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J Van Roy

Publications and source records attributed to J Van Roy.

18 recordsLinked to original sources
PubMed2000

Molecular characterization of the first two enzymes of the pentose-phosphate pathway of Trypanosoma brucei. Glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase.

Trypanosomatids are parasitic protists that have part of their glycolytic pathway sequestered inside peroxisome-like organelles: the glycosomes. So far, at least one enzyme of the pentose-phosphate pathway has been found to be associated partially with glycosomes. Here, we describe how two genes from Trypanosoma brucei, coding for the first two enzymes of the pentose-phosphate pathway, i.e. glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase, were identified by in silico screening of trypanosome genome project data bases. These genes were cloned and sequenced. Analysis of the lactonase sequence revealed that it contained a C-terminal peroxisome targeting signal in agreement with its subcellular localization in the bloodstream form trypanosome (15% glycosomal and 85% cytosolic). However, the dehydrogenase sequence did not reveal any targeting signal, despite its localization inside glycosomes. The corresponding enzymes have been overexpressed in Escherichia coli and purified, and their biochemical characteristics have been determined.

Amino Acid Sequence↗
PubMed2000

Trypanosoma brucei contains a 2,3-bisphosphoglycerate independent phosphoglycerate mutase.

Assays of phosphoglycerate mutase (PGAM) activity in lysates of bloodstream form Trypanosoma brucei appeared not to require exogenous 2,3-bisphosphoglycerate, thus suggesting that this protist contains an enzyme belonging to the class of cofactor-independent PGAMs. A gene encoding a polypeptide with motifs characteristic for this class of enzymes was cloned. The predicted T. brucei PGAM polypeptide contains 549 amino acids, with Mr 60 557 and pI 5.5. Comparison with 15 cofactor-independent PGAM sequences available in databases showed that the amino-acid sequence of the trypanosome enzyme has 59-62% identity with plant PGAMs and 29-35% with eubacterial enzymes. A low 28% identity was observed with the only available invertebrate sequence. The trypanosome enzyme has been expressed in Escherichia coli, purified to homogeneity and subjected to preliminary kinetic analysis. Previous studies have shown that cofactor-dependent and -independent PGAMs are not homologous. It has been inferred that the cofactor-independent PGAMs are in fact homologous to a family of metalloenzymes containing alkaline phosphatases and sulphatases. Prediction of the secondary structure of T. brucei PGAM and threading the sequence into the known crystal structure of E. coli alkaline phosphatase (AP) confirmed this homology, despite the very low sequence identity. Generally, a good match between predicted (PGAM) and actual (AP) secondary structure elements was observed. In contrast to trypanosomes, glycolysis in all vertebrates involves a cofactor-dependent PGAM. The presence of distinct nonhomologous PGAMs in the parasite and its human host offers great potential for the design of selective inhibitors which could form leads for new trypanocidal drugs.

2,3-Diphosphoglycerate↗
PubMed1995

Molecular analysis of glyceraldehyde-3-phosphate dehydrogenase in Trypanoplasma borelli: an evolutionary scenario of subcellular compartmentation in kinetoplastida.

In Trypanoplasma borelli, a representative of the Bodonina within the Kinetoplastida, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was detected in both the cytosol and glycosomes. This situation is similar to that previously found in Trypanosomatidae, belonging to a different Kinetoplastida suborder. In Trypanosomatidae different isoenzymes, only distantly related, are responsible for the activity in the two cell compartments. In contrast, immunoblot analysis indicated that the GAPDH activity in cytosol and glycosomes of T. borelli should be attributed to identical or at least very similar proteins related to the glycosomal GAPDH of Trypanosomatidae. Moreover, only genes related to the glycosomal GAPDH genes of Trypanosomatidae could be detected. All attempts to identify a gene related to the one coding for the trypanosomatid cytosolic GAPDH remained unsuccessful. Two tandemly arranged genes were found which are 95% identical. The two encoded polypeptides differ in 17 residues. Their sequences are 72-77% identical to the glycosomal GAPDH of the other Kinetoplastida and share with them some characteristic features: an excess of positively charged residues, specific insertions, and a small carboxy-terminal extension containing the sequence -AKL. This tripeptide conforms to the consensus signal for targeting of proteins to glycosomes. One of the two gene copies has undergone some mutations at positions coding for highly conserved residues of the active site and the NAD(+)-binding domain of GAPDH. Modeling of the protein's three-dimensional structure suggested that several of the substitutions compensate each other, retaining the functional coenzyme-binding capacity, although this binding may be less tight. The presented analysis of GAPDH in T. borelli gives further support to the assertion that one isoenzyme, the cytosolic one, was acquired by horizontal gene transfer during the evolution of the Kinetoplastida, in the lineage leading to the suborder Trypanosomatina (Trypanosoma, Leishmania), after the divergence from the Bodonina (Trypanoplasma). Furthermore, the data clearly suggest that the original GAPDH of the Kinetoplastida has been compartmentalized during evolution.

