摘要:

Mycobacterium tuberculosisinfection continues to be a daunting clinical challenge. Although it may well be one of the most studied bacteria in history, several aspects of its pathology remain a mystery. The resurgence of drug-resistantM. tuberculosisstrains and with its unusual pathology have promoted a renewed basic and clinical research interest in developing new therapies to combat this pathogen. The primary localization site forM. tuberculosisis within alveolar macrophages. Drug delivery strategies and novel therapeutic agents designed to target alveolar macrophages may lead to efficient destruction ofM. tuberculosis. Oligodeoxynucleotides (ODN) are short segments of nucleic acids that can interfere with transcription and translation processes. In this report, a monocyte-macrophage cell line was characterized in regard to ODN transport in the presence or absence ofM. tuberculosisinfection. The cells accumulated ODN in a time-dependent and concentration-dependent manner, regardless of the presence of serum. After 4 hours of incubation withM. tuberculosis(multiplicity of infection [MOI] 10:1), infected NR8383 cells demonstrated 1.5-7-fold increase in fluorescein isothiocyanate (FITC)-labeled phosphorothioate ODN accumulation as measured by flow cytometry. The increase in uptake was associated only with fluorescent-labeled ODN and not labeled markers of fluid phase endocytosis (e.g., tetramethylrhodamine isothiocyanate [TRITC], FITC-labeled dextran). NR8383 cells activated by phytohemagglutinin (PHA) did not demonstrate a significant increase in the uptake of either FITC-labeled dextran or FITC-labeled ODN. These studies demonstrate that NR8383 cells that have been infected withM. tuberculosiscan specifically accumulate ODN, and this route of accumulation may lead to a means of drug targeting to mycobacteria-containing cells.

ISSN:1087-2906    

DOI:10.1089/oli.1.2000.10.1

年代:2000

数据来源: MAL

摘要:

Morpholino antisense oligomers directed against the cytokine tumor necrosis factor (TNF-α)can specifically inhibit production of TNF-α by macrophagesin vitro. To evaluate the efficacy of morpholino antisensein vivo, we characterized a mouse model of increased pulmonary TNF-α production and inflammation in response to aerosolized endotoxin. Pretreatment of mice by intranasal (i.n.) insufflation of oligomers (30 μl of 100 μ M/ml ) 12 hours prior to lipopolysaccharide (LPS) exposure resulted in specific and consistent inhibition of TNF-α production by the oligomer MAS-2, whereas no effect was observed with a sequence-scrambled control (% inhibition 31.5 ± 3.5 vs. 1.3 ± 8.0, respectively,p<0.005). Dose-response analysis showed similar efficacy for MAS-2 at 25-100 μM/ml and diminished effects with lower concentrations. Inhibition of TNF-α did not alter the increase in neutrophils seen in bronchoalveolar lavage (BAL) fluid, a result consistent with observations using i.n. administration of neutralizing anti-TNF-α antibody or TNF receptor knockout mice. The results establish that morpholino oligomers directed against cytokine targets can functionin vivo. Additional studies of other targets and administration protocols to improve efficacy are

ISSN:1087-2906    

DOI:10.1089/oli.1.2000.10.11

年代:2000

数据来源: MAL

摘要:

The p53 tumor suppressor is regulated by the MDM2 oncoprotein through a negative feedback mechanism. MDM2 promotes the ubiquitination and proteasome-dependent degradation of p53, possibly by acting as a ubiquitin ligase. In cervical cancer cells containing high-risk human papillomaviruses (HPV), p53 is also targeted for degradation by the HPV E6 oncoprotein in combination with the cellular E6-AP ubiquitin ligase. In this report, we describe the identification of efficient antisense oligonucleotides against human E6-AP. The roles of MDM2 and E6-AP in p53 regulation were investigated using a novel E6-AP antisense oligonucleotide and a previously characterized MDM2 antisense oligonucleotide. In HPV16-positive and HPV-18 positive cervical cancer cells, inhibition of E6-AP, but not MDM2, expression results in significant induction of p53. In HPV-negative tumor cells, p53 is activated by inhibition of MDM2 but not E6-AP. Furthermore, treatment with both E6-AP and MDM2 antisense oligonucleotides in HPV-positive cells does not lead to further induction of p53 over inhibition of E6-AP alone. Therefore, E6-AP-mediated degradation is dominant over MDM2 in cervical cancer cells but does not have a significant role in HPV-negative cells.

