Antimalarials increase vesicle pH in Plasmodium falciparum.

The asexual erythrocytic stage of the malarial parasite ingests and degrades the hemoglobin of its host red cell. To study this process, we labeled the cytoplasm of uninfected red cells with fluorescein-dextran, infected those cells with trophozoite- and schizont-rich cultures of Plasmodium falciparum, and harvested them 110-120 h later in the trophozoite stage. After lysis of the red cell cytoplasm with digitonin, the only fluorescence remaining was in small (0.5-0.9 micron) vesicles similar to the parasite's food vacuole. As measured by spectrofluorimetry, the pH of these vesicles was acid (initial pH 5.2-5.4), and they responded to MgATP with acidification and to weak bases such as NH4Cl with alkalinization. These three properties are similar to those obtained with human fibroblasts and suggest that the endocytic vesicles of plasmodia are similar to those of mammalian cells. Each of the antimalarials tested (chloroquine, quinine, and mefloquine) as well as NH4Cl inhibited parasite growth at concentrations virtually identical to those that increased parasite vesicle pH. These results suggest two conclusions: (a) The increases in vesicle pH that we have observed in our digitonin-treated parasite preparation occur at similar concentrations of weak bases and antimalarials in cultures of parasitized erythrocytes, and (b) P. falciparum parasites are exquisitely dependent on vesicle pH during their asexual erythrocytic cycle, perhaps for processes analogous to endocytosis and proteolysis in mammalian cells, and that antimalarials and NH4Cl may act by interfering with these events.

Efflux of chloroquine from Plasmodium falciparum: mechanism of chloroquine resistance.

Chloroquine-resistant Plasmodium falciparum accumulate significantly less chloroquine than susceptible parasites, and this is thought to be the basis of their resistance. However, the reason for the lower accumulation of chloroquine was unknown. The resistant parasite has now been found to release chloroquine 40 to 50 times more rapidly than the susceptible parasite, although their initial rates of chloroquine accumulation are the same. Verapamil and two other calcium channel blockers, as well as vinblastine and daunomycin, each slowed the release and increased the accumulation of chloroquine by resistant (but not susceptible) Plasmodium falciparum. These results suggest that a higher rate of chloroquine release explains the lower chloroquine accumulation, and thus the resistance observed in resistant Plasmodium falciparum.

Plasmodium falciparum maturation abolishes physiologic red cell deformability.

Normal red cells deform markedly as they pass through the spleen and the peripheral capillaries. In these studies, the effects of Plasmodium falciparum infection and maturation on the deformability of parasitized red cells exposed to fluid shear stress in vitro were examined by means of a rheoscope. Red cells containing the early (ring) erythrocytic stage of the parasite have impaired deformability at physiologic shear stresses, and recover their normal shape more slowly. Red cells containing more mature parasites (trophozoites or schizonts) exhibit no deformation under the same conditions. These results provide a mechanism to explain the ability of the spleen to remove parasitized red cells from the circulation of both immune and nonimmune hosts.

Antibiotic-induced lysis of enterococci.

Enterococci are resistant to penicillin killing in vivo and in vitro. Because some bacteria resistant to penicillin killing have reduced autolytic activity, we examined the lysis of clinical enterococcal isolates suspended in buffer (spontaneous lysis), and compared it with their susceptibility to antibiotic-induced lysis and killing. We found significant correlations between spontaneous and antibiotic-induced lysis, using five antibiotics that inhibit cell wall synthesis (penicillin, cephalothin, bacitracin, cycloserine, and vancomycin). Among isolates, strains more rapidly lysed by one antibiotic were more rapidly lysed by the other antibiotics, and more susceptible to spontaneous lysis. In studies involving a single strain grown in different media, spontaneous lysis also correlated closely with antibiotic-induced lysis. These results are consistent with a common mechanism for spontaneous and antibiotic-induced lysis, such as the autolytic enzyme system. Human serum was one of the least permissive media tested for enterococcal growth and antibiotic-induced lysis and killing. We suggest that the inhibitory effect of human serum on growth and the activation of the enterococcal autolytic enzyme system may be a critical factor in the resistance of enterococcal endocarditis to treatment with penicillin alone.

Aminoglycoside-inactivating enzymes in clinical isolates of Streptococcus faecalis. An explanation for resistance to antibiotic synergism.

