Human Brain Lectin: A Soluble Lectin That Binds Actin

A biotinylated probe was used for detection of endogenous ligands of a human brain lectin on blotted human brain soluble proteins. Of the various proteins from brain extract resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, five reacted with the biotinylated probe. After elimination of saccharidic moieties by periodic treatment of the same extract, a single band with Mr approximately 43,000 was recognized by the lectin. This band was identified as actin using an anti-actin antibody. These results were confirmed by binding of biotinylated lectin to purified actin.     

桑寄生凝集素的纯化及部分性质研究

利用DEAE一纤维素柱盐离子浓度梯度洗脱,再经猪胃粘蛋白-Sepharose 4 B亲和柱可以从中药桑寄生中分离纯化出桑寄生凝集素。经pH8.9,Tris-EDTAN_2a-borate的PACE和SDS-PAGE测定均呈现单一蛋白带,测得其分子量为67 500,中性糖含量为14.6％,DNS法测得N-末端氨基酸为缬氨酸。氨基酸组成分析表明,该凝集素富含酸性氨基酸,而碱性氨基酸含量较少,不含精氨酸。当凝集素浓度为15.6μg/mL时,即可凝集兔红细胞,而对人的A、B、O型血细胞,凝集素浓度高达1000μg/mL,也不发生凝集反应。Gal、GalNAc、山梨糖、岩藻糖和松三糖对凝集兔红细胞的能力有抑制作用。桑寄生凝集素是一种促有丝分裂原,对猪血淋巴细胞的转化率达78％,细胞分裂比率为11.2％。     

Biotin Transport in Primary Culture of Astrocytes: Effect of Biotin Deficiency

The uptake of radioactive biotin has been studied in glial cell cultures from dissociated cerebral hemispheres of newborn rats. We describe saturable kinetics for the biotin uptake at biotin concentrations of less than 60 nM and linear at greater than 60 nM. The uptake appeared temperature sensitive, Na+ independent, nonsensitive to valinomycin, and not affected by metabolic inhibitors such as sodium fluoride or azide. Lipoic acid and biocytin were effective in inhibiting the biotin uptake. These findings are consistent with biotin uptake by the primary culture of astrocytes as a process of facilitated diffusion. Moreover, biotin uptake in astrocytes grown in biotin-restricted conditions was significantly higher compared with the control. This increase appeared mediated through a pronounced increase (10-fold) in the Vmax of the biotin uptake without any change in the apparent Km.     

Collagen targeting using multivalent protein-functionalized dendrimers

Collagen is an attractive marker for tissue remodeling in a variety of common disease processes. Here we report the preparation of protein dendrimers as multivalent collagen targeting ligands by native chemical ligation of the collagen binding protein CNA35 to cysteine-functionalized dendritic divalent (AB₂) and tetravalent (AB₄) wedges. The binding of these multivalent protein constructs was studied on collagen-immobilized chip surfaces as well as to native collagen in rat intestinal tissues. To understand the importance of target density we also created collagen-mimicking surfaces by immobilizing synthetic collagen triple helical peptides at various densities on a chip surface. Multivalent display of a weak-binding variant (CNA35-Y175K) resulted in a large increase in collagen affinity, effectively restoring the collagen imaging capacities for the AB₄ system. In addition, dissociation of these multivalent CNA35 dendrimers from collagen surfaces was found to be strongly attenuated.     

Synthesis of tetravalent LacNAc-glycoclusters as high-affinity cross-linker against Erythrina cristagalli agglutinin

Four kinds of tetravalent double-headed glycoclusters [(LacNAc)₄-DHGs] were designed with linkers of varying lengths consisting of alkanedioic carboxyamido groups (C₆, C₁₂, C₁₈ and C₂₄) between two bi-antennary LacNAc-glycosides. These glycoclusters served as high-affinity cross-linking ligands for the LacNAc-binding lectin Erythrina cristagalli agglutinin (ECA). The binding activity and cross-linking between each ligand and ECA were characterized by a hemagglutination inhibition (HI) assay, isothermal titration calorimetry (ITC), a quantitative precipitation assay and dynamic light scattering (DLS). For the precipitation assay and DLS measurement, the synthesized (LacNAc)₄-DHGs were found to be capable of binding and precipitating the ECA as multivalent ligands. ITC analysis indicated the binding of (LacNAc)₄-DHGs was driven by a favorable enthalpy change. Furthermore, the entropy penalty from binding (LacNAc)₄-DHGs clearly decreased in a spacer length-dependent manner. The binding affinities of flexible (LacNAc)₄-DHGs (C₁₈ and C₂₄) with long spacers were found to be more favorable than those of the clusters having short spacers (C₆ and C₁₂). These results were supported by molecular dynamics simulations with explicit water molecules for the tetravalent glycoclusters with ECA. We concluded that the subtle modification in the epitope-presenting scaffolds exerts the significant effect in the recognition efficiency involved in the LacNAc moieties by ECA.     

