Uncharged P-selectin blockers

The blocking potency of P- and L-selectin was studied for certain small molecule mannosides and their polyacrylamide (PAA, 30 kDa) conjugates in comparison to SiaLe^x and fucoidan. Two experimental systems were used: (1) solid phase static assay based on recombinant selectins, and (2) P-selectin dependent rat peritoneal inflammation. βMan-SC₆H₄NO₂-p was four times more potent P-selectin inhibitor as compared to SiaLe^x. Docking of this molecule onto the P-selectin carbohydrate-binding site demonstrated that a nitro group enabled an electrostatic interaction with residue Lys 84, while the phenyl ring and the CH₂ at C-6 contacted the CH₂ groups of the same Lys residue. In vivo, βMan-SC₆H₄NO₂-p blocked experimental inflammation better than SiaLe^x, but significantly lower than fucoidan. In vitro Man-polyacrylic acid conjugates appeared to be very potent inhibitors comparable to fucoidan, uncharged Man-PAA proved rather active, comparable to SiaLe^x-PAA both in vitro, and in vivo, whereas mannan did not display any P-selectin blocking effect. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Uncharged tRNA-phosphofructokinase interaction in amino acid deficiency

Summary When the tRNA of mammalian cells is incompletely charged due to amino acid deficiency or by analogs which cannot be activated, many metabolic events become limited. This rapid demise of cell function appears to be due to the inhibition of phosphofructokinase (PFK) by uncharged tRNA (FEBS Lett 302: 113 (1992)). Charged tRNA has been shown to be sequestered within the protein synthetic machinery, (Negrutskii, B. S. and Deutscher, M. P. (1992) Proc Natl Acad Sci USA 89: 3601) and would therefore be removed from an inhibitory role. Besides the direct demonstration that tRNA inhibits PFK in an assay regarded as indicative of its control mechanism, several reports in the literature support this model. These include 1) The rapid onset of inhibition of glycolysis and glucose uptake by intact cells upon amino acid deficiency and the similar lesion at the 43S ribosomal subunit on glucose or amino acid deprivation. 2) The recognition that unusually high concentrations of cAMP required to stimulate protein synthesis in energy depleted or gel filtered lysates correlates with its action on PFK as an analog of the positive effector, adenosine-5-monophosphate. 3) The often repeated observation that the product of PFK activity, fructose-1,6-diphosphate, is a stimulant of protein synthesis (see Jackson, R. J., et al. (1983) Eur J Biochem 131: 289). This diphosphate has been shown to be the proximate effector binding to eIF-2B, the guanine nucleotide exchange factor (Singh, L. P. Arror, A. R. and Wahba, A. J. (1994), FASEB J. 8: 279) which by releasing GDP bound to the inactive GDP: eIF-2 complex, permits the factor to initiate a new peptide chain. The above information supports the view that the block at the G1 restriction point in the cell cycle of normal cells brought about by amino acid deprivation is a result of inhibition of protein synthesis through the phosphofructokinase-uncharged tRNA mechanism. This is consistent with observations in the literature that tumor and transformed cells, which are more resistant to this block (Pardee, A. B., Proc Natl Acad Sci USA 71: 1286–1291 (1974)) have a higher phosphofructokinase activity or higher levels of fructose-1,6-diphosphate.Presented at the 4th International Congress on Amino Acids, Vienna, Austria, August 7–11, 1995. (Amino Acids (1995) 9: 23, Abstract).     

重组人P-选凝素及P-选凝素糖蛋白配基-1的表达与鉴定

P-选凝素表达于血管内皮细胞及血小板膜上,它可以与白细胞膜表面的P-选凝素糖蛋白配基-1(P-selectin glyco-protein ligand-1,PSGL-1)相互作用,在炎症过程中介导白细胞的滚动并启动随后的白细胞迁移级联过程.我们构建了重组人野生型可溶性P-选凝素及其钙离子结合位点突变体,同时构建了重组PSGL-1免疫球蛋白融合分子(PSGL-1-Rg),并应用昆虫杆状病毒表达系统在Sf9细胞中表达这些重组蛋白,最后用镍金属螯和柱或Protein A亲和柱予以纯化.结果显示,用该系统表达的P-选凝素或PSGL-1是有活性的,但是P-选凝素的4个钙离子结合位点突变体却没有活性.该研究证明了P-选凝素钙离子结合位点在其与配基相互作用中的重要性.     

