Biomedical subjects

D G Spiller

Publications and source records attributed to D G Spiller.

29 records · Page 2Linked to original sources

PubMed1995

Optimization of antisense oligodeoxynucleotide structure for targeting bcr-abl mRNA.

Antisense oligodeoxynucleotides targeted to bcr-abl are potential ex vivo purging agents for use with autologous bone marrow transplantation in the treatment of chronic myeloid leukemia (CML). We investigated, in a cell-free system, the activity and nuclease resistance of phosphodiester, phosphorothioate, chimeric methylphosphonate/phosphodiester, and chimeric methylphosphonate/phosphorothioate antisense octadecamers directed against either b2a2 or b3a2 bcr-abl breakpoint RNAs. Certain chimeric compounds were shown to possess targeted activity broadly equal to the parent phosphodiester or phosphorothioate forms and greater resistance to the nucleases present in cell extracts. Selected chimeric structures were compared with phosphodiester and phosphorothioate analogues for antisense activity in human CML cells containing either b2a2 or b3a2 bcr-abl breakpoint mRNAs. We present results showing that all four structures can suppress bcr-abl mRNA level in vivo. The rank of in vivo activity is chimeric methylphosphonate/phosphodiester > or = phosphodiester > phosphorothioate > methylphosphonate/phosphorothioate. We show that b2a2 breakpoint RNAs can be more effectively targeted than b3a2 sequence RNAs both in vitro and in vivo and suggest that RNA secondary structure may be a possible explanation for this phenomenon.

Base Sequence↗

PubMed1995

Single base discrimination for ribonuclease H-dependent antisense effects within intact human leukaemia cells.

We have previously demonstrated, in vitro, that phosphodiester and phosphorothioate antisense oligodeoxynucleotides could direct ribonuclease H to cleave non-target RNA sites and that chimeric methylphosphonodiester/phosphodiester analogue structures were substantially more specific. In this report we show that such chimeric molecules can promote point mutation-specific scission of target mRNA by both Escherichia coli and human RNases H in vitro. Intact human leukaemia cells 'biochemically microinjected' with antisense effectors demonstrated efficient suppression of target mRNA expression. It was noted that the chimeric methylphosphonodiester/phosphodiester structures showed single base discrimination, whereas neither the phosphodiester nor phosphorothioate compounds were as stringent. Finally, we show that the antisense effects obtained in intact cells were due to endogenous RNase H activity.

Bacterial Proteins↗

PubMed1995

Nuclear delivery of antisense oligodeoxynucleotides through reversible permeabilization of human leukemia cells with streptolysin O.

Most mammalian cell types appear to take up antisense oligonucleotides and oligonucleotide analogs from the bathing medium by highly inefficient endocytic mechanisms, and most if not all intracellular oligomer is sequestered in vesicles, still separated by a membrane from the target mRNA. On the other hand, oligonucleotides introduced directly into the cytoplasm by microinjection rapidly accumulate in the cell nucleus. Poor delivery to the designated site of action of antisense oligonucleotides is a major problem limiting their routine use in genetic research and their development as potential therapeutic agents. In view of this difficulty, various means of membrane permeabilization were applied to cultured human leukemia cells in an attempt to enhance intracytoplasmic delivery of fluorescein-tagged oligodeoxynucleotides. The outcome of the manipulations was monitored by flow cytometry and fluorescence microscopy. This work has directly confirmed the conclusion suggested by reported antisense effects, that streptolysin O reversibly permeabilizes the plasma membrane toward oligonucleotides and may be utilized to effect biochemical "microinjection" of these molecules directly into the cytoplasm. KY01 myelogenous leukemia cells treated in this way accumulated over 100-fold higher intracellular levels of oligodeoxynucleotides than in the absence of streptolysin O and, in contrast to the latter case, were observed to concentrate internalized molecules in their nuclei.

Bacterial Proteins↗

PubMed1995

Detection of ribonuclease H-generated mRNA fragments in human leukemia cells following reversible membrane permeabilization in the presence of antisense oligodeoxynucleotides.

