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Biomedical subjects

J L Clarke

Publications and source records attributed to J L Clarke.

At least 19 recordsLinked to original sources
PubMed2002

Scrotal calcification: ultrasound appearances, distribution and aetiology.

This pictorial review illustrates the ultrasound appearances of scrotal calcification, distinguishing between intratesticular and extratesticular calcification. Intratesticular calcification may be due to phleboliths, spermatic granulomas or vascular calcification, or it may occur in association with tumours. Extratesticular calcification is more frequently encountered and is usually related to previous inflammatory disease of the epididymis. Testicular microlithiasis, a rare condition characterized by multiple scattered echogenic foci within the testis, is produced by the formation of microliths from degenerating cells in the seminiferous tubules. Testicular microlithiasis has been demonstrated as an incidental finding as well as in association with both benign and malignant tumours of the testis.

Calcinosis↗
PubMed2001

The genetic regulation of fucosylated and sialylated antigens on developing myeloid cells.

The first part of this article reviews the stages of normal development of haemopoietic cells committed to the myeloid lineage, properties of leukaemic cell lines that are arrested at specific maturation stages along the granulocytic pathway, the structures of carbohydrate antigenic markers that appear on myeloid cell surfaces, with especial reference to sialyl-Le(x) (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3]GlcNAc), and the role of this antigen on mature granulocytes as a ligand for selectin molecules. The families of fucosyl- and sialyltransferase genes encoding enzymes responsible for the biosynthesis of sialyl-Le(x), and the pathways leading to the formation of this antigen, and more complex related structures, are described. The second part of the article outlines the work carried out in the authors' laboratory with leukaemic cell lines in an attempt to ascertain the biochemical and genetic basis of the lowering of sialyl-Le(x) expression that occurs at intermediate stages of normal haemopoietic development. Analysis of enzyme levels and mRNA expression of the fucosyl- and sialyltransferase genes has led to the conclusion that depletion of substrate resulting from high levels of enzyme activity from co-expressed genes FUT4 and ST6Gal1 probably accounts for the dip in expression of sialyl-Le(x), rather than a change in the level of expression of FUT7, the gene in myeloid cells encoding the enzyme ultimately responsible for the synthesis of sialyl-Le(x). The possible significance of this change in relation to normal cell maturation is discussed.

Animals↗
PubMed2001

Glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase. A novel bifunctional enzyme in malaria parasites.

Plasmodium falciparum glucose 6-phosphate dehydrogenase (Pf Glc6PD), compared to other Glc6PDs has an additional 300 amino acids at the N-terminus. They are not related to Glc6PD but are similar to a family of proteins (devb) of unknown function, some of which are encoded next to Glc6PD in certain bacteria. The human devb homologue has recently been shown to have 6-phosphogluconolactonase (6PGL) activity. This suggests Pf Glc6PD may be a bifunctional enzyme, the evolution of which has involved the fusion of adjacent genes. Further functional analysis of Pf Glc6PD has been hampered because parts of the gene could not be cloned. We have isolated and sequenced the corresponding Plasmodium berghei gene and shown it encodes an enzyme (Pb Glc6PD) with the same structure as the P. falciparum enzyme. Pb Glc6PD is 950 amino acids long with significant sequence similarity in both the devb and Glc6PD domains with the P. falciparum enzyme. The P. berghei enzyme does not have an asparagine-rich segment between the N and C halves and it contains an insertion at the same point in the Glc6PD region as the P. falciparum enzyme but the insertion in the P. berghei is longer (110 versus 62 amino acids) and unrelated in sequence to the P. falciparum insertion. Though expression of this enzyme in bacteria produced largely insoluble protein, conditions were found where the full-length enzyme was produced in a soluble form which was purified via a histidine tag. We show that this enzyme has both Glc6PD and 6PGL activities. Thus the first two steps of the pentose phosphate pathway are catalysed by a single novel bifunctional enzyme in these parasites.

Amino Acid Sequence↗
PubMed2000

A randomised crossover trial of chemotherapy in the home: patient preferences and cost analysis.

