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Biomedical subjects

P Greenwell

Publications and source records attributed to P Greenwell.

At least 19 recordsLinked to original sources
PubMed2005

Trichomonas vaginalis: paradigm of a successful sexually transmitted organism.

Trichomonas vaginalis (TV) is one of the most successful protozoan pathogens and one of the most common sexually transmitted organism in females, yet it is also one of the most poorly investigated. By producing a wide array of glycosidases and cysteine proteinase enzymes, the organism can easily adapt to the environment, harvesting host proteins and DNA for metabolism. With the ability to cause lesions, vaginitis and acute inflammatory disease of the genital mucosa, TV acts as a potential catalyst in the acquisition of secondary infections including human immunodeficiency virus (HIV) and human papillomavirus (HPV), the organism responsible for the pathogenesis of cervical cancer. Treatment of TV infection is relatively easy and could dramatically reduce the transmission of HIV in areas where TV is endemic.

AIDS-Related Opportunistic Infections↗
PubMed2004

Detection of human papillomavirus from liquid-based cytology specimens by in-house PCR: a pilot study.

The Papanicolaou smear remains the most common method for the detection of precancerous changes in cervical cytology. However, the introduction of a liquid-based cytology (LBC) technique expands the possibility of cervical intraepithelial neoplasia (CIN) diagnosis, and permits detection of precancerous changes and human papillomavirus (HPV) simultaneously. In the pilot study reported here, using an in-house polymerase chain reaction (PCR) method, high-grade HPV was detected in 32% of a cohort of 38 patients. This conventional PCR method could be developed for use on a real-time PCR platform or in a microtitre-well format and subsequently automated.

Adolescent↗
PubMed2001

Isoforms of Na+, K+-ATPase in human prostate; specificity of expression and apical membrane polarization.

The cellular distribution of Na+, K+-ATPase subunit isoforms was mapped in the secretory epithelium of the human prostate gland by immunostaining with antibodies to the alpha and beta subunit isoforms of the enzyme. Immunolabeling of the alpha1, beta1 and beta2 isoforms was observed in the apical and lateral plasma membrane domains of prostatic epithelial cells in contrast to human kidney where the alpha1 and beta1 isoforms of Na+, K+-ATPase were localized in the basolateral membrane of both proximal and distal convoluted tubules. Using immunohistochemistry and PCR we found no evidence of Na+, K+-ATPase alpha2 and alpha3 isoform expression suggesting that prostatic Na+, K+-ATPase consists of alpha1/beta1 and alpha1/beta2 isozymes. Our immunohistochemical findings are consistent with previously proposed models placing prostatic Na+, K+-ATPase in the apical plasma membrane domain. Abundant expression of Na+, K+-ATPase in epithelial cells lining tubulo-alveoli in the human prostate gland confirms previous conclusions drawn from biochemical, pharmacological and physiological data and provides further evidence for the critical role of this enzyme in prostatic cell physiology and ion homeostasis. Na+, K+-ATPase most likely maintains an inwardly directed Na+ gradient essential for nutrient uptake and active citrate secretion by prostatic epithelial cells. Na+, K+-ATPase may also regulate lumenal Na+ and K+, major counter-ions for citrate.

Aged↗
PubMed2000

Analysis of microsatellite mutations in the mitochondrial DNA of Saccharomyces cerevisiae.

In the nuclear genome of Saccharomyces cerevisiae, simple, repetitive DNA sequences (microsatellites) mutate at rates much higher than nonrepetitive sequences. Most of these mutations are deletions or additions of repeat units. The yeast mitochondrial genome also contains many microsatellites. To examine the stability of these sequences, we constructed a reporter gene (arg8(m)) containing out-of-frame insertions of either poly(AT) or poly(GT) tracts within the coding sequence. Yeast strains with this reporter gene inserted within the mitochondrial genome were constructed. Using these strains, we showed that poly(GT) tracts were considerably less stable than poly(AT) tracts and that alterations usually involved deletions rather than additions of repeat units. In contrast, in the nuclear genome, poly(GT) and poly(AT) tracts had similar stabilities, and alterations usually involved additions rather than deletions. Poly(GT) tracts were more stable in the mitochondria of diploid cells than in haploids. In addition, an msh1 mutation destabilized poly(GT) tracts in the mitochondrial genome.

DNA Mutational Analysis↗
PubMed2000

The application of databases and PCR in the cloning of glycosidase genes from the protozoan Tritrichomonas foetus.

