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41.
《Bioorganic & medicinal chemistry》2020,28(9):115437
NDM-1 can hydrolyze nearly all available β-lactam antibiotics, including carbapenems. NDM-1 producing bacterial strains are worldwide threats. It is still very challenging to find a potent NDM-1 inhibitor for clinical use. In our study, we used a metal-binding pharmacophore (MBP) enriched virtual fragment library to screen NDM-1 hits. SPR screening helped to verify the MBP virtual hits and identified a new NDM-1 binder and weak inhibitor A1. A solution NMR study of 15N-labeled NDM-1 showed that A1 disturbed all three residues coordinating the second zinc ion (Zn2) in the active pocket of NDM-1. The perturbation only happened in the presence of zinc ion, indicating that A1 bound to Zn2. Based on the scaffold of A1, we designed and synthesized a series of NDM-1 inhibitors. Several compounds showed synergistic antibacterial activity with meropenem against NDM-1 producing K. pneumoniae. 相似文献
42.
S. Han M. H. U. Khan Y. Yang K. Zhu H. Li M. Zhu O. Amoo S. U. Khan C. Fan Y. Zhou 《Plant biology (Stuttgart, Germany)》2020,22(4):709-721
- The CLE (CLAVATA3/ESR) gene family, encoding a group of small secretory peptides, plays important roles in cell‐to‐cell communication, thereby controlling a broad spectrum of development processes. The CLE family has been systematically characterized in some plants, but not in Brassica napus.
- In the present study, 116 BnCLE genes were identified in the B. napus genome, including seven unannotated, six incorrectly predicted and five multi‐CLE domain‐encoding genes. These BnCLE members were separated into seven distinct groups based on phylogenetic analysis, which might facilitate the functional characterization of the peptides.
- Further characterization of CLE pre‐propeptides revealed 31 unique CLE peptides from 45 BnCLE genes, which may give rise to distinct roles of BnCLE and expansion of the gene family. The biological activity of these unique CLE dodecamer peptides was tested further through in vitro peptide assays. Variations in several important residues were identified as key contributors to the functional differentiation of BnCLE and expansion of the gene family in B. napus. Expression profile analysis helped to characterize possible functional redundancy and sub‐functionalization among the BnCLE members.
- This study presents a comprehensive overview of the CLE gene family in B. napus and provides a foundation for future evolutionary and functional studies.
43.
The membrane-bound atrial natriuretic peptide receptor (GCA) catalyzes the formation of cGMP from GTP in response to natriuretic peptide hormones. Previous structural studies have focused on the extra-cellular hormone binding domain of this receptor whereas its intra-cellular domain has not yet been amenable to such studies. We report here the baculovirus expression and purification of the GCA intra-cellular domain construct GCAID comprising the complete intra-cellular region which includes the kinase-homology domain, coiled-coil region, and catalytic cyclase domain. The intra-cellular domain was enzymatically characterized in terms of guanylyl cyclase activity and the effects of ATP, manganese, and Triton X-100. Our results indicate that the activity of the intra-cellular domain of the ANP receptor is about 2 fold less active compared to a truncated cyclase domain construct lacking the kinase-like domain that was also expressed and purified. In addition, unlike the full length receptor, the intra-cellular domain could not be activated by Triton X-100/Mn2+ or its activity stimulated by ATP. These data therefore indicate that the major part of the transition from the basal state to the fully, ANP/ATP-dependent, activated state as well its stimulation/enhancement by Triton X-100/Mn2+ requires the full length receptor. These receptor insights could aid in the development of novel therapeutics as the GCA receptor is a key drug target for cardiovascular diseases. 相似文献
44.
