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Biomedical subjects

P Hendry

Publications and source records attributed to P Hendry.

At least 19 recordsLinked to original sources
PubMed2006

Radiation induced angiosarcoma a sequela of radiotherapy for breast cancer following conservative surgery.

Radiation induced angiosarcomas (RIA) can affect breast cancer patients who had radiotherapy following conservative breast surgery. They are very rare tumors and often their diagnosis is delayed due to their benign appearance and difficulty in differentiation from radiation induced skin changes. Therefore it is very important that clinicians are aware of their existence. We report here a case of RIA followed by discussion and review of literature.

Journal Article↗
PubMed2005

Li-Fraumeni syndrome: a case report and discussion.

Li-Fraumeni syndrome is a rare cause of breast cancer. It should be considered in cancer cases where a genetic link is suspected. It impacts dramatically on treatment and has major implications for the patient and their family.

Breast Neoplasms↗
PubMed2001

Identifying ribozyme-accessible sites using NUH triplet-targeting gapmers.

Accurately identifying accessible sites in RNA is a critical prerequisite for optimising the cleavage efficiency of hammerhead ribozymes and other small nucleozymes. Here we describe a simple RNase H-based procedure to rapidly identify hammerhead ribozyme-accessible sites in gene length RNAS: Twelve semi-randomised RNA-DNA-RNA chimeric oligonucleotide probes, known as 'gapmers', were used to direct RNase H cleavage of transcripts with the specificity expected for hammerhead ribozymes, i.e. after NUH sites (where H is A, C or U). Cleavage sites were identified simply by the mobility of RNase H cleavage products relative to RNA markers in denaturing polyacrylamide gels. Sites were identified in transcripts encoding human interleukin-2 and platelet-derived growth factor. Thirteen minimised hammerhead ribozymes, miniribozymes (Mrz), were synthesised and in vitro cleavage efficiency (37 degrees C, pH 7.6 and 1 mM MgCl2) at each site was analysed. Of the 13 Mrz, five were highly effective, demonstrating good initial rate constants and extents of cleavage. The speed and accuracy of this method commends its use in screening for hammerhead-accessible sites.

Base Sequence↗
PubMed2000

Small, efficient hammerhead ribozymes.

The hammerhead ribozyme is able to cleave RNA in a sequence-specific manner. These ribozymes are usually designed with four basepairs in helix II, and with equal numbers of nucleotides in the 5' and 3' hybridizing arms that bind the RNA substrate on either side of the cleavage site. Here guidelines are given for redesigning the ribozyme so that it is small, but retains efficient cleavage activity. First, the ribozyme may be reduced in size by shortening the 5' arm of the ribozyme to five or six nucleotides; for these ribozymes, cleavage of short substrates is maximal. Second, the internal double-helix of the ribozyme (helix II) may be shortened to one or no basepairs, forming a miniribozyme or minizyme, respectively. The sequence of the shortened helix + loop II greatly affects cleavage rates. With eight or more nucleotides in both the 5' and the 3' arms of a miniribozyme containing an optimized sequence for helix + loop II, cleavage rates of short substrates are greater than for analogous ribozymes possessing a longer helix II. Cleavage of gene-length RNA substrates may be best achieved by miniribozymes.

Base Sequence↗
PubMed1999

Selected classes of minimised hammerhead ribozyme have very high cleavage rates at low Mg2+ concentration.

In vitro selection was used to enrich for highly efficient RNA phosphodiesterases within a size-constrained (18 nt) ribonucleotide domain. The starting population (g0) was directed in trans against an RNA oligonucleotide substrate immobilised to an avidin-magnetic phase. Four rounds of selection were conducted using 20 mM Mg2+to fractionate the population on the basis of divalent metal ion-dependent phosphodiesterase activity. The resulting generation 4 (g4) RNA was then directed through a further two rounds of selection using low concentrations of Mg2+. Generation 6 (g6) was composed of sets of active, trans cleaving minimised ribozymes, containing recognised hammerhead motifs in the conserved nucleotides, but with highly variable linker domains (loop II-L.1-L.4). Cleavage rate constants in the g6 population ranged from 0.004 to 1.3 min-1at 1 mM Mg2+(pH 8.0, 37 degrees C). Selection was further used to define conserved positions between G(10.1) and C(11.1) required for high cleavage activity at low Mg2+concentration. At 10 mM MgCl2the kinetic phenotype of these molecules was comparable to a hammerhead ribozyme with 4 bp in helix II. At low Mg2+concentration, the disparity in cleavage rate constants increases in favour of the minimised ribozymes. Favourable kinetic traits appeared to be a general property for specific selected linker sequences, as the high rates of catalysis were transferable to a different substrate system.

