We have cloned, sequenced and expressed a gene of Haemonchus contortus that encodes a protein (te... more We have cloned, sequenced and expressed a gene of Haemonchus contortus that encodes a protein (termed HcCYP) consisting of a cyclophilin domain and an RNA recognition motif (RRM). An antiserum raised against the recombinant protein showed that HcCYP was present in the insoluble fraction (mostly nuclear) of the parasite homogenate. The recombinant protein possessed the typical cis-trans peptidyl-prolyl isomerase activity of cyclophilins and this activity was inhibited by the immunosuppressant cyclosporin A. The N-terminal portion of the molecule, carrying the RRM, was able to bind to nucleic acids, whereas the C-terminal portion did not have any binding activity. The possible function of HcCYP in the parasite is discussed on the basis of information available on similar proteins in other organisms.
Molecular and Biochemical Parasitology, Nov 1, 2002
Rapamycin (RAPA) is a well-known immunosuppressant, the action of which is mediated by the immuno... more Rapamycin (RAPA) is a well-known immunosuppressant, the action of which is mediated by the immunophilin FKBP12. Upon RAPA binding, FKBP12 forms ternary complexes with phosphatidyl inositol related kinases known as the target of RAPA (TOR), which can lead to a mitotic block at the G1 Á/S phase transition. Such an antiproliferative effect makes RAPA an attractive anticancer, antifungal or antiparasitic compound. In this study, we found the helminth parasite Schistosoma mansoni to be insensitive to the drug. In order to elucidate the mechanism underlying RAPA resistance, the S. mansoni drug receptor FKBP12 (SmFKBP12) was cloned for functional analysis. Western blot experiments showed that the protein is constitutively expressed in all life cycle stages and in both male and female parasites. The Escherichia coli-synthesised recombinant protein possessed enzymatic activity, which was inhibitable by RAPA. Moreover, SmFKBP12 was able to complement mutant Saccharomyces cerevisiae cells lacking FKBP12 in their RAPA sensitivity phenotype, leading us to conclude that SmFKBP12 is expressed in yeast in a functional form and capable of interacting with the drug and yeast TOR kinase. Even though the wild type SmFKBP12 appeared to restore a large part of RAPA sensitivity, a mutation of Asp 89 Á/Lys 90 to Pro 89 Á/Gly 90 in the schistosome protein was found to be more effective and restored drug sensitivity to the same level as the endogenous yeast protein. Despite ternary complex formation, our results suggest that additional unknown factors other than a functional drug receptor are implicated in drug resistance mechanisms.
mansoni: Relationship between parasite age and time of spontaneous elimination from the rat. Expe... more mansoni: Relationship between parasite age and time of spontaneous elimination from the rat. Experimental Parasitology 45, 74-80. Laboratory rats infected with Schistosoma munsoni invariably reject the majority of parasites between the fourth and sixth week after infection (self-cure). This investigation was designed to determine whether the timing of rejection is dictated by the rat or by the parasite. Schistosomes were perfused from rats infected 2, 3, or 4 weeks previously and were transferred into the mesenteric veins of normal rats. Recipient animals were perfused at weekly intervals after transfer, and the timing of worm elimination was determined in recipients. It was found that e-week-old worms were rejected 2 weeks after transfer, 3-week-old worms I week after transfer, and 4-week-old worms immediately after transfer. Schistosomes perfused from mice or hamsters and transferred into rats showed the same pattern of worm elimination. It is concluded that at the fourth week of normal schistosome development there is a critical event which makes virtually impossible any further survival of the parasite in laboratory rats.
