强力霉素抗性基因目标供体菌、受体菌和接合子的筛选及鉴定

目的为进一步研究环境中病原微生物目标供体菌强力霉素抗性基因(Dox)水平传播机制奠定基础。方法以强力霉素抗性基因为筛选指标,通过药敏试验从7个菌株中筛选出遗传型分别为Dox~RX~S的候选供体菌和Dox~SX~R的候选受体菌(X代表不是强力霉素的抗生素),将供、受体菌共培养后筛选出遗传型为Dox~RX~R的接合子。分别测定X对目标供体菌、Dox对目标受体菌的最小抑菌浓度。对目标供、受体菌从形态学、生理生化、分子生物学和BIOLOG方面进行鉴定。结果研究的7个病原菌均为多重耐药,最多能耐受15种抗生素,对链霉素、萘啶酮酸和磺胺二甲嘧啶的耐药率最高(100%),对庆大霉素、诺氟沙星和环丙沙星的耐药率最低(0%)。目标供体菌、受体菌和接合子Con-Ⅱ基因型分别为Dox~RKan~S、Dox~SKan~R和Dox~RKan~R。Kan对目标供体菌的最小抑菌浓度为0.310 0μg/mL,Dox对目标受体菌的最小抑菌浓度为0.048 8μg/mL。目标供体菌(Dox~RKan~S)鉴定为Escherichia coli O157:H7(E.coli O157:H7),目标受体菌TR-M30-1(Dox~SKan~R)鉴定为产酸克雷伯菌。结论目标供体菌为E.coli O157:H7(Dox~RKan~S),目标受体菌为TR-M30-1(Dox~SKan~R),接合子为Con-Ⅱ(Dox~RKan~R)。     

Degradation of 17β-estradiol and its conjugates: Effects of initial concentration and MLSS concentration

There have been many strong retrospective observations regarding adverse effects in reproductive and fertile developments in human and the wildlife caused by the endocrine disrupting compounds (EDCs) present in the aquatic environment recently. Degradation by biomass is one of the main removal mechanisms for 17β-estradiol (E2), the most important EDC, and its conjugates from sewage in wastewater treatment plants. In this batch study, the degradation rates of E2 and its conjugates were found to be affected by initial concentration of concerned pollutants and mixed liquor suspended solids (MLSS) concentration of activated sludge tested. Quantitative analysis was performed by liquid chromatography tandem mass spectrometry (LC-MS-MS). Overall, Michaelis–Menten Model adequately described the degradation of E2 and its conjugates over the range of the initial concentrations and MLSS concentrations tested. The degradation rates increased with the increase of initial concentration and MLSS concentration.     

Synthesis and antibacterial evaluation of amino acid–antibiotic conjugates

Amino acid conjugates of quinolone, metronidazole and sulfadiazine antibiotics were synthesized in good yields using benzotriazole methodology. All the conjugates were screened for their antibacterial activity using methods adapted from the Clinical and Laboratory Standards Institute. Antibiotic conjugates were tested for activity in four medically relevant organisms; Staphylococcus aureus (RN4220), Escherichia coli (DH5α), Pseudomonas aeruginosa (PAO1), and Bacillus subtilis (168). Several antibiotic conjugates show promising results against several of the strains screened.     

铁载体-抗生素耦合物:一种新型的抗菌制剂

随着细菌对抗生素耐药性的增强,寻找一种新型抗菌制剂越来越重要。细菌细胞外膜对药物分子的通透性降低是引起致病菌产生耐药性的一个重要因素,克服膜介导耐药性的方法之一是利用铁载体-抗生素耦合物。铁载体是细菌分泌的一种小分子铁离子螯合物,与铁离子螯合后被特定的外膜受体识别并转运至胞浆内供细菌利用。人工合成的铁载体-抗生素耦合物被特定外膜受体识别后主动转运跨过外膜进入胞质内。当铁载体-抗生素耦合物到达细胞质,它们通过释放药物杀死微生物,这可以阻止进一步获取铁离子,并且耦合物自身也可以作为一种抗菌剂。本文综述了铁载体-抗生素耦合物作为一种新型抗菌制剂的研究进展,有助于为进一步研发新型抗菌药物提供理论基础,对治疗耐药性细菌性疾病具有潜在的重要意义。     

