Biomedical subjects

Mark A Lindsay

Publications and source records attributed to Mark A Lindsay.

14 recordsLinked to original sources

PubMed2007

Maternally imprinted microRNAs are differentially expressed during mouse and human lung development.

MicroRNAs (miRNAs) are a recently discovered class of noncoding genes that regulate the translation of target mRNA. More than 300 miRNAs have now been discovered in humans, although the function of most is still unknown. A highly sensitive, semiquantitative real-time polymerase chain reaction method was used to reveal the differential expression of several miRNAs during the development of both mouse and human lung. Of note was the up-regulation in neonatal mouse and fetal human lung of a maternally imprinted miRNA cluster located at human chromosome 14q32.31 (mouse chromosome 12F2), which includes the miR-154 and miR-335 families and is situated within the Gtl2-Dio3 domain. Conversely, several miRNAs were up-regulated in adult compared with neonatal/fetal lung, including miR-29a and miR-29b. Differences in the spatial expression patterns of miR-154, miR-29a, and miR-26a was demonstrated using in situ hybridization of mouse neonatal and adult tissue using miRNA-specific locked nucleic acid (LNA) probes. Of interest, miR-154 appeared to be localized to the stroma of fetal but not adult lungs. The overall expression profile was similar for mouse and human tissue, suggesting evolutionary conservation of miRNA expression during lung development and demonstrating the importance of maternally imprinted miRNAs in the developmental process.

Animals↗

PubMed2006

MALDI-TOF mass spectral analysis of siRNA degradation in serum confirms an RNAse A-like activity.

Synthetic siRNA duplexes are used widely as reagents for silencing of mRNA targets in cells and are being developed for in vivo use. Serum stability is a major concern if siRNA is to be used for therapeutic delivery within blood circulation. We have developed the use of MALDI-TOF mass spectrometry as a rapid and convenient analytical tool to identify the most vulnerable sites within siRNA to serum degradation. Using this approach, we found that one siRNA duplex (Dh3) with UpA sequences close to one end was particularly vulnerable to rapid cleavage. This produced a fragment of mass consistent with the presence of a 2',3'-cyclic phosphate that was slowly hydrolysed to a 2'-(3'-)phosphate on extended incubation. Substitution of these sites with 2'-O-methyl U residues prevented cleavage and confirmed that the major pathway for initial degradation is via cleavage by an RNAse A-like activity. Mass spectral analysis was used to follow the serum degradation of siRNA over more prolonged periods to show the accumulation of many fragments, almost all showing cleavage following pyrimidine nucleoside residues. Overall, the MALDI-TOF mass spectral analysis technique should prove useful for preliminary screening of the serum stability of siRNA duplexes and for identification of the most vulnerable cleavage sites.

Base Sequence↗

PubMed2005

Apoptosis as a therapeutic target for the treatment of lung disease.

It has been speculated that the destruction of lung tissue, associated with several acute and chronic diseases, is in part mediated through apoptosis or programmed cell death. Thus, it has been shown that both increased endothelial/epithelial cell apoptosis and decreased cell death of inflammatory cells are associated with acute lung injury/acute respiratory distress syndrome, chronic obstructive pulmonary disease, asthma and pulmonary fibrosis. In the light of these observations, it might be possible to use apoptosis modulation as a novel therapeutic approach to the treatment of lung disease.

Animals↗

PubMed2005

Finding new drug targets in the 21st century.

The past 30 years have witnessed a steady decline in the number of new drug targets. This review concentrates on the initial process of target identification and argues that current problems have resulted from a decrease in clinical research, an overemphasis on the discovery of new targets through an understanding of the molecular causes of disease and the adoption of cell and animal models that are poor predictors of human disease. To resolve this situation, we argue for increased clinical research and show that an intervention at the physiological level, using drugs to target at the extracellular signalling pathways, will facilitate identification of novel drug targets in the 21st century.

Animals↗

PubMed2005

Characterisation of cell-penetrating peptide-mediated peptide delivery.

Cell-penetrating peptides such as antennapedia, TAT, transportan and polyarginine have been extensively employed for in vitro and in vivo delivery of biologically active peptides. However, little is known of the relative efficacy, toxicity and uptake mechanism of individual protein transduction domain-peptide conjugates, factors that will be critical in determining the most effective sequence. In the present study, we show by FACS analysis that unconjugated antennapedia, TAT, transportan and polyarginine demonstrate similar kinetic uptake profiles, being maximal at 1-3 h and independent of cell type (HeLa, A549 and CHO cell lines). A comparison of the magnitude of uptake of cell-penetrating peptide conjugates demonstrated that polyarginine=transportan>antennapedia>TAT. However, examination of cellular toxicity showed that antennapedia<TAT<transportan< intersectionpolyarginine, with antennapedia-peptide conjugates having no significant toxicity even at 100 microM. Confocal studies of the mechanism of antennapedia- and TAT-peptide uptake showed that the time course of uptake and their cellular distribution did not correlate with transferrin, a marker of clathrin-mediated endocytosis. In contrast, the peptides co-localised with a marker of lipid rafts domains, cholera toxin, which was attenuated following the disruption of these domains using methyl-beta-cyclodextrin. Overall, comparison of the uptake and toxicity suggests that antennapedia provides the optimal cell-penetrating peptide for peptide delivery in vitro and that both antennapedia- and TAT-mediated peptide delivery occurs predominantly via lipid raft-dependent but clathrin-independent endocytosis.