Tropical Medicine & International Health, Feb 1, 2010
To determine whether praziquantel (PZQ) has retained its efficacy against Schistosoma haematobium... more To determine whether praziquantel (PZQ) has retained its efficacy against Schistosoma haematobium on Pemba Island after 20 years of mass administration--albeit discontinuous--and to analyse retrospectively the performance of schistosomiasis control programmes. A sample of Pemba schoolchildren was examined before and after PZQ treatment by urine filtration, macro- and micro-haematuria and viability of excreted eggs. Although 5% of treated children continued to pass some eggs in the urine up to the seventh week after PZQ administration, none of these eggs was viable, indicating an effective schistosomicidal activity followed by a slow release of dead eggs from host tissues. No signs of PZQ resistance could be detected in the population under study. An overall retrospective analysis of schistosomiasis control activities in Pemba Island revealed that mass drug administration is clearly effective in reducing infection prevalence, but soon after interruption of drug distribution prevalence returns rapidly to pre-intervention levels.
the larger size of the parasites themselves, by the number of schistosome eggs and their rate of ... more the larger size of the parasites themselves, by the number of schistosome eggs and their rate of deposition in the host liver. In addition, Ab/L mice could develop an acquired resistance t o schistosome re-infection which was as good as, or possibly even bettek than the resistance developed by Ab/H mice. These findings suggest that humoral antibodies per se may not play a critical role in schistosome immunity, and at the same time call attention t o the possible importance of macrophages in determining the results observed.
Two drug-resistant strains of Schistosoma mansoni were compared in this study in order to decide ... more Two drug-resistant strains of Schistosoma mansoni were compared in this study in order to decide whether they are both mutated in the same gene with respect to drug-sensitive schistosomes. One of the two strains was isolated in the laboratory, while the other one originated from a treated uncured patient and was subsequently drug selected in the laboratory. The approach consisted in a genetic complementation test performed essentially by crossing the two strains and assessing resistance in the progeny. Since no reappearance of drug sensitivity was detected in the progeny, it was concluded that the two strains failed to complement and were therefore mutated in the same gene. This finding suggests that a single step of drug activation operates in sensitive schistosomes and is ineffective in resistant worms.
Testing the hypothesis This is the essence of the scienti ic approach and when I do this I am con... more Testing the hypothesis This is the essence of the scienti ic approach and when I do this I am conscious of a highly exciting intellectual activity. I am also conscious that I may be attaching some emotion to my commitment to this hypothesis. I may feel "certain" that I am on the right track-this can mislead the scientist and often scientists are unwilling to renounce their own ideas. In this activity, an "Act of faith" is necessary for the immense intellectual and physical effort required to complete the necessary work. The formulation of the hypothesis often has involved a creative intuition and the choice of subject may involve a great feeling of compassion and emotion. This idea has been well described and discussed in Michael Polanyi's "Personal Knowledge" (especially under "Intellectual Passions" Polanyi) So the scientist is certainly using spiritual qualities, committing the whole of him/her self. Scienti ic colleagues The interaction with friends and colleagues is an essential activity during the agony of 2 INTRODUCTION 06
The chemotherapeutic effect of praziquantel against Schistosoma mansoni is dependent on host anti... more The chemotherapeutic effect of praziquantel against Schistosoma mansoni is dependent on host antibody response.
ABSTRACT Administration of the drug oxamniquine to mice with mature infections of Schistosoma man... more ABSTRACT Administration of the drug oxamniquine to mice with mature infections of Schistosoma mansoni results in differential elimination of male worms (Foster and Cheetham 1973; Popiel and Erasmus ~982). In the surviving females reduction in overall worm size took place. Electron microscope observations of the reproductive system (Popiel and Erasmus 1983) revealed regression of the ovary and vitelline gland to a stage resembling the structure of immature worms. A second dose of oxamniquine had no further visible effect on these worms, which resembled immature adults. Although a direct effect of the drug could not be ruled out, it was concluded that normal reproductive function in females is dependent on the continued presence of male worms and that the regression of the residual females was largely the result of discontinued male stimulation. To elucidate whether this phenomenon was an irreversible effect of drug action or a reversible effect of cessation of male stimulation, such "regressed" females were reunited in vivo with normal male worms. Oxamniquine, as a suspension in chremophor, was administered to mice with sexually mature (49 days) infections of S. mansoni (Puerto Rican strain) as a single oral dose of 25 or 50 mg/kg. Worms were recovered by perfusion with citrate saline 67 days later and the females to be transplanted were resuspended in medium RMI-1640 buffered with 20 mM Hepes, containing 10% horse serum. Nile rats (Arvicanthis niloticus) received such females together with normal sexually mature males, similarly obtained from a 16-week bisexual infection, using the transplantation technique of Cioli (1976). Controls consisted of regressed females transplanted alone. Worms
International Journal for Parasitology, Feb 1, 1980
... SCHRIBER RA & ZUCKER-FRANKLIN D. 1975. Induction of blood eosinophilia by pulmonary embol... more ... SCHRIBER RA & ZUCKER-FRANKLIN D. 1975. Induction of blood eosinophilia by pulmonary embolization of antigen-coated particles: The relationship to cell-mediated immunity. Journal of Immunology 114: 1348-1353. SHER A., SMITHERS SR & MACKENZIE P. 1975. ...
