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Biomedical subjects

H Ginsburg

Publications and source records attributed to H Ginsburg.

At least 19 recordsLinked to original sources
PubMed2006

Can biochemical properties serve as selective pressure for gene selection during inter-species and endosymbiotic lateral gene transfer?

During the evolution of endosymbiosis, only one orthologous gene, either from the invader or the invaded genome, is preserved. Genetic and environmental factors are usually invoked to explain this gene preference. How biochemical parameters can play a role in the selection of genes that code for enzymes that constitute a metabolic pathway is explored. Simple Michaelis-Menten-like enzymes are considered whose kinetic parameters are randomly generated to construct two parallel homologous pathways to account for the contributions of the invaded and the invader. Steady-state fluxes as targets of natural selection are focused. Enzymes are eliminated one by one so that the total flux through the pathway is least disturbed. Analysis of the results, done by different criteria, indicate that the maximal velocities, both forward and backward, are more influential in selection than the respective Michaelis constants. This inclination disappears as metabolite concentrations are increased. It is shown that kinetic selection criteria can result in a mosaicism of enzymes in the same pathway in terms of their genetic origin. Analysis of the results using the control coefficient paradigm disclosed an expected robust correlation between flux control coefficients of enzymes and their selective elimination. Similar analyses, performed for the case of single gene transfer or for gene replication with subsequent mutation, yielded essentially similar results. The results conform with the phenomenon of genetic mosaicism found in phylogenetic analyses of single or double endosymbioses and lateral gene transfer.

Animals↗
PubMed2004

The new permeability pathways induced by the malaria parasite in the membrane of the infected erythrocyte: comparison of results using different experimental techniques.

The membrane of erythrocytes infected with malaria parasites is highly permeable to a large variety of solutes, including anions, carbohydrates, amino acids, nucleosides, organic and inorganic cations and small peptides. The altered permeability is presumed to be due to the activation of endogenous dormant channels, the new permeability pathways. The latter have been studied by different techniques-isosmotic lysis and tracer fluxes-and recently by patch-clamping. Here we analyze all available published data and we show that there is generally a good agreement between the two first methods. From the fluxes we calculate the number of channels per cell using reasonable assumptions as to the radius of the channel, and assuming that penetration through the channel is by diffusion through a water-filled space. The number of channels so calculated is <10 for most solutes, but approximately 400 for anions and the nucleosides thymidine and adenosine. This latter number is not far from that calculated from patch-clamp experiments. However, the anion flux measured directly by tracer is an order of magnitude larger than expected from conductance measurements. We conclude that the new permeability pathways consist of two types of channels; one is present in small number, and is charge- and size-selective. The other type is about 100-fold more abundant and is anion-selective, but does not admit non-electrolytes other than perhaps nucleosides.

Animals↗
PubMed2003

The treatment of Plasmodium falciparum-infected erythrocytes with chloroquine leads to accumulation of ferriprotoporphyrin IX bound to particular parasite proteins and to the inhibition of the parasite's 6-phosphogluconate dehydrogenase.

Ferriprotoporphyrin IX (FPIX) is a potentially toxic product of hemoglobin digestion by intra-erythrocytic malaria parasites. It is detoxified by biomineralization or through degradation by glutathione. Both processes are inhibited by the antimalarial drug chloroquine, leading to the accumulation of FPIX in the membranes of the infected cell and their consequent permeabilization. It is shown here that treatment of Plasmodium falciparum-infected erythrocytes with chloroquine also leads to the binding of FPIX to a subset of parasite proteins. Parasite enzymes such as aldolase, pyrimidine nucleaside monophosphate kinase and pyrimidine 5'-nucleotidase were inhibited by FPIX in vitro, but only the activity of 6-phosphogluconate dehydrogenase was reduced significantly in cells after drug treatment. Additional proteins were extracted from parasite cytosol by their ability to bind FPIX. Sequencing of these proteins identified heat shock proteins 90 and 70, enolase, elongation factor 1-alpha, phoshoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, L-lactate dehydrogenase and gametocytogenesis onset-specific protein. The possible involvement of these proteins in the antimalarial mode of action of chloroquine is discussed. It is concluded that drug-induced binding of FPIX to parasite glycolytic enzymes could underlie the demonstrable inhibition of glycolysis by chloroquine. The inhibition of 6-phosphogluconate dehydrogenase could explain the reduction of the activity of the hexose monophosphate shunt by the drug. Inhibition of both processes is deleterious to parasite survival. Binding of FPIX to other proteins is probably inconsequential to the rapid killing of the parasite by chloroquine.

