High yield expression and purification of full-length Neurotensin with pyroglutamate modification.

Neurotensin (NT) is a 13-residue endogenous peptide found in mammals, with neurotransmission and hormonal roles in the central nervous system and gastrointestinal tract, respectively. The first residue of NT is a pyroglutamate (pGlu) that makes the expression and purification of large amounts of NT with native modification challenging. Here, we describe a simple and efficient procedure for expression and purification of large amounts of NT based on using the small ubiquitin-like modifier (SUMO) as a fusion partner and subsequent enzymatic conversion of the N-terminal glutamine to pGlu. Yields of 13 mg/L and 8 mg/L of pure peptide were obtained from expression in rich and minimal media, respectively. The method is adaptable to expression and purification of proteins and peptides with pGlu modification in a wide range of eukaryotic and prokaryotic expression hosts.

Antiplatelet antibody-induced thrombocytopenia does not correlate with megakaryocyte abnormalities in murine immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by increased peripheral immune platelet destruction and megakaryocyte defects in the bone marrow. Although ITP was originally thought to be primarily due to antibody-mediated autoimmunity, it is now clear that T cells also play a significant role in the disease. However, the exact interplay between platelet destruction, megakaryocyte dysfunction and the elements of both humoral and cell-mediated immunity in ITP remains incompletely defined. While most studies have focused on immune platelet destruction in the spleen, an additional possibility is that the antiplatelet antibodies can also destroy bone marrow megakaryocytes. To address this, we negated the effects of T cells by utilizing an in vivo passive ITP model where BALB/c mice were administered various anti-αIIb, anti-ß3 or anti-GPIb antibodies or antisera and platelet counts and bone marrow megakaryocytes were enumerated. Our results show that after 24 hours, all the different antiplatelet antibodies/sera induced variable degrees of thrombocytopenia in recipient mice. Compared with naïve control mice, however, histological examination of the bone marrow revealed that only 2 antibody preparations (mouse-anti-mouse ß3 sera and an anti- αIIb monoclonal antibody (MWReg30) could affect bone marrow megakaryocyte counts. Our study shows that while most antiplatelet antibodies induce acute thrombocytopenia, the majority of them do not affect the number of megakaryocytes in the bone marrow. This suggests that other mechanisms may be responsible for megakaryocyte abnormalities seen during immune thrombocytopenia.

Buckling of an Elastic Ridge: Competition between Wrinkles and Creases.

We investigate the elastic buckling of a triangular prism made of a soft elastomer. A face of the prism is bonded to a stiff slab that imposes an average axial compression. We observe two possible buckling modes which are localized along the free ridge. For ridge angles ϕ below a critical value ϕ^{⋆}≈90°, experiments reveal an extended sinusoidal mode, while for ϕ above ϕ^{⋆}, we observe a series of creases progressively invading the lateral faces starting from the ridge. A numerical linear stability analysis is set up using the finite-element method and correctly predicts the sinusoidal mode for ϕ≤ϕ^{⋆}, as well as the associated critical strain ε_{c}(ϕ). The experimental transition at ϕ^{⋆} is found to occur when this critical strain ε_{c}(ϕ) attains the value ε_{c}(ϕ^{⋆})=0.44 corresponding to the threshold of the subcritical surface creasing instability. Previous analyses have focused on elastic crease patterns appearing on planar surfaces, where the role of scale invariance has been emphasized; our analysis of the elastic ridge provides a different perspective, and reveals that scale invariance is not a sufficient condition for localization.

Rafflesia spp.: propagation and conservation.

