Susceptibility of an Insect Leptomonas and Crithidia fasciculata to Several Established Antitrypanosomatid Agents

Growth inhibition of the lower trypanosomatids Crithidia fasciculata and a Leptomonas from a hemipteron by several established trypanocides and leishmanicides were compared in four complex and one defined media. The Leptomonas was more susceptible than C. fasciculata in all media, especially to phenanthridines (ethidium, prothidium, isometamidium) and diamidines (pentamidine, diminazene diaceturate [Berenil], hydroxystilbamidine, stilbamidine); concentrations of these drugs required for 50% inhibition of the Leptomonas were <5 μg/ml. In contrast, C. fasciculata was uninhibited by <20 μg of diamidines per ml and was three- to sixfold less susceptible than the Leptomonas to isometamidium and prothidium. Both trypanosomatids were susceptible to nucleoside antibiotics, e.g., nucleocidin. Neither was inhibited by suramin, melarsen, melarsen oxide, or tryparsamide. The Leptomonas was more susceptible to standard trypanocides than five other insect trypanosomatids in a complex medium; it was the only one inhibited by <20 μg of stilbamidine and hydroxystilbamidine per ml.     

Inhibition of growth and purine-metabolizing enzymes of trypanosomid flagellates by N6-methyladenine.

N6-methyladenine (6-methylaminopurine [6-MA]), a plant growth regulator and a normal constituent of nucleic acids, has been found to inhibit the growth of Trypanosoma cruzi, Leishmania braziliensis, L. donovani, L. tarentolae, L. mexicana, and Crithidia fasciculata. The extent of growth inhibition in these organisms is related to the sensitivity of guanine deaminase (guanine aminohydrolase, EC 3.5.4.3), adenine deaminase (adenine aminohydrolase, EC 3.5.4.2), and adenosine hydrolase and phosphorylase. 6-MA was not an inhibitor of the purine phosphoribosyltransferases. Of the trypanosomid flagellates tested. Trypanosoma cruzi was most susceptible to 6-MA. Neither adenine deaminase (as found in the leishmaniae and C. fasciculata) nor adenosine deaminase (as found in mammalian cells) could be demonstrated in T. cruzi. Guanine deaminase, which is strikingly inhibited by 6-MA in T. cruzi, appears to play a major role in the purine salvage pathway of this organism, as judged from growth experiments and enzyme inhibition studies. Enzyme sensitivities to 6-MA vary greatly depending upon the organism. Rabbit liver guanine deaminase was shown to be insensitive to 6-MA at the concentrations used in this study.     

Selective uptake of intact parathyroid hormone by the liver: differences between hepatic and renal uptake.

Hepatic and renal extraction of immunoreactive parathyroid hormone (i-PTH) was studied in awake dogs with explanted kidneys and chronic indwelling hepatic vein catheters. After a single injection of bovine PTH 1-84 (b-PTH 1-84), hepatic arteriovenous (A-V) differences for immunoreactive PTH (i-PTH) was 39% at 2 min after injection but decreased to 0% by 25 min, despite high levels of i-PTH in the arterial circulation. Gel filtration of arterila and hepatic venous samples obtained when hepatic A-V differences for i-PTH were demonstrable revealed hepatic uptake of the intact hormone and addition of a smaller COOH-terminal fragment, eluting just after the intact hormone, to the hepatic venous blood. Gel filtration of samples obtained 20-30 min after injection of b-PTH was demonstrable) revealed no detectable intact hormone in the circulation. Levels of COOH-terminal fragments of the hormone at the time were identical in arterial and hepatic venous samples. In additional experiemtns no hepatic A-V difference was observed after the injection of the synthetic bovine PTH 1-34 (syn b-PTH 1-34). By comparison there was a demonstrable A-V difference of 20% across the kidney for both intact PTH and COOH-terminal fragments that persisted until i-PTH disappeared from the circulation. The kidney also demonstrated an A-V difference of 22% after injection of syn b-PTH 1-34. These studies demonstrate selective extraction of intact PTH but not of its fragments by the liver. The kidney, on the other hand, extracted the intact hormone and both COOH and NH2 terminal fragments. The studies demonstrate that the kidney was the only organ of those examined that detectably removed the fragments of PTH from the circulation.     