Animals↗
PubMed2002

Is the development of falciparum malaria in the human host limited by the availability of uninfected erythrocytes?

BACKGROUND: The development and propagation of malaria parasites in their vertebrate host is a complex process in which various host and parasite factors are involved. Sometimes the evolution of parasitaemia seems to be quelled by parasite load. In order to understand the typical dynamics of evolution of parasitaemia, various mathematical models have been developed. The basic premise ingrained in most models is that the availability of uninfected red blood cells (RBC) in which the parasite develops is a limiting factor in the propagation of the parasite population. PRESENTATION OF THE HYPOTHESIS: We would like to propose that except in extreme cases of severe malaria, there is no limitation in the supply of uninfected RBC for the increase of parasite population. TESTING THE HYPOTHESIS: In this analysis we examine the biological attributes of the parasite-infected RBC such as cytoadherence and rosette formation, and the rheological properties of infected RBC, and evaluate their effects on blood flow and clogging of capillaries. We argue that there should be no restriction in the availability of uninfected RBC in patients. IMPLICATION OF THE HYPOTHESIS: There is no justification for the insertion of RBC supply as a factor in mathematical models that describe the evolution of parasitaemia in the infected host. Indeed, more recent models, that have not inserted this factor, successfully describe the evolution of parasitaemia in the infected host.

Animals↗
PubMed2002

A non-radiolabelled ferriprotoporphyrin IX biomineralisation inhibition test for the high throughput screening of antimalarial compounds.

Intraerythrocytic malaria parasites produce large amounts of toxic ferriprotoporphyrin IX (FP) during their digestion of host cell haemoglobin. The inhibition of biomineralisation of FP to haemozoin (or beta-haematin) by antimalarial drugs underlies their mode of action. We have developed an in vitro microassay for testing the inhibition of biomineralisation by drugs. It is based on the detection by optical density measurement of solubilised beta-haematin remaining after contact with drugs. The assay uses a 192-microM haemin chloride solution in dimethyl sulfoxide, 96-well filtration microplates as well as normal microplates; it lasts 18-24h and requires a spectrophotometer. We determined by this assay the IC(50) of chloroquine phosphate (28microM) and quinine base (324microM) and showed that unlike previous methods it is insensitive to inorganic anions. We also determined the activity of synthetic dyes and plant extract to determinate the interference of coloured compounds on the accuracy of the test. We found that methylene blue, thionine (IC(50) 38 and 87microM, respectively), and an extract of plants that contains quinoline derivatives, inhibited the biomineralisation of FP regardless of their intrinsic colour.

Animals↗
PubMed2002

Mathematical modelling of malaria chemotherapy: combining artesunate and mefloquine.

Clinical data on the use of artesunate combined with mefloquine in a variety of treatment regimens and parasite loads in Thailand were modelled on the basis of experimentally determined pharmacokinetic data. The model assumed no pharmacodynamic interaction between artesunate and mefloquine, but that the parasites were already resistant to mefloquine. Predictions of the model accorded well with the data. In articular, in accordance with clinical observations, the model showed that monotherapy with either drug failed to cure at moderate parasitaemia, yet such patients could be treated effectively with the combination of 3 days of artesunate + mefloquine. For high levels of parasitaemia, 5 days of artesunate + mefloquine were needed. Simulations were also performed for situations of lower resistance to mefloquine and for the immune human populations found in Africa. The importance of mathematical modelling of combination therapy is borne out by this study and suggests its wider application for other drug combinations.

Animals↗
PubMed2001

Transglutaminase in Plasmodium parasites: activity and putative role in oocysts and blood stages.

