Modifications to toxic CUG RNAs induce structural stability,rescue mis-splicing in a myotonic dystrophy cell model and reduce toxicity in a myotonic dystrophy zebrafish model

CUG repeat expansions in the 3′ UTR of dystrophia myotonica protein kinase (DMPK) cause myotonic dystrophy type 1 (DM1). As RNA, these repeats elicit toxicity by sequestering splicing proteins, such as MBNL1, into protein–RNA aggregates. Structural studies demonstrate that CUG repeats can form A-form helices, suggesting that repeat secondary structure could be important in pathogenicity. To evaluate this hypothesis, we utilized structure-stabilizing RNA modifications pseudouridine (Ψ) and 2′-O-methylation to determine if stabilization of CUG helical conformations affected toxicity. CUG repeats modified with Ψ or 2′-O-methyl groups exhibited enhanced structural stability and reduced affinity for MBNL1. Molecular dynamics and X-ray crystallography suggest a potential water-bridging mechanism for Ψ-mediated CUG repeat stabilization. Ψ modification of CUG repeats rescued mis-splicing in a DM1 cell model and prevented CUG repeat toxicity in zebrafish embryos. This study indicates that the structure of toxic RNAs has a significant role in controlling the onset of neuromuscular diseases.     

In vivo and in vitro studies with agents that cause quasi-morphine withdrawal syndromes

Compounds that elicit a "quasi-morphine withdrawal syndrome" (QMWS) after acute administration to rats constitute a new class of behavior-modifying agent. In studying the pharmacological bases of quasi-opiate withdrawal syndromes, we found that the dihydrocodeinone, RX 336-M, a standard QMWS-inducing agent, caused a naloxone-insensitive increase in twitch tension on the field-stimulated rat was deferens preparation. In contrast, IBMX (3-isobutyl-1-methylxanthine), the historical inducer of quasi-abstinence in rats, gave a naloxone-insensitive decrease in twitch tension. Tolerance developed to the "wet-dog" shakes elicited by twice-daily s.c. injections of RX 336-M or IBMX in rats for 5 or 15 days, respectively. The parallel development of tolerance to the in vitro effects of these compounds could not be demonstrated.     

Folate-liposome-mediated antisense oligodeoxynucleotide targeting to cancer cells: evaluation in vitro and in vivo

The objective of this study was to investigate the use of folate-targeted liposomes for the delivery of encapsulated oligonucleotides to folate receptor (FR)-positive tumor cells in vitro and in vivo. This project involved the synthesis and biological evaluation of many folate-PEG-lipid conjugates, where the chemical form of the folate moiety (pteroate) and the length of the PEG linker chain were varied widely. Folate-targeted oligonucleotide-containing liposomes were prepared using conventional methods, and the extent of cell uptake was evaluated using, among others, the FR positive KB cell line. Oligonucleotide-loaded folate-targeted liposomes were found to rapidly associate with the KB cells, and saturation was typically reached within the first hour of incubation at 37 degrees C. Nearly 100,000 liposomes per cell were bound or internalized at saturation. Importantly, cell association was blocked by a large excess folic acid, thus reflecting the FR-specific nature of the cell interaction. Full targeting potential was achieved with PEG linkers as low as 1000 in molecular weight, and pteroates bearing glycine or gamma-aminobutyryl residues juxtaposed to the pteroic acid moiety were also effective for targeting, provided that a terminal cysteine moiety was present at the distal end of the PEG chain for added hydrophilicity. When tested in vivo, folate-targeted liposomes were found to deliver approximately 1.8-fold more oligonucleotide to the livers of nude mice (relative to the nontargeted PEG-containing formulations); however, no improvement in KB tumor uptake was observed. We conclude from these results that folate liposomes can effectively deliver oligonucleotides into folate receptor-bearing cells in vitro, but additional barriers exist in vivo that prevent or decrease effective tumor uptake and retention.     