Antibiotic uptake by alveolar macrophages of smokers.

Cigarette smoking, particularly when associated with chronic pulmonary disease, increases the risk of respiratory tract infection. Thus, we elevated the uptake of antibiotics by alveolar macrophages (AM) obtained by bronchoalveolar lavage from persons who smoke and have associated pulmonary abnormalities, circumstances which adversely affect certain macrophage functions. The entry of radiolabeled drugs into AM was determined by a velocity-gradient centrifugation technique, and uptake was expressed as the ratio of cellular to extracellular antibiotic concentration (C/E). Cefamandole and penicillin G were taken up poorly by the AM obtained from smokers (C/E less than or equal to 1). Cellular levels of isoniazid, gentamicin, and tetracycline were similar to their extracellular concentrations. The lipid-soluble drugs lincomycin, chloramphenicol, and rifampin were concentrated severalfold by the AM from smokers (C/E = 3 to 11). Ethambutol also entered macrophages readily (C/E = 11). Erythromycin and clindamycin were massively concentrated by the AM from smokers (C/E = 23 to 56). The AM of smokers accumulated a lipid-soluble antibiotic (rifampin) and actively transported agents (erythromycin propionate, clindamycin) more avidly than did the AM of nonsmokers. Augmented uptake of these antibiotics by the AM of smokers may be related to structural and functional alterations induced by smoking.     

Effect of calcium on oxalate uptake and transport by the rat intestine.

The effect of calcium concentration (0-10 mmol 1(-1)) on oxalate uptake and transport was investigated in vitro using everted gut segments and sacs. Increase in calcium concentration in the incubation medium led to an increase in the amounts of precipitated oxalate on the intestine; however, the net oxalate flux to the serosal side decreased. The ions, i.e. Ca2+, Ox2-, H2PO4-, HPO4(2-), present in the incubation medium favoured formation of hydroxyapatite and calcium oxalate crystals, as evidenced by Equil II analysis and free energy of the system. The nature of precipitates was confirmed by elemental analysis, X-ray diffraction spectrometry and electron microscopy. Oxalate precipitated on the intestine following incubation with calcium could be released into a calcium- and oxalate-free medium. Animals fed oxalate in the absence and presence of calcium revealed that, during 1 h in the absence of calcium, oxalate moved down the intestinal tract as a distinct peak of greater than 50% (70-90 cm in the intestine), leaving less than 10% in the stomach and first 50 cm of the intestine. In the case of animals fed calcium along with oxalate, 35% of the oxalate was still present in the stomach, and the amounts of oxalate in the intestinal segments gradually increased from 4.5% to 21.7% (0-90 cm) and dropped to 2.1% in the next 20 cm. Since oxalaemia and oxaluria appear to be influenced by intestinal oxalate absorption, the present observations may help to improve understanding of the pathophysiology of disorders exhibiting altered oxalate metabolism.     

Differing uptake of emulsion triglyceride by the fed and fasted rat liver.