Conserved sequence amplification (CSA) has been used to obtain sequence data for two glycosidase genes from the primitive eukaryote Tritrichomonas foetus. Few genes have been cloned from this organism, and there is little information concerning protein sequence. CSA is reliant on the use of database searches to identify short sequences of 3-9 amino acids conserved within a protein across a wide range of species. PCR primers are then constructed based on this sequence data and the DNA is amplified and sequenced. In the case of the beta-galactosidase gene, N-terminal amino acid sequence data were used to construct a primer that replaced the upstream primer to ensure the amplified product was related to beta-D-galactosidase. CSA was also applied to the gene encoding the enzyme beta-N-acetyl-D-glucosaminidase from T. foetus, but in this case a segment of DNA was amplified, which, if correct, should contain a third conserved motif. The products of the CSA were sequenced, and the data obtained were compared to data in the SwissProt database. The results obtained suggest that this approach is useful for the cloning of genes to obtain novel sequence data from organisms where little genetic information is available.

Acetylglucosaminidase↗
PubMed1998

An Israeli family with six cisAB members: serologic and enzymatic studies.

BACKGROUND: The cisAB blood type is a rare phenomenon in which both the A and B blood types are inherited from a single parent. Several forms of this phenotype have been characterized that differ with respect to serologic reactions and the activities of the gene-encoded blood group A and B transferases. STUDY DESIGN AND METHODS: The cisAB blood type was suspected when a baby typed as blood group O was born to a mother whose blood group was AB. Family studies revealed four generations in whom the pattern of inheritance could be explained only by the inheritance of the cisAB genotype. Blood and saliva samples from the family were tested serologically and assayed for the relevant glycosyltransferases. Samples suitable for DNA analysis were not available. RESULTS: Six family members were shown serologically to be of the cisAB type. The A and B transferases in the sera of these individuals were 20 to 35 and 25 to 50 percent of those obtained for A and B individuals, respectively. The enzymic characteristics of the A and B transferases were determined. The A transferase in the sera of the cisAB persons did not bind to agarose beads. CONCLUSION: The family described carries the cisAB gene, which encodes production of A and B transferases that differ from those of "normal" A, AB, and B controls. This variant has properties that are distinctly different from those described in other reports.

ABO Blood-Group System↗
PubMed1997

Purification and partial characterization of beta-galactosidase from Tritrichomonas foetus.

The work presented in this paper describes the purification and properties of a beta-galactosidase from the protozoan Tritrichomonas foetus. An inexpensive and straightforward method for extraction of the enzyme involving ammonium sulphate precipitation, ion exchange and affinity chromatography resulted in a high level of purification. After purification beta-N-acetylglucosaminidase was the only enzyme present as a contaminant at a significant level. The beta-galactosidase isolated had a pH optimum of 5.8. The Km determined at pH 5.8 was found to be 2.2 mM. Interesting results were obtained when studies were carried out to determine the effect of various metal ions on enzyme activity. Of the metal ions used in this study only manganese ions were found to activate the enzyme. This seems to be a characteristic of trichomonad enzymes, as N-acetyl-beta-glucosaminidase, alpha-galactosidase and N-acetyl-alpha-galactosaminidase are also activated by manganese ions. The strongest inhibition was recorded with lead and to a lesser extent by zinc. The result with lead is not unexpected as the heavy metal is known to cause irreversible inhibition by binding to the amino-acid backbone of the enzyme. The result with zinc is interesting as high levels of zinc are present and trichomonads are known to be apathogenic in semen. The purified beta-galactosidase was found to have the capacity to hydrolyse lactose (Gal beta1-4 Glc), lacto-N-biose 1 (Gal beta1-3 GlcNAc) and N-acetyllactosamine (Gal beta1-4 GlcNAc). When the enzyme was applied to a non-denaturing polyacrylamide gel a single band was observed when stained with Coomassie brilliant blue. This band coincided with that obtained when the gel was stained with p-nitrophenyl beta-galactopyranoside. When the same gel was incubated with p-nitrophenyl N-acetyl beta-glucopyranoside a band was detected which did not coincide with that of beta-galactosidase. Since the beta-N-acetylglucosaminidase enzyme does not move to the same position on a non-denaturing gel as the beta-galactosidase, we will use this technique to isolate the latter enzyme and determine the N-terminal sequence as a prelude to cloning and further study of the gene.

Animals↗
PubMed1997

Glycosidases in mucin-dwelling protozoans.