MAIN CONCLUSION: The propagation of Rafflesia spp. is considered to be important for future development of ornamental and other applications. Thus far, the only successful propagation technique has been grafting. This mini-review succinctly emphasizes what is known about Rafflesia species. Members of the genus Rafflesia (Rafflesiaceae), which are holoparasitic plants known to grow on a host vine, Tetrastigma sp., are widely spread from the Malayan Peninsula to various islands throughout Indonesia. The plant's geographical distribution as well as many other aspects pertaining to the basic biology of this genus have still not been studied. The young flower buds and flowers of wild Rafflesia hasseltii Suringar, Rafflesia keithii Meijer and Rafflesia cantleyi Solms-Laubach are used in local (Malaysia and Indonesia) traditional ethnomedicine as wound-healing agents, but currently no formal published research exists to validate this property. To maintain a balance between its ethnomedicinal and ornamental use, and conservation, Rafflesia spp. must be artificially cultivated to prevent overexploitation. A successful method of vegetative propagation is by host grafting using Rafflesia-impregnated Tetrastigma onto the stem of a normal Tetrastigma plant. Due to difficulties with culture contamination in vitro, callus induction was only accomplished in 2010 for the first time when picloram and 2,4-D were added to a basal Murashige and Skoog medium, and the tissue culture of holoparasitic plants continues to be extremely difficult. Seeds harvested from fertile fruit may serve as a possible method to propagate Rafflesia spp. This paper provides a brief synthesis on what is known about research related to Rafflesia spp. The objective is to further stimulate researchers to examine, through rigorous scientific discovery, the mechanisms underlying the ethnomedicinal properties, the flowering mechanisms, and suitable in vitro regeneration protocols that would allow for the fortification of germplasm conservation.

Shapes of a suspended curly hair.

We investigate how natural curvature affects the configuration of a thin elastic rod suspended under its own weight, as when a single strand of hair hangs under gravity. We combine precision desktop experiments, numerics, and theoretical analysis to explore the equilibrium shapes set by the coupled effects of elasticity, natural curvature, nonlinear geometry, and gravity. A phase diagram is constructed in terms of the control parameters of the system, namely the dimensionless curvature and weight, where we identify three distinct regions: planar curls, localized helices, and global helices. We analyze the stability of planar configurations, and describe the localization of helical patterns for long rods, near their free end. The observed shapes and their associated phase boundaries are then rationalized based on the underlying physical ingredients.

Identification of a Novel Subtype-Selective α1B-Adrenoceptor Antagonist.

α1A-, α1B-, and α1D-adrenoceptors (α1-ARs) are members of the adrenoceptor G protein-coupled receptor family that are activated by adrenaline (epinephrine) and noradrenaline. α1-ARs are clinically targeted using antagonists that have minimal subtype selectivity, such as prazosin and tamsulosin, to treat hypertension and benign prostatic hyperplasia, respectively. Abundant expression of α1-ARs in the heart and central nervous system (CNS) makes these receptors potential targets for the treatment of cardiovascular and CNS disorders, such as heart failure, epilepsy, and Alzheimer's disease. Our understanding of the precise physiological roles of α1-ARs, however, and their involvement in disease has been hindered by the lack of sufficiently subtype-selective tool compounds, especially for α1B-AR. Here, we report the discovery of 4-[(2-hydroxyethyl)amino]-6-methyl-2H-chromen-2-one (Cpd1), as an α1B-AR antagonist that has 10-15-fold selectivity over α1A-AR and α1D-AR. Through computational and site-directed mutagenesis studies, we have identified the binding site of Cpd1 in α1B-AR and propose the molecular basis of α1B-AR selectivity, where the nonconserved V19745.52 residue plays a major role, with contributions from L3146.55 within the α1B-AR pocket. By exploring the structure-activity relationships of Cpd1 at α1B-AR, we have also identified 3-[(cyclohexylamino)methyl]-6-methylquinolin-2(1H)-one (Cpd24), which has a stronger binding affinity than Cpd1, albeit with reduced selectivity for α1B-AR. Cpd1 and Cpd24 represent potential leads for α1B-AR-selective drug discovery and novel tool molecules to further study the physiology of α1-ARs.

Geometry-induced rigidity in nonspherical pressurized elastic shells.

We present results from an experimental investigation of the indentation of nonspherical pressurized elastic shells with a positive Gauss curvature. A predictive framework is proposed that rationalizes the dependence of the local rigidity of an indented shell on the curvature in the neighborhood of the locus of indentation, the in-out pressure differential, and the material properties. In our approach, we combine classic theory for spherical shells with recent analytical developments for the pressurized case, and proceed, for the most part, by analogy, guided by our own experiments. By way of example, our results elucidate why an eggshell is significantly stiffer when compressed along its major axis, as compared to doing so along its minor axis. The prominence of geometry in this class of problems points to the relevance and applicability of our findings over a wide range of length scales.