Schistosoma mansoni fatty acid binding protein (Sm14) was crystallized with bound oleic acid (OLA... more Schistosoma mansoni fatty acid binding protein (Sm14) was crystallized with bound oleic acid (OLA) and arachidonic acid (ACD), and their structures were solved at 1.85 and 2.4 A resolution, respectively. Sm14 is a vaccine target for schistosomiasis, the second most prevalent parasitic disease in humans. The parasite is unable to synthesize fatty acids depending on the host for these nutrients. Moreover, arachidonic acid (ACD) is required to synthesize prostaglandins employed by schistosomes to evade the host's immune defenses. In the complex, the hydrocarbon tail of bound OLA assumes two conformations, whereas ACD adopts a unique hairpin-looped structure. ACD establishes more specific interactions with the protein, among which the most important is a pi-cation bond between Arg78 and the double bond at C8. Comparison with homologous fatty acid binding proteins suggests that the binding site of Sm14 is optimized to fit ACD. To test the functional implications of our structural data, the affinity of Sm14 for 1,8-anilinonaphthalenesulfonic acid (ANS) has been measured; moreover the binding constants of six different fatty acids were determined from their ability to displace ANS. OLA and ACD exhibited the highest affinities. To determine the rates of fatty acid binding and dissociation we carried out stopped flow kinetic experiments monitoring displacement by (and of) ANS. The binding rate constant of ligands is controlled by a slow pH dependent conformational change, which we propose to have physiological relevance.
Molecular and Biochemical Parasitology, Oct 1, 1992
Crude extracts of hycanthone sensitive Schistosoma mansoni incubated at 37 degrees C in the prese... more Crude extracts of hycanthone sensitive Schistosoma mansoni incubated at 37 degrees C in the presence of ATP and Mg2+ induced the covalent binding of tritiated hycanthone (HC) to macromolecules. The same behavior was shown by the HC sensitive species, Schistosoma rodhaini, whereas two independently isolated HC resistant S. mansoni strains had no detectable activity. Sensitive male schistosomes had more activity than females or immature worms. Virtually no activity was present in mouse liver, in human liver, in HeLa cells or in the naturally resistant species Schistosoma japonicum. The activity was destroyed by boiling or by Proteinase K treatment. Covalent binding of tritiated HC to macromolecules could be inhibited by cold HC, oxamniquine or IA-4, while none of the in vitro ineffective analogs, like lucanthone, UK-3883 or 4-desmethyl lucanthone, were inhibitory. These results strongly support the previously advanced suggestion that HC is activated by enzymatic mechanisms which are present only in drug sensitive schistosomes.
The effect of methyl substitution on the biological properties of the ellipticines was reexamined... more The effect of methyl substitution on the biological properties of the ellipticines was reexamined. 9-Hydroxy-6H-pyrido[4,3-b]carbazole was synthesized and shown to be devoid of antitumor activity in murine P388 lymphocytic leukemia in mice. 5-(Hydroxymethyl)-11-methyl-6H-pyrido[4,3-b]carbazole (46) and its N-methylcarbamate (48) were synthesized and their effect on macromolecular synthesis in HeLa cells and their antitumor properties were compared with those of ellipticine. In contrast to the alkaloid 1 and the hydroxymethyl derivative 46, which produced partially reversible inhibition of [3H]thymidine incorporation, the carbamate ester irreversibly blocked incorporation of the tritiated pyrimidine. The ester was also a more potent antitumor agent in P388 lymphocytic leukemia than 1 or 46.
