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D J Krogstad

Publications and source records attributed to D J Krogstad.

At least 55 records · Page 3Linked to original sources
PubMed1987

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.

Animals↗
PubMed1987

Inoculum effect with chloroquine and Plasmodium falciparum.

In the studies reported here, we examined the inoculum effect observed with chloroquine and Plasmodium falciparum. The 50% effective doses observed with both chloroquine-susceptible and -resistant parasites increased five- to sevenfold from their baseline values as the inoculum was increased from 2 X 10(5) to 2 X 10(7) parasitized erythrocytes per ml (parasitemias of 0.1 to 10% with a hematocrit of 2%). Increasing the inoculum also decreased the chloroquine uptake per parasitized erythrocyte 15- to 20-fold with both chloroquine-susceptible and -resistant parasites. However, because of the 100-fold increase in the inoculum, the total amount of chloroquine taken up actually increased sufficiently to reduce the extracellular chloroquine concentration in vitro by 60 to 90%. These studies suggest that a chloroquine uptake of greater than or equal to 2.0 pmol/10(6) parasitized erythrocytes is necessary for chloroquine to inhibit parasite growth. More marked reductions in the amount of chloroquine uptake per parasitized erythrocyte were observed with a hematocrit of 40% using similar parasitemias of 0.1 to 10% (inocula of 4 X 10(6) to 4 X 10(8) parasitized erythrocytes per ml). Thin-layer chromatography of [3H]chloroquine taken up by chloroquine-resistant P. falciparum revealed no evidence of drug alteration by the parasite. These studies define the mechanism responsible for the inoculum effect observed with chloroquine and P. falciparum in vitro.

Animals↗
PubMed1987

Ion pair high-performance liquid chromatographic assay for ceftriaxone.

We developed a high-performance liquid chromatographic assay to measure ceftriaxone in serum, urine, and cerebrospinal fluid. Ion pairing was used because ceftriaxone is a relatively polar compound which is poorly retained on C18 columns in standard reverse-phase high-performance liquid chromatography and which produces trailing peaks in the absence of ion-pairing agents. The mobile phase was a combination of acetonitrile and water (46:54), adjusted to pH 9.0 with 10 mM K2HPO4, which contained 10 mM hexadecyltrimethylammonium bromide as the ion-pairing agent. Moxalactam (200 micrograms/ml) was used as the internal standard. A silica-packed precolumn (3 cm long) was used to prevent rapid deterioration of the analytical column (30 by 0.4 cm) by the alkaline pH of the mobile phase, and it significantly extended the life of the analytical column. The assay was linear with ceftriaxone concentrations of 1 to 250 micrograms/ml (r = 0.999) and correlated well with an agar diffusion bioassay (r = 0.990). Reproducibility was good, with intrarun coefficients of variation from 2.3 to 6.4% and interrun coefficients of variation from 3.2 to 21.4%. The absolute recoveries of ceftriaxone and moxalactam were 91 to 97 and 96 to 98%, respectively. No interferences were observed with more than 40 commonly prescribed drugs, including 10 cephalosporins (cefotaxime, cefoperazone, ceftazidime, ceftizoxime, cefoxitin, cefamandole, cephalothin, cefazolin, cephapirin, and cephalexin), or with sera from patients with renal or hepatic disease.

Ceftriaxone↗
PubMed1987

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.

Animals↗
PubMed1986

Acid intracellular vesicles and the cytolysis of mammalian target cells by Entamoeba histolytica trophozoites.

Entamoeba histolytica kills mammalian target cells in a multi-step sequential process with separate adherence, cytolytic, and phagocytic events. In the studies reported here, we used fluorescein isothiocyanate linked to dextran to label the endocytic vesicles of the HM1 strain of E. histolytica and measure vesicle pH (5.1 +/- 0.2 by spectrofluorimetry). Concentrations of NH4Cl (1.0-10.0 mM) sufficient to increase vesicle pH to greater than or equal to 5.7 inhibited amebic killing of target Chinese hamster ovary (CHO) cells as assayed by trypan blue staining, by the release of 3H-thymidine previously incorporated into CHO cell monolayers, and by the release of 111indium oxine from radiolabeled CHO cells. Similar effects were also observed with two other weak bases, primaquine and chloroquine (both 50 microM). In contrast, NH4Cl (10 mM) did not affect either the adherence or phagocytic events, as measured by amebic adherence to CHO cells at 4 degrees C and by the binding and ingestion of 3H-leucine-labeled bacteria. In the presence of NH4Cl and the carbohydrate ligand asialofetuin, there was no evidence of intracellular trapping of the amebic galactose-inhibitable lectin; inhibition of adherence by cycloheximide (10 micrograms/ml for 3 h) suggested rapid turnover of the surface lectin. Prolonged exposure to NH4Cl for 48 h (which had no effect on amebic protein synthesis) or shorter exposure to cycloheximide (10 micrograms for 3 h) produced persistent inhibition of cytolysis. These results indicate that an uninterrupted acid pH in intracellular endocytic vesicles is necessary for the cytolysis of target cells by E. histolytica trophozoites.

Adhesiveness↗
PubMed1986

Sensitive high-pressure liquid chromatographic assay for amphotericin B which incorporates an internal standard.

We developed a rapid, sensitive, high-pressure liquid chromatographic (HPLC) procedure which incorporates a commercially available internal standard, 1-amino-4-nitronaphthalene, to measure amphotericin B in serum. Recovery was quantitative (greater than or equal to 90%), and the standard curve was linear from 0.04 to at least 10.0 micrograms/ml. The reproducibility of the assay was good, with intrarun coefficients of variation from 2.0 to 6.8% and interrun coefficients of variation from 4.9 to 10.0%. Comparison by linear regression analysis of the HPLC assay with an agar well diffusion bioassay gave a correlation coefficient of 0.942, with the HPLC assay exhibiting greater precision and sensitivity. No interference was encountered from over 20 drugs and three amphotericin B analogs. However, serum specimens that contained high concentrations of conjugated bilirubin (greater than 3 mg/dl) produced interfering peaks in both this assay and other previously reported HPLC assays for amphotericin B. We also describe a solid-phase extraction procedure which effectively removes this interference and uses an alternative internal standard (N-acetyl amphotericin B).

Amphotericin B↗
PubMed1985

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.

Ammonia↗
PubMed1984

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.

Animals↗
PubMed1984

Clindamycin activity against chloroquine-resistant Plasmodium falciparum.

The clindamycin dose-response curves observed with both chloroquine-resistant and chloroquine-susceptible strains of Plasmodium falciparum in vitro demonstrated a plateau region that extended from 10(-2) to 10(1) micrograms/ml of drug (22 nM to 22 microM). Similar dose-response curves were also observed with the three major metabolites of clindamycin (clindamycin sulfoxide, de-N-methyl clindamycin, and de-N-methyl clindamycin sulfoxide). The position of this plateau was time dependent and rose from 20%-25% to 90%-95% inhibition of parasite growth between 24 and 72 hr of exposure to the drug. Clinidamycin treatment reduced plasmodial protein and nucleic acid synthesis (as measured by the incorporation of [3H]isoleucine and [3H]hypoxanthine, respectively) but did not interfere with knob formation. The combination of quinine plus a fixed concentration of clindamycin (0.1 microgram/ml) inhibited growth of the quinine-resistant Indochina I strain, although most of the antiplasmodial activity observed at quinine concentrations less than 50 ng/ml (154 nM) could be attributed to clindamycin alone.

Chloroquine↗