Novel Biotin Phosphoramidites with Super-long Tethering Arms

Abstract

A number of novel biotin phosphoramidites, possessing exceptionally long and uncharged tethering arms, were synthesized from methoxyoxalamido (MOX) and succinimido (SUC) precursors. Included among these monomers is a uridine derivative with the biotin moiety attached through the 2′-position. Some of these phosphoramidites were used to make 5′-biotinylated primers, which were applied in direct sequencing of genomic DNA and capture of Sanger fragment pools.     

A Sensitive Radiometric Assay for Tryptophan Hydroxylase Applicable to Crude Extracts

Abstract: We describe here a simple and convenient method for assay of tryptophan 5-monooxygenase (hydroxylase), applicable to enzyme in all states of purification. It is based on the enzyme-catalysed formation of 5-hydroxy-[4-³H]tryptophanfrom [5-³H]tryptophan, and the subsequent acid-dependent quantitative release of ³H as ³H₂O; unreacted substrate is removed with activated charcoal. The assay is linear with respect to both protein concentration and time, and gives results similar to those in a standard fluorimetric assay.     

Synthesis of lactoside glycodendrons using photoaddition and reductive amination methodologies

Carbohydrate-based divalent and tetravalent lactoside glycodendrons were constructed in a convergent manner. The dendrons were synthesized beginning with the photoaddition of hepta-O-acetyl-1-thio-beta-lactose, in an anti-Markovnikov manner, to a bis-allyl AB2 trisaccharide to form a divalent dendron. Following two nearly quantitative deprotection steps, the divalent lactoside was coupled to another AB2 trisaccharide by reductive amination to afford a tetravalent dendron. These paucivalent compounds were characterized by NMR spectroscopy and mass spectrometry.     

乳腺癌ConA、LCA、PSA免疫组化研究

运用ＡＢＣ法研究５０例乳腺癌三种凝集素受体（Ｌｅｃｔｉｎ－Ｒ）的表达,并以乳腺纤维瘤和乳腺小叶增生症各１０例作为对照。结果表明：该三种凝集素受体在乳腺良、恶性病变中的表达率无显著性差异。ＣＯｎＡ－Ｒ在乳腺癌以胞浆型定位为主,在纤维腺瘤则以胞膜型多见（Ｐ＜０．０５）。ＬＣＡ－Ｒ和ＰＳＡ－Ｒ在乳腺三种病变中均以胞浆型为主。ＣｏｎＡ－Ｒ与乳腺癌分化程度具良好的相关性;ＬＣＡ－Ｒ与乳腺癌瘤体大小有关。该三种凝集素受体与ＥＲ、ＰＲ及ＣＥＡ的表达未显示密切的关联。     

化学法合成生物素标记寡核苷酸探针

 生物素可通过氨烷基磷酰胺基团连接到寡核苷酸5'末端,反应在水溶液中进行,核苷酸的侧链基团不用保护。我们以这种化学法合成了生物素标记的寡核苷酸探针,其显色灵敏度达50pg,杂交灵敏度达0.4fmol,并与酶标生物素寡核苷酸探针进行了比较,也对两种不同显色体系作了比较。     

Probing multivalent carbohydrate-lectin interactions by an enzyme-linked lectin assay employing covalently immobilized carbohydrates

We report here the synthesis of a series of mono- to trivalent N-acetylglucosamine (GlcNAc) derivatives as ligands for the plant lectin wheat germ agglutinin (WGA). Their WGA binding potencies were determined by an established enzyme-linked lectin assay (ELLA) employing microtiter plates with non-covalently immobilized porcine stomach mucin (PSM) as reference ligand and an ELLA with a new GlcNAc derivative covalently immobilized via a thiourea linkage. Comparison of both assays revealed that the type of presentation of GlcNAc residues on the microtiter plates either as part of a glycoprotein or as a covalently immobilized monosaccharide derivative strongly influences the outcome of the assay. Although the apparent dissociation constants K(D)(ELLA) for the interaction of peroxidase-labeled WGA with the microtiter plates are comparable for both surfaces, IC(50) values obtained with the PSM-free ELLA were substantially lower. Even more strikingly, this ELLA displayed a better differentiation between ligands of different valency leading to significantly higher relative inhibitory potencies of multivalent ligands compared to monovalent. Additionally, problems associated with the use of PSM, such as maximum inhibition at considerably less than 100% and poor reproducibility of IC(50) values could be overcome with this type of ELLA.     

Di- and heptavalent nicotinic analogues to interfere with α7 nicotinic acetylcholine receptors

In the field of nicotinic acetylcholine receptors (nAChRs), recognized as important therapeutic targets, much effort has been dedicated to the development of nicotinic analogues to agonize or antagonize distinct homo- and heteropentamers nAChR subtypes, selectively. In this work we developed di- and heptavalent nicotinic derivatives based on ethylene glycol (EG) and cyclodextrin cores, respectively. The compounds showed a concentration dependent inhibition of acetylcholine-induced currents on α7 nAChR expressed by Xenopus oocytes. Interesting features were observed with the divalent nicotinic derivatives, acting as antagonists with varied inhibitory concentrations (IC₅₀) in function of the spacer arm length. The best divalent compounds showed a 16-fold lowered IC₅₀ compared to the monovalent reference (12 vs 195?µM). Docking investigations provide guidelines to rationalize these experimental findings.     