Crystal structure of the α1B-adrenergic receptor reveals molecular determinants of selective ligand recognition.

α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α1BAR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α1BAR structure allows the identification of two unique secondary binding pockets. By structural comparison of α1BAR with α2ARs, and by constructing α1BAR-α2CAR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α1BAR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype.

Antigen-induced B lymphocyte activation involves the p21ras and ras.GAP signaling pathway.

Ligation of a B lymphocyte surface immunoglobulin (sIg) antigen receptor (AgR) by its specific Ag ligand initiates a signaling pathway that culminates in B cell activation. However, many events of this pathway have not been elucidated. Here we present three novel findings that demonstrate directly that AgR-mediated signaling in B cells functions by the p21ras/ras.GAP-dependent pathway. First, stimulation of TA3 7.9 Ag-specific murine B lymphoma cells for 2 min with either Ag or F(ab')2 anti-IgM induces p21ras activation as measured by an increase in the GTP/GDP ratio of its bound nucleotides. This activation of p21ras does not occur via a change in its guanine nucleotide exchange rate. Second, Ag stimulation results in the inhibition of activity of p120 ras.GAP, a protein that regulates p21ras activation. Tyrosine phosphorylation of ras.GAP occurs within 1 min after Ag stimulation but is no longer detectable at 20 min after stimulation, at which time ras.GAP activity remains inhibited. Thus, tyrosine phosphorylation of ras.GAP is not required for the inhibition of its activity. Third, despite the role proposed for a ras.GAP-associated p190 protein in the control of ras.GAP activity in B cells, p190 was not detectable either in anti-ras.GAP immunoprecipitates of [35S]methionine labeled lysates of Ag-stimulated or -unstimulated 7.9 cells or as a tyrosine phosphoprotein in Western blots of anti-ras.GAP immunoprecipitates of Ag-stimulated 7.9 cell lysates. Inasmuch as the TA3 7.9 B lymphoma is representative of a mature, sIgM-bearing B cell, our observations raise the intriguing possibility that the capacity of p190 to associate with ras.GAP and regulate the activities of ras.GAP and p21ras in a B cell is dependent on the stage of differentiation of the B cell.

Thymic T cell anergy in autoimmune nonobese diabetic mice is mediated by deficient T cell receptor regulation of the pathway of p21ras activation.

Thymic T cell anergy, as manifested by thymocyte proliferative unresponsiveness to antigens expressed in the thymic environment, is commonly believed to mediate the acquisition of immunological self-tolerance. However, we previously found that thymic T cell anergy may lead to the breakdown of tolerance and predispose to autoimmunity in nonobese diabetic (NOD) mice. Here, we show that NOD thymic T cell anergy, as revealed by proliferative unresponsiveness in vitro after stimulation through the T cell receptor (TCR), is associated with defective TCR-mediated signal transduction along the PKC/p21ras/p42mapk pathway of T cell activation. PKC activity is reduced in NOD thymocytes. Activation of p21ras is deficient in quiescent and stimulated NOD T cells, and this is correlated with a significant reduction in the tyrosine phosphorylation of p42mapk, a serine/threonine kinase active downstream of p21ras. Treatment of NOD T cells with a phorbol ester not only enhances their p21ras activity and p42mapk tyrosine phosphorylation but also restores their proliferative responsiveness. Since p42mapk activity is required for progression through to S phase of the cell cycle, our data suggest that reduced tyrosine phosphorylation of p42mapk in stimulated NOD T cells may abrogate its activity and elicit the proliferative unresponsiveness of these cells.

Interleukin 4 reverses T cell proliferative unresponsiveness and prevents the onset of diabetes in nonobese diabetic mice.