The recycling perfusion of a fasted rat liver with an apoprotein E-enriched synthetic triglyceride emulsion revealed a significantly greater hepatic uptake of both the apoprotein and the triglyceride than did the liver of a chow-fed animal. This greater hepatic triglyceride uptake by the perfused fasted liver in comparison to the fed was also noted for emulsions containing no added apoprotein or supplemented with both the E and CIII-1 proteins. However, no difference in the uptake of the triglyceride emulsion was seen for the fed and fasted livers when evaluated by a nonrecycling single pass perfusion. The isolated hepatocyte plasma membranes from fasted rats failed to demonstrate enhanced binding of apoprotein or lipid when compared to those from fed animals. If the residual E loaded triglyceride emulsion was recovered from the recycling perfusates of fed and fasted livers and evaluated in a non-recycling single-pass system, the emulsion from the fasted perfusion was cleared as facilely as previously, whereas that from the fed was less actively cleared. The emulsions retrieved from the perfusion of the fed liver contained significantly more protein than did the fasted; in particular apo C. This apparent alteration of emulsion apoproteins by the fed liver possibly results in a less active hepatic retrieval and may be important in downregulating the entry of lipoprotein triglyceride in the postabsorptive liver.     

Mutation analysis of genetic diseases by asymmetric-PCR SSCP and ethidium bromide staining: application to neurofibromatosis and cystic fibrosis.

The Single Strand Conformation Polymorphism (SSCP) technique is widely used in mutation analysis. We have introduced several modifications to the SSCP method, which overcome the problem of incomplete denaturation or reannealing of DNA during electrophoresis. The modifications consist of asymmetrical PCR amplification of the sequence of interest, electrophoresis with a higher concentration of acrylamide, and the analysis of the DNA fragments under u.v. light. We have applied this method to the analysis of two specific diseases: neurofibromatosis type 1 (NF1) and cystic fibrosis (CF) from PCR amplified exons. Two single nucleotide changes were observed with this method.     

Palmitate uptake by hepatocyte monolayers. Effect of albumin binding.

The uptake of 14C-palmitate by rat liver cell monolayers is depressed by binding of the fatty acid to albumin. When the uptake flux is divided by the concentration of free palmitate in the bathing medium, however, the resulting clearance is approximately 14 times greater in the presence of albumin than in its absence. These findings are not accounted for by the different diffusion rates of free and bound palmitate across an unstirred fluid layer, nor attributable to nonequilibrium binding. Instead we argue that the most plausible explanation is accelerated dissociation of albumin-palmitate complexes mediated by the cell surface--an interpretation that also explains the uptake kinetics of other albumin-bound organic anions by perfused rat liver.     

Transferrin and iron uptake by isolated rat liver mitochondria.

Isolated rat liver mitochondria accumulate iron from the suspending medium when [59Fe]transferrin is used as a model compound. The accumulation proceeds by two different mechanisms, i.e. by an energy-independent and an energy-dependent (uncoupler sensitive) mechanism, which have different time, pH, and temperature dependencies. The energy-dependent accumulation, which is inhibited by ruthenium red and sulphydryl reagents, reaches a saturation level of approx. 30 pmoles iron/mg protein during 30 min incubation. The energy-independent accumulation of iron-transferrin reveals no saturation kinetics, it is inhibited neither by ruthenium red nor by N-ethylmaleimide, and it proceeds linearly for at least 90 min. With [125I]transferrin as a model compound, quantitatively the energy-independent accumulation is as reported for [59Fe]transferrin. There is, however, no energy-dependent accumulation of [125I]transferrin. The results indicate that the energy-dependent accumulation of [59Fe]transferrin represents a process by which mitochondria accumulate iron from transferrin.     

Relation of intracellular K+ and steroidogenesis in isolated adrenal zona glomerulosa and fasciculata cells.

