建立快速定量检测猪瘟兔化弱毒苗的荧光定量PCR技术

在猪瘟病毒兔化弱毒疫苗株的5’非编码区设计一对引物和一条荧光探针,利用荧光定量PCR原理,结合LightCycler检测系统,首次建立了定量检测猪瘟兔化弱毒苗方法。结果表明,该方法的灵敏度为10^2拷贝数,线性范围为10^7—10^2,达6个数量级;标准样品的变异系数为2．3％—5．1％(n＝10),疫苗样品组内实验变异系数为0．85％—2．8％(n＝5)、组间实验为2．5％—7．3％(n＝5),对同一样品分5次RNA提取和逆转录,其变异系数为5．0％;对9份疫苗样品进行了检测,与免体定型热反应方法相比较,有很好的相关性;整个检测过程仅需4h。该法可望取代传统的兔体定型热反应用于疫苗生产过程中的效价测定及指导疫苗的配制,也为猪瘟病毒分子生物学研究提供了一种新的、简捷有效的工具。     

Metallo-β-lactamase: Inhibitors and reporter substrates

Metallo-β-lactamases represent an emerging clinical threat due to their ability to render ineffective an entire class of antibiotics. Accordingly, this family of enzymes has been suggested as an attractive target for drug design. Progress toward developing effective inhibitors as well as the development of reporter substrates is reviewed. Inhibitors are classified into six classes and known binding interactions with metallo-β-lactamases are summarized. The development of chromogenic and fluorogenic reporter substrates is also reviewed with respect to current and prospective applications to future inhibitor and diagnostic discovery, mechanistic studies, and biological imaging. Despite progress in molecular probe development, the sequence and structural diversity within the metallo-β-lactamase family continue to present substantial hurdles for rational ligand design.     

An integrated approach to the ligand binding specificity of Neisseria meningitidis M1 alanine aminopeptidase by fluorogenic substrate profiling,inhibitory studies and molecular modeling

Neisseria meningitides is a gram-negative diplococcus bacterium and is the main causative agent of meningitis and other meningococcal diseases. Alanine aminopeptidase from N. meningitides (NmAPN) belongs to the family of metallo-exopeptidase enzymes, which catalyze the removal of amino acids from the N-terminus of peptides and proteins, and are found among all the kingdoms of life. NmAPN is suggested to be mostly responsible for proteolysis and nutrition delivery, similar to the orthologs from other bacteria.     

Two fluorogenic substrates for purine nucleoside phosphorylase,selective for mammalian and bacterial forms of the enzyme

Two nontypical nucleosides, 7-β-d-ribosyl-2,6-diamino-8-azapurine and 8-β-d-ribosyl-2,6-diamino-8-azapurine, have been found to exhibit moderately good, and selective, substrate properties toward calf and bacterial (Escherichia coli) forms of purine nucleoside phosphorylase (PNP). The former compound is effectively phosphorolysed by calf PNP and the latter by PNP from E. coli. Both compounds are fluorescent with λ_max ∼ 425 to 430 nm, but the reaction product, 2,6-diamino-8-azapurine, emits in a different spectral region (λ_max ∼ 363 nm) with nearly 40% yield, providing a strong fluorogenic effect at 350 to 360 nm.     

Gelatin degradation assay reveals MMP-9 inhibitors and function of O-glycosylated domain

AIM: To establish a novel, sensitive and high-throughput gelatinolytic assay to define new inhibitors and compare domain deletion mutants of gelatinase B/matrix metalloproteinase (MMP)-9. METHODS: Fluorogenic Dye-quenched (DQ)TM-gelatin was used as a substrate and biochemical parameters (substrate and enzyme concentrations, DMSO solvent concentrations) were optimized to establish a highthroughput assay system. Various small-sized libraries (ChemDiv, InterBioScreen and ChemBridge) of hetero-cyclic, drug-like substances were tested and compared with prototypic inhibitors. RESULTS: First, we designed a test system with gelatin as a natural substrate. Second, the assay was validated by selecting a novel pyrimidine-2,4,6-trione (barbitu- rate) inhibitor. Third, and in line with present structural data on collagenolysis, it was found that deletion of the O-glycosylated region significantly decreased gelatinolytic activity (kcat/kM ± 40% less than full-length MMP-9). CONCLUSION: The DQTM-gelatin assay is useful in high-throughput drug screening and exosite targeting. We demonstrate that flexibility between the catalytic and hemopexin domain is functionally critical for gelatinolysis.     