Clinical isolates of enterococci (Streptococcus faecalis) with high-level resistance to both streptomycin and kanamycin (minimal inhibitory concentration >2,000 mug/ml), and resistant to synergism with penicillin and streptomycin or kanamycin were examined for aminoglycoside-inactivating enzymes. All of the 10 strains studied had streptomycin adenylyltransferase and neomycin phosphotransferase activities; the latter enzyme phosphorylated amikacin as well as its normal substrates, such as kanamycin. Substrate profiles of the neomycin phosphotransferase activity suggested that phosphorylation occurred at the 3'-hydroxyl position, i.e., aminoglycoside 3'-phosphotransferase. A transconjugant strain, which acquired high-level aminoglycoside resistance and resistance to antibiotic synergism after mating with a resistant clinical isolate, also acquired both enzyme activities. Quantitative phosphorylation of amikacin in vitro by a sonicate of the transconjugant strain inactivated the antibiotic, as measured by bioassay, and the phosphorylated drug failed to produce synergism when combined with penicillin against a strain sensitive to penicillin-amikacin synergism.No differences were found in the sensitivity of ribosomes from a sensitive and resistant strain when examined in vitro using polyuridylic acid directed [(14)C]-phenylalanine incorporation in the presence of streptomycin, kanamycin, or amikacin. Therefore, we conclude that aminoglycoside-inactivating enzymes are responsible for the aminoglycoside resistance, and resistance to antibiotic synergism observed in these strains.

Plasmid-mediated resistance to antibiotic synergism in enterococci.

Mating experiments have shown that high-level resistance (minimal inhibitory concentration greater than 2,000 microgram/ml) to streptomycin and kanamycin, and resistance to penicillin-streptomycin and penicillin-kanamycin synergism are transferable by conjugation from resistant clinical isolates of enterococci to a sensitive recipient strain. Cesium chloride-ethidium bromide ultracentrifugation revealed a satellite (plasmid) band in resistant clinical isolates and the transconjugant strains but not in the sensitive recipient. Examination of these satellite bands by agarose gel electrophoresis and electron microscopy demonstrated a common plasmid with a weight of 45 megadaltons. Novobiocin treatment of a resistant clinical isolate produced simultaneous loss of high-level resistance to streptomycin and kanamycin, and of resistance to penicillin-aminoglycoside synergism. These results suggest that (a) high-level resistance to streptomycin and kanamycin among some clinical isolates of enterococci is associated with a 45 megadalton plasmid, and (b) the same plasmid is also responsible for the resistance to penicillin-aminoglycoside synergism observed in these strains.

Physiologic rate of carrier-mediated Ca2+ entry matches active extrusion in human erythrocytes.

The intracellular Ca2+ concentration of nearly all cells is kept at submicromolar levels. The magnitudes of transmembrane Ca2+ movement that maintain this steady state in the human red blood cell have long been debated. Although there is agreement that the physiologic extrusion of Ca2+ by the well-characterized Ca2+. ATPase amounts to 45 mumol/liter cells per h (1982. Nature (Lond.). 298:478-481), the reported passive entry rates in physiological saline (2-20 mumol/liter cells per h) are all substantially lower. This discrepancy could be due to incomplete inhibition of the pump in the previous measurements of Ca2+ entry. We therefore examined both rate and mechanism of entry after completely inactivating the pump. This required pretreatment with iodoacetamide (to lower the intracellular ATP concentration) and vanadate (to inhibit any residual Ca2+ pump activity). The rate of Ca2+ entry (53 mumol/liter cells per h) was now found to be comparable to the accepted extrusion rate. Entry closely obeyed Michaelis-Menten kinetics (Vmax = 321 +/- 17 nmol Ca/g dry wt per h, Km = 1.26 +/- 0.13 mM), was competitively inhibited by external Sr2+ (Ki = 10.8 +/- 1.2 mM), and was accelerated by intracellular Ca2+. 45Ca2+ efflux from these pump-inactivated cells was also accelerated by either external Ca2+ or Sr2+. These accelerating effects of divalent cations on the opposite (trans) face of the membrane rule out a simple channel. Substrate-gated channels are also ruled out: cells equilibrated with 45Ca2+ lost the isotope when unlabeled Ca2+ or Sr2+ was added externally. Thus, passive Ca2+ movements occur predominantly by a reversible carrier-mediated mechanism for which Sr2+ is an alternate substrate. The carrier's intrinsic affinity constants for Ca2+ and Sr2+, 1.46 and 0.37 mM-1, respectively, indicate that Ca2+ is the preferred substrate.

Accumulation of chloroquine by membrane preparations from Plasmodium falciparum.

