Bovine Nucleus Caudatus Acetylcholinesterase: Active Site Determination and Investigation of a Dimeric Form Obtained by Selective Proteolysis

Abstract: The number of catalytic subunits of purified bovine nucleus caudatus acetylcholinesterase (E.C. 3.1.1.7) has been determined by active site labelling with [³H]diisopropyl fluorophosphate ([³H]DFP). The 10.5 S, 16 S, and 20 S forms were estimated to contain two, four, and six active sites, respectively, per molecule. A 4.8 S form, which showed a weak amphiphile-dependent activity behavior, was obtained by selective proteolytic digestion with pronase. The inability of the purified 4.8 S form to aggregate after detergent removal, and the molecular mass in the range of 130-165 kD under nondenaturating conditions, indicate that this form is a dimeric form, lacking those hydrophobic regions responsible for aggregation.     

人类免疫缺陷病毒1／2型抗体检测酶联免疫试剂盒的研制

采用二聚体合成肽（ＨＩＶ－１ｇｐ４１、ｇｐ１２０、ｐ２４和ＨＩＶ－２ｇｐ３６）包被酶标板条制备成固相抗原,与鼠抗人ＩｇＧ单克隆抗体酶标记物、底物ＴＭＢ及阴阳性参考血清配套制备成ＨＩＶ抗体ＥＩＡ试剂盒,专供检测人血清或血浆ＨＩＶ１／２抗体之用。以荷兰、韩国及万泰试剂作为对照,用该试剂盒对检定所的Ｐａｎｅｌ标准及献血员１５５５０例（其中ＨＣＶ抗体阳性１２８例,ＨＢｓＡｇ阳性４６例）进行检测,四种试剂对检定所Ｐａｎｅｌ标准的１３份阳性血清均呈阳性反应,２８份阴性血清均为阴性;献血员１５５５０例,四种试剂对其中１份血清均呈阳性反应,经Ｗｅｓｔｅｒｎｂｌｏｔ试验证实为阴性,四种试剂的阴性检出率均为９９．９９％。连续制备三批试剂经中国药品生物制品检定所检定,所检项目全部合格;同时委托检定所进行临床考核,４７份阳性血清全部呈阳性反应,１５０份阴性血清全部为阴性。说明该试剂盒具有很好的敏感性和特异性。     

Studies on the evolutionary relationships between hemoglobins inChironomus pallidivittatus andC. Tentans

Summary The monomeric hemoglobins ofChironomus tentans andC. pallidivittatus have been isolated and separated into their respective components by gel chromatography on Sephadex G-75 and ion-exchange chromatography on DEAE-Sephacel. The amino acid compositions of the purified components are given. The sequence of the 30 N-terminal amino acid residues of one of the monomeric components (Hb I fromC. pallidivittatus) was determined and found to be identical in almost all of its parts with the monomeric hemoglobins ofC. thummi (CTT III and CTT IV).Antibodies against the monomeric hemoglobins Hb I and Hb IIc and the dimeric fraction were highly specific and no cross reaction between dimeric and monomeric hemoglobins could be demonstrated. The antibodies against the monomers crossreact with the monomeric hemoglobins CTT III and CTT IV ofC. thummi. Taken together with genetic data, the immunological results indicate that divergence of monomeric from dimeric forms was an early event in the evolution of the various hemoglobins inChironomus.     

Dimeric argininamide-type neuropeptide Y receptor antagonists: Chiral discrimination between Y1 and Y4 receptors

The structurally related peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) are endogenous agonists of the NPY receptor (YR) family, which in humans comprises four functionally expressed subtypes, designated Y₁R, Y₂R, Y₄R and Y₅R. Nonpeptide antagonists with high affinity and selectivity have been described for the Y₁R, Y₂R and Y₅R, but such compounds are still lacking for the Y₄R. In this work, the structures of the high affinity selective (R)-argininamide-type Y₁R antagonists BIBP3226 and BIBO3304 were linked via the guanidine or urea moieties to give homo-dimeric argininamides with linker lengths ranging from 31 to 41 atoms. Interestingly, the twin compounds proved to be by far less selective for the Y₁R than the R-configured monovalent parent compounds. The decrease in selectivity ratio was most pronounced for Y₁R versus Y₄R subtype, resulting in comparable affinities of bivalent ligands for Y₁R and Y₄R (e.g. UR-MK177 ((R,R)-49): K_i = 230 nM (Y₁R) and 290 nM (Y₄R)). With a K_i value of 130 nM and a K_b value of 20 nM, UR-MK188 ((R,R)-51) was superior to all Y₄R antagonists known to date. The S,S-configured optical antipodes of UR-MK177 and UR-MK188 (UR-MEK381 ((S,S)-49) and UR-MEK388 ((S,S)-51)) were synthesized to investigate the stereochemical discrimination by the different receptor subtypes. Whereas preference for R,R-configured argininamides was characteristic of the Y₁R, stereochemical discrimination by the Y₄R was not observed. This may pave the way to selective Y₄R antagonists.     