We have cloned, sequenced and expressed a gene of Haemonchus contortus that encodes a protein (te... more We have cloned, sequenced and expressed a gene of Haemonchus contortus that encodes a protein (termed HcCYP) consisting of a cyclophilin domain and an RNA recognition motif (RRM). An antiserum raised against the recombinant protein showed that HcCYP was present in the insoluble fraction (mostly nuclear) of the parasite homogenate. The recombinant protein possessed the typical cis-trans peptidyl-prolyl isomerase activity of cyclophilins and this activity was inhibited by the immunosuppressant cyclosporin A. The N-terminal portion of the molecule, carrying the RRM, was able to bind to nucleic acids, whereas the C-terminal portion did not have any binding activity. The possible function of HcCYP in the parasite is discussed on the basis of information available on similar proteins in other organisms.
Molecular and Biochemical Parasitology, Nov 1, 2002
Rapamycin (RAPA) is a well-known immunosuppressant, the action of which is mediated by the immuno... more Rapamycin (RAPA) is a well-known immunosuppressant, the action of which is mediated by the immunophilin FKBP12. Upon RAPA binding, FKBP12 forms ternary complexes with phosphatidyl inositol related kinases known as the target of RAPA (TOR), which can lead to a mitotic block at the G1 Á/S phase transition. Such an antiproliferative effect makes RAPA an attractive anticancer, antifungal or antiparasitic compound. In this study, we found the helminth parasite Schistosoma mansoni to be insensitive to the drug. In order to elucidate the mechanism underlying RAPA resistance, the S. mansoni drug receptor FKBP12 (SmFKBP12) was cloned for functional analysis. Western blot experiments showed that the protein is constitutively expressed in all life cycle stages and in both male and female parasites. The Escherichia coli-synthesised recombinant protein possessed enzymatic activity, which was inhibitable by RAPA. Moreover, SmFKBP12 was able to complement mutant Saccharomyces cerevisiae cells lacking FKBP12 in their RAPA sensitivity phenotype, leading us to conclude that SmFKBP12 is expressed in yeast in a functional form and capable of interacting with the drug and yeast TOR kinase. Even though the wild type SmFKBP12 appeared to restore a large part of RAPA sensitivity, a mutation of Asp 89 Á/Lys 90 to Pro 89 Á/Gly 90 in the schistosome protein was found to be more effective and restored drug sensitivity to the same level as the endogenous yeast protein. Despite ternary complex formation, our results suggest that additional unknown factors other than a functional drug receptor are implicated in drug resistance mechanisms.
mansoni: Relationship between parasite age and time of spontaneous elimination from the rat. Expe... more mansoni: Relationship between parasite age and time of spontaneous elimination from the rat. Experimental Parasitology 45, 74-80. Laboratory rats infected with Schistosoma munsoni invariably reject the majority of parasites between the fourth and sixth week after infection (self-cure). This investigation was designed to determine whether the timing of rejection is dictated by the rat or by the parasite. Schistosomes were perfused from rats infected 2, 3, or 4 weeks previously and were transferred into the mesenteric veins of normal rats. Recipient animals were perfused at weekly intervals after transfer, and the timing of worm elimination was determined in recipients. It was found that e-week-old worms were rejected 2 weeks after transfer, 3-week-old worms I week after transfer, and 4-week-old worms immediately after transfer. Schistosomes perfused from mice or hamsters and transferred into rats showed the same pattern of worm elimination. It is concluded that at the fourth week of normal schistosome development there is a critical event which makes virtually impossible any further survival of the parasite in laboratory rats.