Novel conjugates of podophyllotoxin and coumarin: Synthesis,cytotoxicities, cell cycle arrest,binding CT DNA and inhibition of Topo IIβ

A series of conjugates of podophyllotoxin and coumarin were prepared using the click reaction, and their cytotoxicities against A549, HepG2, HeLa, and LoVo cells were evaluated. Among them, compound 14e exhibited the strongest cytotoxicities against these cancer cells with IC₅₀ values of 4.9–17.5 μM. Furthermore, 14e disrupted microtubules and induced cell cycle arrest at G1 phase by regulating P21 and Cyclin D1 in LoVo cells. In addition, 14e bond CT DNA and selectively inhibited Topo IIβ over Topo IIα. Molecular docking model showed that 14e appeared to form stable hydrogen bonds with several DNA bases and residue Gln778. Taken together, these conjugates have the potential to be developed as anti-tumor drugs.     

Conjugates of Oligo(2′-O-Methylribonucleotides) with Minor Groove Binders as New Sequence-Specific Agents Recognizing Both Grooves of Double-Stranded DNA

Abstract

Design, synthesis and physico-chemical studies of new pyrimidine oligo(2′-O-methylribonucleotide) conjugates with one or two oligo(pyrrolecarboxamide) minor groove binders (MGB) are described.     

Oligonucleotide—Minor Groove Binder 1:2 Conjugates: Side by Side Parallel Minor Groove Binder Motif in Stabilization of DNA Duplex

Synthetic polycarboxamides consisting of N‐methylpyrrole (Py), N‐methylimidazole (Im), N‐methyl‐3‐hydroxypyrrole (Hp) and β‐alanine (β) show strong and sequence‐specific interaction with the DNA minor groove when they form hairpin structures with side‐by‐side antiparallel motifs. In the present paper, new conjugates containing two ligands linked to the same terminal phosphate of DNA strand were constructed. The paper describes optimized synthesis and properties of oligonucleotide‐linked polyamide strands that insert into the minor groove of a duplex in a parallel or antiparallel orientation. Strong stabilization of DNA duplexes by two attached minor groove ligands is demonstrated by the thermal denaturation method. The unmodified duplex 5′‐CGTTTATTp‐3′/5′‐AATAAACG‐3′ melts at 20°C. When one tetra(Py) residue was attached to the first strand of this duplex, denaturation temperature was increased to 46°C; attachment of the second tetra(Py) in a parallel orientation resulted in denaturation temperature of 60°C. It is even higher than in case of “classic” octapyrrole hairpin ligand (Tm = 58°C). Sequence‐specific character of stabilization by two conjugated ligands was demonstrated for G:C‐containing oligonucleotides attached to tetracarboxamide and octacarboxamide ligands constructed from Py, Im and β units according to established recognition rules (ΔTm = 20°C). The two‐strand parallel minor groove binder constructions attached to addressing oligonucleotides could be considered as site‐specific ligands recognizing single‐ and double‐stranded DNA similarly to already described hairpin MGB structures with antiparallel orientation of carboxamide units.     

