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P Upcroft

Publications and source records attributed to P Upcroft.

At least 55 records · Page 3Linked to original sources
PubMed1994

Multiple drug resistance in the pathogenic protozoa.

Evidence for the phenomenon of multiple drug resistance (MDR) in the well studied pathogenic protozoa has been examined. This has been placed in the more familiar context of the MDR efflux transporters and the cloned mdr genes of mammalian cells. Homologues of the mdr gene family in protozoa and their possible role in drug efflux have been compared with their mammalian counterparts. Possible mechanisms and models for drug efflux have been considered. The unusual and extensive range of substrates transported by the ATP-binding cassette (ABC) family of transporters which includes the MDRs has been raised. The impact of kinetics, structure and bioenergetics of the MDR family members on mechanisms of transport has been accentuated to argue that MDR efflux considered in isolation appears bizarre but may be better understood in a broader context.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Comparison of properties of agarose for electrophoresis of DNA.

Agarose as a medium for separation of DNA was first introduced in 1962 and since the early 1970s agarose submarine gel electrophoresis has been synonymous with separations of DNA molecules larger than 1 kilobase pair (kb). The large pore size, low electroendosmosis and strength of the matrix have advantages over other media such as polyacrylamide for many applications. The variety of grades of agarose, developed by chemical manipulation of the substitutions on the agarose polymer, provides a range of matrices for separation of DNA molecules from a few base pairs (bp) to over 5 megabase pairs (Mb) in length. The introduction of low-melting-temperature agarose has revolutionised the extraction and manipulation of chromosome-sized molecules. On the other hand, the demand for analysis of very small quantities of DNA will most likely lead to the increasing importance of capillary electrophoresis. Many theories have been propounded to explain the electrophoretic migration of DNA in agarose. The most popular of these has been reptation theory but none can account for all of the reported anomalies in migration. However, anomalous migration has been exploited to study DNA structure, topology and catenation. An example of the use of two-dimensional electrophoresis to demonstrate the complexity of DNA migration through agarose is given. Generally, for molecules smaller than 50 kb, electrophoretic separation is a function of length. By alternately electrophoresing DNA in two different directions, molecules as large as 5.7 Mb have been effectively separated, although with such large molecules DNA structure as well as size may determine migration. In the case of separations of chromosomes from the intestinal protozoan, Giardia duodenalis, for example, a discrepancy of 1 Mb in the size of one chromosome, with an apparent size of 0.7-2.0 Mb, depended on the boundary conditions of separation. Major challenges for the molecular biologist are separation of larger chromosomal sized molecules, greater number of samples and smaller formats. Towards this challenge computer-aided technology is a key component in the control of electrophoresis parameters and analysis.

Animals↗
PubMed1993

The 3' terminal region of a gene encoding a cysteine-rich surface protein in Giardia duodenalis.

DNA derived from chromosome band 3 of the cloned Giardia duodenalis line, WB-1B was used to construct a cloned library in E. coli. One of these clones, C3/23, has been identified as the 3' coding region of a G. duodenalis cysteine-rich variable surface protein (CRVSP) gene by homology with other published CRVSPs and also contains 720 bp of the 3' flanking region. The sequence of C3/23, was derived from genomic DNA independently of cDNA, or expression copies of the CRVSP genes. The 3' flanking region is not homologous to the 3' untranslated regions of published CRVSPs which probably reflects its genomic origin. Subclones of C3/23 were used to show that the 3' flanking region was conserved in all strains examined in this study and was repeated many times in the genome. The 3' flanking repeats were located on three chromosome bands and were not always associated with the coding sequence of C3/23 which was represented, although not equally, on all chromosome bands. The highly conserved nature of the 3' flanking region and its multiple representation in the genome emphasize the probable role of this sequence in the localization or regulation of expression of the CRVSPs in G. duodenalis.

Amino Acid Sequence↗
PubMed1993

Chromosomal duplication in Giardia duodenalis.

Strains of Giardia duodenalis from diverse parts of the world have three or four chromosomal bands in the range of 650-800 kb as defined by field inversion gel electrophoresis. The extra chromosome band in this range defined a group of strains which are geographically distinct from other strains missing this band. The cloned line WB-1B has three chromosome bands in this size range and chromosome band 3 was used to construct a library of chromosome-specific probes. In some strains examined, including BRIS/83/HEPU/106, a subset of these WB-1B probes hybridized to chromosome band 3 and to the extra chromosome band 4, indicating a partial duplication of chromosome 3 in BRIS/83/HEPU/106. This duplication was estimated to be at least 500 kb when the sizes of NotI chromosome segments which hybridized with chromosome band 3-specific probes were added. A second subset of WB-1B chromosome 3-specific probes hybridized to a fifth chromosome of strain BRIS/83/HEPU/106, in the size range 650-800 kb, which was not visible by ethidium bromide staining. The two subsets of WB-1B probes hybridized to a variety of chromosome combinations in this size range in other Giardia strains and included apparent reversal of chromosome 3 and 4 mobility as well as identification of other minor chromosomes. These data indicate that chromosome band 3 of the line WB-1B is a cluster of at least two different chromosomes that cannot be electrophoretically separated but genetic rearrangements in other strains allow separation of linkage groups carried by chromosome band 3 of WB-1B.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Drug resistance and Giardia.