Transglutaminase was identified in malaria parasites by immunofluorescence microscopy using alpha-transglutaminase antiserum. Functional enzyme was demonstrated in vivo and in vitro using labeled polyamines that become incorporated into protein substrates through TGase activity. In Plasmodium falciparum intraerythrocytic parasites, transglutaminase activity was stage-dependent: it was weak in ring-forms but much stronger in trophozoites and schizonts. High levels of activity were detected in P. gallinaceum zygotes and ookinetes and in capsules of oocysts developing on mosquito midguts. Unlike most known transglutaminases, the enzymatic activity in Plasmodium was Ca(2+)-independent. Furthermore, levels of activity were similar at 37 and 26 degrees C. Parasite transglutaminase may be responsible for the modification of erythrocytic cytoskeleton in infected cells and it may facilitate the construction of oocyst capsules by cross-linking mosquito-derived basement membrane components with Plasmodium-derived proteins.

Aedes↗
PubMed2000

Effect of ferriprotoporphyrin IX and non-heme iron on the Ca(2+) pump of intact human red cells.

Previous studies have shown that ferriprotoporphyrin IX (FP) and non-heme iron have a marked inhibitory effect on the Ca(2+)-Mg(2+)-ATPase activity of isolated red cell membranes, the biochemical counterpart of the plasma membrane Ca(2+) pump (PMCA). High levels of membrane-bound FP and non-heme iron have been found in abnormal red cells such as sickle cells and malaria-infected red cells, associated with a reduced life span. It was important to establish whether sublytic concentrations of FP and non-heme iron would also inhibit the PMCA in normal red cells, to assess the possible role of these agents in the altered Ca(2+) homeostasis of abnormal cells. Active Ca(2+) extrusion by the plasma membrane Ca(2+) pump was measured in intact red cells that had been briefly preloaded with Ca(2+) by means of the ionophore A23187. The FP and nonheme iron concentrations used in this study were within the range of those applied to the isolated red cell membrane preparations. The results showed that FP caused a marginal inhibition ( approximately 20%) of pump-mediated Ca(2+) extrusion and that non-heme iron induced a slight stimulation of the Ca(2+) efflux (11-20%), in contrast to the marked inhibitory effects on the Ca(2+)-Mg(2+)-ATPase of isolated membranes. Thus, FP and non-heme iron are unlikely to play a significant role in the altered Ca(2+) homeostasis of abnormal red cells.

Biological Transport↗
PubMed2000

Optimisation of flow cytometric measurement of parasitaemia in plasmodium-infected mice.

Mouse malaria is often used as a model for drug testing. The results of drug trials are monitored by tedious (and consequently, sometimes inaccurate) microscopic counting of blood smears, or by flow cytometry. We suggest an improved, accurate and time-saving flow cytometric method for determination of parasitaemias in mice infected with Plasmodium vinckei petteri or Plasmodium berghei. The method involves collection of drops of blood from the tail vein, fixation, storage, permeabilisation, staining and analysis with a visible range flow cytometer. Three nucleic acid dyes, YOYO-1, propidium iodide and acridine orange were compared. YOYO-1 was found to be the best stain for the discrimination of parasitised erythrocytes from non-infected ones. A good direct correlation was obtained between parasitaemia determined by conventional microscopy and parasitaemia measured by flow cytometry. Drug effects could be assessed by the cytometric method. For the detection of low level of parasitemia, parasitised cells were treated with RNAse to completely cancel RNA-derived signals originating from host reticulocytes. This procedure also revealed discrete peaks arising from red cells infected with multiple parasites or from parasites with different numbers of nuclei.

Animals↗
PubMed2000

Potent antimalarial activity of clotrimazole in in vitro cultures of Plasmodium falciparum.