Tropical Medicine & International Health, Feb 1, 2010
To determine whether praziquantel (PZQ) has retained its efficacy against Schistosoma haematobium... more To determine whether praziquantel (PZQ) has retained its efficacy against Schistosoma haematobium on Pemba Island after 20 years of mass administration--albeit discontinuous--and to analyse retrospectively the performance of schistosomiasis control programmes. A sample of Pemba schoolchildren was examined before and after PZQ treatment by urine filtration, macro- and micro-haematuria and viability of excreted eggs. Although 5% of treated children continued to pass some eggs in the urine up to the seventh week after PZQ administration, none of these eggs was viable, indicating an effective schistosomicidal activity followed by a slow release of dead eggs from host tissues. No signs of PZQ resistance could be detected in the population under study. An overall retrospective analysis of schistosomiasis control activities in Pemba Island revealed that mass drug administration is clearly effective in reducing infection prevalence, but soon after interruption of drug distribution prevalence returns rapidly to pre-intervention levels.
the larger size of the parasites themselves, by the number of schistosome eggs and their rate of ... more the larger size of the parasites themselves, by the number of schistosome eggs and their rate of deposition in the host liver. In addition, Ab/L mice could develop an acquired resistance t o schistosome re-infection which was as good as, or possibly even bettek than the resistance developed by Ab/H mice. These findings suggest that humoral antibodies per se may not play a critical role in schistosome immunity, and at the same time call attention t o the possible importance of macrophages in determining the results observed.
Two drug-resistant strains of Schistosoma mansoni were compared in this study in order to decide ... more Two drug-resistant strains of Schistosoma mansoni were compared in this study in order to decide whether they are both mutated in the same gene with respect to drug-sensitive schistosomes. One of the two strains was isolated in the laboratory, while the other one originated from a treated uncured patient and was subsequently drug selected in the laboratory. The approach consisted in a genetic complementation test performed essentially by crossing the two strains and assessing resistance in the progeny. Since no reappearance of drug sensitivity was detected in the progeny, it was concluded that the two strains failed to complement and were therefore mutated in the same gene. This finding suggests that a single step of drug activation operates in sensitive schistosomes and is ineffective in resistant worms.
Testing the hypothesis This is the essence of the scienti ic approach and when I do this I am con... more Testing the hypothesis This is the essence of the scienti ic approach and when I do this I am conscious of a highly exciting intellectual activity. I am also conscious that I may be attaching some emotion to my commitment to this hypothesis. I may feel "certain" that I am on the right track-this can mislead the scientist and often scientists are unwilling to renounce their own ideas. In this activity, an "Act of faith" is necessary for the immense intellectual and physical effort required to complete the necessary work. The formulation of the hypothesis often has involved a creative intuition and the choice of subject may involve a great feeling of compassion and emotion. This idea has been well described and discussed in Michael Polanyi's "Personal Knowledge" (especially under "Intellectual Passions" Polanyi) So the scientist is certainly using spiritual qualities, committing the whole of him/her self. Scienti ic colleagues The interaction with friends and colleagues is an essential activity during the agony of 2 INTRODUCTION 06
The chemotherapeutic effect of praziquantel against Schistosoma mansoni is dependent on host anti... more The chemotherapeutic effect of praziquantel against Schistosoma mansoni is dependent on host antibody response.
ABSTRACT Administration of the drug oxamniquine to mice with mature infections of Schistosoma man... more ABSTRACT Administration of the drug oxamniquine to mice with mature infections of Schistosoma mansoni results in differential elimination of male worms (Foster and Cheetham 1973; Popiel and Erasmus ~982). In the surviving females reduction in overall worm size took place. Electron microscope observations of the reproductive system (Popiel and Erasmus 1983) revealed regression of the ovary and vitelline gland to a stage resembling the structure of immature worms. A second dose of oxamniquine had no further visible effect on these worms, which resembled immature adults. Although a direct effect of the drug could not be ruled out, it was concluded that normal reproductive function in females is dependent on the continued presence of male worms and that the regression of the residual females was largely the result of discontinued male stimulation. To elucidate whether this phenomenon was an irreversible effect of drug action or a reversible effect of cessation of male stimulation, such "regressed" females were reunited in vivo with normal male worms. Oxamniquine, as a suspension in chremophor, was administered to mice with sexually mature (49 days) infections of S. mansoni (Puerto Rican strain) as a single oral dose of 25 or 50 mg/kg. Worms were recovered by perfusion with citrate saline 67 days later and the females to be transplanted were resuspended in medium RMI-1640 buffered with 20 mM Hepes, containing 10% horse serum. Nile rats (Arvicanthis niloticus) received such females together with normal sexually mature males, similarly obtained from a 16-week bisexual infection, using the transplantation technique of Cioli (1976). Controls consisted of regressed females transplanted alone. Worms
International Journal for Parasitology, Feb 1, 1980
... SCHRIBER RA & ZUCKER-FRANKLIN D. 1975. Induction of blood eosinophilia by pulmonary embol... more ... SCHRIBER RA & ZUCKER-FRANKLIN D. 1975. Induction of blood eosinophilia by pulmonary embolization of antigen-coated particles: The relationship to cell-mediated immunity. Journal of Immunology 114: 1348-1353. SHER A., SMITHERS SR & MACKENZIE P. 1975. ...
