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J Conaty

Publications and source records attributed to J Conaty.

12 recordsLinked to original sources
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

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↗
PubMed1997

Identification of a novel tissue-specific processed HPRT gene and comparison with X-linked gene transcription in the Australian marsupial Macropus robustus.

The genome of the Australian marsupial Macropus robustus contains a highly conserved processed hypoxanthine phosphoribosyltransferase homologue, HPRT-2. Using the techniques of reverse transcriptase-polymerase chain reaction (RT-PCR) and protein isoelectric focusing (IEF) we have shown this processed gene to be fully functional, but liver specific. In contrast, the unprocessed X-linked parent gene HPRT-1 was expressed in all somatic tissues. Expression of the HPRT-2 gene effectively doubles the total HPRT enzyme activity in liver compared to other tissues. Analysis of the 5'-flanking sequence of HPRT-2 revealed regions with homology to the liver-specific regulatory motifs C/EBP, NF-IL6, LF-A1 and LF-B1, although the functional significance of these regions remains unknown. Consistent with X chromosome inactivation in female mammals, transcript levels of the unprocessed X-linked gene HPRT-1 were similar in males and females in all tissues examined. No HPRT-2 activity was detected in testes, indicating that this gene does not compensate for sex chromosome inactivation during spermatogenesis. Moreover, the demonstration of very high HPRT-1 enzyme levels in testes indicated that such a compensatory mechanism may not be required. Phylogenetic analyses attribute considerable antiquity to the processed gene and PCR with conserved primers spanning exons 4-8 of genomic DNA from several different kangaroo species inferring the existence of a conserved processed HPRT-2 homologue in these marsupial species. However, no such conserved PCR product was obtained with DNA from eutherian species, suggesting that integration of HPRT-2 occurred after the separation of the metatherian and eutherian lineages.

Amino Acid Sequence↗
PubMed1992

Physiotherapy with alcohol and drug dependent patients: implications for general settings.

This article focuses on some of the problems presented by alcohol and drug dependent patients and how physiotherapists can respond to them. Approaches used on the Medical Ward of the Clinical Institute of the Addiction Research Foundation (ARF) are presented. The attitude of the physiotherapist toward addictions is important, both in the rehabilitation of the patient's physical problem and in the treatment of his/her alcohol or drug problem. Questions about drug or alcohol use in a physiotherapy assessment can reveal important information about how the patient manages pain and add to understanding of other psycho-social considerations. A brief overview of the types of patient conditions commonly seen on the Medical Ward is given. This is followed by an outline of the ways in which withdrawal from drugs and alcohol is managed. Information is included about pathology related to alcohol abuse. Evidence that many alcohol-related neurological conditions are often reversible suggests that physiotherapists should be optimistic in their expectations about successful alcohol and drug treatment outcomes and should convey this optimism to their patients.

Adult↗
PubMed1987

The potential use of implanted radiolabelled bovine nasal cartilage in dialysis tubing to evaluate agents affecting cartilage degradation in vivo.

Cartilage which undergoes extensive autolysis in vitro (spontaneous or stimulated) is characterized by proteoglycan loss. Experimental conditions and inhibitor profils studies suggest neutral metalloproteinases induce the autolysis. In these preliminary studies we compared the degradation of Na2 35SO4 labeled bovine nasal cartilage (BNC) plugs placed in dialysis tubing in vitro and in vivo. The dialysis tubing was used to exclude large molecules (molecular weights greater than 2000) like proteinases, and proteinase inhibitors (e.g. alpha 2-macroglobulin) but not potential test agents from the implanted cartilage. Cartilage autolysis occurred with live tissue but not with heat-killed tissue in both the in vitro and in vivo systems. In addition retinoic acid and phenanthroline were effective when placed inside or outside the dialysis tubing. A potentially useful procedure to evaluate agents which affect cartilage degradation is described.

Animals↗
PubMed1986

Proteoglycan- and collagen-degrading enzymes from human interleukin 1-stimulated chondrocytes from several species: proteoglycanase and collagenase inhibitors as potentially new disease-modifying antiarthritic agents.

Human IL-1-stimulated chondrocytes derived from rabbit, bovine, and human articular cartilage produce proteoglycan- and collagen-degrading enzymes. These studies demonstrate that the biological activity of IL-1 is not species specific. Several thiol, carboxyalkyl, and hydroxamic acid peptide inhibitors showed differential effects. The thiols were equipotent inhibitors of both the collagen- and proteoglycan-degrading enzymes whereas the carboxyalkyls appear to inhibit solely the proteoglycan-degrading enzyme(s). The hydroxamic acid peptides, the most potent inhibitors, appear to be more active against the proteoglycan-degrading enzymes. These synthetic inhibitors of proteoglycan- and/or collagen-degrading enzymes may represent a new class of disease-modifying antiarthritic agents.

Animals↗
PubMed1986

Lack of response of rat chondrosarcoma chondrocytes to interleukin-1 to produce proteoglycan- and collagen-degrading enzymes.

Chondrocytes obtained from rat articular cartilage and Swarm rat chondrosarcoma were maintained in culture. Human interleukin-1 (IL-1) was added at confluency, and three days later the spent media was evaluated for proteoglycan- and collagen-degrading enzymes. Unlike chondrocytes from articular cartilage from several species, chondrocytes from Swarm rat chondrosarcoma did not respond to IL-1 stimulation. This lack of sensitivity to IL-1 may be a useful biochemical marker to classify various types of chondrosarcomas. However, it appears that the use of chondrosarcoma chondrocyte cultures are not useful as a source for metalloproteinases.

Animals↗
PubMed1983

Population estimates from longitudinal records in otherwise data-deficient settings.

Existing methods for estimating population parameters in settings of data deficiency do not provide techniques for analysis of commonly available longitudinal data. In setting where complete population data is unavailable, longitudinal data recorded for only a subset of the total population are often available (e.g., event registers, genealogies). In this article we present and evaluate models which derive population parameters for the population subgroup underlying such longitudinal data. Using the distribution of individual times until first recorded event within a measurement interval, population parameters are estimated which provide basic denominator data for analyzing event occurrence. The models which we derive are especially suited to records which may include migration and population growth trends. The use of the models is demonstrated and evaluated through an application to genealogical records for a nineteenth-century population. Possible extensions of these models and their major limitations are also discussed.

Adult↗
PubMed1982

Comparative potency and pharmacology of isomers of leukotriene D4 on guinea-pig trachea: requirement for a 5(S)6(R) configuration.

The relative contractile activity of C5 and C6 diastereomers of Leukotriene D4 (LTD4), as well as 11-trans stereoisomers were evaluated in guinea-pig tracheal smooth muscle. 5(S)6(R) LTD4 was 1000 times more potent than histamine as a contractile agent. While a change of the 11-ethylenic bond from cis to trans resulted in a four fold decrease in potency, a change in configuration of the 5-hydroxyl group and/or the 6-peptide adduct resulted in a decrease in potency of at least 2 to 3 orders of magnitude. The contractile activity of all LTD4 isomers was inhibited by FPL 55712, whereas indomethacin markedly enhanced the contractile activity of 5(S)6(R) LTD4, but appeared to have less of an effect on the other diastereomers. The results demonstrate the critical nature of configuration of the 5-hydroxyl and the 6-peptide adduct of eicosatetraenoic acid for maintenance of high affinity for receptors.

Animals↗