A Novel Flow Cytometric Hemozoin Detection Assay for Real-Time Sensitivity Testing of Plasmodium falciparum

Resistance of Plasmodium falciparum to almost all antimalarial drugs, including the first-line treatment with artemisinins, has been described, representing an obvious threat to malaria control. In vitro antimalarial sensitivity testing is crucial to detect and monitor drug resistance. Current assays have been successfully used to detect drug effects on parasites. However, they have some limitations, such as the use of radioactive or expensive reagents or long incubation times. Here we describe a novel assay to detect antimalarial drug effects, based on flow cytometric detection of hemozoin (Hz), which is rapid and does not require any additional reagents. Hz is an optimal parasite maturation indicator since its amount increases as the parasite matures. Due to its physical property of birefringence, Hz depolarizes light, hence it can be detected using optical methods such as flow cytometry. A common flow cytometer was adapted to detect light depolarization caused by Hz. Synchronized in vitro cultures of P. falciparum were incubated for 48 hours with several antimalarial drugs. Analysis of depolarizing events, corresponding to parasitized red blood cells containing Hz, allowed the detection of parasite maturation. Moreover, chloroquine resistance and the inhibitory effect of all antimalarial drugs tested, except for pyrimethamine, could be determined as early as 18 to 24 hours of incubation. At 24 hours incubation, 50% inhibitory concentrations (IC50) were comparable to previously reported values. These results indicate that the reagent-free, real-time Hz detection assay could become a novel assay for the detection of drug effects on Plasmodium falciparum.     

Prenylated flavonoids from seeds of Calopogonium mucunoides

The seeds of Calopogonium mucunoides furnished 7-O-γ,γ-dimethylallyl-8-methoxy-3′,4′-dioxymethylene-isoflavone, 7-O-γ,γ-dimethylallyl-3′-hydroxy-4′-methoxyisoflavone, 7-O-γ,γ-dimethylallyl-3′,4′-dimethoxyisoflavone and 2S-di[6′',6′'-dimethylpyrano (2′',3′':7,8;2′',3′':4′,3′)]-flavanone whose structures were established by spectroscopic means involving the use of 400 MHz ¹H NMR with double irradiation and INDOR techniques.     

Acrosomal reaction and early events at fertilization in Marthasterias glacialis (Echinodermata: Asteroidea)

When the spermatozoon of M glacialis contacts the mature oocyte jelly it adheres to it. Following this, there is a slight tumefaction of the acrosome, which is followed by the disruption of the apical acrosomal vesicle and cytoplasmic membranes. Acrosomal vesicle contents are liberated and spread along the outer surface of the oocyte jelly. Meanwhile, the acrosomal process begins to extend, penetrates all the jelly extension, then the vitelline layer, and finally contacts the cytoplasmic egg membrane. Nevertheless, the sperm cell continues lying at the outer border of the jelly. From the beginning of the acrosome reaction the dense and finely fibrillar subacrosomal material is connected, by some expansions, to the basal acrosomal vesicle membrane. Both nuclear and mitochondrial diameters have diminished.     

Inhibition of cytochrome P-450 activity in rat liver microsomes by the naturally occurring flavonoid, quercetin

The kinetic characteristics and mechanism of flavonoid inhibition of cytochrome P-450-mediated reactions were examined in rat liver microsomes, using the naturally occurring flavonoid, quercetin (3,3',4',5,7-pentahydroxyflavone). Quercetin inhibited the O-deethylation of ethoxyresorufin in beta-naphthoflavone-induced microsomes by 15-80% at concentrations of 10-250 nM. The pattern of inhibition was dependent on quercetin concentration. Quercetin also inhibited p-nitroanisole demethylation and benzo(a)-pyrene hydroxylation, but did not change the proportions of the individual benzo(a)pyrene metabolites in comparison to controls. Specific steps in the P-450 reaction pathway were tested for sensitivity to quercetin inhibition. The Km values of the P-450 substrates tested were increased in the presence of quercetin; competition for and/or alteration of the substrate binding site contributes to the mechanism of inhibition. In experiments under anaerobic, carbon monoxide-saturated conditions, quercetin did not inhibit cytochrome P-450 reduction by NADPH-cytochrome P-450 reductase. The cumene hydroperoxide-supported O-deethylation of ethoxyresorufin was inhibited by quercetin (15-60% inhibition at concentrations of 50-300 nM), suggesting that quercetin may interfere with the formation or breakdown of the oxygenated heme complex. Stoichiometry experiments established that quercetin is a potent uncoupler of P-450 reactions, elevating the rates of H2O2 formation almost twofold. Structure/activity studies indicated that certain other naturally occurring flavonoids were at least as potent inhibitors of ethoxyresorufin deethylation as quercetin. These findings are of interest in light of the significant dietary exposure of the human population to the flavonoids.     

Dietary factors affecting the urinary mutagenicity assay system. I. Detection of mutagenic activity in human urine following a fried beef meal

Studies have been conducted to determine whether the mutagens in fried beef ingested by human subjects are excreted in the urine. Urine samples were collected from individuals on liquid or regular diets before and after a fried beef meal. The mutagenic activity of the samples was tested in the Ames Salmonella/microsome assay system. The results showed that in individuals on liquid diets, most of the urinary mutagenic activity is recovered within 2-6 h after consuming a fried beef meal. In one individual tested, mutagenic activity was found in urine samples obtained 6-15 h after the fried beef meal. No mutagenic activity was detected in any of the urine samples obtained 15-24 h following the meal. In individuals on a regular diet, however, mutagenic activity was frequently observed in urine samples obtained 16-24 h following the fried beef meal, although the mutagenic activity was not as great as that in the preceding 16 h. It appears that the mutagenic agents generated by the frying of beef are ingested, absorbed, and excreted by the human body in biologically detectable quantities. These results suggest that subjects should abstain from fried beef at least one day prior to and during urine mutagenicity screening.     