Yasuo Kato Yasuhisa Asano Akiko Nakazawa Kiyosi Kondo 《Biocatalysis and Biotransformation》1990,3(3):207-215
Synthesis of D-alanine oligopeptides from D-alanine methylester hydrochloride has been demonstrated by use of immobilized D-aminopeptidase from Ochrobactrum anthropi (Achromobacter sp.) in non-aqueous media. D-Alanine dimer and trimer were obtained in 56% and 6% yield, respectively, when 250 mM of the substrate was incubated for 3 hours with urethane-prepolymer immobilized D-aminopeptidase (1.5 U/ml) and 3 equivalents of triethylamine in water-saturated toluene. The kcat of this reaction was calculated to be 19,500 (min-1), which is several ten thousand times greater than that of the known enzymatic syntheses of amino acid oligomers. 相似文献
45.
46.
Astrocytes from Forebrain, Cerebellum, and Spinal Cord Differ in Their Responses to Vasoactive Intestinal Peptide 总被引:3,自引:2,他引:1
Astrocytes from cortex, cerebellum, and spinal cord responded to isoproterenol and vasoactive intestinal peptide (VIP) with increases in intracellular cyclic AMP levels. The response to VIP was as great as that to isoproterenol in cortical astrocytes (180-fold and 185-fold, respectively), and the effect of VIP in combination with isoproterenol was partially additive. Spinal cord astrocytes also responded to VIP and isoproterenol with equal potency (seven- to ninefold and eight- to 13-fold, respectively), but the level of response was much smaller than in cortex. Spinal cord astrocytes were synergistic in their response to VIP and isoproterenol. The response to VIP was lowest in cerebellar astrocytes (only threefold), and no additivity was observed when VIP was added together with isoproterenol. A small response to alpha-melanocyte stimulating hormone (alpha-MSH) was also observed in cortex and cerebellum, but not in spinal cord. Somatostatin inhibited the response to isoproterenol in cortex and cerebellum, but had no effect in spinal cord. The results from the above study show that astrocytes obtained from these three regions of the rat CNS express quite different responses to VIP and alpha-MSH and further point to possible astrocyte heterogeneity. 相似文献
47.
To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen. 相似文献
48.
C. Steven McDaniel Taghi Manshouri M. Zouhair Atassi 《Journal of Protein Chemistry》1987,6(5):455-461
A peptide corresponding to residues 26–41 of α-bungarotoxin, and closed by a disulfide bond between two cysteine residues
at the amino and C terminal ends of the peptide, was synthesized and the monomeric form was purified. The peptide, which represents
the exposed part of the long central loop of the toxin molecule, was examined for binding to acetylcholine receptor. The peptide
was shown by radiometric titrations to bind radiolabeled receptor, and radiolabeled peptide was bound by receptor. The specificity
of the binding was confirmed by inhibition with the parent toxin. A synthetic analog of the peptide in which Trp-28 was replaced
by glycine had very little (10%) of the original activity. Succinylation of the amino groups of the peptide resulted in virtually
complete (98%) loss of the binding activity. These results indicate that a shortened loop peptide corresponding to the region
26–41 of α-bungarotoxin exhibits binding activities mimicking those of the parent molecule. In this region, Trp-28, and one
or both of Lys-26 and Lys-38, are essential contact residues in the binding to receptor. 相似文献
49.
Peptidomimetic analogs of the peptide RRASVA, known as the “minimal substrate” of the catalytic subunit of the cAMP-dependent protein kinase (PKA), were synthesized by consecutive replacement of natural amino acids by their aza-β3 analogs. The peptidomimetics were tested as PKA substrates and the kinetic parameters of the phosphorylation reaction were determined. It was found that the interaction of these peptidomimetics with the enzyme active center was sensitive to the location of the backbone modification, while the maximal rate of the reaction was practically not affected by the structure of substrates. The pattern of molecular recognition of peptidomimetics was in agreement with the results of structure modeling and also with the results of computational docking study of peptide and peptidomimetic substrates with the active center of PKA. It was concluded that the specificity determining factors which govern substrate recognition by the enzyme should be grouped along the phosphorylatable substrate, and such clustering might open new perspectives for pharmacophore design of peptides and peptide-like ligands. 相似文献
50.