Animals↗
PubMed1999

In vitro plasmid DNA cleavage by chromium(V) and -(IV) 2-hydroxycarboxylato complexes.

The ability of relatively stable Cr(V) and Cr(IV) complexes with 2-hydroxycarboxylato ligands [2-ethyl-2-hydroxybutanoate(2-) = ehba; (1R,3R,4R,5R)-1,3,4,5-tetrahydroxycyclohexanecarboxylate(2-) = quinate = qa] to induce single-strand breaks in plasmid DNA has been studied under a wide range of reaction conditions. The Cr(V) complex, Na[CrVO(ehba)2], causes substantial DNA cleavage at pH 4.0-8.0 [[Cr(V)]0 = 0.010-0.75 mM, phosphate buffer, and 37 degrees C]. The DNA cleavage is inhibited by the presence of excess ligand, by exclusion of O2, or by addition of organic compounds, such as alcohols, carboxylic acids, or DMSO, but it is not affected by traces of catalytic metals [Fe(III) or Cu(II)] or by addition of catalase. The Cr(IV)-qa complexes, unlike the Cr(V) complexes, are able to cleave DNA in the presence of the ligand in a large excess [[Cr(IV)]0 = 0.50 mM, [qa] = 20-100 mM, pH 3.5-6.0, and 37 degrees C]. This is the first direct evidence for DNA cleavage induced by well-characterized Cr(IV) complexes. The proposed mechanism for DNA cleavage includes the following: (i) partial aquation of the bis-chelated Cr(V) and -(IV) complexes with the formation of reactive monochelated forms, (ii) binding of the Cr(V) and -(IV) monochelates to the phosphate backbone of DNA, (iii) one- or two-electron oxidations at the deoxyribose moieties of DNA by Cr(V) and -(IV), and (iv) cleavage of the resulting DNA radicals or cations with or without participation of O2. The patterns of DNA damage by Cr(V) and -(IV) can include strand breaks, generation of abasic sites, and the formation of Cr(III)-DNA complexes.

Chromium↗
PubMed1999

RNA hairpin loops repress protein synthesis more strongly than hammerhead ribozymes.

A general study has been carried out to determine how well hammerhead ribozymes might reduce levels of specific protein synthesis in living cells, compared with RNA hairpin loops as stable but noncleaving controls. Four different experiments are described. First, a wide variety of hammerhead ribozymes, as well as hairpin loops, was cloned into a gene-expression cassette for beta-galactosidase, upstream of the coding sequences for that reporter gene, and expressed from plasmids in several strains of Escherichia coli. The results show that ribozymes, when acting intramolecularly in E. coli, do not significantly reduce the amount of protein synthesized from any construct. As a control, long RNA hairpin loops do greatly reduce the amount of protein made. Secondly, we studied the transcription-translation of these same plasmids in a cell extract from E. coli. Once again, hammerhead ribozymes show no effect on levels of beta-galactosidase, whereas long RNA hairpin loops produce a strong reduction, by apparent attentuation at the level of translation. Thirdly, we added an SV40 promoter to each plasmid, in order to study the effects of these gene-regulators on protein synthesis in Chinese hamster ovary cells. Here active intramolecular ribozymes produce a slight reduction in beta-galactosidase, whereas long RNA hairpin loops produce an even stronger reduction than before. Those hairpin loops apparently induce degradation of their own mRNA in Chinese hamster ovary cells, by a mechanism not seen in E. coli. Finally, analyses of total RNA by S1-trimming show that hammerhead ribozymes will self-cleave a mRNA by a total of no more than 45-50% in E. coli, compared with 70-80% in vitro. Other analyses using Northern blotting were unable to detect any ribozyme cleavage in E. coli or Chinese hamster ovary cells. In summary, the ability of hammerhead ribozymes to reduce protein synthesis appears weak or nonexistent in all the cellular systems tested. By comparison, long RNA hairpin loops reduce protein synthesis strongly: by an apparent attentuation mechanism in E. coli or by a novel degradation of their own mRNA in Chinese hamster ovary cells.