Dietary factors affecting the urinary mutagenicity assay system. II. The absence of mutagenic activity in human urine following consumption of red wine or grape juice

The mutagenic activity of urine samples from nonsmoking individuals before and after the consumption of either red wine or grape juice was determined. Urine samples collected from individuals on liquid or regular diets were concentrated using XAD-2 resin. No mutagenic activity of urine concentrates was detected with Salmonella tester strains TA98 or TA100 with or without microsomal activation. The addition of 1000 units of beta-glucuronidase into the agar overlay did not show any mutagenic activity. The mutagens in red wine and grape juice, however, were extracted using the XAD-2 column. Concentrates of urine samples spiked with either of the two extracts exhibited mutagenic activity.     

Ultrastuctural and cytochemical study of spermatogenesis in Scrobicularia plana (Mollusca,Bivalvia)

The ultrastructure of the mature spermatozoa and spermatogenesis of the bivalve Scrobicularia plana are described. Support cells extend from the basal lamina to the lumen of the testis and are laterally connected to the germinal epithelium. Germ cells present intercellular bridges and flagella since the spermatogonial stage. While spermatogonia and spermatocytes appear connected to support cells by desmosome-like junctions, elongated spermatids are held at the acrosomal region by support cell finger-like processes. During spermiogenesis, the acrosomal vesicle differentiates from a golgian saccule and then migrates to the nuclear apex. A microtubular manchette arising from centrioles surrounds the acrosomal vesicle, the nucleus, and the mitochondria at the time these three organelles start their elongation, disappearing after that. The mature spermatozoon of S. plana lacks a distinct midpiece because the mitochondria extend from the region of the pericentriolar complex along the nucleus anteriorly for approximately 1.4 μm. The features of this bivalve type of modified spermatozoon are compared with those of other animal groups having similar modifications.     

Transfer factor therapy in a case of complex immunodeficiency

A young woman with unclassified complex immune deficiency disease characterized by relapsing skin ulcerations, greatly depressed cellular immunity, absent serum IgA, and inability to develop circulating antibodies to typhoid vaccine, was treated twice with the dialyzable transfer factor from normal persons. Following such therapy she showed clinical improvement and strong delayed skin sensitivity to Candida, streptokinase-streptodornase, and moderate, rather short-lived hypersensitivity to PPD. In addition, she could be sensitized to dinitrochlorobenzene and her leucocytes responded normally to phytohaemagglutinin; all these manifestions of cellular immunity had been absent prior to therapy. She acquired the capacity to develop anti-typhoid antibodies.These findings appear to indicate that dialyzable transfer factor not only transfers delayed-type hypersensitivities preexisting in the donor but is also able to remedy hitherto unspecific defects existing in certain forms of immune deficiency.     

Excitation of System I and System II in Photosynthesis as a Possible Control Mechanism of Glycolate Metabolism in the Blue-Green Alga Anacystis nidulans

Photosynthetic enhancement studies performed at 619 nm (excitation of Systems I and II) and at 446 nm (mainly excitation of System I) revealed an 18% photosynthetic enhancement simultaneously with a 31% reduction in glycolate excretion. This observation supports the hypothesis that some glycolate may be consumed in an oxidation process associated with System I when System II is poorly excited and the supply of electrons from the water splitting process of photosynthesis is low.     

Bacterial mesosomes: Real structures of artifacts?

The ultrastructural study of membrane organization in gram-positive bacteria related to the OsO₄ fixation conditions revealed that large, complex mesosomes are observed only when the bacteria are subjected to an initial fixation with 0.1% OsO₄ in the culture broth, as in the prefixation step of the Ryter-Kellenberger procedure. Evidence was obtained suggesting that the large mesosomes are produced by this prefixation. The kinetic study of the membrane morphological alterations occurring during the prefixation of Bacillus cereus with 0.1% OsO₄ in the culture broth showed that the amount of mesosome material increases linearly from zero to a maximum observed at 1.7 min of prefixation and that at about this time a maximum is reached for the number of mesosomes per unity of cell area and for the average individual mesosome area. The large mesosomes observed in gram-positives fixed by the complete Ryter-Kellenberger procedure would be the result of the membrane-damaging action of 0.1% OsO₄. Such damaging action was deduced from the observation that 0.1% OsO₄ quickly lyses protoplasts and induces a quick and extensive leakage of intracellular K⁺ from B. cereus and Streptococcus faeculis. In support of that interpretation is the observation that in bacteria subjected to several membrane-damaging treatments, mesosome-like structures are seen after three different fixation procedures. In bacteria initially fixed with 1% OsO₄, 4% OsO₄ or 2.5% glutaraldehyde, no large, complex mesosomes are observed, small and simple invaginations of the cytoplasmic membrane being present. The size of these minute mesosomes is inversely proportional that causes of fixation. Uranyl acetate was found among the studied fixatives the one to the rate the least damage to bacterial membranes. This fixative satisfactorily preserves protoplasts. In bacteria initially fixed with uranyl acetate no mesosomes were found. The results of the present work throw serious doubts on the existence of mesosomes, both large and small, as real structures of bacterial cells. It is proposed that a continuous cytoplasmic membrane without infoldings (mesosomes) would be the real pattern of membrane organization in gram-positives.