Rifampicin-loaded liposomes for the passive targeting to alveolar macrophages: in vitro and in vivo evaluation

Mycobacterium avium complex (MAC), the most frequent cause of opportunistic nontuberculous pulmonary infection, is made up of a group of intracellular pathogens that are able to survive and multiply inside lung alveolar macrophages. As nebulized liposomes are reported to be effective to target antibacterial agents to macrophages, in this work we have prepared and characterized re-dispersible freeze-dried rifampicin (RFP)-loaded vesicles by using soy lecithin (SL) and a commercial, enriched mixture of soy phosphatidylcholine (Phospholipon 90, P90) with or without cholesterol. The obtained results showed that RFP could be loaded stably in SL vesicles only when cholesterol was not present in the film preparation, whereas with P90 vesicles, the highest stability was obtained with formulations prepared with P90/cholesterol 7:1 or 4:1 molar ratios. RFP-liposome aerosols were generated using an efficient high-output continuous-flow nebulizer, driven by a compressor. After the experiments, nebulization efficiency (NE%) and nebulization efficiency of the encapsulated drug (NEED%) were evaluated. The results of our study indicated that nebulization properties and viscosity of formulations prepared with the low-transition-temperature phospholipids, SL and P90, are affected by vesicle composition. However, all formulations showed a good stability during nebulization and they were able to retain more than 65% of the incorporated drug. The effect of liposome encapsulation on lung levels of RFP following aerosol inhalation was determined in rats. The in vitro intracellular activity of RFP-loaded liposomes against MAC residing in macrophage-like J774 cells was also evaluated. Results indicated that liposomes are able to inhibit the growth of MAC in infected macrophages and to reach the lower airways in rats.     

The apolipoprotein CII gene: Subchromosomal localisation and linkage to the myotonic dystrophy locus

Summary The human apolipoprotein CII gene probe detects a restriction fragment length polymorphism located on chromosome 19. We have investigated the linkage of this polymorphism to the myotonic dystrophy locus in families. The two lici are closely linked with a maximum Lod score of 7.877 at 4% recombination. The close linkage and informativeness of the APOC2 polymorphism suggest that this probe may be of use for presymptomatic diagnosis of the myotonic dystrophy gene. The APOC2 gene was localised to the region 19p13–19q13 using somatic cell hybrids, providing further evidence that the myotonic dystrophy locus is situated in the central region of chromosome 19.     

Diffusible and volatile compounds produced by an antagonistic Bacillus subtilis strain cause structural deformations in pathogenic fungi in vitro

An efficient antagonistic strain of Bacillus subtilis, originally isolated from the rhizosphere of established tea bushes, was found to cause structural deformities in six pathogenic fungi under in vitro culture conditions. This effect was attributed to the production of diffusible and volatile antifungal compounds. Out of the selected test fungi four were phytopathogenic, while the remaining two were of clinical importance. The bacterial strain successfully restricted the growth of all test fungi in dual cultures, and induced morphological abnormalities such as mycelial and conidial deviations. The inhibitory effect caused by volatiles was greater than that by diffusible compounds.     

Peptide inhibitors of HIV-1 integrase: From mechanistic studies to improved lead compounds

The HIV-1 integrase enzyme (IN) catalyzes integration of viral DNA into the host genome. We previously developed peptides that inhibit IN in vitro and HIV-1 replication in cells. Here we present the design, synthesis and evaluation of several derivatives of one of these inhibitory peptides, the 20-mer IN1. The peptide corresponding to the N-terminal half of IN1 (IN1 1–10) was easier to synthesize and much more soluble than the 20-mer IN1. IN1 1–10 bound IN with improved affinity and inhibited IN activity as well as HIV replication and integration in infected cells. While IN1 bound the IN tetramer, its shorter derivatives bound dimeric IN. Mapping the peptide binding sites in IN provided a model that explains this difference. We conclude that IN1 1–10 is an improved lead compound for further development of IN inhibitors.     

靶向铜绿假单胞菌(Pseudomonas aeruginosa)粘肽合成酶PBP3的抗菌先导化合物的虚拟筛选及活性研究

【目的】开发出与铜绿假单胞菌粘肽合成酶PBP3有高亲和力,具有全新结构的先导化合物。【方法】以铜绿假单胞菌粘肽合成酶PBP3为靶点,通过分子对接软件DOCK6.5,对含有104万个小分子化合物的数据库进行了大规模虚拟筛选,选取结构相对简单的中选化合物进行合成,得到先导化合物,并验证其抑菌活性。【结果】通过grid score进行第一轮初筛,筛选出grid score分值小于–30 kcal/mol的6万个化合物,接着以amber score进行第二轮筛选,筛出amber score分值小于–20 kcal/mol的化合物约200个。最终,经过观察分析,从中挑选出4种打分高并且结构新颖的小分子化合物作为先导化合物。合成出的先导化合物及其衍生物对铜绿假单胞菌等常见微生物的最小抑菌浓度(MIC)在175–275μg/m L之间,对革兰氏阴性菌和阳性菌均有效,MIC值比作为阳性对照的磺胺嘧啶更低,说明先导化合物具有较好的抗菌活性。【结论】这些先导化合物可以进一步开发为新型抗菌药物,用于解决铜绿假单胞菌的耐药性问题。     

A combinatorial library of indinavir analogues and its in vitro and in vivo studies

A combinatorial library of 300HIV protease inhibitors has been synthesized. The library was screened against recombinant wild-type and mutant HIV-1 protease enzymes. The pharmacokinetics of the library was evaluated by dosing in dogs. Compounds that are notably more potent than indinavir and have favorable pharmacokinetic properties were identified.     