Design and synthesis of new substrates of HtrA2 protease

HtrA2 belongs to the HtrA (high temperature requirement A) family of ATP-independent serine proteases. The primary function of HtrA2 includes maintaining the mitochondria homeostasis, cell death (by apoptosis, necrosis, or anoikis), and contribution to the cell signaling. Several recent reports have shown involvement of HtrA2 in development of cancer and neurodegenerative disorders. Here, we describe the profiling of HtrA2 protease substrate specificity via the combinatorial chemistry approach that led to the selection of novel intramolecularly quenched substrates. For all synthesized compounds, the highest HtrA2-mediated hydrolysis efficiency and selectivity among tested HtrA family members was observed for ABZ-Ile-Met-Thr-Abu-Tyr-Met-Phe-Tyr(3-NO₂)-NH₂, which displayed a specificity constant k_cat/K_M value of 14,535 M⁻¹ s⁻¹.     

Development of benzothiazole 'click-on' fluorogenic dyes

‘Click-on’ fluorogenic reaction: a non-fluorescent benzothiazole with an electron-deficient alkyne group at 2-position reacts with azide containing molecules could form fluorescent adducts.     

Aromatic phosphonates inhibit the lysophospholipase D activity of autotaxin

Autotaxin (ATX) is an attractive target for the anticancer therapeutics that inhibits angiogenesis, invasion and migration. ATX is an extracellular lysophospholipase D that hydrolyzes lysophosphatidylcholine to form the bioactive lipid lysophosphatidic acid. The aromatic phosphonate S32826 was the first described nanomolar inhibitor of ATX. However, the tridecylamide substituent on aromatic ring contributed to its poor solubility and bioavailability, severely limiting its utility in vivo. c Log P calculations revealed that the lipophilicity of S32826 could be lowered by shortening its hydrophobic chain and by introducing substituents alpha to the phosphonate. Herein, we describe the synthesis of a small set of α-substituted phosphonate analogs of S32826, and we show that shortening the chain and adding α-halo or α-hydroxy substituents increased solubility; however, ATX inhibition was reduced by most substitutions. An optimal compound was identified for examination of biological effects of ATX inhibition in vivo.     

Fluorogenic substrates of glycogen debranching enzyme for assaying debranching activity

Glycogen debranching enzyme (GDE) degrades glycogen in concert with glycogen phosphorylase. GDE has two distinct active sites for maltooligosaccharide transferase and amylo-1,6-glucosidase activities. Phosphorylase limit dextrin from glycogen is debranched by cooperation of the two activities. Fluorogenic branched dextrins were prepared as substrates of GDE from pyridylaminated maltooctaose (PA-maltooctaose) and maltotetraose, taking advantage of the synthetic action of Klebsiella pneumoniae pullulanase. Their structures were as follows: Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4GlcPA (B3), Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B4), Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B5), Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B6), Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B7), and Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B8). These dextrins were incubated with porcine skeletal muscle GDE. No fluorogenic product was found in the digest of B8. The fluorogenic products from B3, B4, and B5 were PA-maltooctaose only. PA-maltooctaose, PA-maltoundecaose, and 6(7)-O-alpha-glucosyl-PA-maltooctaose were from B7. PA-maltooctaose and 6(6)-O-alpha-glucosyl-PA-maltooctaose were from B6. These results indicate that the maltooligosaccharide transferase removed the maltotriosyl residues from the maltotetraosyl branches by hydrolysis or intramolecular transglycosylation to expose 6-O-alpha-glucosyl residues, and then the amylo-1,6-glucosidase hydrolyzed the alpha-1,6-glycosidic linkages of the products rapidly. Probably, 6-O-alpha-glucosyl-PA-maltooctaoses from B7 and B6 were less susceptible to the amylo-1,6-glucosidase than were those from B3, B4, and B5. Taking this into account, B3, B4, and B5 are suitable substrates for GDE assay.