Animals↗
PubMed1998

Simultaneous synthesis of partially homologous oligonucleotide sequences.

A technique is described that allows the concurrent synthesis of homologous regions of separate oligonucleotides. The technique utilizes synthesis columns that are readily interconverted between single-chambered and dual-chambered. The regions of the oligonucleotides that differ are synthesized separately with single-chamber columns, and with the single molecules joined, the homologous regions synthesized simultaneously. After synthesis, the chambers are opened separately, the solid-phase-bound oligonucleotides are placed in deprotection vials and treated as normal. Compared with standard syntheses, no decrease in yield or oligonucleotide quality was observed. This technique can result in significant savings in time and reagent costs when synthesizing a series of homologous oligonucleotides. This technique could be extended to the simultaneous synthesis of more than two oligonucleotides, possibly up to four or five oligonucleotides in an appropriate multichambered column.

Electrophoresis, Capillary↗
PubMed1997

A minimised hammerhead ribozyme with activity against interleukin-2 in human cells.

A "minizyme" is a smaller version of the hammerhead ribozyme, in which stem-loop II has been replaced by a short linker. Here, we have synthesised a DNA-containing minizyme and a ribozyme, which are designed to cut within a 15-nucleotide sequence in human interleukin-2 mRNA, and have tested for their activity in vitro and in cells. In vitro at 37 degrees C, a minizyme with linker of sequence d(GTTTT) cleaves a 15-ribonucleotide synthetic substrate 5-fold slower than does the full-sized ribozyme. In human cells, the minizyme inhibits the production of interleukin-2 protein to a similar extent as does the ribozyme. Also, the minizyme and the ribozyme are more effective in cells than any of three controls: an inactive minizyme, a 15-nucleotide antisense DNA, or DNA of random sequence. The positive effect observed in cells indicates that minizymes may be useful as pharmaceuticals.

Animals↗
PubMed1996

Unexpected anisotropy in substrate cleavage rates by asymmetric hammerhead ribozymes.

RNA substrates which form relatively short helices I and III with hammerhead ribozymes are generally cleaved more rapidly than substrates which create longer binding helices. We speculated that for optimum cleavage rates, one of the helices needed to be relatively weak. To identify this helix, a series of ribozymes and substrates of varying lengths were made such that in the complex, helices I and III consisted of 5 and 10 bp respectively or vice versa. In two independent systems, substrates in the complexes with the shorter helix I and longer helix III were cleaved one to two orders of magnitude more rapidly than those in the complexes with the longer helix I and shorter helix III. Similar results were obtained whether the numbers of base pairs in helices I and III were limited either by the length of the hybridizing arms of the ribozyme or the length of the substrate. The phenomenon was observed for both all-RNA and DNA armed ribozymes. Thus, a relatively short helix I is required for fast cleavage rates in pre-formed hammer-head ribozyme-substrate complexes. When helix III has 10 bp, the optimum length for helix I is approximately 5 bp.

Animals↗
PubMed1996

Nonthoracotomy implantation of cardioverter defibrillators: preliminary experience with a defibrillation lead placed at the right ventricular outflow tract.