P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin

Neutrophils roll on P-selectin expressed by activated platelets or endothelial cells under the shear stresses in the microcirculation. P- selectin glycoprotein ligand-1 (PSGL-1) is a high affinity ligand for P- selectin on myeloid cells. However, it has not been demonstrated that PSGL-1 contributes to the rolling of neutrophils on P-selectin. We developed two IgG mAbs, PL1 and PL2, that appear to recognize protein- dependent epitopes on human PSGL-1. The mAbs bound to PSGL-1 on all leukocytes as well as on heterologous cells transfected with PSGL-1 cDNA. PL1, but not PL2, blocked binding of 125-I-PSGL-1 to immobilized P-selectin, binding of fluid-phase P-selectin to myeloid and lymphoid leukocytes, adhesion of neutrophils to immobilized P-selectin under static conditions, and rolling of neutrophils on P-selectin-expressing CHO cells under a range of shear stresses. PSGL-1 was localized to microvilli on neutrophils, a topography that may facilitate its adhesive function. These data indicate that (a) PSGL-1 accounts for the high affinity binding sites for P-selectin on leukocytes, and (b) PSGL- 1 must interact with P-selectin in order for neutrophils to roll on P- selectin at physiological shear stresses.     

Permeation of Uncharged Organic Molecules and Water Through Tomato Roots

Permeation of ethylene glycol, D-mannitol, L glucose, and raffinosethrough excised tomato roots was investigated. Solute transportfrom external solution to the xylem sap was determined at variousrates of exudate flow achieved in turn by application of differentpressures to the solution surrounding the roots. At an applied pressure of two bar, steady-state solute concentrationsexpressed as a percentage of external concentrations were, 52per cent for ethylene glycol, 3–4 per cent for mannitoland glucose, and I per cent for raffinose. Such relationshipsbetween the chemical structure of the solute and its concentrationin the xylem sap are similar to those demonstrated by otherworkers for solute permeation into single cells. Thus in thepresent experiments most of these solutes presumably flowedthrough at least one membrane before reaching the xylem. The data also indicate that the flow of water via wholly extracellularpathways was slight, at most, I per cent of the total flow reachingthe xylem via this route.     

Uncharged tRNA, protein synthesis, and the bacterial stringent response

Uncharged tRNA has been shown in vivo to have an active role both in the stringent response, and in modulating the rate of translational elongation. Both of these effects appear to be mediated by codon-anticodon interactions on the ribosome. Although the involvement of uncharged tRNA in the stringent response was expected from in vitro experiments, it has only recently been confirmed in vivo. Inhibition of translation by cognate uncharged tRNA was not expected, and a model is proposed in which excess uncharged tRNA competes with charged tRNA (in ternary complex) for the 30S component of the ribosomal A site. When uncharged tRNA is in sufficient excess over charged tRNA, interaction of uncharged tRNA with the 50S component of the A site occurs as well, leading to a stringent response. The cell has a continuum of responses to decreasing aminoacyl-tRNA levels: in moderately limited conditions, the proportion of uncharged tRNA increases, and the translation rate is slowed; under more severe limitations, uncharged tRNA provokes a stringent response, with pleiotropic consequences for the cell.     

A novel glycosulfopeptide binds to P-selectin and inhibits leukocyte adhesion to P-selectin.

