Pharmacologic identification of muscarinic receptors in the pylorus of the cat by binding of [3H]-quinuclidinyl benzilate

Muscarinic receptors in the smooth muscle of the cat pylorus (pyloric sphincter) were identified by binding of the ligand (±) [³H]-quinuclidinyl benzilate ([³H]-QNB). Receptor related binding of [³H]-QNB reached steady-state in thirty minutes at 37°C, was saturable, showed pharmacologic specificity and was stereoselective. An apparent equilibrium dissociation constant, K_D, of 1.9 ± 0.3 nM and maximum receptor concentration of 122 ± 13 femtomoles per mg of protein (means ± S.E.M.) were determined from Scatchard plots of [³H]-QNB binding. Hill coefficients of 0.99 and 1.01 indicated the absence of cooperative interactions. The muscarinic antagonists atropine and propantheline inhibited binding with IC₅₀ values in the nanomolar range, whereas bethanechol was over four orders of magnitude less potent. Noncholinergic agents had little or no effect on [³H]-QNB binding. The $\text{levo}$ isomer of QNB was about seventy times more effective at inhibiting binding than its $\text{dextro}$ isomer while $\text{dextro}$ benzetimide was greater than two thousand fold more active than $\text{levo}$ benzetimide. The isomers of another anticholinergic compound, tropicamide, also competed for [³H]-QNB binding sites in a stereoselective manner, the $\text{levo}$ isomer being eighty-five times more potent than the $\text{dextro}$ isomer.     

19-hydroxycoronaridin und 19-hydroxyibogamin,zwei antibiotisch wirksame alkaloide vom ibogamin-typ

During the separation of an extract from Tabernaemontana glandulosa, small amounts of a new alkaloid were obtained for which the structure 19-hydroxycoronaridine was deduced. This new alkaloid and 19-hydroxyibogamine, which can be obtained from it, show marked antibiotic activity in the agar plate diffusion test.     

Protein phosphokinase activity of rat liver nuclear membrane

The presence of protein phosphokinase activity in a purified nuclear-membrane preparation from adult rat liver was demonstrated by measuring the incorporation of ³²P from γ-³²P-ATP into endogenous nuclear-membrane proteins as well as into the exogenous protein substrates, dephosphophosvitin (DPV) and lysine-rich histone (LRH). The activity of this enzyme toward DPV was 60 times greater than that toward LRH. cAMP and cGMP did not appear to affect the phosphorylation of endogenous-membrane proteins.     

A facile method for detection of the genuine sapogenin from a saponin extract

We present a simple, efficient system for preparative polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate. The instrument features a novel sample collection assembly which is fitted directly onto the bottom of the gel cylinder. Buffer pumped uniformly upward through a porous plastic disk from the lower tray sweeps the emerging sample into a collection system with high efficiency. Measures which prevent the destruction of tryptophan residues and the blockage of α-amino groups observed in electrophoresed polypeptides are described.     

Change of concanavalin A induced agglutinability during preimplantation mouse development

Mouse embryos during early cleavage (zygote to eight-cell stage) were agglutinable with a low concentration (10 μg/ml) of concanavalin A (ConA). This agglutinability was reduced during the first mitotic division. Morulae were agglutinable with a slightly higher concentration (100 μg/ml), whereas blastocysts were not agglutinable even with ConA at a concentration of 5000 μg/ml; however, isolated inner cell masses agglutinated readily at 10 μg/ml of ConA. Embryos grown in vitro behaved as did those isolated directly from the genital tract. Treatment with proteolytic enzymes did not induce agglutinability of mouse blastocyst. The change in agglutinability of trophoblastic cells reflects dramatic changes in the cell surface.     

A sedimentation procedure for the detection of binding to concanavalin A and heparin to lipoproteins.

Specific enzymatic bands in disc gel electrophoresis are generally determined by either of two methods: (i) Gel is sliced and the enzymatic activity is assayed on each slice or (ii) gel is stained histochemically, if the product of the enzymatic reaction and the dye can form an insoluble precipitate, and the activity band is located on the gel by a color band. The former is laborious and often inaccurate in the calculation of electrophoretic mobility. The latter, often nonspecific, is not applicable when the enzymatic product cannot form an insoluble precipitate with the dye. Staining with tetrazolium salt has been widely employed for amine oxidase (1–6). However, this method has limitations: (i) Tetrazolium salt is nonspecific for amine oxidase and may show artifacts (6,7), and (ii) the use of tetrazolium salt is limited only to substrates containing indolamine such as tryptamine or serotonin (8). Other substrates, like benzylamine, the most active substrate for plasma amine oxidase, do not form a color band with tetrazolium salt.This communication reports a simple spectrophotometric method for the identification of the enzymatic activity band for amine oxidase on disc gel electrophoresis. Neither slice and assay nor staining is needed. This method may possibly also be used generally for other enzyme systems which have a specific absorption at ultraviolet or visible range.     

Engineering defensin α‐helix to produce high‐affinity SARS‐CoV‐2 spike protein binding ligands

The binding of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein to the angiotensin‐converting enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high‐affinity and cost‐effective ACE2 mimetic ligands that disrupt this protein–protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small (2–5 kDa) and highly stable proteins containing solvent‐exposed alpha‐helix, conformationally constrained by two disulfide bonds. Therefore, we engineered the amino acid residues on the constrained alpha‐helix of defensins to mimic the critical residues on the ACE2 helix 1 that interact with the SARS‐CoV‐2 spike protein. The engineered proteins (h‐deface2, p‐deface2, and p‐deface2‐MUT) were soluble and purified to homogeneity with a high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability (Tm 70.7°C), high‐affinity binding to the spike protein with apparent K _d values of 54.4 ± 11.3, 33.5 ± 8.2, and 14.4 ± 3.5 nM for h‐deface2, p‐deface2, and p‐deface2‐MUT, respectively, and were used in a diagnostic assay that detected SARS‐CoV‐2 neutralizing antibodies. This work addresses the challenge of developing helical ACE2 mimetics by demonstrating that defensins provide promising scaffolds to engineer alpha‐helices in a constrained form for designing of high‐affinity ligands.     

基于网络药理学与分子对接技术的清瘟护肺颗粒防治新型冠状病毒肺炎(COVID-19)的潜在药效物质研究

本文通过网络药理学和分子对接技术探讨清瘟护肺颗粒防治新型冠状病毒肺炎(COVID-19)的潜在药效物质.首先,通过TCMSP数据库,BATMAN-TCM数据库及TCMIP数据库检索清瘟护肺颗粒中十六味药的化学成分及作用靶点,利用GeneCards和OMIM数据库检索COVID-19的相关疾病靶点.然后,通过venny2...