Amino Acid Sequence↗

PubMed2004

siRNA for gene silencing: a route to drug target discovery.

The identification of RNA interference in mammalian cells, mediated via both virally-derived short interference RNA (siRNA) and endogenously produced microRNA, has revolutionised our understanding of the translational control of gene expression. Indeed, since its initial discovery, siRNA has been rapidly deployed for the elucidation of gene function and the identification of potential drug targets, a process often known as target discovery. In this review, we briefly discuss the mechanism of RNA interference and then critically examine the use of siRNA in target discovery, with a particular emphasis upon issues such as efficacy, selectivity, delivery and application in high-throughput studies.

Animals↗

PubMed2004

Overview of target validation and the impact of oligonucleotides.

During target validation, researchers attempt to modulate the activity of potential drug targets in relevant cell and/or animal disease models in order to identify those that might be expected to have therapeutic benefit in human patients. This has become increasingly important with the large expansion of potential targets identified by the human genome project and as a result of the spiralling costs of bringing drugs to market. This review will present an overview of the target-validation mechanism and examine the strengths and weaknesses of using oligonucleotide-based technologies such as antisense, short interfering RNA and aptamers.

Animals↗

PubMed2003

Protein transduction domains: are they delivering?

Early studies with protein transduction domains (PTDs), such as those derived from Tat and Drosophila Antennapedia, showed rapid, receptor- and endosomal-independent uptake of conjugated biological tools into all cell types. However, recent mechanistic studies suggest that these observations were artefacts of the positively charged nature of PTDs and that uptake is instead via electrostatic binding to the plasma membrane and subsequent endocytosis. Given these observations, we assess the future utility of PTDs for in vitro and in vivo cellular delivery.

Animals↗

PubMed2003

Target discovery.

Target discovery, which involves the identification and early validation of disease-modifying targets, is an essential first step in the drug discovery pipeline. Indeed, the drive to determine protein function has been stimulated, both in industry and academia, by the completion of the human genome project. In this article, we critically examine the strategies and methodologies used for both the identification and validation of disease-relevant proteins. In particular, we will examine the likely impact of recent technological advances, including genomics, proteomics, small interfering RNA and mouse knockout models, and conclude by speculating on future trends.

Animals↗

PubMed2003

Regulation of eosinophil apoptosis by nitric oxide: Role of c-Jun-N-terminal kinase and signal transducer and activator of transcription 5.

BACKGROUND: Reduced eosinophil apoptosis is considered to be a key mechanism for eosinophilia in allergic diseases such as asthma, rhinitis, and eczema. OBJECTIVE: The aim of our study was to investigate the possible modulatory effect of nitric oxide (NO) in human eosinophils. METHODS: Apoptosis in isolated eosinophils was assessed by relative DNA fragmentation assay, annexin-V binding, and morphologic analysis. The activation of c-Jun-N-terminal kinase (JNK) and signal transducer and activator of transcription 5 (STAT5) was assessed by immunoblot analysis. RESULTS: The NO donor S-nitroso-N -acetylpenicillamine (SNAP) reversed the survival-prolonging effect of IL-5 by inducing apoptosis. This effect was blocked by the NO scavenger (2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxide.potassium salt), indicating that reversal of IL-5-mediated eosinophil survival was due to NO. The effect of NO on IL-5-afforded cell survival was not mediated by cyclic guanosine 3': 5'-monophosphate (cGMP), because neither an inhibitor of guanylyl cyclase nor inhibitors of phosphodiesterases had any effect on SNAP-induced eosinophil apoptosis in IL-5-treated cells. SNAP induced a time-dependent increase in the activity of JNK, and an inhibitor peptide specific for JNK, L-JNKI1, completely reversed SNAP-induced apoptosis in IL-5-treated eosinophils. In contrast, SNAP did not inhibit IL-5-induced STAT5 activation. Inhibition of the activity of caspases by Z-Asp-CH(2)-DCB reversed the effect of SNAP, suggesting that NO promotes apoptosis in IL-5-treated human eosinophils in a caspase-dependent manner. However, this effect of NO was not mediated by means of activation of caspases 3, 8, or 9. CONCLUSIONS: Our results suggest that exogenous NO reverses IL-5-mediated eosinophil survival by inducing apoptosis, and this is mediated by means of activation of JNK in a cGMP-independent manner.

Apoptosis↗

PubMed2002

Divergent effect of mometasone on human eosinophil and neutrophil apoptosis.