ISSN:1087-2906    

DOI:10.1089/oli.1.2000.10.17

年代:2000

数据来源: MAL

摘要:

A fission yeast model was employed to investigate the influence of antisense gene location on the efficacy of antisense RNA-mediated target gene suppression. Fission yeast transformants were generated that contained the targetlacZgene at a fixed position and a single copy antisenselacZgene integrated into various genomic locations, including the same locus as the target gene. No significant difference inlacZsuppression was observed when the antisense gene was integrated in close proximity to the target gene locus compared with other genomic locations, indicating that target and antisense gene colocalization is not a critical factor for efficient antisense RNA-mediated gene expressionin vivo. Instead, increasedlacZdownregulation correlated with an increase in antisense dose, with the steady-state levels of antisense RNA being dependent on genomic position effects and transgene copy number.

ISSN:1087-2906    

DOI:10.1089/oli.1.2000.10.29

年代:2000

数据来源: MAL

摘要:

Anin situsingle-pass perfusion model was used to assess the effect of chemical modification and length on permeability and absorption of various oligonucleotides in rat intestine. Phosphorothioate oligodeoxynucleotides (PS-ODN) were compared with oligoribonucleotides with 2‵-methoxyethyl (MOE) or 2‵-0-methyl (OMe) modifications. A 25-mer PS-OMe-modified oligonucleotide showed relatively poor permeability in this model, as did unmodified 20-mer PS-ODN (permeability coefficient [Peff] = 2-8 X 10-6cm/sec). Modifying some or all of the oligonucleotides with 2‵-MOE groups on deoxyribose and 5‵-methylation of the cytosines substantially increased intestinal permeability of oligonucleotides. Both partially and fully modified PS-MOE oligonucleotides showed a 2–4-fold increase in permeability as compared with unmodified PS-ODN. The presence of a phosphodiester backbone in MOE-modified compounds led to further increases in intestinal permeability. PS-MOE composed of 6,8,10,12,14,16,18,20, and 22 nucleotides were also examined. It was found that the permeability of these oligonucleotides increased linearly with decreasi

ISSN:1087-2906    

DOI:10.1089/oli.1.2000.10.35

年代:2000

数据来源: MAL

摘要:

A method is described for monitoring the cleavage of an oligoribonucleotide substrate by the 2-5A-dependent RNase L based on fluorescence resonance energy transfer (FRET). The oligoribonucleotide, rC11U2C7, was labeled covalently at its 5‵-terminus with fluorescein and at its 3‵-terminus with rhodamine to provide a substrate for RNase L. On cleavage, the fluorescence at 538 nm (with 485 nm excitation) increased by a factor of 2.8, allowing real-time quantitation of the reaction progress. The method was performed easily in a 96-well plate format and allowed quantitative high throughput analyses of RNase L activity with different activat

ISSN:1087-2906    

DOI:10.1089/oli.1.2000.10.45

年代:2000

数据来源: MAL

摘要:

In the past, the use of delta ribozyme as a therapeutic tool was limited because substrate specificity was thought to be determined by only 8 nucleotides. Recently, we have accumulated evidence suggesting that the substrate sequence upstream of the cleavage site, which is not involved in the binding with the delta ribozyme, appears to be essential in the selection of an appropriate cleavage site. To understand the role of this region in efficient cleavage, we synthesized a collection of small substrates that possessed single and multiple mutations in positions -1 to -4 and determined the kinetic parameters of their cleavage using a model antigenomic delta ribozyme. Some substrates were found to be uncleavage, whereas others showed>60-fold difference in relative specificity between the least and most efficiently cleaved substrates. The base at each position from -1 to -4 contributes differently to the ability of a substrate to be cleaved. An optimal sequence for positions -1 to -4 was determined to be-1HRHY-4(H = U, C, or A). These results shed light on new features that contribute to the substrate requirement of delta ribozyme cleavage and should increase interest in the use of this unique ribozyme.

ISSN:1087-2906    

DOI:10.1089/oli.1.2000.10.53

年代:2000

数据来源: MAL