Isolation and characterization of a novel lectin from the mushroom Armillaria luteo-virens

From the dried fruiting bodies of the mushroom Armillaria luteo-virens, a dimeric lectin with a molecular mass of 29.4 kDa has been isolated. The purification procedure involved (NH(4))(2)SO(4) precipitation, ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The hemagglutinating activity of the lectin could not be inhibited by simple sugars but was inhibited by the polysaccharide inulin. The activity was stable up to 70 degrees C but was acid- and alkali-labile. Salts including FeCl(3), AlCl(3), and ZnCl(2) inhibited the activity whereas MgCl(2), MnCl(2), and CaCl(2) did not. The lectin stimulated mitogenic response of mouse splenocytes with the maximal response achieved by 1microM lectin. Proliferation of tumor cells including MBL2 cells, HeLa cells, and L1210 cells was inhibited by the lectin with an IC(50) of 2.5, 5, and 10 microM, respectively. However, proliferation of HepG2 cells was not affected. The novel aspects of the isolated lectin include a novel N-terminal sequence, fair thermostability, acid stability, and alkali stability, together with potent mitogenic activity toward spleen cells and antiproliferative activity toward tumor cells.     

Facile synthesis of 1-thio-beta-lactoside clusters scaffolded onto p-methoxyphenyl,beta-D-galactopyranoside,beta-D-glucopyranoside,and lactoside

The free-radical addition of 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-acetyl-1-thio-beta-D-glucopyranose to the allyl ether functions of p-methoxyphenyl per-O-allyl-D-galactopyranoside, D-glucopyranoside, and lactoside provides a concise and effective route for synthesis of glycoside clusters, of use for exploring anti-metastatic activity.     

Clusters of ligands on dendrimer surfaces

The development of methodology that is designed to allow a significant increase in the patterning and in the functionalization of the dendrimer is the ultimate goal of the research described here. Glycoside clusters based on TRIS were formed using click chemistry and were attached to PAMAM dendrimers. A series of dendrimers bearing tris-mannoside and an ethoxyethanol group was synthesized, and the binding interactions of these dendrimers with Concanavalin A were evaluated using inhibition ELISAs. The results of the inhibition ELISAs suggest that tris-mannoside clusters can replace individual sugars on the dendrimer without loss of function. Since tris-mannoside clustering allows for a redistribution of the dendrimers’ surface functionalities, from this chemistry one can envision patterned dendrimers that incorporate multiple groups to increase the function and utility of the dendrimer.     

雪山豆(Phaseolus sp.)凝集素的亲和层析纯化与其部分性质

 用猪甲状腺球蛋白-对氨基苯砜乙基-交联琼脂作亲和吸附剂,可对雪山豆(Phaseolus sp.)凝集素进行亲和层析纯化。纯化后的凝集素在聚丙烯酰胺凝胶电泳中显示单一蛋白质带。Sephadex G-100凝胶层析法测得分子量为65,000道尔顿,SDS-聚丙烯酰胺凝胶电泳表明该凝集素分子仅有一个分子量为65,000道尔顿的亚基,酚-硫酸法测得总糖含量为4.6％,氨基酸组成分析表明雪山豆凝集素富含门冬氨酸,而甲硫氨酸含量甚少。该凝集素是强促有丝分裂原,对人外周血中淋巴细胞的转化率大于90％,细胞分裂比率达12％。雪山豆凝集素不仅能凝集多种动物红细胞,还能凝集人精细胞。经体外实验表明,雪山豆凝集素对人肝癌细胞有抑制作用。     

OXIDATION OF BIOTIN DURING OLIGONUCLEOTIDE SYNTHESIS

A new “polystyrene biotin support” has been synthesized for the solid support synthesis of the 3′-biotinylated oligonucleotides. Several oligos were synthesized and were analyzed by the HPLC and Mass Spec. Oligo analysis revealed that the biotin gets oxidized to “biotin sulfoxide” during the synthesis.     

Interaction Between the Oxygen and Tryptophan Dependence of Synaptosomal Tryptophan Hydroxylase

Abstract: The substrate dependence of tryptophan hydroxylase activity in rat striatal synaptosomes was examined. The K _m for tryptophan in air is 8-13 μM, comparable to the concentration present in cerebrospinal fluid. The reaction is inhibited by amino acids with large nonpolar side chains. For leucine the inhibition appears competitive; it results from a decrease in the steady state levels of intrasynaptosomal tryptophan. The oxygen K_m at 10 μM-tryptophan is 3-4 mm Hg, which is increased when the reaction is assayed with low levels of tryptophan in the medium, and in the presence of amino acids such as leucine. Similarly, the tryptophan K _m is increased at low oxygen tensions; at 9 mm Hg of oxygen it is 23 μM. These interactions between tryptophan and oxygen dependence of the reaction are discussed in terms of likely physiological significance and implications for the pharmacological use of tryptophan.