UNLABELLED: Although morbidity and mortality associated with defibrillator implantation using a nonthoracotomy approach have decreased as compared with a thoracotomy approach, defibrillation thresholds have been higher and fewer patients satisfied implant criteria. It may be possible to improve on the success of nonthoracotomy defibrillator implantation by the placement of a right ventricular (RV) outflow defibrillation lead. Implantable cardioverter defibrillator implantation data of 30 consecutive patients with clinical VT or VF were reviewed. Three defibrillation leads were routinely used. When either pacing threshold at the RV apex was inadequate (n = 2) or 18-J shocks were not successful in terminating VF in 3 of 4 trials (n = 8), the RV apex lead was positioned to the RV outflow tract attaching to the septum. Defibrillation testing was first performed with the RV apex lead in combination with CS, SVC, and/or subcutaneous leads. Twenty patients satisfied implant criteria with a defibrillation threshold of 13.5 +/- 3.6 J. In 7 of the 10 patients, whose RV lead was repositioned to the RV outflow tract, this lead in combination with SVC, CS, or subcutaneous leads produced successful defibrillation at < or = 18 J or in 3 of 4 trials. This approach improved the overall success of nonthoracotomy implantation of defibrillators from 69% to 90%. After a follow-up of 27 +/- 6 months, there was no dislodgment of the RV outflow tract defibrillation leads. CONCLUSIONS: This article reports the preliminary observation that placement of defibrillation leads to the RV outflow tract in humans was possible and without dislodgment. RV outflow tract offers an alternative for placement of defibrillation leads, which may improve on the success of nonthoracotomy defibrillator implantation.

Adult↗
PubMed1996

Detection and quantitation of unlabeled nucleic acids in polyacrylamide gels.

A method for the detection and quantitation of unlabeled nucleic acids in polyacrylamide gels is presented. The technique is a variation on UV shadowing. The technique is based on the observation that some storage phosphor screens (Kodak in this case) are sensitive to UV light in the range where nucleic acids absorb most strongly. Nucleic acid present in a gel resting on the screen absorbs the UV light and casts an ultraviolet shadow on the storage phosphor screen. The amount of nucleic acid in a particular area of the polyacrylamide gel is inversely proportional to the average pixel intensity in the corresponding area of the image. The technique therefore provides an electronic record of the gel in addition to the ability to quantitate the amount of nucleic acid present in any particular band. The storage phosphor screen images are erased by visible light. If the passage of that light to the screen is blocked by the presence of solid objects or images, the reverse process can be achieved, i.e., the recording of positive images by visible light etching of a uniformly charged background. By this method, we are able to transform old autoradiographic data into digital form for easy storage, computerized manipulation and distribution.

Autoradiography↗
PubMed1995

In vitro activity of minimised hammerhead ribozymes.

A number of minimised hammerhead ribozymes (minizymes) which lack stem II have been kinetically characterised. These minizymes display optimal cleavage activity at temperatures around 37 degrees C. The cleavage reactions of the minizymes are first order in hydroxide ion concentration up to around pH 9.3 above which the cleavage rate constants decline rapidly. The reactions show a biphasic dependence on magnesium-ion concentration; one of the interactions has an apparent dissociation constant of around 20 mM while the other appears to be very weak, showing no sign of saturation at 200 mM MgCl2. The minizymes are significantly less active than comparable, full-size ribozymes when cleaving short substrates. However, at a particular site in a transcribed TAT gene from HIV-1, minizymes are more effective than ribozymes.

Animals↗
PubMed1995

A comparison of the in vitro activity of DNA-armed and all-RNA hammerhead ribozymes.

Hammerhead ribozymes targeted against two unrelated RNA substrates have been prepared. For each substrate, four ribozymes, differing in their hybridising arm length and composition (DNA or RNA), have been synthesised and kinetically characterised. The presence of DNA in the hybridising arms had little effect on the overall cleavage rate when the cleavage step was rate determining. Shortening each of the hybridising arms of ribozymes from 10 to 6 nucleotides generally resulted in modest changes in rate constants for cleavage of the same 13mer substrate. In one case the presence of long RNA hybridising arms significantly impeded the cleavage reaction. Cleavage rates displayed first order dependence on hydroxide ion concentration at low pHs. At higher pH, some ribozymes deviated from this first order dependence because of a change in the rate-determining step, possibly due to a requirement for a conformation change in the ribozyme-substrate complex prior to cleavage. Ribozyme cleavage was strongly dependent on temperature in the range 5-45 degrees C, with an activation energy for the reaction of approximately 60 kJ mol-1. The ribozymes displayed biphasic dependence on magnesium ion concentration; evidence of strong apparent binding (Kd approximately 10 mM) as well as a looser interaction was observed for all ribozymes.

Base Sequence↗