Beginning at the time of insulitis (7 wk of age), CD4+ and CD8+ mature thymocytes from nonobese diabetic (NOD) mice exhibit a proliferative unresponsiveness in vitro after T cell receptor (TCR) crosslinking. This unresponsiveness does not result from either insulitis or thymic involution and is long lasting, i.e., persists until diabetes onset (24 wk of age). We previously proposed that it represents a form of thymic T cell anergy that predisposes to diabetes onset. This hypothesis was tested in the present study by further investigating the mechanism responsible for NOD thymic T cell proliferative unresponsiveness and determining whether reversal of this unresponsiveness protects NOD mice from diabetes. Interleukin 4 (IL-4) secretion by thymocytes from > 7-wk-old NOD mice was virtually undetectable after treatment with either anti-TCR alpha/beta, anti-CD3, or Concanavalin A (Con A) compared with those by thymocytes from age- and sex-matched control BALB/c mice stimulated under identical conditions. NOD thymocytes stimulated by anti-TCR alpha/beta or anti-CD3 secreted less IL-2 than did similarly activated BALB/c thymocytes. However, since equivalent levels of IL-3 were secreted by Con A-activated NOD and BALB/c thymocytes, the unresponsiveness of NOD thymic T cells does not appear to be dependent on reduced IL-2 secretion. The surface density and dissociation constant of the high affinity IL-2 receptor of Con A-activated thymocytes from both strains are also similar. The patterns of unresponsiveness and lymphokine secretion seen in anti-TCR/CD3-activated NOD thymic T cells were also observed in activated NOD peripheral spleen T cells. Exogenous recombinant (r)IL-2 only partially reverses NOD thymocyte proliferative unresponsiveness to anti-CD3, and this is mediated by the inability of IL-2 to stimulate a complete IL-4 secretion response. In contrast, exogenous IL-4 reverses the unresponsiveness of both NOD thymic and peripheral T cells completely, and this is associated with the complete restoration of an IL-2 secretion response. Furthermore, the in vivo administration of rIL-4 to prediabetic NOD mice protects them from diabetes. Thus, the ability of rIL-4 to reverse completely the NOD thymic and peripheral T cell proliferative defect in vitro and protect against diabetes in vivo provides further support for a causal relationship between this T cell proliferative unresponsiveness and susceptibility to diabetes in NOD mice.

Intravenous immunoglobulins induce potentially synergistic immunomodulations in autoimmune disorders.

The increase in platelets in patients with immune thrombocytopenia (ITP) by intravenous administration of human immunoglobulin concentrates (IVIG) reflects a therapeutic immunomodulatory intervention targeted at the disturbed immune response in many inflammatory and autoimmune disorders. These immunoglobulin concentrates contain large numbers of antibodies as well as trace levels of various other immunologically active molecules. Clinical and laboratory studies have documented various mechanisms of action of IVIG. The complex network of immunological reactions resulting from the infusion of IVIG includes changes in several cytokines, interactions with dendritic cells, T- and B- lymphocyte effects, macrophage effects, mediated by distinct Fc-gamma receptors. In addition, effects on complement components and apoptosis have also been observed. Synergism between the different elements of the immune response characterizes the beneficial effects of IVIG in inflammatory and autoimmune disorders. They have immunopathogeneses and clinical manifestations which are difficult to define and therefore IVIG treatment indications remain heterogeneous. Dose finding studies are missing for most of the indications of the drug. In future research, defining the appropriate subgroups of patients should be undertaken. This may be accomplished by prospective registries collecting data on large numbers of patients with long-term follow-up. Controlled clinical and laboratory studies may follow based on new, validated patient selection criteria and focused on mechanisms of action, leading to more evidence-based indications.

Mariner V: Plasma and Magnetic Fields Observed near Venus.

Abrupt changes in the amplitude of the magnetic fluctuations, in the field strength, and in the plasma properties, were observed with Mariner V near Venus. They provide clear evidence for the presence of a bow shock around the planet, similar to, but much smaller than, that observed at Earth. The observations appear consistent with an interaction of the solar wind with the ionosphere of Venus. No planetary field could be detected, but a steady radial field and very low plasma density were found 10,000 to 20,000 kilometers behind Venus and 8,000 to 12,000 kilometers from the Sun-Venus line. These observations may be interpreted as relating to an expansion wave tending to fill the cavity produced by Venus in the solar wind. The upper limit to the magnetic dipole moment of Venus is estimated to be within a factor of 2 of 10(-3) items that of Earth.