Amino Acid Sequence↗
PubMed1993

Selective interaction of glycosomal enzymes from Trypanosoma brucei with hydrophobic cyclic hexapeptides.

Hydrophobic cyclic hexapeptides have been reported to selectively inhibit glycosomal triosephosphate isomerase from Trypanosoma brucei (Kuntz et al, 1992, Eur. J. Biochem., 207, 441-447). Here it is shown that this inhibition is not due to a specific interaction between the enzyme and soluble hydrophobic cyclic hexapeptides, but that it is the result of a coprecipitation of trypanosome triosephosphate isomerase with cyclic hexapeptides when the solubilities of the latter are exceeded. A study of the interaction of these hexapeptides with other glycosomal enzymes revealed that several of them, such as phosphoglycerate kinase and hexokinase, also coprecipitated with these peptides, whereas most of the homologous enzymes from other organisms did not coprecipitate, nor were they inactivated.

Amino Acid Sequence↗
PubMed1988

Demonstration of glycosomes (microbodies) in the Bodonid flagellate Trypanoplasma borelli (Protozoa, Kinetoplastida).

Homogenates of Trypanoplasma borelli were subjected to subcellular fractionation by sequential differential and isopycnic centrifugation in sucrose. Glycerol-3-phosphate dehydrogenase and the glycolytic enzymes, glucosephosphate isomerase and triosephosphate isomerase, as well as the peroxisomal marker enzyme catalase were mainly, or in part, associated with sedimentable particles that had a buoyant density in sucrose of 1.22 g cm-3. Moreover, triosephosphate isomerase exhibited latency, both in total homogenates and in the particulate fraction. Electron microscopy of thin sections of T. borelli revealed the presence of microbodies that gave a positive reaction for catalase. Pyruvate kinase behaved as a typical soluble enzyme. It was stimulated by micromolar concentrations of fructose 2,6-bisphosphate and this stimulation was counteracted by inorganic phosphate in the millimolar range. The enzymes involved in the synthesis and degradation of fructose 2,6-bisphosphate, 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase, were both present in T. borelli and behaved as soluble enzymes. We conclude that in T. borelli the glycolytic pathway is compartmentalized in a way similar to that found in another Kinetoplastid family, the Trypanosomatidae, where seven glycolytic enzymes and two enzymes of glycerol metabolism are associated with glycosomes. Apparently the presence of glycosomes is a characteristic of all Kinetoplastida.

Animals↗
PubMed1986

Localization of 3'-nucleotidase and calcium-dependent endoribonuclease in the plasma-membrane of Trypanosoma brucei.

We have characterized a 3'-nucleotidase activity of T. brucei. The enzyme has a pH optimum of 8.7, is inactivated by chelating agents and stimulated by divalent cations. It is inhibited by Zn2+, Mn2+, pyrophosphate and the trypanocidal drug suramin for which it has a Ki of 3 microM. From cell fractionation experiments it is concluded that the enzyme is located in the plasma membrane. Alkaline 3'-endoribonuclease is also located in the plasma membrane of T. brucei and this activity shares a great number of properties with the 3'-nucleotidase activity, including its sensitivity to suramin. The possibility that both 3'-nucleotidase and endonuclease activities are catalyzed by the same enzyme cannot be excluded.

Animals↗
PubMed1982

Involvement of lysosomes in the uptake of macromolecular material by bloodstream forms of Trypanosoma brucei.