OBJECTIVES: To determine patient preferences and cost differences between home-based and hospital-based chemotherapy. DESIGN: Randomised crossover trial. SETTING: A tertiary cancer hospital in Melbourne, Victoria. PARTICIPANTS: 20 patients who required chemotherapy suitable for administration at home. INTERVENTIONS: Patients were assigned at random to receive their first chemotherapy treatment in either the home or the hospital and the second treatment in the alternative setting. MAIN OUTCOME MEASURES: Patient preference, costs. RESULTS: There was universal agreement by the 20 patients in the randomised trial that home-based chemotherapy was the preferred option (P < 0.0001). No problems were nominated by the patients as being associated with home-based chemotherapy. Home-based treatment was estimated to result in an increased cost of $83 (P = 0.0002) for each chemotherapy treatment compared with hospital-based treatment. Reported advantages for chemotherapy in the home included the elimination of travel, reduction in treatment-associated anxiety, reduction in the burden on carers and family, and the ability to continue other duties. There were no significant complications associated with administration of chemotherapy in the home. CONCLUSIONS: Patients prefer home-based chemotherapy to hospital-based treatment. The future of chemotherapy-in-the-home programs in Australia will depend on whether patient preferences are deemed to offset any potential increase in costs.

Aged↗
PubMed2000

Meckel's diverticulitis in an elderly man diagnosed by computed tomography.

Meckel's diverticulum is the most common congenital abnormality of the gastrointestinal tract. Complications most frequently arise in children younger than 2 years who present with gastrointestinal bleeding. The diagnosis is usually made via radionuclide scintigraphy or intraoperatively. The authors report a 71-year-old man who developed a sudden onset of right lower quadrant abdominal pain, without bleeding, and was diagnosed as having Meckel's diverticulitis via computed tomography. The presence of Meckel' s diverticulitis was confirmed at surgery. Complications of a Meckel's diverticulum must be considered at any age. Computed tomography is another modality that may be helpful in the preoperative diagnosis.

Acute Disease↗
PubMed1999

Expression of human alpha-l-fucosyltransferase gene homologs in monkey kidney COS cells and modification of potential fucosyltransferase acceptor substrates by an endogenous glycosidase.

Previous investigations on the monkey kidney COS cell line demonstrated the weak expression of fucosylated cell surface antigens and presence of endogenous fucosyltransferase activities in cell extracts. RT-PCR analyses have now revealed expression of five homologs of human fucosyltransferase genes, FUT1, FUT4, FUT5, FUT7, and FUT8, in COS cell mRNA. The enzyme in COS cell extracts acting on unsialylated Type 2 structures is closely similar in its properties to the alpha1,3-fucosyltransferase encoded by human FUT4 gene and does not resemble the product of the FUT5 gene. Although FUT1 is expressed in the COS cell mRNA, it has not been possible to demonstrate alpha1,2-fucosyltransferase activity in cell extracts but the presence of Le(y) and blood-group A antigenic determinants on the cell surface imply the formation of H-precursor structures at some stage. The most strongly expressed fucosyltransferase in the COS cells is the alpha1,6-enzyme transferring fucose to the innermost N -acetylglucosamine unit in N -glycan chains; this enzyme is similar in its properties to the product of the human FUT8 gene. The enzymes resembling the human FUT4 and FUT8 gene products both had pH optima of 7.0 and were resistant to 10 mM NEM. The incorporation of fucose into asialo-fetuin was optimal at 5.5 and was inhibited by 10 mM NEM. This result initially suggested the presence of a third fucosyltransferase expressed in the COS cells but we have now shown that triantennary N- glycans with terminal nonreducing galactose units, similar to those present in asialo-fetuin, are modified by a weak endogenous beta-galactosidase in the COS cell extracts and thereby rendered suitable substrates for the alpha1,6-fucosyltransferase.

ABO Blood-Group System↗
PubMed1997

Three different endogenous alpha-L-fucosyltransferases expressed in COS cells.

The monkey kidney COS cell line is frequently used for the transient expression of cloned human fucosyltransferase cDNAs in the belief that negligible endogenous expression of fucosyltransferase genes occurs in these cells. In the course of transfection experiments we observed weak cell surface expression of sialyl-Lex and weak fucosyltransferase activity in extracts of control untransfected cells. Since these activities could complicate interpretation of the results with the transfected genes, a more detailed examination was undertaken that has now revealed expression of three different fucosyltransferases in the cells. One enzyme, which utilises N-acetyllactosamine as substrate, has a pH optimum of 7.0, is resistant to heat inactivation, and has been tentatively identified as an alpha1,3-fucosyltransferase. A second enzyme which acts on asialo-fetuin has a pH optimum of 5.5 and is rapidly inactivated by heat; the acceptor sugar and positional linkage of the transferred fucose are not yet established. A third enzyme that utilises asialo-agalacto-fetuin as acceptor is provisionally identified as an alpha1,6-fucosyltransferase.