Chloroquine susceptibility and resistance have been associated respectively with the uptake and efflux of chloroquine by Plasmodium falciparum. We made membrane preparations from parasitized and unparasitized red cells in order to study chloroquine accumulation in a cell-free system. The accumulation of [3H]chloroquine by these preparations is inhibited by unlabeled chloroquine and thus is specific. Only membranes from parasitized red cells demonstrate time-dependent chloroquine accumulation; membranes from unparasitized red cells do not. Chloroquine accumulation is eliminated by detergent (0.05% Triton X-100) and reduced by a hypertonic medium, consistent with accumulation inside membrane vesicles rather than binding to membranes. Accumulation is energy dependent; it has a specific requirement for ATP, which cannot be replaced with GTP, CTP, UTP, TTP or ADP, an apparent Km of 21 microM and an apparent Vmax of 4.6 pmol (mg protein)-1 h-1. Vesicle acidification is MgATP dependent, and is reversed by NH4Cl. Chloroquine accumulation is inhibited by reduced medium pH, N-ethylmaleimide or oligomycin, but not by vanadate or ouabain. These studies demonstrate that membrane vesicles prepared from parasitized red cells provide a model system for the study of chloroquine accumulation by P. falciparum.

Structure-activity relationships for antiplasmodial activity among 7-substituted 4-aminoquinolines.

Aminoquinolines (AQs) with diaminoalkane side chains (-HNRNEt2) shorter or longer than the isopentyl side chain [-HNCHMe(CH2)3NEt2] of chloroquine are active against both chloroquine-susceptible and -resistant Plasmodium falciparum. (De, D.; et al. Am. J. Trop. Med. Hyg. 1996, 55, 579-583). In the studies reported here, we examined structure-activity relationships (SARs) among AQs with different N, N-diethyldiaminoalkane side chains and different substituents at the 7-position occupied by Cl in chloroquine. 7-Iodo- and 7-bromo-AQs with diaminoalkane side chains [-HN(CH2)2NEt2, -HN(CH2)3NEt2, or -HNCHMeCH2NEt2] were as active as the corresponding 7-chloro-AQs against both chloroquine-susceptible and -resistant P. falciparum (IC50s of 3-12 nM). In contrast, with one exception, 7-fluoro-AQs and 7-trifluoromethyl-AQs were less active against chloroquine-susceptible P. falciparum (IC50s of 15-50 nM) and substantially less active against chloroquine-resistant P. falciparum (IC50s of 18-500 nM). Furthermore, most 7-OMe-AQs were inactive against both chloroquine-susceptible (IC50s of 17-150 nM) and -resistant P. falciparum (IC50s of 90-3000 nM).

Prophylactic acyclovir effectively reduces herpes simplex virus type 1 reactivation after exposure of latently infected mice to ultraviolet B.

PURPOSE: To determine the potential efficacy and anatomic sites of action of prophylactic oral acyclovir using a murine model of ultraviolet-B-induced reactivation of herpes simplex 1 keratitis. METHODS: Latent infection with herpes simplex 1 (McKrae) was established in 80 National Institutes of Health inbred strain of mice. Forty of the mice were given acyclovir orally and the other 40 latently infected mice served as controls. Mice were exposed to 250 mJ/cm2 of ultraviolet-B radiation and killed on days 1, 2, 3, and 4 after ultraviolet-B radiation. Trigeminal ganglia and eyes from these mice were homogenized and incubated on Vero cell monolayers for recovery of reactivated virus. RESULTS: Based on the recovery of infectious virus after ultraviolet-B in treated versus control groups, acyclovir effectively reduced detectable viral reactivation at both the ocular level (P = 0.003) and the ganglionic level (P = 0.025). The numbers of viral culture-positive eye and trigeminal ganglia homogenates in the control group were 11 and 6 out of 40, respectively, compared to 1 and 0 out of 40 culture-positive eye and trigeminal ganglia homogenates in the acyclovir treated mice. Therapeutic serum levels of acyclovir were confirmed by high performance liquid chromatography. In the acyclovir-tested group, the single case of viral break-through at the ocular surface was not an acyclovir-resistant mutant. CONCLUSION: Prophylactic acyclovir effectively reduces the incidence of herpes simplex virus-1 reactivation after ultraviolet-B-induced reactivation in National Institutes of Health inbred strain of mice.

Energy dependence of chloroquine accumulation and chloroquine efflux in Plasmodium falciparum.