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin on leukocytes that binds selectins. The molecular features of PSGL-1 that determine this high affinity binding are unclear. Here we demonstrate the in vitro synthesis of a novel glycosulfopeptide (GSP-6) modeled after the extreme N terminus of PSGL-1, which has been predicted to be important for P-selectin binding. GSP-6 contains three tyrosine sulfate (TyrSO(3)) residues and a monosialylated, core 2-based O-glycan with a sialyl Lewis x (C2-O-sLe(x)) motif at a specific Thr residue. GSP-6 binds tightly to immobilized P-selectin, whereas glycopeptides lacking either TyrSO(3) or C2-O-sLe(x) do not detectably bind. Remarkably, an isomeric glycosulfopeptide to GSP-6, termed GSP-6', which contains sLe(x) on an extended core 1-based O-glycan, does not bind immobilized P-selectin. Equilibrium gel filtration analysis revealed that GSP-6 binds to soluble P-selectin with a K(d) of approximately 350 nM. GSP-6 (<5 microM) substantially inhibits neutrophil adhesion to P-selectin in vitro, whereas free sLe(x) (5 mM) only slightly inhibits adhesion. In contrast to the inherent heterogeneity of post-translational modifications of recombinant proteins, glycosulfopeptides permit the placement of sulfate groups and glycans of precise structure at defined positions on a polypeptide. This approach should expedite the probing of structure-function relationships in sulfated and glycosylated proteins, and may facilitate development of novel drugs to treat inflammatory diseases involving P-selectin-mediated leukocyte adhesion.     

Initiation of Positive Streamers near Uncharged Ice Hydrometeors in the Thundercloud Field

Since the threshold electric field required for breakdown of air is much higher than the maximum field strength measured in thunderstorm clouds, the problem of lightning initiation still remains unsolved. According to the popular hypothesis, lightning can be initiated by a streamer discharge in the field enhanced near a hydrometeor. To verify the adequacy of this hypothesis, the development of a positive streamer propagating along the thunderstorm electric field in the vicinity of an ice needle at an air pressure corresponding to an altitude of 5 km (which is typical of the lightning initiation conditions) was simulated numerically. The hydrometeor dimensions are determined at which streamers can be initiated at different strengths of the thunderstorm electric field.     

Noncovalent association of P-selectin glycoprotein ligand-1 and minimal determinants for binding to P-selectin

P-selectin glycoprotein ligand-1 (PSGL-1) is a disulfide-bonded, homodimeric mucin ( approximately 250 kDa) on leukocytes that binds to P-selectin on platelets and endothelial cells during the initial steps in inflammation. Because it has been proposed that only covalently dimerized PSGL-1 can bind P-selectin, we investigated the factors controlling dimerization of PSGL-1 and re-examined whether covalent dimers are required for binding its P-selectin. Recombinant forms of PSGL-1 were created in which the single extracellular Cys (Cys(320)) was replaced with either Ser (C320S-PSGL-1) or Ala (C320A-PSGL-1). Both recombinants migrated as monomeric species of approximately 120 kDa under both nonreducing and reducing conditions on SDS-polyacrylamide gel electrophoresis. P-selectin bound similarly to cells expressing either wild type or mutated forms of PSGL-1 in both flow cytometric and rolling adhesion assays. Unexpectedly, chemical cross-linking studies revealed that both C320S- and C320A-PSGL-1 noncovalently associate in the plasma membrane and cross-linking generates dimeric species. Chimeric recombinants of PSGL-1 in which the transmembrane domain in PSGL-1 was replaced with the transmembrane domain of CD43 (CD43TMD-PSGL-1) could not be chemically cross-linked, suggesting that residues within the transmembrane domain of PSGL-1 are required for noncovalent association. Cells expressing CD43TMD-PSGL-1 bound P-selectin. To further address the ability of P-selectin to bind monomeric derivatives of PSGL-1, intact HL-60 cells were trypsin-treated, which generated a soluble approximately 25-kDa NH(2)-terminal fragment of PSGL-1 that bound to immobilized P-selectin. Because N-glycosylation of PSGL-1 hinders trypsin cleavage, a recombinant form of PSGL-1 was generated in which all three potential N-glycosylation sites were mutated (DeltaN-PSGL-1). Cells expressing DeltaN-PSGL-1 bound P-selectin, and trypsin treatment of the cells generated NH(2)-terminal monomeric fragments (<10 kDa) of PSGL-1 that bound to P-selectin. These results demonstrate that Cys(320)-dependent dimerization of PSGL-1 is not required for binding to P-selectin and that a small monomeric fragment of PSGL-1 is sufficient for P-selectin recognition.     