Animals↗
PubMed1997

Developmental regulation of alpha 1,3-fucosyltransferase expression in CD34 positive progenitors and maturing myeloid cells isolated from normal human bone marrow.

The adhesive interactions of hemopoietic cells within the bone marrow regulate their distribution, growth, and development. Fucosylated structures, of which sialyl Lewis x has been most extensively studied, are important ligands for selectins, but little is known about their function or regulation during normal hemopoietic development. We have studied alpha 1,3-fucosyltransferase activity in CD34 positive progenitors and myeloid cells at different stages of maturation isolated form normal human bone marrow, together with mRNA levels of Fuc-TIV and Fuc-TVII. Enzyme activity measured with H type 2 acceptor was present at all stages but was markedly elevated in fractions of early myeloid cells enriched for promyelocytes, correlating with the appearance of Lewis x on these cells, and thereafter fell progressively as cells matured. Activity measured with 3'sialyllactosamine was present in CD34+ cells and at all stages of maturation. Levels were low in promyelocyte/myelocyte transitional cells and increased, relative to those measured with H type 2, during the later stages of maturation; these changes correlate directly with a maturation-related increase in sialyl Lewis x expression. Using competitive quantitative RT-PCR, mRNA levels of Fuc-TIV and Fuc-TVII were similar in CD34+ cells, early myeloid and late myeloid cells. The significance of these findings in relation to fucosyltransferase activity, the synthesis of selectin ligands and differences between normal cells and leukemic cell lines is discussed.

Antigens, CD34↗
PubMed1997

Independent regulation of Fuc-TIV and Fuc-TVII genes leading to modulation of cell surface antigen expression in developing myeloid cells.

Fucosylated antigen expression, fucosyltransferase activities and expression of Fuc-TIV and Fuc-TVII genes have been measured in the human leukemic cell lines KG1a, arrested at the undifferentiated myeloblast stage of maturation and KG1, arrested at the myeloblast and early promyelocytic stage. The results are compared with those we earlier found for the later promyelocytic cell line HL-60 and the myelocyte form into which HL-60 cells can be induced to differentiate. These leukemic cell lines, and the differentiated HL-60 cells, are believed to correspond to four successive stages of myeloid maturation in the bone marrow. Fuc-TVII mRNA was strongly expressed in the myeloblastic KG1a cells but expression was less in KG1 and HL-60 cells. In contrast to the sharp fall in Fuc-TIV expression observed on differentiation of HL-60 cells, the expression of Fuc-TIV mRNA showed a progressive increase from KG1a to HL-60 cells; thus the peak of expression of this gene was at the HL-60 promyelocyte stage. This peak correlated with an increase in fucosyltransferase activity with nonsialylated acceptors and the transitory downregulation of cell surface sialyl-Le(x) expression and upregulation of Le(x), VIM-2 and Le(y) expression. The variations in levels of expression of the fucosylated antigens on the surface of the developing myeloid cells therefore correlate with variations in mRNA expression arising from the independent regulation of Fuc-TIV and Fuc-TVII genes.

Animals↗
PubMed1996

Alpha1,3-L-fucosyltransferase expression in developing human myeloid cells. Antigenic, enzymatic, and mRNA analyses.