Chloroquine inhibits the growth of susceptible malaria parasites at low (nanomolar) concentrations because of an energy-requiring drug-concentrating mechanism in the parasite secondary lysosome (food vacuole) which is dependent on the acidification of that vesicle. Chloroquine resistance results from another energy-requiring process: efflux of chloroquine from the resistant parasite with a half-time of 2 min. Chloroquine efflux is inhibited reversibly by the removal of metabolizable substrate (glucose); it is also reduced by the ATPase inhibitor vanadate. These results suggest that chloroquine efflux is an energy-requiring process dependent on the generation and hydrolysis of ATP. Chloroquine efflux cannot be explained by differences in drug accumulation between chloroquine-susceptible and -resistant parasites because the 40-50-fold difference in initial efflux rates between -susceptible and -resistant parasites is unchanged when both parasites contain the same amount of chloroquine. Although chloroquine efflux is phenotypically similar to the efflux of anticancer drugs from multidrug-resistant (mdr) mammalian cells, it is not linked to either of the mdr-like genes of the parasite.

Pd(DIPHOS)2-catalyzed cross-coupling reactions of organoborons with free or polymer-bound aryl halides.

Bis[1,2-bis(diphenylphosphino)ethane]palladium(0) [Pd(DIPHOS)2] catalyzes cross-coupling reactions of free or polymer-bound aryl halides with organoboron compounds to produce biaryls in overall yields of 60-96%.

The basis of antimalarial action: non-weak base effects of chloroquine on acid vesicle pH.

Biologically active concentrations of chloroquine increase the pH of the parasite's acid vesicles within 3-5 min. This increase in pH results from two mechanisms, one of which is markedly reduced in chloroquine-resistant parasites. Because chloroquine is a weak base, it increases vesicle pH by that mechanism in chloroquine-susceptible and resistant parasites and mammalian cells (based on its two pKs and on the delta pH between the acid vesicle and the extracellular environment). In chloroquine-susceptible parasites, but not resistant parasites or mammalian cells, chloroquine increases the pH of acid vesicles 700- to 800-fold more than can be accounted for by its properties as a weak base. The increase in acid vesicle pH caused by these non-weak base effects of nanomolar chloroquine in susceptible parasites suggests that chloroquine acts by interfering with acid vesicle functions in the parasite such as the endocytosis and proteolysis of hemoglobin, and the intracellular targeting of lysosomal enzymes. The non-weak base effects of nanomolar chloroquine on parasite vesicle pH are also responsible for its safety because these chloroquine concentrations do not affect mammalian cells.

Doctoral training of African scientists.

There are two principal rationales for doctoral training of African scientists in health: 1) these scientists are essential for the nations of sub-Saharan Africa to define and implement their own health priorities, and 2) the research they perform is essential for development. However, this training is difficult because of its expense (> $20,000 per year), because many developed country mentors are unaware of the realities of research in sub-Saharan Africa, and because major differences in salary provide a financial disincentive to return. We describe a training strategy that reduces attrition because it is linked to the investigators' responsibilities before and after training, and to home country priorities. This strategy requires a close relationship between the developing country (on-site) and developed country (off-site) mentors, with joint participation in the selection and funding process, followed by course work and short-term, independent projects off-site that lead to a thesis project in the developing country, and subsequently to a defined professional position in the developing country after completion of the doctoral degree. For this strategy to succeed, the developed country mentor must have both field experience and investigative expertise; the developing country mentor must have an understanding of modern biology, as well as clinical and epidemiologic experience. In addition, we would like to emphasize that the long-term retention of these talented, highly-trained individuals requires a similar long-term commitment by their developed country mentors, well beyond the short term of most research funding.

Oxygen enhances the antimalarial activity of the imidazoles.

We have previously reported the antimalarial activity of imidazoles and amphotericin B against chloroquine-resistant Plasmodium falciparum. We now report the enhancement of imidazole activity in an atmosphere with 17-18% oxygen (the candle jar) vs. 3% or 0.3% oxygen. Based on both morphologic and radiometric testing, smaller amounts of the imidazoles were required to inhibit parasite growth by 50% in the candle jar vs. 3% or 0.3% oxygen. The use of older (more oxidant-sensitive) red cells also enhanced the antimalarial activity of ketoconazole. Neither increased concentrations of oxygen nor the use of older red cells affected the activity of amphotericin B. These results suggest that the imidazoles may exert their antimalarial effect by increasing the oxidant stress on the red cell-parasite complex.

Aminoquinolines that circumvent resistance in Plasmodium falciparum in vitro.