The increasing resistance of the malaria parasite Plasmodium falciparum to currently available drugs demands a continuous effort to develop new antimalarial agents. In this quest, the identification of antimalarial effects of drugs already in use for other therapies represents an attractive approach with potentially rapid clinical application. We have found that the extensively used antimycotic drug clotrimazole (CLT) effectively and rapidly inhibited parasite growth in five different strains of P. falciparum, in vitro, irrespective of their chloroquine sensitivity. The concentrations for 50% inhibition (IC(50)), assessed by parasite incorporation of [(3)H]hypoxanthine, were between 0.2 and 1.1 microM. CLT concentrations of 2 microM and above caused a sharp decline in parasitemia, complete inhibition of parasite replication, and destruction of parasites and host cells within a single intraerythrocytic asexual cycle (approximately 48 hr). These concentrations are within the plasma levels known to be attained in humans after oral administration of the drug. The effects were associated with distinct morphological changes. Transient exposure of ring-stage parasites to 2.5 microM CLT for a period of 12 hr caused a delay in development in a fraction of parasites that reverted to normal after drug removal; 24-hr exposure to the same concentration caused total destruction of parasites and parasitized cells. Chloroquine antagonized the effects of CLT whereas mefloquine was synergistic. The present study suggests that CLT holds much promise as an antimalarial agent and that it is suitable for a clinical study in P. falciparum malaria.

Animals↗
PubMed2000

Experimental conditions for testing the inhibitory activity of chloroquine on the formation of beta-hematin.

Some antimalarial drugs act by inhibiting the process of ferriprotoporphyrin IX polymerization which protects the parasite against the noxious effect of this product of host cell hemoglobin digestion. As the quest for new drugs with a similar mode of action continues, high-throughput screening methods are needed. We demonstrate herein that such a recently described screening technique (Basilico et al., J. Antimicrob. Chemother. 42, 55-60, 1998) is considerably disturbed by certain ions. Thus, at the assay's pH 2.6, the phosphate ions are responsible for the inhibitory activity of chloroquine phosphate, rather than chloroquine itself. Using a combination of solubility tests and Fourier transform infrared spectrometry we also show that two alternative methods using higher pH's are also prone to salt effects and demonstrate that these can be minimized by extensive washing of the product with DMSO.

Antimalarials↗
PubMed2000

Antimalarial drug development and new targets.

The Molecular Approaches to Malaria (MAM2000) conference, Lorne, Australia, 2-5 February 2000, brought together world-class malaria research scientists. The development of new tools and technologies - transfection, DNA microarrays and proteomic analysis - and the availability of DNA sequences generated by the Malaria Genome Project, along with more classic approaches, have facilitated the identification of novel drug targets, the development of new antimalarials and the generation of a deeper understanding of the molecular mechanism(s) of drug resistance in malaria. It is hoped that combinations of these technologies could lead to strategies that enable the development of effective, efficient and affordable new drugs to overcome drug-resistant malaria, as discussed at MAM2000 and outlined here by Ian Macreadie and colleagues.

Animals↗
PubMed2000

A search for natural bioactive compounds in Bolivia through a multidisciplinary approach. Part IV. Is a new haem polymerisation inhibition test pertinent for the detection of antimalarial natural products?

The search for new antimalarial agents in plant crude extracts using traditional screening tests is time-consuming and expensive. New in vitro alternative techniques, based on specific metabolic or enzymatic process, have recently been developed to circumvent testing of antimalarial activity in parasite culture. The haem polymerisation inhibition test (HPIA) was proposed as a possible routine in vitro assay for the detection of antimalarial activity in natural products. A total of 178 plant extracts from the Pharmacopeia of the Bolivian ethnia Tacana, were screened for their ability to inhibit the polymerisation of haematin. Five extracts from Aloysia virgata (Ruíz & Pavón) A.L. Jussieu (Verbenaceae), Bixa orellana L. (Bixaceae), Caesalpinia pluviosa D.C. (Caesalpiniaceae), Mascagnia stannea (Griseb) Nied. (Malpighiaceae) and Trichilia pleenea (Adr. Jussieu) (Meliaceae) demonstrated more than 70% inhibition of haematin polymerisation at 2.5 mg/ml. The extracts were also tested for antimalarial activity in culture against F32 strain (chloroquine-sensitive) and D2 strain (chloroquine-resistant) of Plasmodium falciparum and in vivo against P. berghei. The extract from Caesalpinia pluviosa was the only one that showed activity in HPIA and in the classical test in culture. The accuracy and pertinence of HPIA, applied to natural products is discussed.