The ATP Costs and Time Required to Degrade Ubiquitinated Proteins by the 26 S Proteasome

The degradation of ubiquitinated proteins by 26 S proteasomes requires ATP hydrolysis. To investigate if the six proteasomal ATPases function independently or in a cyclic manner, as proposed recently, we used yeast mutants that prevent ATP binding to Rpt3, Rpt5, or Rpt6. Although proteasomes contain six ATPase subunits, each of these single mutations caused a 66% reduction in basal ATP hydrolysis, and each blocked completely the 2–3-fold stimulation of ATPase activity induced by ubiquitinated substrates. Therefore, the ATPase subunits must function in a ordered manner, in which each is required for the stimulation of ATPase activity by substrates. Although ATP is essential for multiple steps in proteasome function, when the rate of ATP hydrolysis was reduced incrementally, the degradation of Ub₅-DHFR (where Ub is ubiquitin and DHFR is dihydrofolate reductase) decreased exactly in parallel. This direct proportionality implies that a specific number of ATPs is consumed in degrading a ubiquitinated protein. When the ubiquitinated DHFR was more tightly folded (upon addition of the ligand folate), the rate of ATP hydrolysis was unchanged, but the time to degrade a Ub₅-DHFR molecule (∼13 s) and the energy expenditure (50–80 ATPs/Ub₅-DHFR) both increased by 2-fold. With a mutation in the ATPase C terminus that reduced gate opening into the 20 S proteasome, the energy costs and time required for conjugate degradation also increased. Thus, different ubiquitin conjugates activate similarly the ATPase subunit cycle that drives proteolysis, but polypeptide structure determines the time required for degradation and thus the energy cost.     

Synthesis and bioactivity of new Finasteride conjugate

Finasteride is a synthetic 4-azasteroid compound that acts by inhibiting type II 5α-reductase, the enzyme that converts the androgen testosterone to 5α-dihydrotestosterone. It was approved by the US FDA for the treatment of benign prostatic hyperplasia and male pattern baldness. Here the acylation product of Finasteride C-18 amide N-polimod was synthesized by employing acylation reaction with polimod amide as a pivotal intermediate. The structure of the key intermediate and target molecule was confirmed by infrared spectrum, ¹H NMR and ¹³C NMR spectra and mass spectrum, and the inhibition of the steroid 5α-reductase and the rats’ benign prostatic hyperplasia by the new Finasteride conjugate and Finasteride was also determined. The inhibition of the Finasteride conjugate on 5α-reductase was stronger than that of Finasteride. Prostate hyperplasia of rats was reduced by Finasteride conjugate treatment similar to the Finasteride treatment. However, the Finasteride conjugate treated animals showed better viable condition than the Finasteride treated ones, suggesting the new compound may have improved toxicity profile than Finasteride.     

Criteria to distinguish between natural situations and illegal use of boldenone, boldenone esters and boldione in cattle: 2. Direct measurement of 17β-boldenone sulpho-conjugate in calf urine by liquid chromatography–high resolution and tandem mass spectrometry

Boldenone is banned in the European Union (Directive 96/22/EC) as growth promoter for meat producing animals. Boldione (ADD), boldenone and boldenone esters (mainly the undecylenate form) are commercially available as anabolic preparations, either to the destination of human, horse or cattle. Since the late 90s, the natural occurrence of boldenone metabolites has been reported in cattle. According to EU regulation, the unambiguous demonstration of boldenone administration in bovine urine should be provided on the basis of boldenone identification in the corresponding conjugate fraction. An analytical method has been developed and validated according to current standards with main concern to the measurement of intact 17β-boldenone-sulphate. The analytical procedure included direct extraction–purification of target analyte on octadecylsilyl cartridges and direct detection of phase II metabolite by liquid chromatography (negative electrospray), tandem mass spectrometry (QqQ) or high resolution mass spectrometry (Orbitrap™). Decision limit (CCα) and detection capability (CCβ) were respectively 0.2 μg L⁻¹ and 0.4 μg L⁻¹ on triple quadrupole and 0.1 μg L⁻¹ and 0.2 μg L⁻¹ on hybrid system. The method was successfully applied to the analysis of incurred samples collected in different experiments. 17β-Boldenone-sulphate was measurable up to 36 h after oral administration of boldione, and 30 days after 17β-boldenone undecylenate intra-muscular injection. This conjugate form was never detected in non-treated animals, confirming its status of definitive candidate marker for boldenone administration in calf.