Mometasone is a potent synthetic glucocorticoid, which is under development as an inhaled preparation for the treatment of asthma. Previous studies have suggested that glucocorticoids have direct effects on human eosinophil and neutrophil apoptosis. The present study was designed to characterize the effects of mometasone on constitutive apoptosis and cytokine-afforded survival in isolated human eosinophils and neutrophils. The isolated eosinophils or neutrophils were cultured in vitro, and apoptosis was assessed by flow cytometric analysis of relative DNA content, by annexin-V binding and morphological analysis. Mometasone enhanced constitutive human eosinophil apoptosis in a concentration-dependent manner. The maximal enhancement of eosinophil apoptosis was 2.1-fold with an EC(50) value of 5.63 +/- 2.33 nM. This enhancing effect was reversed by the glucocorticoid receptor antagonist, mifepristone. In the presence of added cytokines, mometasone reversed tumor necrosis factor -alpha-induced eosinophil survival but not that afforded by interleukin -5. In contrast, mometasone inhibited human neutrophil apoptosis in a concentration-dependent manner. The maximal inhibition of neutrophil apoptosis was 50% with an EC(50) value of 0.17 +/- 0.03 nM. The inhibitory effect was partly reversed by mifepristone. In the presence of added cytokines, mometasone further enhanced neutrophil survival induced by the granulocyte-macrophage colony-stimulating factor and leukotriene B(4). The present data suggests that mometasone has opposite effects on apoptosis of human eosinophils and neutrophils at clinically relevant drug concentrations via an effect on glucocorticoid receptor.

Apoptosis↗

PubMed2002

Caspase-catalyzed cleavage and activation of Mst1 correlates with eosinophil but not neutrophil apoptosis.

We have examined the role of caspase-mediated cleavage of the Ste20-like kinases, mammalian sterile 20-like 1 and 2 (Mst1/Mst2), in the mechanism of human eosinophil and neutrophil apoptosis. Initial measurements of kinase activity, using myelin basic protein (MBP) as a substrate in "in-gel" renaturation assays, showed that constitutive eosinophil and neutrophil apoptosis were associated temporally with the activation of a 36-kd MBP kinase (p36 MBPK) and a 34-kd MBP kinase (p34 MBPK), respectively. A constitutively active 63-kd MBP kinase (p63 MBPK) was also detected in freshly prepared eosinophils but not neutrophils, whose activity was transiently augmented during spontaneous apoptosis. Immunoblotting studies demonstrated the expression of Mst1 and Mst2 in eosinophils but not neutrophils whereas immunoprecipitation studies identified the p63 MBPK activity as being Mst1 and Mst2 and showed that the p36 MBPK activity represented the N-terminal catalytic fragment of Mst1. A role for the p36 MBPK in eosinophil cell death was supported by studies showing increased activation upon exposure to the proapoptotic Fas/CD95-activating antibody, CH-11, and attenuation in the presence of the survival-promoting cytokine, interleukin-5. Furthermore, spontaneous and Fas-induced activation of p36 MBPK was inhibited by catalase and the general caspase inhibitor, z-Asp-CH(2)-DCB, at concentrations that suppressed eosinophil apoptosis. These studies therefore implicate a role for caspase- and H(2)O(2)-mediated cleavage of the Mst1 and the subsequent release of the 36-kd catalytic fragment in the mechanism of eosinophil apoptosis. In contrast, neutrophil apoptosis occurs independently of Mst1 and Mst2 but instead is correlated with the activation of an as-yet-unidentified 34-kd MBPK.

Apoptosis↗

PubMed2002

Peptide-mediated cell delivery: application in protein target validation.

Recent reports have suggested that conjugation of peptides, proteins and antisense to short highly basic peptides, such as TAT, antennapedia and transportan, results in their rapid translocation into cells. Importantly, these conjugates have been shown to exert actions in a number of animal models suggesting their general utility for the determination of protein function in vitro and in vivo.

Animals↗

PubMed2002

Hydrogen peroxide reverses IL-5 afforded eosinophil survival and promotes constitutive human eosinophil apoptosis.

BACKGROUND: Eosinophils play a central role in the induction and perpetuation of allergic inflammatory responses. The present study was performed to investigate the effects of reactive oxygen intermediates on constitutive apoptosis as well as on interleukin (IL)-5 afforded human eosinophil survival. METHODS: Peripheral blood eosinophils were isolated by CD16-negative selection to >99% purity and were cultured for 48 h. The number of apoptotic eosinophils in the culture was assessed by flow cytometric analysis of relative DNA content in propidium-iodide-stained cells, annexin-V binding or by morphological analysis. Apoptosis was confirmed by the appearance of a typical ladder pattern in the DNA fragmentation assay by agarose gel electrophoresis. RESULTS: Exogenous H(2)O(2) reversed IL-5-afforded eosinophil survival by inducing apoptosis. Constitutive eosinophil apoptosis was inhibited by a reduction of intracellular levels of H(2)O(2) by catalase. Exogenous H(2)O(2) increased the rate of constitutive apoptosis. CONCLUSIONS: Our results suggest that H(2)O(2) may play a role in the downregulation of eosinophilic inflammation by inducing eosinophil apoptosis.

Apoptosis↗