The involvement of ribonuclease H (RNase H) in antisense phenomena in intact cells has, to date, only been adequately demonstrated for microinjected Xenopus systems. The significance of RNase H for the antisense effects of oligodeoxynucleotides observed in human and other mammalian cell cultures has remained obscure, in part because of inadequate analytic methods. In this report we show that the "reverse ligation-mediated PCR" (RL-PCR) procedure permits amplification of RNA fragments produced by oligodeoxynucleotide-directed RNase H activity. We have used this procedure to demonstrate RNase H-dependent antisense effects in irreversibly permeabilized (dead) cells and reversibly permeabilized (live) cells.

Animals↗

PubMed1993

Chimeric oligodeoxynucleotide analogues: enhanced cell uptake of structures which direct ribonuclease H with high specificity.

A previous report has demonstrated that normal phosphodiester oligodeoxynucleotides could direct extensive non-targeted ribonuclease (RNase) H-dependent effects, and that greatly enhanced specificity could be achieved upon methylphosphonodiester substitution of terminal phosphodiester residues. In this report, we extend our previous observations to show that phosphorothioate oligodeoxynucleotides also direct substantial inappropriate RNase H-mediated hydrolysis of non-targeted RNA. Chimeric methylphosphonodiester/phosphodiesters were found to be capable of efficiently directing RNase H when the central phosphodiester section was reduced to just two contiguous internucleoside linkages. Furthermore, cleavage of non-target RNA sites was found to be undetectable, or minimal in extent, when RNase H was directed by such chimeras. In addition, we show that analogue structures which contain three, or fewer, phosphodiester residues in otherwise methylphosphonodiester molecules were imported into cells via the comparatively more efficient route taken by methylphosphonates, rather than by receptor-mediated endocytosis, which is generally characteristic for polyanionic structures. Evidence is presented that the primary process responsible for enhanced uptake is an active mechanism. Nevertheless, a proportion of the applied oligodeoxynucleotide analogues, which demonstrate augmented uptake, appear to have penetrated into the cytoplasmic cellular compartment. The present results suggest that chimeric molecules of the type we describe here may show considerable utility as antisense effectors due to their increased cellular import, access to the intracellular compartments, and their highly efficient and specific direction of RNase H.

Base Sequence↗

PubMed1992

Abrogation of c-MYC protein degradation in human lymphocyte lysates by prior precipitation with perchloric acid.

Conventional lysis buffers, though containing cocktails of protease inhibitors, did not prevent the degradation of c-MYC recombinant protein added immediately prior to lysis to cell pellets from human mixed lymphocyte cultures. Treatment of the cells with 4.2% perchloric acid, however, prevented protein degradation and facilitated the detection of c-MYC protein by Western blotting even in unstimulated lymphocytes, where previously it had been reported to be undetectable or barely detectable using this technique. PHA stimulation of lymphocytes induced an approximately six fold increase in measured c-MYC protein within 5 h if cell extracts were prepared using perchloric acid precipitation. However, using conventional lysis buffer the proto-oncogene protein was undetectable until 48-72 h after mitogen addition. Pretreatment with perchloric acid may be useful for Western blotting analysis of protein in other systems where it may be desirable to dispense with the use of toxic protease inhibitors or where these may be incompletely effective.

Blotting, Western↗

PubMed1992

The uptake kinetics of chimeric oligodeoxynucleotide analogues in human leukaemia MOLT-4 cells.

Chimeric oligodeoxynucleotides with terminal nonionic methylphosphonate analogue sections and internal phosphodiester regions offer several advantages as antisense effectors over either structure alone. These include enhanced biological stability relative to all-phosphodiester molecules, increased activity in directing ribonuclease H mediated destruction of target RNA, increased specificity and reduced non-specific toxicity. However, another important parameter, the ability of these molecules to enter intact mammalian cells, has not previously been investigated. Therefore, oligodeoxynucleotides were tagged at their 5'-termini with fluorescein reporter groups and a detailed study of uptake kinetics in human leukaemia MOLT-4 cells undertaken by calibrated flow cytometry. Baseline measurements with all-phosphodiester and all-methylphosphonate molecules confirmed that uptake of oligodeoxynucleotides by intact cells is a highly inefficient process. The kinetic data were in agreement with previous reports of mechanisms of cell uptake involving receptor mediated endocytosis in the case of phosphodiester molecules and simple diffusion for methylphosphonates. Chimeric oligodeoxynucleotides exhibited saturable cell uptake kinetics similar to all-phosphodiester oligodeoxynucleotides, suggesting that uptake was receptor-mediated and distinct from concentration-dependent uptake of all-methylphosphonate molecules. Similarly, chimeric molecules were apparently confined to the endosomal compartment within cells. These results imply that reversible masking of the negative charges of the phosphodiester sections of chimeric oligodeoxynucleotides may be required to change the uptake mechanism back to simple diffusion and allow intracellular delivery to the site of the target RNA.