To investigate whether the lysosomes of Trypanosoma brucei are capable of uptake of macromolecules after internalization by the cell, we used Triton WR-1339, a non-digestible macromolecular compound, which is known to cause a marked decrease in the density of hepatic lysosomes due to massive intralysosomal storage. Intraperitoneal administration of 0.4 g/kg Triton WR-1339 to rats infected with T. brucei led to the development of a large vacuole in the trypanosomes between nucleus and kinetoplast within 22 h. Higher doses (2 g/kg) led to the disappearance of the trypanosomes from the blood and resulted in permanent cures (greater than 100 days). Lysosomes isolated from the trypanosomes of animals treated with a sub-curative dose showed a decrease in equilibrium density of 0.03 g/cm3 in sucrose gradients. These lysosomes were partly damaged as evidenced by a reduction in latency and an increase in the non-sedimentable part of lysosomal enzymes. We conclude that acid proteinase and alpha-mannosidase-containing organelles of T. brucei take up exogenous macromolecules and must therefore be considered as true lysosomes and that Triton WR-1339 acts in T. brucei as a true lysosomotropic drug. Its trypanocidal action probably results from an interference with lysosomal function.

Animals↗
PubMedDate not supplied

Standardized model for diagnosing cervical carcinoma in situ based on the cytologic signs.

An attempt was made to optimize the recognition of cervical intraepithelial carcinoma in situ (CIS) by the adoption of a standard set of rules derived from a case-control study of 50 histologically confirmed incidence cases of CIS and a simple random sample of 100 controls from the same mass screening population examined between 1977 and 1984. One cytotechnologist screened the slides for all occurrences of a standard set of classic cytopathologic signs. Chromatin clumping, dyskeratosis, anisonucleosis and an increased nuclear-cytoplasmic ratio in cells from the deep layer and chromatin clumping and anisonucleosis in cells from the superficial layer showed an association with the diagnosis of CIS, in decreasing order of magnitude. These variables make up a binary discriminant model; adding vacuolization, anisocytosis, prominent nucleoli and multinucleation to this model did not appear to improve its discriminatory power. The discrimination function accommodates any a priori judgment regarding the probability and utility of diagnostic outcomes. Cytologists normally manipulate the probabilities and utilities intuitively; the model requires the translation of intuitive judgments into quantitative estimates. For example, in our cytology laboratory, the CIS pick-up rate is approximately 1 per 1,000 smears; a 1:1,000 negative utility ratio of a false positive versus a false negative is thus used in our model. Given this, our model has a sensitivity of 86% and a specificity of 96% upon reclassification, and its predictive power (true positives/false positives) is 21.5. This standardized cytodiagnosis model is relevant to any local diagnosis-specific probability and utility estimates.

Carcinoma in Situ↗
PubMedDate not supplied

Classification of cervical smears with discordance between the cytologic and/or histologic ratings.

The problems of diagnostic variability between certified cytotechnologists was studied. Three cytology laboratories submitted a total of 28 cervical smears that had a discordance between the cytologic and/or histologic ratings. Eight independent cytotechnologists provided blind readings on each slide, expressed as "absence of cervical intraepithelial neoplasia (CIN)" to "CIN III." The median rating was absence of CIN or CIN I for 8 slides, CIN II for 5 and CIN III for 15. With a kappa value greater than 0 reflecting agreement beyond chance expectation and a value of 0.40 indicating fair agreement, the kappa value for 8 X 28 ratings was 0.36 (P = .0001), with a 90% confidence interval (CI) between 0.34 and 0.37. The kappa value was 0.14 (P = .10), with a 90% CI between 0.10 and 0.18, on a subsample of nine smears with two or more positive cytology diagnoses but a negative histology. Sixteen of the 28 slides represented cases of histologically proven cancer. Treating cytologic diagnoses of CIN II and CIN III as positive, the sensitivity of the cytologist with reference to histology varied between 71% and 86% while the specificity ranged from 18% to 62%. The positive predictive value was 1/2.5 to 1/1 and the negative predictive value was 1/6 to 1/1. The predictive power (true positives/false positives) ranged from 1.0 to 2.2. The cytodiagnosis of these cervical smears from cases of discordance thus exhibited limited reliability. Standardization of the relevant cytologic knowledge and its routine application is needed to improve the level of performance.

Adult↗