A range of protozoans were tested for the presence of glycosidases using p-nitrophenyl sugars as substrates. Some of the organisms were mucin dwellers whereas others were blood borne parasites. It had been hypothesized that glycosidase production would be significantly higher in the mucin dwellers. The results obtained demonstrated that the urogenital protozoans Tritrichomonas foetus and Trichomonas vaginalis produced a vast range of glycosidases which included those required for mucin breakdown. The gut dwelling protozoans Giardia lamblia and Entamoeba histolytica both produced beta-N-acetylglucosaminidase. G.lamblia also had detectable beta N-acetylgalactosaminidase activity, and small amounts of beta mannosidase were found in the extracts from E. histolytica. In contrast, little or no glycosidase activity was detected under the same experimental conditions in Leishmania donovani, Trypanosoma brucei or T. cruzi. The mucin dwelling protozoans all produce beta-N-acetylglucosaminidase but only the Trichomonads produced the range of enzymes required for complete breakdown of mucin. This seems to suggest that mucin breakdown is not a characteristic of all mucin dwelling protozoans.

Animals↗
PubMed1997

Blood group antigens: molecules seeking a function?

The blood group antigens have been dismissed by some researchers as merely 'icing on the cake' of glycoprotein structures. The fact that there are no lethal mutations and individuals have been described lacking ABO, H and Lewis antigens seems to lend weight to the argument. This paper reviews the research which suggests that these antigens do indeed have function and argues that blood group antigens play important roles in modulation of protein activity, infection and cancer. It explores the evidence and poses questions as to the relevance and implications of the results.

ABO Blood-Group System↗
PubMed1997

Microsatellite instability in yeast: dependence on repeat unit size and DNA mismatch repair genes.

We examined the stability of microsatellites of different repeat unit lengths in Saccharomyces cerevisiae strains deficient in DNA mismatch repair. The msh2 and msh3 mutations destabilized microsatellites with repeat units of 1, 2, 4, 5, and 8 bp; a poly(G) tract of 18 bp was destabilized several thousand-fold by the msh2 mutation and about 100-fold by msh3. The msh6 mutations destabilized microsatellites with repeat units of 1 and 2 bp but had no effect on microsatellites with larger repeats. These results argue that coding sequences containing repetitive DNA tracts will be preferred target sites for mutations in human tumors with mismatch repair defects. We find that the DNA mismatch repair genes destabilize microsatellites with repeat units from 1 to 13 bp but have no effect on the stability of minisatellites with repeat units of 16 or 20 bp. Our data also suggest that displaced loops on the nascent strand, resulting from DNA polymerase slippage, are repaired differently than loops on the template strand.

Chromosome Mapping↗
PubMed1995

A Bombay individual lacking H and Le antigens but expressing normal levels of alpha-2- and alpha-4-fucosyltransferases.

BACKGROUND: The rare Bombay phenotype is usually due to a primary genetic defect in an alpha-2- or alpha-4-fucosyltransferase. The present study was done to investigate a patient with normal transferases, who exhibits the Bombay phenotype. CASE REPORT: Red cells of the patient, his parents, and siblings were phenotyped for A, B, and H antigens. The presence of B, H, and Le transferases in serum and saliva was measured. RESULTS: The parents and siblings were all group B, Le(a-b-). The propositus was typed as Oh, Le(a-b-). His serum contained anti-A, anti-B, and anti-H. Normal levels of B, H, and Le transferases were found in all family members including the patient. CONCLUSION: In an unusual case, a person has the Bombay phenotype, but normal levels of transferases in serum and saliva. A general defect in fucose metabolism seems to be the primary abnormality in this case.

ABO Blood-Group System↗
PubMed1991

Genetic analysis of a meiotic recombination hotspot on chromosome III of Saccharomyces cerevisiae.

In a previous study, we analyzed meiotic recombination events that occurred in the 22-kb region (LEU2 to CEN3) of chromosome III of Saccharomyces cerevisiae. We found one region with an enhanced level of crossovers (a hotspot) and one region with a depressed level of crossovers. In this study, we show that about one-third of the crossovers that occur between LEU2 and CEN3 are initiated in a 1.3-kb region located approximately 6 kb from the centromere. Both crossovers and gene conversion events are initiated at this site. Events initiated at this position can be resolved as crossovers in regions located either centromere-distally or centromere-proximally from the initiation site.

Base Sequence↗
PubMed1991

Regulation of the oncodevelopmental expression of type 1 chain ABH and Lewis(b) blood group antigens in human colon by alpha-2-L-fucosylation.