Observations at venus encounter by the plasma science experiment on mariner 10.

Preliminary results from the rearward-looking electrostatic analyzer of the plasma science experiment during the Mariner 10 encounter with Venus are described. They show that the solar-wind interaction with the planet probably involves a bow shock rather than an extended exosphere, but that this is not a thin boundary at the point where it was crossed by Mariner 10. An observed reduction in the flux of electrons with energies greater than 100 electron volts is interpreted as evidence for somne direct interaction with the exosphere. Unusual intermittent features observed downstream of the planet indicate the presence of a comet-like tail hundreds of scale lengths in length.

Observations at mercury encounter by the plasma science experiment on mariner 10.

A fully developed bow shock and magnetosheath were observed near Mercury, providing unambiguous evidence for a strong interaction between Mercury and the solar wind. Inside the sheath there is a distinct region analogous to the magnetosphere or magnetotail of Earth, populated by electrons with lower density and higher temperature than the electrons observed in the solar wind or magnetosheath. At the time of encounter, conditions were such that a perpendicular shock was observed on the inbound leg and a parallel shock was observed on the outbound leg of the trajectory, and energetic plasma electron events were detected upstream from the outbound shock crossing. The interaction is most likely not atmospheric, but the data clearly indicate that the obstacle to solar wind flow is magnetic, either intrinsic or induced. The particle fluxes and energy spectra showed large variations while the spacecraft was inside the magnetosphere, and these variations could be either spatial or temporal.

Plasma observations near jupiter: initial results from voyager 2.

The first of at least nine bow shock crossings observed on the inbound pass of Voyager 2 occurred at 98.8 Jupiter radii (R(J)) with final entry into the magnetosphere at 62 R(J). On both the inbound and outbound passes the plasma showed a tendency to move in the direction of corotation, as was observed on the inbound pass of Voyager 1. Positive ion densities and electron intensities observed by Voyager 2 are comparable within a factor of 2 to those seen by Voyager 1 at the same radial distance from Jupiter; the composition of the magnetospheric plasma is again dominated by heavy ions with a ratio of mass density relative to hydrogen of about 100/1. A series of dropouts of plasma intensity near Ganymede may be related to a complex interaction between Ganymede and the magnetospheric plasma. From the planetary spin modulation of the intensity of plasma electrons it is inferred that the plasma sheet is centered at the dipole magnetic equator out to a distance of 40 to 50 R(J) and deviates from it toward the rotational equator at larger distances. The longitudinal excursion of the plasma sheet lags behind the rotating dipole by a phase angle that increases with increasing radial distance.

Plasma observations near jupiter: initial results from voyager 1.

Extensive measurements of low-energy positive ions and electrons were made throughout the Jupiter encounter of Voyager 1. The bow shock and magneto-pause were crossed several times at distances consistent with variations in the upstream solar wind pressure measured on Voyager 2. During the inbound pass, the number density increased by six orders of magnitude between the innermost magnetopause crossing at approximately 47 Jupiter radii and near closest approach at approximately 5 Jupiter radii; the plasma flow during this period was predominately in the direction of corotation. Marked increases in number density were observed twice per planetary rotation, near the magnetic equator. Jupiterward of the Io plasma torus, a cold, corotating plasma was observed and the energylcharge spectra show well-resolved, heavy-ion peaks at mass-to-charge ratios A/Z* = 8, 16, 32, and 64.

Plasma observations near neptune: initial results from voyager 2.