Schistosoma mansoni fatty acid binding protein (Sm14) was crystallized with bound oleic acid (OLA... more Schistosoma mansoni fatty acid binding protein (Sm14) was crystallized with bound oleic acid (OLA) and arachidonic acid (ACD), and their structures were solved at 1.85 and 2.4 A resolution, respectively. Sm14 is a vaccine target for schistosomiasis, the second most prevalent parasitic disease in humans. The parasite is unable to synthesize fatty acids depending on the host for these nutrients. Moreover, arachidonic acid (ACD) is required to synthesize prostaglandins employed by schistosomes to evade the host's immune defenses. In the complex, the hydrocarbon tail of bound OLA assumes two conformations, whereas ACD adopts a unique hairpin-looped structure. ACD establishes more specific interactions with the protein, among which the most important is a pi-cation bond between Arg78 and the double bond at C8. Comparison with homologous fatty acid binding proteins suggests that the binding site of Sm14 is optimized to fit ACD. To test the functional implications of our structural data, the affinity of Sm14 for 1,8-anilinonaphthalenesulfonic acid (ANS) has been measured; moreover the binding constants of six different fatty acids were determined from their ability to displace ANS. OLA and ACD exhibited the highest affinities. To determine the rates of fatty acid binding and dissociation we carried out stopped flow kinetic experiments monitoring displacement by (and of) ANS. The binding rate constant of ligands is controlled by a slow pH dependent conformational change, which we propose to have physiological relevance.
Molecular and Biochemical Parasitology, Oct 1, 1992
Crude extracts of hycanthone sensitive Schistosoma mansoni incubated at 37 degrees C in the prese... more Crude extracts of hycanthone sensitive Schistosoma mansoni incubated at 37 degrees C in the presence of ATP and Mg2+ induced the covalent binding of tritiated hycanthone (HC) to macromolecules. The same behavior was shown by the HC sensitive species, Schistosoma rodhaini, whereas two independently isolated HC resistant S. mansoni strains had no detectable activity. Sensitive male schistosomes had more activity than females or immature worms. Virtually no activity was present in mouse liver, in human liver, in HeLa cells or in the naturally resistant species Schistosoma japonicum. The activity was destroyed by boiling or by Proteinase K treatment. Covalent binding of tritiated HC to macromolecules could be inhibited by cold HC, oxamniquine or IA-4, while none of the in vitro ineffective analogs, like lucanthone, UK-3883 or 4-desmethyl lucanthone, were inhibitory. These results strongly support the previously advanced suggestion that HC is activated by enzymatic mechanisms which are present only in drug sensitive schistosomes.
The effect of methyl substitution on the biological properties of the ellipticines was reexamined... more The effect of methyl substitution on the biological properties of the ellipticines was reexamined. 9-Hydroxy-6H-pyrido[4,3-b]carbazole was synthesized and shown to be devoid of antitumor activity in murine P388 lymphocytic leukemia in mice. 5-(Hydroxymethyl)-11-methyl-6H-pyrido[4,3-b]carbazole (46) and its N-methylcarbamate (48) were synthesized and their effect on macromolecular synthesis in HeLa cells and their antitumor properties were compared with those of ellipticine. In contrast to the alkaloid 1 and the hydroxymethyl derivative 46, which produced partially reversible inhibition of [3H]thymidine incorporation, the carbamate ester irreversibly blocked incorporation of the tritiated pyrimidine. The ester was also a more potent antitumor agent in P388 lymphocytic leukemia than 1 or 46.
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Papers by Donato Cioli