Giardiasis is a worldwide disease that can cause serious morbidity. Metronidozole is the current recommended drug for treatment, and is mostly still effective. However, Giardia duodenalis, the causative agent, is capable of developing resistance to high levels of metronidozole and other drugs, in vitro, via a number of mechanisms. Resistance, in vivo, has been reported and many cases of treatment failure have been variously attributed to a number of causes, including resistance. Here, Jacqueline and Peter Upcroft ask: is this the beginning of another chapter of drug resistance? or is the situation likely to remain as a 'few refractory cases'? Should we wait to find out or can we act positively to avert the possibility of yet another valuable drug in our limited pharmacopoeia becoming obsolete?

Journal Article↗
PubMed1992

A gene associated with cell division and drug resistance in Giardia duodenalis.

A 3000 bp cDNA insert (G6/1) from Giardia duodenalis, cloned into Escherichia coli is located on chromosome 3 of Giardia stocks which have 3 chromosomes detectable by field-inversion gel electrophoresis in the range 650-800 kb and on chromosome 3 and/or 4 of stocks with 4 chromosomes in this size range. The loss of this sequence from chromosome 4 but not chromosome 3 was associated with the induction of drug resistance in a previously sensitive laboratory stock. G6/1 appears to represent a single copy gene in Giardia as determined by hybridization of the probe to cleaved genomic DNA. Furthermore, the sequences flanking at least 12 kb of G6/1 are the same when G6/1 appears on both chromosomes 3 and 4. The cDNA encodes a protein associated with the nuclei of trophozoites during some stages of growth of the parasite. In a non-dividing culture, the antigen is associated with the nuclei of about 30% of trophozoites and fewer in a dividing culture. Three Giardia stocks with obvious chromosome rearrangements, which grow poorly and fail to divide normally, apparently lack the DNA sequence G6/1.

Animals↗
PubMed1991

DNA fingerprinting of the human intestinal parasite Giardia intestinalis with hypervariable minisatellite sequences.

Individual isolates of the Giardia duodenalis group of protozoan intestinal parasites were identified by DNA fingerprinting with hypervariable minisatellite sequences. A morphologically identical parasite is found in some forty different animal species. Although the species name intestinalis is reserved for the human isolates, electrophoretic karyotyping suggests that most duodenalis isolates fall into the same species grouping. Distinction based upon morphology, restriction endonuclease cleavage of genomic DNA or isoenzyme analysis has not been adequate to identify individual strains. The successful use of hypervariable sequences in the identification of individual human genomes encouraged us to examine the use of these same sequences for the possible identification of parasite isolates. We initially use as a fingerprinting probe the genome of the bacteriophage M13, which has repeated sequences recognising homologous hypervariable sequences in the human genome. The M13 probe recognises a weakly homologous set of hypervariable sequences in Giardia. The number of informative bands is comparable to those seen in mammals, since the lower molecular weight bands are also useful. There is considerable divergence in the sequences of individual Giardia minisatellites. Some cloned Giardia hypervariable sequences are more homologous to M13 than they are to each other. Similar results were observed with the hypervariable repeat sequences 3' to the human alpha-globin gene when they were used as a probe to distinguish Giardia isolates. The poly(dA-dC).poly(dG-dT) probe which recognises frequent TG tracts in a number of organisms also detects a few variable bands amidst a hybridisation background in the Giardia genome. Thus Giardia isolates which could not be distinguished by restriction endonuclease cleavage, antibody typing or isoenzyme analysis have been identified by DNA fingerprinting procedures. Detailed analysis of strain movement, resurgence, variation, host range and drug resistance is now possible. Similar families of sequences may be widespread in lower eukaryotes and useful for generating individual specific fingerprints. A procedure for detecting individual parasites is also presented. Since Giardia is regarded as the most ancient eukaryote before the occurrence of symbiosis with purple non-sulphur bacteria to generate mitochondria, the identification of hypervariable sequences in the Giardia genome should also aid in understanding the mechanism of generation and evolution of these sequences.

Animals↗
PubMed1990

Antigen expression from the ribosomal DNA repeat unit of Giardia intestinalis.

The amitochondrial human intestinal parasite Giardia intestinalis is regarded to be the most ancient living example of single-celled eukaryotes and should display primitive features of pre-metazoan gene regulation. Characterization of E. coli clones which express Giardia antigens from plasmid vectors has revealed that an antigen is encoded by the rDNA repeat unit from the strand complementary to that encoding the rRNAs. The open reading frame (ORF) originates in the spacer region between the small (SS) and large (LS) subunit rRNA genes and terminates within the LS rRNA gene. The promoter region of this ORF has characteristics of both RNA polymerase (pol) II and pol III regulatory sequences, suggestive of gene regulation before these different promoter types evolved. The rDNA repeat unit is located on multiple chromosomal sites which are different in each isolate, although the electrophoretic karyotypes appear very stable in Giardia from both human and animal sources.