Aminoquinoline (AQ) resistance is one of the most important factors in the worldwide resurgence of malaria due to Plasmodium falciparum. We synthesized a series of AQs to define the structure-activity relationships responsible for AQ action against chloroquine-susceptible and -resistant P. falciparum. The AQs with ethyl, propyl, isopropyl, butyl, pentyl, isopentyl (chloroquine), hexyl, octyl, decyl, or dodecyl side chains were equally active against chloroquine-susceptible P. falciparum (50% inhibitory concentrations [IC50s] = 5-15 nM). The AQs with ethyl, propyl, isopropyl, decyl, or dodecyl side chains were also active against chloroquine-, mefloquine- and multiply-resistant P. falciparum (IC50s = 5-20 nM). Verapamil, which enhances the activity of chloroquine against chloroquine-resistant parasites, had no effect on the activity of AQs that were active against resistant parasites. These results indicate that AQs with 2-12 carbon side chains are as active as chloroquine against chloroquine-susceptible P. falciparum, and that AQs with side chains shorter or longer than chloroquine are often active against chloroquine-, mefloquine-, and multiply-resistant P. falciparum.

A prospective study of the effects of ultralow volume (ULV) aerial application of malathion on epidemic Plasmodium falciparum malaria. IV. Epidemiologic aspects.

In the Miragoane Valley of Haiti a consistent pattern in the incidence of Plasmodium falciparum malaria over a 10-year period made it possible to predict an annual outbreak and perform a prospective study to test the effects of aerial ultralow volume (ULV) malathion on epidemic levels of this disease. At the end of October 1972, after epidemic levels (100 cases/month/10,000 population) had been reached, spray operations were begun. The first spray cycle produced a sharp and immediate drop in populations of the vector Anopheles albimanus, followed 4 weeks later by a decrease in the incidence of malaria throughout the valley. Although the incidence of malaria was similar in sprayed and unsprayed areas prior to the effect of ULV malathion (176.1 and 198.7 cases/month/10,000 population, respectively), it was significantly different during the subsequent 3 months (16.8 cases/month/10,000 population in sprayed areas and 65.4 in unsprayed; p less than 0.001). Travel histories indicated that only 4% of all cases had spent a night away from home during the 4 weeks prior to onset of symptoms; therefore, we concluded that these incidence data represent malaria transmission in the valley. Results of the study indicate that aerial spraying of ULV malathion can interrupt epidemic transmission of P. falciparum malaria by a susceptible vector.

A novel pathway for Ca++ entry into Plasmodium falciparum-infected blood cells.

Growth of the human malaria parasite, Plasmodium falciparum, within the red blood cell (RBC) requires external Ca++ and is associated with a markedly elevated intracellular Ca++ concentration, [Ca++]i. We used 45Ca++ flux studies and patch clamp recordings to examine the mechanisms responsible for this increased [Ca++]i. The 45Ca++ flux studies indicated that net Ca++ entry into parasitized RBCs (PRBCs) is 18 times faster than into unparasitized ATPase that keeps the [Ca++]i of unparasitized RBCs exceedingly low. Acceleration of the preexisting Ca++ entry, ATPase that keeps the [Ca++] of unparasitized RBCs exceedingly low. Acceleration of the preexisting Ca++ entry, mediated by a divalent cation carrier, also cannot explain Ca++ accumulation in PRBCs: there are fundamental differences in substrate preference and in the effects of external Ca++ on 45Ca++ efflux between unparasitized RBCs and PRBCs. Patch clamp of intact PRBC surface membranes revealed rare unitary channel openings not observed on unparasitized RBCs. With 80 mM of CaCl2 in the patch pipette, this channel carried inward current, suggesting Ca++ entry at a rate comparable with the observed 45Ca++ flux. These data indicate that the malaria parasite induces a novel pathway in the host RBC membrane for Ca++ entry and suggest that this pathway is a Ca++-permeable channel.

Drug discovery, development and deployment: a report from the 28th Joint Conference of the U.S.-Japan Parasitic Diseases Panels, Baltimore, Maryland, July 1993.

The 28th Joint Conference of the Parasitic Diseases Panels of the U.S.-Japan Cooperative Medical Sciences Program held in Baltimore, Maryland focused on current research within both countries on antiparasitic chemotherapy. This meeting report summarizes presentations of work in progress on antiparasitic drugs currently in use and drugs under development or in clinical trials, as well as reports on potentially unique parasite characteristics that may provide targets for development of future therapeutics.