Animals↗
PubMed2000

Mathematical modelling of the chemotherapy of Plasmodium falciparum malaria with artesunate: postulation of 'dormancy', a partial cytostatic effect of the drug, and its implication for treatment regimens.

Although artesunate, one of the potent derivatives of the qinghaosu family of drugs for treating falciparum malaria, is already in use in the field, its therapeutic protocol has only been developed empirically by hit-or-miss. A pharmacokinetic-pharmacodynamic (PK-PD) model, required for creating such a protocol, is not straightforward. Artesunate presents extremely fast pharmacokinetics. As a result the stage specificity of its action must be treated explicitly. Also, use of standard PK-PD modelling fails to explain the clinical results. Our PK-PD modelling of its activity leads us to the postulation of the existence of a novel effect: a small fraction of the parasites, as a result of chemotherapeutic pressure, become cytostatic, or 'dormant'. At this stage, the parasite cycle is halted, making them unsusceptible to further dosing until wakening. This slows down the antimalarial activity of the drug, entailing either many frequent doses or an extended period of treatment and surveillance. Based on our modelling, we suggest a method for deciding on rational models of chemotherapy against falciparum malaria.

Animals↗
PubMed2000

Mathematical modelling of the within-host dynamics of Plasmodium falciparum.

The development of malaria due to Plasmodium falciparum is a complex, multi-stage process. It is usually characterized by an exponential growth in the number of parasite-infected erythrocytes, followed by marked oscillations in this number with a period of 48 h, which are eventually dampened. This course of events has been the subject of various mathematical models. In this paper we propose a new mathematical model for the in-host asexual erythrocytic development of P. falciparum malaria. Synchronicity of the infection is shown to be an inherent feature of infection, irrespective of the duration of merozoite release from the liver. It will, therefore, cause periodic symptoms, as known in malaria patients. We also simulate the effects of an induced host immune response and show how the level of immunity affects the development of disease. The simulations fit well with the clinical observations. We show how infection can become asynchronous and discuss the effect of desynchronization on the circulating and total parasitaemia and demonstrate that synchronized broods will show parasitaemia fluctuations.

Animals↗
PubMed2000

Antimalarial activities of dermaseptin S4 derivatives.

The hemolytic antimicrobial peptide dermaseptin S4 was recently shown to exert antimalarial activity. In this study, we attempted to understand the underlying mechanism(s) and identify derivatives with improved antimalarial activity. A number of dermaseptin S4 derivatives inhibited parasite growth with a 50% inhibitory concentration (IC(50)) in the micromolar range. Among these, the substituted S4 analog K(4)K(20)-S4 was the most potent (IC(50) = 0.2 microM), while its shorter version, K(4)-S4(1-13)a, retained a considerable potency (IC(50) = 6 microM). Both K(4)K(20)-S4 and K(4)-S4(1-13)a inhibited growth of the parasites more at the trophozoite stage than at the ring stage. Significant growth inhibition was observed after as little as 1 min of exposure to peptides and proceeded with nearly linear kinetics. The peptides selectively lysed infected red blood cells (RBC) while having a weaker effect on noninfected RBC. Thus, K(4)K(20)-S4 lysed trophozoites at concentrations similar to those that inhibited their proliferation, but trophozoites were >30-fold more susceptible than normal RBC to the lytic effect of K(4)K(20)-S4, the most hemolytic dermaseptin. The same trend was observed with K(4)-S4(1-13)a. The D isomers of K(4)K(20)-S4 or K(4)-S4(1-13)a were as active as the L counterparts, indicating that antimalarial activity of these peptides, like their membrane-lytic activity, is not mediated by specific interactions with a chiral center. Moreover, dissipation of transmembrane potential experiments with infected cells indicated that the peptides induce damage in the parasite's plasma membrane. Fluorescence confocal microscopy analysis of treated infected cells also indicated that the peptide is able to find its way through the complex series of membranes and interact directly with the intracellular parasite. Overall, the data showed that dermaseptins exert antimalarial activity by lysis of infected cells. Dermaseptin derivatives are also able to disrupt the parasite plasma membrane without harming that of the host RBC.

Amphibian Proteins↗