Uncharged Gemini-Amphiphiles as Components of Cationic Liposomes for Delivery of Nucleic Acids

Russian Journal of Bioorganic Chemistry - New uncharged gemini-amphiphiles have been synthesized. A series of cationic liposomes based on the polycationic amphiphile...     

Functional role of P-selectin glycoprotein ligand 1/P-selectin interaction in the generation of tolerogenic dendritic cells

Dendritic cells (DCs) have a key role in both the generation of the immune response and the induction of tolerance to self-Ags. In this work, the possible role of P-selectin glycoprotein ligand 1 (PSGL-1) on the tolerogenic activity of human DCs was explored. We found that the engagement of PSGL-1 by P-selectin on DCs induced the expression of c-Fos, IDO, IL-10, and TGF-beta genes. Remarkably, stimulation of DCs through PSGL-1 with P-selectin enhanced their capability to generate CD4(+)CD25(+)Foxp3(+) regulatory T cells, which expressed high levels of TGF-beta1 mRNA, synthesized IL-10, and suppressed the proliferation of autologous CD4(+)CD25(-) T cells. Accordingly, we found that DCs from PSGL-1(-/-) mice expressed higher levels of MHC class II molecules, and exhibited an enhanced immunogenicity compared with wild-type mice. In addition, the percentage of CD4(+)CD25(+)Foxp3(+) regulatory T cells in the thymus of PSGL-1-deficient animals was significantly reduced. Our data reveal an unexpected role of PSGL-1 on the tolerogenic function of DCs, and the regulation of the immune response.     

Impairment of physical performance after treatment with beta blockers and alpha blockers.

An investigation was made into the effect of various types of beta blockers, an alpha blocker, a combined alpha and beta blocker, and a diuretic on physical performance in a normotensive man. Beta blockers, the alpha blocker, and the combined alpha and beta blocker significantly (p less than 0.001) reduced physical performance. Further studies are needed to confirm these findings in a larger series of subjects.     

Uncharged thioflavin-T derivatives bind to amyloid-beta protein with high affinity and readily enter the brain

In vivo assessment of the beta-sheet proteins deposited in amyloid plaques (A beta peptide) or neurofibrillary tangles (tau protein) presents a target for the development of biological markers for Alzheimer's disease (AD). In an effort to develop in vivo beta-sheet imaging probes, derivatives of thioflavin-T (ThT) were synthesized and evaluated. These compounds lack the positively charged quaternary heterocyclic nitrogen of ThT and are therefore uncharged at physiological pH. They are 600-fold more lipophilic than ThT. These ThT derivatives bind to A beta(1-40) fibrils with higher affinity (Ki = 20.2 nM) than ThT (Ki = 890 nM). The uncharged ThT derivatives stained both plaques and neurofibrillary tangles in post-mortem AD brain, showing some preference for plaque staining. A carbon-11 labeled compound, [N-methyl-11C]6-Me-BTA-1, was prepared, and its brain entry and clearance were studied in Swiss-Webster mice. This compound entered the brain at levels comparable to commonly used neuroreceptor imaging agents (0.223 %ID-kg/g or 7.61 %ID/g at 2 min post-injection) and showed good clearance of free and non-specifically bound radioactivity in normal rodent brain tissue (brain clearance t(1,2) = 20 min). The combination of relatively high affinity for amyloid, specificity for staining plaques and neurofibrillary tangles in post-mortem AD brain, and good brain entry and clearance makes [N-methyl-11C]6-Me-BTA-1 a promising candidate as an in vivo positron emission tomography (PET) beta-sheet imaging agent.     

alpha-Neurotoxins and alpha-conotoxins--nicotinic cholinoreceptor blockers

The review is devoted to the competitive blockers of different nicotinic acetylcholine receptors, alpha-neurotoxins from snake venoms, and alpha-conotoxins from marine snails of the Conidae family. The relationship between the structure and function of these toxins is discussed. Recent data on the mechanism of alpha-neurotoxin and alpha-conotoxin interaction with the nicotinic acetylcholine receptor are presented.     