Amino Acid Sequence↗

PubMed1990

Lymphocyte subpopulations in peripheral blood of lambs experimentally infected with bovine respiratory syncytial virus.

The lymphocyte subpopulations of peripheral blood of normal lambs and lambs experimentally infected with bovine respiratory syncytial virus (RSV) were analysed by flow cytometry, using a panel of monoclonal antibodies against specific lymphocyte epitopes. Experimental infection with bovine RSV was characterized by a significant rise in SBU-T8+ (CD8+ or cytotoxic) T cells and a significant reduction in SBU-T4+ (CD4+ or helper) T cells and B (LCA p220+) lymphocytes (P less than 0.05). The helper/suppressor (CD4/CD8) ratio was reduced from 3.91 on the day of experimental infection to 1.13 on 10 days after experimental infection (P less than 0.001). The total number of SBU-T4+ (CD4+) and B cells returned to pre-inoculation values 14 days after experimental infection but the helper/suppressor ratio remained depressed up to 21 days post-inoculation.

Animals↗

PubMed1987

Scanning electron microscopy of the wall of the ovine cervix uteri in relation to tensile strength at parturition.

There was a progressive loosening of the collagenous fibre bundles in the stroma of the ovine cervix during spontaneous parturition. The change from an organ with predominantly thick collagen fibre bundles to one with a loose network of collagen fibrils was associated in time with a threefold increase in softness as measured by the Stretch modulus. Infusion of oestradiol 17 beta (E2 beta) for 18 hours elicited similar changes in the structure and mechanical properties of the cervix. Infusion of sodium meclofenamate for 18 hours significantly (P less than 0.005) reduced the effect of E2 beta administered simultaneously.

Animals↗

PubMedDate not supplied

Mechanical properties of the ovine cervix during pregnancy, labour and immediately after parturition.

The stretch modulus (stress/strain) of strips of ovine cervical tissue in vitro was measured at different stages of gestation. A decrease in stretch modulus implies softening of the tissue. There was a gradual but progressive reduction in the stretch modulus with advancing pregnancy between 30 and 140 days gestation with a marked increase between 140 days gestation and the onset of labour (P less than 0.005). After the first trimester, the uterine end of the cervix was usually more distensible than the vaginal end. No further reduction in the stretch modulus of the cervix occurred between labour and delivery of the fetus. The results of this study suggest that while there is some softening of the ovine cervix during pregnancy, most of the softening takes place immediately prior to the onset of labour and delivery of the fetus.

Animals↗

PubMedDate not supplied

Precursor cystatin C in cultured retinal pigment epithelium cells: evidence for processing through the secretory pathway.

Evidence was recently reported that the cysteine proteinase inhibitor, cystatin C, is highly expressed by cultured human retinal pigment epithelial (RPE) cells. As a step towards understanding possible functions of this protein associated with the RPE, the localization, targetting and trafficking of cystatin C were investigated. Constructs encoding an enhanced variant of green fluorescent protein (EGFP) fused to precursor cystatin C and to mature cystatin C were made and transfected into cultured human RPE cells. Expression of fusion proteins was monitored in vivo by fluorescence confocal microscopy. In cells transfected with precursor cystatin C-EGFP, fluorescence was initially targetted to the perinuclear zone, co-localizing with the Golgi apparatus. Transfected cells were observed at intervals over a period of up to 3 weeks, during which time fluorescent vesicles developed peripherally and basally while fluorescence continued to be detected in the Golgi region. Immunochemical analysis of cell lysates confirmed the expression of a fusion protein recognized by antibodies to both cystatin C and EGFP. Cells transfected with the construct lacking the leader peptide of precursor cystatin C presented a diffuse and weak fluorescence. Together, these results imply a leader sequence-dependent processing of cystatin C through the secretory pathway of RPE cells. This was confirmed by the detection, by Western blotting, of the chimaeric protein alongside endogenous cystatin C in the medium of transfected RPE cells.

Amino Acid Sequence↗