Blood group antigen expression in the distal human colon is related to the development of the organ and is modified by malignant transformation. To elucidate the biochemical basis for these changes, we have (a) analysed the activity of glycosyltransferases coded for by the H, Se, Le, X, and A genes, in tissue biopsy specimens from normal and malignant proximal and distal human colon; (b) characterised the glycosphingolipids expressed in the various regions of normal and malignant colon by immunostaining of high performance thin layer chromatography plates; and (c) located the antigens on tissue sections from the same subjects by immunohistochemistry. In both secretors and non-secretors we found a significantly higher activity of alpha-2-L-fucosyltransferases in carcinomatous rectal tissue than in tissue from normal subjects, whereas the other transferase activities studied showed no significant differences. The acceptor substrate specificity suggested that both the Se and the H gene dependent alpha-2-L-fucosyltransferases are increased in carcinomas. In non-malignant tissue the only enzyme which showed appreciably higher activity in caecum than in rectum was alpha-2-L-fucosyltransferase. Immunochemistry and immunohistochemistry showed alpha-2-L-fucosylated structures in normal caecum from secretors and in tumour tissue from both secretors and non-secretors. We conclude that the alpha-2-L-fucosyltransferases control the expression of ABH, and Lewis(b) structures in normal and malignant colon.

ABO Blood-Group System↗
PubMed1988

Regulation of expression of carbohydrate blood group antigens.

The carbohydrate antigens associated with the human ABO and Lewis blood group systems are excellent models for the study of the genetic regulation of glycoconjugate biosynthesis because their expression on erythrocytes and in saliva has been thoroughly investigated in terms of classical genetics and the chemical structures and pathways for the formation of the antigens are now well understood. The primary protein products of the blood group genes are believed to be the glycosyltransferase enzymes that complete the biosynthesis of the determinants. The important controlling factors still to be elucidated are the genetic and environmental influences leading to the tissue specific expression of these antigens. The 3 types of regulation mechanisms discussed in this review are those arising: 1) from the specificity requirements of the glycosyltransferases encoded by the blood group genes; 2) from the competition or co-operation of glycosyltransferases encoded by genes at the same or independent loci; and 3) from the existence and tissue distribution of glycosyltransferases with related, but not identical, substrate specificities.

ABO Blood-Group System↗
PubMed1988

Zulu XX/XX dispermic chimaera from Natal with two populations of red blood cells and patchy skin pigmentation.

The chimaera is female and has two children. Her blood contains 99% group O, type AcP:BA, Pep-A:8-2 and 1% group A2B, type AcP:RA, Pep-A:1 red cells. H-, A- and B-transferase activities were demonstrated in her serum. The level of the H enzyme activity is low but is at the lower end of the normal range for group O persons. The levels of the A and B enzymes are also low but are higher than expected in a person with 1% A2B red cells in the blood. The levels of the A and B enzymes indicate that tissues other than the chimaera's haemopoietic tissue carry her genetically A2B cell line and are contributing the corresponding transferases to her plasma. Gross patchy skin pigmentation is present on the upper part of her body. The chimaera has evidently inherited two dissimilar germ nuclei from each parent.

Blood Group Antigens↗
PubMed1988

Immunolocalization of blood group A gene specified alpha 1,3N-acetylgalactosaminyltransferase and blood group A substance in the trans-tubular network of the Golgi apparatus and mucus of intestinal goblet cells.

The subcellular distribution of blood group A gene specified alpha 1,3N-acetylgalactosaminyltransferase and its product was studied in human intestinal goblet cells by immunoelectron microscopy. The O-glycosylation step yielding blood group A-active glycoconjugates occurred in the trans region of the Golgi apparatus as indicated by the presence of immunolabel for both antigens. In the Golgi apparatus, immunoreactive alpha 1,3N-acetylgalactosaminyltransferase was detectable in trans cisternae and in the trans-tubular network which was found to be continuous with the cisternal stack and exhibited acid phosphatase activity. This demonstrates that in intestinal goblet cells (i) the trans-tubular network does not constitute a compartment distinct from trans cisternae, and (ii) structures corresponding to GERL are structurally and functionally part of the Golgi apparatus. In addition to immunolabel for transferase at the inner surface of the cisternal membranes, luminally located immunolabel indicating the presence of free, not membrane-associated transferase became first detectable in the trans-tubular network and early forming mucus droplets contained therein. Further, the content of mature mucus droplets as well as the extracellular mucus layer were labeled. Absence of immunolabel in blood group 0 subjects lacking the blood group A gene specified transferase and the apparent non-reactivity of the antibodies with carbohydrate epitopes indicates that free alpha 1,3N-acetylgalactosaminyltransferase is present in mucus droplets and becomes secreted by intestinal goblet cells.

ABO Blood-Group System↗