Uncharged tRNA activates GCN2 by displacing the protein kinase moiety from a bipartite tRNA-binding domain

Protein kinase GCN2 regulates translation in amino acid-starved cells by phosphorylating elF2. GCN2 contains a regulatory domain related to histidyl-tRNA synthetase (HisRS) postulated to bind multiple deacylated tRNAs as a general sensor of starvation. In accordance with this model, GCN2 bound several deacylated tRNAs with similar affinities, and aminoacylation of tRNAphe weakened its interaction with GCN2. Unexpectedly, the C-terminal ribosome binding segment of GCN2 (C-term) was required in addition to the HisRS domain for strong tRNA binding. A combined HisRS+ C-term segment bound to the isolated protein kinase (PK) domain in vitro, and tRNA impeded this interaction. An activating mutation (GCN2c-E803V) that weakens PK-C-term association greatly enhanced tRNA binding by GCN2. These results provide strong evidence that tRNA stimulates the GCN2 kinase moiety by preventing an inhibitory interaction with the bipartite tRNA binding domain.     

Structurally distinct requirements for binding of P-selectin glycoprotein ligand-1 and sialyl Lewis x to Anaplasma phagocytophilum and P-selectin

Colonization of neutrophils by the bacterium Anaplasma phagocytophilum causes the disease human granulocytic ehrlichiosis. The pathogen also infects mice, its natural host. Like binding of P-selectin, binding of A. phagocytophilum to human neutrophils requires expression of P-selectin glycoprotein ligand-1 (PSGL-1) and alpha1-3-fucosyltransferases that construct the glycan determinant sialyl Lewis x (sLex). Binding of A. phagocytophilum to murine neutrophils, however, requires expression of alpha1-3-fucosyltransferases but not PSGL-1. To further characterize the molecular features that A. phagocytophilum recognizes, we measured bacterial binding to microspheres bearing specific glycoconjugates or to cells expressing human PSGL-1 and particular glycosyltransferases. Like P-selectin, A. phagocytophilum bound to purified human PSGL-1 and to glycopeptides modeled after the N terminus of human PSGL-1 that presented sLex on an O-glycan. Unlike P-selectin, A. phagocytophilum bound to glycopeptides that contained sLex but lacked tyrosine sulfation or a specific core-2 orientation of sLex on the O-glycan. A. phagocytophilum bound only to glycopeptides that contained a short amino acid sequence found in the N-terminal region of human but not murine PSGL-1. Unlike P-selectin, A. phagocytophilum bound to cells expressing PSGL-1 in cooperation with sLex on both N-and O-glycans. Moreover, bacteria bound to microspheres coupled independently with glycopeptide lacking sLex and with sLex lacking peptide. These results demonstrate that, unlike P-selectin, A. phagocytophilum binds cooperatively to a nonsulfated N-terminal peptide in human PSGL-1 and to sLex expressed on PSGL-1 or other glycoproteins. Distinct bacterial adhesins may mediate these cooperative interactions.     

肝素对P选择素介导的肿瘤转移的抑制作用

肝素是一种常见的抗凝药,临床治疗和动物试验中发现肝素还具有抗肿瘤转移的作用,能显著提高肿瘤患者的生存率。在肝素多种抗肿瘤相关的生物活性中,竞争性抑制P选择素介导的肿瘤细胞的黏附作用最为关键,决定了肝素抗肿瘤转移能力的大小,这种与肝素的抗凝机制不同。通过对肝素分子基团进行化学修饰可以消除其副作用的危险,得到具有抗肿瘤转移活性的肝素衍生物。