In an attempt to correlate the cell surface expression of Lex and sialyl-Lex structures in immature and mature myeloid cells with the genes expressing alpha1,3-fucosyltransferase(s) we have examined: 1) the properties of the cellular alpha1,3-fucosyltransferases and the mRNA transcripts corresponding to the five cloned genes, Fuc-TIII, Fuc-TIV, Fuc-TV, Fuc-TVI, and Fuc-TVII, in mature granulocytes and in the myeloid cell line HL-60, before and after dimethyl sulfoxide-induced differentiation and 2) the properties of the alpha1,3-fucosyltransferases expressed in COS-7 cells transfected with plasmids containing Fuc-TIV and Fuc-TVII cDNAs. The previously shown increase in cell surface expression of sialyl-Lex on differentiation of HL-60 cells (Skacel P. O., Edwards A. J., Harrison C. T., and Watkins W. M. (1991) Blood 78, 1452-1460) is accompanied by a sharp fall in expression of Fuc-TIV mRNA and a persistence of expression of Fuc-TVII mRNA. The properties of the alpha1,3-fucosyltransferase expressed in COS-7 cells transfected with Fuc-TIV are consistent with this being the major gene responsible for the expression of Lex in the immature myeloid cells. In Northern blot analyses, no transcripts of Fuc-TIII, Fuc-TV, or Fuc-TVI were detected in total RNA from mature granulocytes or mRNA from HL-60 cells before or after differentiation. In total RNA from mature granulocytes, Fuc-TIV transcripts were only faintly visible, whereas Fuc-TVII transcripts were quite definitely expressed. The specificity properties of Fuc-TVII expressed in COS-7 cells are consistent with this gene being the major candidate alpha1, 3-fucosyltransferase controlling the expression of sialyl-Lex on mature cells. However, Lex continues to be expressed on the surface of mature granulocytes and cell extracts retain the capacity to transfer fucose to non-sialylated acceptor substrates. The question therefore remains as to whether these properties result from the weakly expressed Fuc-TIV gene or whether another alpha1, 3-fucosyltransferase gene remains to be identified.

Animals↗
PubMed1995

Purification, properties and possible gene assignment of an alpha 1,3-fucosyltransferase expressed in human liver.

alpha 1,3-Fucosyltransferase solubilized from human liver has been purified 40,000-fold to apparent homogeneity by a multistage process involving cation exchange chromatography on CM-Sephadex, hydrophobic interaction chromatography on Phenyl Sepharose, affinity chromatography on GDP-hexanolamine Sepharose and HPLC gel exclusion chromatography. The final step gave a major protein peak that co-chromatographed with alpha 1,3-fucosyltransferase activity and had a specific activity of approximately 5-6 mumol min-1 mg-1 and an M(r) approximately 44,000 deduced from SDS-PAGE and HPLC analysis. The purified enzyme readily utilized Gal beta 1-4GlcNAc, NeuAc alpha 2-3Gal beta 1-4GlcNAc and Fuc alpha 1-2Gal beta 1-4GlcNAc, with a preference for sialylated and fucosylated Type 2 acceptors. Fuc alpha 1-2Gal beta 1-4Glc and the Type 1 compound Gal beta 1-3GlcNAc were very poor acceptors and no incorporation was observed with NeuAc alpha 2-6Gal beta 1-4GlcNAc. A polyclonal antibody raised against the liver preparation reacted with the homologous enzyme and also with the blood group Lewis gene-associated alpha 1,3/1,4-fucosyltransferase purified from the human A431 epidermoid carcinoma cell line. No cross reactivity was found with alpha 1,3-fucosyltransferase(s) isolated from myeloid cells. Examination by Northern blot analysis of mRNA from normal liver and from the HepG2 cell line, together with a comparison of the specificity pattern of the purified enzyme with that reported for the enzyme expressed in mammalian cells transfected with the Fuc-TVI cDNA, suggests a provisional identification of Fuc-TVI as the major alpha 1,3-fucosyltransferase gene expressed in human liver.

Ammonium Sulfate↗
PubMed1989

Immunoselection techniques for cloning DNA-encoding parasite-specific antigens.

New techniques sometimes generate a 'band- waggon' effect, with research workers keen to jump on and apply the technique in their own favourite field without always pausing to consider why. Michael Miles and Louise Clarke describe a new technique which they are applying to a well-defined and valuable end - improvement in the differential diagnosis of parasitic infections.

Journal Article↗
PubMed1989

Multiple peptide synthesis (Pepscan method) for the systematic analysis of B- and T-cell epitopes: Application to parasite proteins.

In 1984 Mario Geysen and his colleagues described a technique for the simultaneous synthesis of hundreds of peptides on polyethylene rods. The peptides, still on the rods, could be used directly in enzyme-linked immunosorbent assays (ELISAs) and in this way linear parts of B-cell epitopes could be mapped. For the analysis of T-cell epitopes, peptides can be cleaved from the rods and incorporated into proliferation assays. This method, called the 'Pepscan' procedure, has been used for the detailed characterization of epitopes of viruses, Chlamydia and Mycobacteria: it is a powerful new approach to the epitope mapping of parasite proteins.

Journal Article↗