Selective inhibition of cytosolic epoxide hydrolase activity in vitro by compounds that inhibit catalase

The ability of a number of known inhibitors of catalase activity to affect cytosolic and microsomal epoxide hydrolase activities in vitro, measured as enzymatic trans-stilbene oxide hydrolysis and styrene oxide hydrolysis, respectively, was investigated. Catalase and cytosolic epoxide hydrolase activities are inhibited by hydroxylated metabolites of 2-amino-4,5-diphenylthiazole (DPT). The metabolite hydroxylated on the 4-phenyl ring (4OH-DPT) and the metabolite hydroxylated on both phenyl rings (4,5-DIOH-DPT) are potent inhibitors of both enzymes; the metabolite hydroxylated on the 5-phenyl ring (5OH-DPT) is less potent. Unmetabolized DPT has no effect on either enzyme. 4OH-DPT inhibits, but 5OH-DPT enhances, microsomal epoxide hydrolase activity. 4,5-DIOH-DPT and DPT have no effect on this enzyme. Other compounds that inhibit both catalase and cytosolic epoxide hydrolase activities, but do not inhibit microsomal epoxide hydrolase activity, are nordihydroguaiaretic acid and 2-aminothiazole. Microsomal epoxide hydrolase activity is enhanced by 2-aminothiazole and levamisole in vitro. Thus these inhibitors of catalase are selective epoxide hydrolase inhibitors in that they inhibit cytosolic epoxide hydrolase activity in vitro, but have either no effect on, or increase the activity of, microsomal epoxide hydrolase in vitro. Conversely, the selective cytosolic epoxide hydrolase inhibitors 4-phenylchalcone oxide and 4'-phenylchalcone oxide do not inhibit catalase activity, nor does trichloropropene oxide, a selective microsomal epoxide hydrolase inhibitor.     

In vitro and in vivo application of active compounds with anti-yeast activity to improve the shelf life of ready-to-eat table grape

The anti-yeast effects of several compounds at different concentrations were screened in vitro against main table grape spoilage yeasts. The compounds showing the most significant anti-yeast activity were applied by dipping to table grape, to evaluate the sensory perception. In a subsequent final step, dipping treatments with potassium sorbate, eugenol, citrus extract and ethanol, were applied to ready-to-eat seedless table grape, packaged in air or under modified atmosphere packaging (MAP). The in vitro test highlights good effects of cinnamon bark oil and citrus extract, even at the lowest concentrations used in this work. From a sensory point of view, the preliminary panel test selected potassium sorbate, citrus extract, eugenol and ethanol as most suitable substances. The in vivo application of active compounds showed that dipping in eugenol solution and ethanol (20 and 50 %) in combination with MAP increased shelf life of fruit if compared to the control sample (24.08, 28.47, 35.79 and 14.26 days, respectively).     

Comparison of in vivo and in vitro translation of Cowpea Mosaic Virus RNAs

The translation products from Cowpea Mosaic Virus (CPMV) RNAs obtained in two different cell-free systems were compared with the viral polypeptides synthesized in CPMV-infected cowpea protoplasts. It was shown that in both the wheat germ system and the rabbit reticulocyte lysate CPMV M component RNA was translated into two polypeptides of 105,000 and 95,000 dalton, which were not detected in CPMV-infected protoplasts. B component RNA however, gave different products depending on the system used. In the reticulocyte system this RNA was translated into a 200,000 dalton polypeptide which was further cleaved to give 170,000 and 32,000 dalton polypeptides. In the wheat germ system this processing step was lacking as only the 200,000 dalton product was formed. Since the 170,000 and 32,000 dalton polypeptides were also found in CPMV-infected protoplasts the two in vitro systems used apparently represent different stages of the expression of the B component RNA, thus providing a tool to study the mechanism of CPMV gene expression in vivo.