Design,synthesis, and biological characterization of novel PEG-linked dimeric modulators for CXCR4

CXCR4 dimerization has been widely demonstrated both biologically and structurally. This paper mainly focused on the development of structure-based dimeric ligands that target CXCL12–CXCR4 interaction and signaling. This study presents the design and synthesis of a series of [PEG]_n linked dimeric ligands of CXCR4 based on the knowledge of the homodimeric crystal structure of CXCR4 and our well established platform of chemistry and bioassays for CXCR4. These new ligands include [PEG]_n linked homodimeric or heterodimeric peptides consisting of either two DV3-derived moieties (where DV3 is an all-d-amino acid analog of N-terminal modules of 1–10 (V3) residues of vMIP-II) or hybrids of DV3 moieties and CXCL12_1–8. Among a total of 24 peptide ligands, four antagonists and three agonists showed good CXCR4 binding affinity, with IC₅₀ values of <50 nM and <800 nM, respectively. Chemotaxis and calcium mobilization assays with SUP-T1 cells further identified two promising lead modulators of CXCR4: ligand 4, a [PEG₃]₂ linked homodimeric DV3, was an effective CXCR4 antagonist (IC₅₀ = 22 nM); and ligand 21, a [PEG₃]₂ linked heterodimeric DV3–CXCL12_1–8, was an effective CXCR4 agonist (IC₅₀ = 407 nM). These dimeric CXCR4 modulators represent new molecular probes and therapeutics that effectively modulate CXCL12–CXCR4 interaction and function.     

Synthesis and reactivity of nonstabilized diazo sugars

1,6-Anhydro-4-deoxy-4-diazo-2,3-O-isopropylidene-beta-D-lyxo-hexopyranose (4) is a stable crystalline compound readily accessible by an improved synthetic procedure. It has been used as a model for evaluating the reactivity of the diazo group, when not stabilized by an adjacent carbonyl function, in a rigid chiral matrix. A range of carbene-type, electrophile-promoted, and 1,3-dipolar reactions were evaluated, leading to 4,4'-alkene dimers, 4-deoxy-3-enose and related derivatives, 4,4-dihalo compounds, 4-spirocyclopropane derivatives, 4-spiropyrazole structures, and by skeletal rearrangement, branched-chain anhydropentose structures having a bicyclo[2.2.2] skeleton.     

Terpenoids from Achillea distans Waldst. & Kit. ex Willed

The flower heads of Achillea distans afforded nine new sesquiterpene lactones (one glaucolide, six guaianolides and two dimeric lactones) in addition to 18 known terpenoids. The structures and stereochemistry of the new compounds were elucidated by spectroscopic methods. The stereochemistry of 11,13-dihydroezomontanin was revised.     

One-dimensional coordination polymer and its hydrogen bonded 2D network generated from dimeric Ag(I) building units

A new dimeric silver(I) complex [Ag(PhPPy₂)(CH₃CN)]₂(ClO₄)₂ (1) (PhPPy₂ = bis(2-pyridyl)phenylphosphine) was synthesized by a direct reaction of [Ag(CH₃CN)₄]ClO₄ with ligand PhPPy₂. X-ray crystallographical studies revealed that in 1, two silver atoms are bridged by two PhPPy₂ ligands and bonded to each other. Each Ag(I) adopts a distorted trigonally bi-pyramidal geometry, and axially coordinated acetonitrile molecules are collinear with two silver atoms. By using 1 as a building block precursor, a 1D coordination polymer, [Ag₂(PhPPy₂)₂(1,3,5-C₆H₃(CO₂)₂(CO₂H))]_∞ (2) was prepared by replacing axially coordinated acetonitrile molecules in 1 with two carboxylate groups of a bridging ligand, 1,3,5-benzenetricarboxylate. In solid state, linear polymeric chains are oriented parallel to each other and interestingly interact by hydrogen bonding through their carboxylic/carboxylate groups to construct a novel wave-shaped 2D network. Both 1 and 2 exhibit similar photoluminescent properties in solid state at room temperature.     

Constituents from Chimonanthus praecox (wintersweet)

One new (1) and seven (2–8) known sesquiterpenoids, four dimeric tryptamine-related alkaloids (9–12) and six glycosides (13–18) were isolated from the fruits and leaves of Chimonanthus praecox (wintersweet). Based on its spectroscopic data, the new structure of compound 1, an oppositane-type sesquiterpenoid, was elucidated to be the C-4 epimer of bullatantriol (2). All isolated compounds were evaluated for their cytotoxicities against a small panel of human cancer cell lines, and only the chimonanthine-type alkaloids (10–12) were found to have cytotoxic effects against gastric carcinoma NUGC3 and hepatocarcinoma SNU739 cancer cells, with IC₅₀ values ranging from 10.3 to 19.7 μM.     

Odorant binding initially occurring at the central pocket in bovine odorant-binding protein

Why bovine odorant-binding protein (OBPb), among OBP family, assumes a dimeric structure has been unclear. Here we clarified, by measuring the fluorescence of intrinsic tryptophan and tyrosine residues of intact OBPb and OBPb whose C-terminal 10 amino acids were deleted, that odorant enters the central pocket formed by the dimerization when OBPb first encounters odorant, and odorant with high affinity with OBPb subsequently enters the internal cavity (suggested binding site), releasing the pre-bound odorant. The internal cavity-bound odorant can be released by the binding of other odorants at another internal cavity or at the central pocket, depending on the binding odorants. Due to this mechanism enabled by the dimerization, OBPb is more reactive than other monomeric OBPs.