hpr. Our data may offer an explanation for the distinct distribution of Hpr among HDL subspecies. Rather than protein-protein interactions mediating localization, direct interaction with phospholipids and sensitivity to lipid fluidity may be sufficient for localization of Hpr and may represent a mechanism of HDL subspeciation.     

Inhibition of large-conductance calcium-activated potassium channel by 2-methoxyestradiol in cultured vascular endothelial (HUV-EC-C) cells

2-Methoxyestradiol, an endogenous metabolite of 17β-estradiol, is known to have antitumor and antiangiogenic actions. The effects of 2-methoxyestradiol on ionic currents were investigated in an endothelial cell line (HUV-EC-C) originally derived from human umbilical vein. In the whole-cell patch-clamp configuration, 2-methoxyestradiol (0.3–30 μm) reversibly suppressed the amplitude of K⁺ outward currents. The IC ₅₀ value of the 2-methoxyestradiol-induced decrease in outward current was 3 μm. Evans blue (30 μm) or niflumic acid (30 μm), but not diazoxide (30 μm), reversed the 2-methoxyestradiol-induced decrease in outward current. In the inside-out configuration, application of 2-methoxyestradiol (3 μm) to the bath did not modify the single-channel conductance of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels; however, it did suppress the channel activity. 2-Methoxyestradiol (3 μm) produced a shift in the activation curve of BK_Ca channels to more positive potentials. Kinetic studies showed that the 2-methoxyestradiol-induced inhibition of BK_Ca channels is primarily mediated by a decrease in the number of long-lived openings. 2-Methoxyestradiol-induced inhibition of the channel activity was potentiated by membrane stretch. In contrast, neither 17β-estradiol (10 μm) nor estriol (10 μm) affected BK_Ca channel activity, whereas 2-hydroxyestradiol (10 μm) slightly suppressed it. Under current-clamp condition, 2-methoxyestradiol (10 μm) caused membrane depolarization and Evans blue (30 μm) reversed 2-methoxyestradiol-induced depolarization. The present study provides evidence that 2-methoxyestradiol can suppress the activity of BK_Ca channels in endothelial cells. These effects of 2-methoxyestradiol on ionic currents may contribute to its effects on functional activity of endothelial cells. Received: 27 November 2000/Revised: 13 April 2001     

Somatic Embryogenesis and in vitro Flowering in <Emphasis Type="Italic">Saposhnikovia divaricata</Emphasis>

Efficient somatic embryogenesis (SE) and in vitro flowering and fruiting were achieved in Saposhnikovia divaricata (Turcz.) Schischk. Friable embryogenic callus developed from the root, internode, and leaf explants on Murashige and Skoog medium (MS) with 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D), and subsequently developed into somatic embryos on MS medium containing 4–5% sucrose, 1.74 μM naphthaleneacetic acid (NAA), 4.44 μM 6-benzylaminopurine (BA), and 1.90 μM abscisic acid (ABA). Then the mature embryos were separated and transferred onto MS with 3% sucrose and 0.6% agar for further development and conversion to plantlets. In vitro flowering and fruiting were obtained when the subcultures were carried out for over 15 months. Paclobutrazol (PP333) or ethephon (ETH) at low levels promoted flowering significantly. Also, abnormal rootless somatic embryos of S. divaricata could form flowers and fruits in vitro.     

Differential responses of normal human coronary artery endothelial cells against multiple cytokines comparatively assessed by gene expression profiles

Endothelial cells play an important role in terms of biological functions by responding to a variety of stimuli in the blood. However, little is known about the molecular mechanism involved in rendering the variety in the cellular response. To investigate the variety of the cellular responses against exogenous stimuli at the gene expression level, we attempted to describe the cellular responses with comprehensive gene expression profiles, dissect them into multiple response patterns, and characterize the response patterns according to the information accumulated so far on the genes included in the patterns. We comparatively analyzed in parallel the gene expression profiles obtained with DNA microarrays from normal human coronary artery endothelial cells (HCAECs) stimulated with multiple cytokines, interleukin-1β, tumor necrosis factor-, interferon-β, interferon-γ, and oncostatin M, which are profoundly involved in various functional responses of endothelial cells. These analyses revealed that the cellular responses of HCAECs against these cytokines included at least 15 response patterns specific to a single cytokine or common to multiple cytokines. Moreover, we statistically extracted genes contained within the individual response patterns and characterized the response patterns with the genes referring to the previously accumulated findings including the biological process defined by the Gene Ontology Consortium (GO). Out of the 15 response patterns in which at least one gene was successfully extracted through the statistical approach, 11 response patterns were differentially characterized by representing the number of genes contained in individual criteria of the biological process in the GO only. The approach to dissect cellular responses into response patterns and to characterize the pattern at the gene expression level may contribute to the gaining of insight for untangling the diversity of cellular functions.