1. Intracellular K+ content, water spaces and corticosterone output were measured in isolated zona glomerulosa and zona fasciculata-reticularis cell suspensions of rat adrenal cortex, after incubation in vitro under conditions designed to alter steroidogenesis. 2. Intracellular K+ of unpurified zona glomerulosa cells was not altered after stimulation of corticosterone output with serotonin. Similarly, with zona glomerulosa cells purified by unit gravity sedimentation, no change in intracellular K+ was detected after stimulation of steroidogenesis with serotonin or angiotensin II. 3. In high-potassium medium (final concentration 8.4 mmol/1), parallel increases in intracellular K+ and corticosterone output were observed with both purified zona glomerulosa cells. However, a similar increase in intracellular K+ also occurred in high-potassium medium with zona fasciculata cells, whose steroid output is unresponsive to external potassium concentration ([K+]). 4. Ouabain at 10(-5) mol/1 depressed the intracellular [K+] of glomerulosa cells but did not alter basal or stimulated corticosterone output. Similar results were obtained with fasciculata cells. 5. Ouabain at 5 times 10(-4) mol/1 further depressed intracellular [K-+] of glomerulosa cells and inhibited basal and stimulated corticosterone output. However, this concentration of ouabain also inhibited steroidogenesis in fasciculata cells. 6. These results demonstrate a variety of situations where changes in intracellular [K+] are dissociated from those in corticosterone output and indicate that intracellular [K+] cannot be the sole mechanism regulating steroidogenesis under these conditions.     

Autoradiography of tobramycin uptake by the proximal and distal tubules of normal and endotoxin-treated rats.

Multiple factors may modify the pharmacokinetics of aminoglycosides and increase their nephrotoxic potential. In the present study, the influence of Escherichia coli endotoxin on the renal handling of [3H]tobramycin was investigated. The accumulation of [3H]tobramycin in proximal tubules, distal tubules, and collecting ducts was compared in both normal and endotoxin-injected (0.25 mg/kg) rats. Histological observations were also made. Blood pressure and cardiac frequency were recorded, and renal function was evaluated with labeled inulin and p-aminohippuric acid. Following administration of endotoxin, disturbed intrarenal localization of the drug was noted. Grain counts were affected in both proximal and distal tubules. Increased labeling was observed at all time intervals in the proximal tubules. In the distal tubules of endotoxemic animals we could detect higher amounts of drug at 10 and 60 min in the medulla and at 10 min in the cortex. Not all of the tubules were labeled to the same extent. No histological lesion was noted on light microscopy in animals receiving either normal saline or endotoxin. The dose of endotoxin used resulted in very fine physiological disturbance. Both blood pressure and cardiac frequency were minimally affected by endotoxin. Decreases in glomerular filtration rate and renal plasma flow were observed. However, none of these changes was statistically significant. The present study has shown that low doses of endotoxin alter the renal handling of aminoglycosides in the absence of any major physiological disturbance or histological changes. By increasing the total amount of drug within the kidney, endotoxin might increase the nephrotoxic potential of aminoglycosides.     

Effect of narcotics on the uptake of serotonin precursors by the rat brain.

The extraction of 14C-tryptophan and 14C-hydroxytryptophan (5-HTP) from the blood to the brain was measured using an indicator dilution technique. Acute treatment with morphine caused a dose-related decrease in the extraction of tryptophan by the brain and a increase in that of 5-HTP. Naloxone alone had no effect on the extraction of either tryptophan or 5-HTP but completely blocked the effect of 20 mg/kg of morphine on the extraction of both tryptophan and 5-HTP. In contrast to acute treatment with morphine, the extractions of tryptophan and 5-HTP were not significantly altered 48 hours after chronic treatment with morphine. The extraction of 5-HTP remained unchanged and that of tryptophan increased significantly 72 hours after chronic morphine treatment. In equivalent doses, levorphanol decreased the extraction of tryptophan more than its inactive isomer, dextrorphan, whereas levorphanol increased and dextrorphan had no effect on the extraction of 5-HTP. These results suggest that an increase in the rate of central serotonin synthesis after acute treatment with morphine may be due to an increased uptake of 5-HTP from the blood to the brain while that after chronic treatment with morphine may be due to an increased uptake of tryptophan.     