Amino Acid Sequence↗
PubMed1990

DNA fingerprinting of the intestinal parasite Giardia duodenalis with the M13 phage genome.

A DNA fingerprint procedure has been established for the intestinal parasite, Giardia duodenalis. This permits the identification of individual strains from both human and animal sources. Analysis of strain movement, resurgence and variation is now possible. The fingerprint probe is based on the tandem repetitive sequence found in bacteriophage M13 which hybridizes to a set of hypervariable polymorphic minisatellite sequences found in higher eukaryotes. This probe recognizes a weakly homologous set of hypervariable regions in the Giardia genome to provide a DNA fingerprint comparable to those seen in higher eukaryotes.

Animals↗
PubMed1990

Changing approaches to the study of Giardia epidemiology: 1681-2000.

Since the first description of Giardia by Antony van Leeuwenhoek in 1681, this parasite has proved to be enigmatic with a much more complicated epidemiology than originally envisaged. Even the nomenclature of this organism is confused. Despite numerous community prevalence studies, it was not until 1970 that the first proven report of waterborne transmission occurred. In 1981 the first common source outbreak due to contaminated food was recorded. It is also now recognized that venereal transmission occurs, especially among homosexual males. The faecal-oral route still remains the most important mode of infection despite the elucidation of these other methods of transmission. Recent developments in molecular biology especially DNA 'fingerprinting' and karyotyping which allow individual strain identification are being used to answer key epidemiological questions; for example are there truly pathogenic strains of Giardia, does drug resistance develop in vivo and is giardiasis a zoonotic disease? These and other molecular biological approaches will form the basis of research into the epidemiology of giardiasis and other intestinal protozoal infections until the end of the century and are certain to hold many surprises.

Animals↗
PubMed1990

Drug resistance in Giardia intestinalis.

Evidence for drug resistance in giardiasis is reviewed and biochemical studies undertaken to determine the basis for this resistance are discussed. Metronidazole and furazolidone, which produce toxic radicals within the cell, have different biochemical mechanisms of action. Resistance to metronidazole is negatively correlated with the intracellular concentration of pyruvateferredoxin oxidoreductase leading to a concomitant decrease in the uptake of free metronidazole into the cell, while resistance to furazolidone appears to be due to an increase in thiol cycling enzymes. At the molecular level resistance to metronidazole is associated with DNA changes. DNA probes which hybridize with specific chromosomes and repetitive sequences indicate that rearrangements both at the chromosome and repetitive DNA level occurred concurrently with the development of metronidazole resistance. The problems of cross-resistance and treatment failures that occur in the absence of resistance are additional difficulties which have important implications for the management of individual patients. New drugs such as azithromycin, while showing great variation in activity against different stocks may be useful in treating some refractory cases of giardiasis. In the community, it is important to recognize the occurrence and spread of drug resistant Giardia, and markers, such as DNA probes, provide methods to monitor potential epidemics and the spread of drug resistant Giardia.

Animals↗
PubMed1989

Geographic variation in Giardia karyotypes.

Chromosomes of 41 stocks of Giardia duodenalis derived from humans and 14 stocks from other animal species were analysed by field inversion gel electrophoresis (FIGE). These stocks have two predominant karyotypes as judged by FIGE which appear to fit a geographic distribution. Under FIGE conditions used to optimize the detection of size variation in Giardia chromosomes, five or six major chromosomes could be identified. Most of the stocks derived from North America have three major chromosomes smaller than 800 kb while most of the Australian stocks have four. A few exceptions, and minor variations, of these karyotypes were observed. It was estimated that not all of the DNA entered the gel, the remainder being trapped conformations or very large chromosomes. Karyotypes of Giardia stocks from different animal hosts and human sources within a geographical region are similar.

Animals↗
PubMed1989

Chromosomes of Blastocystis hominis.

Three stocks of Blastocystis hominis were adapted to monophasic culture in minimal essential medium (MEM) and the chromosomes of these stocks separated by field inversion gel electrophoresis (FIGE). Ten-twelve chromosomes were distinguished in the electrophoretic karyotype of these three stocks over the range 200 kilobase pairs to greater than 1 megabase pairs. The karyotype of each stock was different. Three DNA probes, B10, B30 and B31, derived from the Netsky stock isolated in America were used as chromosome markers. Probe B10 hybridized to chromosomes of the same size in two of the stocks, one of which was isolated in the U.S.A. and the other in Queensland. B30 and B31 hybridized to a similar number of chromosomes of different sizes in these two stocks. The third stock, from Australia, did not hybridize at all with probes B10 and B30 and only weakly with probe B31.

Animals↗