The plasma science experiment on Voyager 2 made observations of the plasma environment in Neptune's magnetosphere and in the surrounding solar wind. Because of the large tilt of the magnetic dipole and fortuitous timing, Voyager entered Neptune's magnetosphere through the cusp region, the first cusp observations at an outer planet. Thus the transition from the magnetosheath to the magnetosphere observed by Voyager 2 was not sharp but rather appeared as a gradual decrease in plasma density and temperature. The maximum plasma density observed in the magnetosphere is inferred to be 1.4 per cubic centimeter (the exact value depends on the composition), the smallest observed by Voyager in any magnetosphere. The plasma has at least two components; light ions (mass, 1 to 5) and heavy ions (mass, 10 to 40), but more precise species identification is not yet available. Most of the plasma is concentrated in a plasma sheet or plasma torus and near closest approach to the planet. A likely source of the heavy ions is Triton's atmosphere or ionosphere, whereas the light ions probably escape from Neptune. The large tilt of Neptune's magnetic dipole produces a dynamic magnetosphere that changes configuration every 16 hours as the planet rotates.

Plasma observations near saturn: initial results from voyager 1.

Extensive measurements of low-energy plasma electrons and positive ions were made during the Voyager 1 encounter with Saturn and its satellites. The magnetospheric plasma contains light and heavy ions, probably hydrogen and nitrogen or oxygen; at radial distances between 15 and 7 Saturn-radii (Rs) on the inbound trajectory, the plasma appears to corotate with a velocity within 20 percent of that expected for rigid corotation. The general morphology of Saturn's magnetosphere is well represented by a plasma sheet that extends from at least 5 to 17 Rs, is symmetrical with respect to Saturn's equatorial plane and rotation axis, and appears to be well ordered by the magnetic shell parameter L (which represents the equatorial distance of a magnetic field line measured in units of Rs). Within this general configuration, two distinct structures can be identified: a central plasma sheet observed from L = 5 to L = 8 in which the density decreases rapidly away from the equatorial plane, and a more extended structure from L = 7 to beyond 18 Rs in which the density profile is nearly flat for a distance +/- 1.8 Rs off the plane and falls rapidly thereafter. The encounter with Titan took place inside the magnetosphere. The data show a clear signature characteristic of the interaction between a subsonic corotating magnetospheric plasma and the atmospheric or ionospheric exosphere of Titan. Titan appears to be a significant source of ions for the outer magnetosphere. The locations of bow shock crossings observed inbound and outbound indicate that the shape of the Saturnian magnetosphere is similar to that of Earth and that the position of the stagnation point scales approximately as the inverse one-sixth power of the ram pressure.

Plasma observations near saturn: initial results from voyager 2.

Results of measurements of plasma electrons and poitive ions made during the Voyager 2 encounter with Saturn have been combined with measurements from Voyager 1 and Pioneer 11 to define more clearly the configuration of plasma in the Saturnian magnetosphere. The general morphology is well represented by four regions: (i) the shocked solar wind plasma in the magnetosheath, observed between about 30 and 22 Saturn radii (RS) near the noon meridian; (ii) a variable density region between approximately 17 RS and the magnetopause; (iii) an extended thick plasma sheet between approximately 17 and approximately 7 RS symmetrical with respect to Saturn's equatorial plane and rotation axis; and (iv) an inner plasma torus that probably originates from local sources and extends inward from L approximately 7 to less than L approximately 2.7 (L is the magnetic shell parameter). In general, the heavy ions, probably O(+), are more closely confined to the equatorial plane than H(+), so that the ratio of heavy to light ions varies along the trajectory according to the distance of the spacecraft from the equatorial plane. The general configuration of the plasma sheet at Saturn found by Voyager 1 is confirmed, with some notable differences and additions. The "extended plasma sheet," observed between L approximately 7 and L approximately 15 by Voyager 1 is considerably thicker as observed by Voyager 2. Inward of L approximately 4, the plasma sheet collapses to a thin region about the equatorial plane. At the ring plane crossing, L approximately 2.7, the observations are consistent with a density of O(+) of approximately 100 per cubic centimeter, with a temperature of approximately 10 electron volts. The location of the bow shock and magnetopause crossings were consistent with those previously observed. The entire magnetosphere was larger during the outbound passage of Voyager 2 than had been previously observed; however, a magnetosphere of this size or larger is expected approximately 3 percent of the time.