Evidence of active efflux in resistance to ciprofloxacin and to ethidium bromide by Mycoplasma hominis

The uptake of fluoroquinolones was characterized for the fluoroquinolone-susceptible strain PG21 of Mycoplasma hominis. Accumulation of fluoroquinolones appeared to occur by passive diffusion. Addition of arginine as the energizer significantly reduced the uptake of fluoroquinolones, suggesting the presence of an energy-dependent efflux process. Reserpine and orthovanadate, two multidrug pump inhibitors, increased significantly the ciprofloxacin (CIP) uptake. In contrast, such a strong effect was not observed for moxifloxacin and pefloxacin uptakes. Two ethidium bromide (EtBr)-resistant strains, selected in vitro, showed a resistance profile compatible with a multidrug-resistant phenotype, with increased MICs for the hydrophilic fluoroquinolones, CIP and norfloxacin, EtBr, and acriflavine. Taking the EtBr-resistant strain RB1La as a model, a significant decrease of the CIP and EtBr uptakes was observed compared to the reference strain PG21. In the presence of reserpine and orthovanadate, both inhibitors of ATP-dependent efflux pumps, the CIP uptake increased significantly, reaching approximately the same level as that of the susceptible strain. Similar results were obtained with EtBr uptake and efflux experiments. Our data suggest the presence of an active efflux system, possibly an ABC-type efflux pump, implicated in the resistance to CIP and unrelated compounds like EtBr in the human mycoplasma M. hominis.     

The pharmacodynamics of 5-hydroxytryptamine uptake and metabolism by the isolated perfused rabbit lung.

We have investigated the dynamics for the removal of 5-hydroxytryptamine (5-HT) from the circulation using the isolated perfused rabbit lung. 5-HT was removed from the circulation at a constant rate and metabolized completely to 5-hydroxyindoleacetic acid which effluxed from the lung into the circulation. Two methods were developed to determine the constant rate of removal of 5-HT: (1) the constant rate is equal to the difference between the 5-HT concentration flowing into the lung and the 5-HT concentration in the effluent times the flow rate and (2) extrapolation of the rate of appearance of radioactivity in the effluent to zero time. With these methods, we have confirmed the 5-HT is removed by the lung by a carrier-mediated Na+-dependent transport system. Studies of transport systems in perfused organs required an adequate supply of the chemical to the lung. Supply rates less than removal rate will result in erroneous measurements of the constant removal rate. The relationships between the rate of removal, perfusate concentration and perfusion rate were analyzed.     

Inhibition of tumor cell glutamine uptake by isolated neutrophils.

Antitumor activity of phorbol myristate acetate-(PMA) stimulated neutrophils was measured against CCRF-CEM cells. Neutrophils and tumor cells were incubated (a) as a suspension with continuous mixing to maximize the availability of oxygen or (b) after centrifugation as a pellet to maximize cell-cell contact. The cells were then incubated briefly as a suspension with [14C]glutamine under conditions that blocked further damage to the tumor cells. When cells were incubated as a suspension, inhibition of tumor-cell glutamine uptake was mediated by the myeloperoxidase/hydrogen peroxide/chloride system of stimulated neutrophils. Inhibition was blocked by adding catalase, an inhibitor of myeloperoxidase, or compounds that scavenge hypochlorous acid or chloramines. When cells were incubated as a pellet, a portion of the inhibition could not be blocked in this way, indicating that a nonoxidative mechanism contributed to inhibition. In both systems, inhibition of glutamine uptake was rapid and was obtained at effector-cell/target-cell ratios as low as 0.5:1. This inhibition was obtained under conditions that did not result in 51Cr release from cells labeled with [51Cr]-chromate, indicating that inhibition of glutamine uptake measured cytotoxicity rather than cytolysis. 51Cr release was observed only when cells were incubated together for an hour or more as a pellet at high E/T ratios. This cytolysis was mediated by the myeloperoxidase system, and a nonoxidative contribution to cytolysis was not observed. The results indicate that stimulated neutrophils are potent antitumor effectors cells when cytotoxicity rather than cytolysis is the measure of activity. Because glutamine is required for growth of many tumor cells, inhibition of glutamine uptake may represent a significant tumoristatic or tumoricidal effect.