Cellular uptake of chloroquine is dependent on binding to ferriprotoporphyrin IX and is independent of NHE activity in Plasmodium falciparum.
(57/16882)
Here we provide definitive evidence that chloroquine (CQ) uptake in Plasmodium falciparum is determined by binding to ferriprotoporphyrin IX (FPIX). Specific proteinase inhibitors that block the degradation of hemoglobin and stop the generation of FPIX also inhibit CQ uptake. Food vacuole enzymes can generate cell-free binding, using human hemoglobin as a substrate. This binding accounts for CQ uptake into intact cells and is subject to identical inhibitor specificity. Inhibition of CQ uptake by amiloride derivatives occurs because of inhibition of CQ-FPIX binding rather than inhibition of the Na+/H+ exchanger (NHE). Inhibition of parasite NHE using a sodium-free medium does not inhibit CQ uptake nor does it alter the ability of amilorides to inhibit uptake. CQ resistance is characterized by a reduced affinity of CQ-FPIX binding that is reversible by verapamil. Diverse compounds that are known to disrupt lysosomal pH can mimic the verapamil effect. These effects are seen in sodium-free medium and are not due to stimulation of the NHE. We propose that these compounds increase CQ accumulation and overcome CQ resistance by increasing the pH of lysosomes and endosomes, thereby causing an increased affinity of binding of CQ to FPIX. (+info)
Faecal peritonitis causes oedema and neuronal injury in pig cerebral cortex.
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Encephalopathy is a common complication of sepsis. However, little is known about the morphological changes that occur in the brain during sepsis. Faecal peritonitis was induced in pigs that were killed 8 h later and frontal cortex samples were taken immediately after death. The tissue was investigated using light and electron microscopy and compared with frontal cortex samples taken from sham-operated controls. Septic pigs had 49.5% more perimicrovessel oedema than sham pigs. However, the tight junctions between cerebral microvessel endothelial cells appeared morphologically intact in both septic and sham pigs. Sepsis also resulted in neuronal injury, disruption of astrocytic end-feet and swollen, rounded erythrocytes. These morphological changes may be sufficient to underlie the clinical features seen in septic encephalopathy. (+info)
The independent contribution of bone and erythrocyte lead to urinary lead among middle-aged and elderly men: the normative aging study.
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Plasma is the component of blood from which lead is free to cross cell membranes and cause organ toxicity. Plasma lead levels, however, are extremely low and difficult to measure. Urinary lead originates from plasma lead that has been filtered at the glomerular level; thus, urinary lead adjusted for glomerular filtration rate serves as a proxy for plasma lead levels. In this investigation we examined the interrelationships of lead levels in whole blood corrected by hematocrit [i.e., erythrocyte lead (EPb)], trabecular bone (TBoPb), cortical bone (CBoPb), and urine excreted over 24 hr (UPb); all samples were obtained from 71 middle-aged and elderly men with no known occupational lead exposures. Lead was measured by graphite furnace atomic absorption spectroscopy (blood), K-X-ray fluorescence (bone), and inductively coupled plasma mass spectroscopy (urine). Lead levels were generally low, with mean EPb, TBoPb, and CBoPb values of 13.8, 31.1, and 21.7 microg/g, respectively, and a median UPb value of 6.15 microg/day. In generalized additive models adjusted for body weight and creatinine clearance rate, both EPb and bone lead variables remained independently and significantly associated with UPb. This finding suggests that bone influences plasma lead in a manner that is independent of the influence of erythrocytic lead on plasma lead. Thus, the superiority of bone lead over blood lead in predicting some chronic forms of toxicity may be mediated through bone's influence on plasma lead. In addition, this study suggests that measurement of urinary lead might be useful as a proxy for plasma lead levels in studies of lead toxicity. (+info)
Correction for erythroid cell contamination in microassay for immunophenotyping of neonatal lymphocytes.
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Immunophenotyping of blood lymphocyte subpopulations in neonates and young infants is hampered by the limited amount of blood that can be collected. Contamination of the flow cytometric "lympho-gate" by normoblasts and analysed erythrocytes, and therefore the underestimation of the relative frequencies of lymphocyte subpopulations, interferes with the precise calculation of absolute counts. A microassay was developed by adapting the lysed whole blood technique. Triple immunostaining in a single antibody staining step was used to reduce washing steps and cell loss. Introduction of a triple staining for CD71 (expressed by erythroid precursors), glycophorin A (GpA, expressed by all erythroid cells), and CD45 (expressed by all leucocytes) permitted the relative frequencies of normoblasts (CD71(+)/GpA+/CD45(-) population) and unlysed erythrocytes (CD71(-)/GpA+/CD45(-) population)to be identified and measured within the "lympho-gate" of neonatal cord blood samples. Particularly high frequencies were found (median: 31%) in cord blood samples from preterm neonates. These erythroid cells disappear rapidly by 1 week of age The relative frequencies of erythroid cells can be used to calculate correct lymphocyte subpopulation values. Using only 0.5-0.8 ml of blood, this micro- assay would also be suitable for rapid prenatal immunodiagnosis of congenital immunodeficiencies. (+info)
Pre-steady-state reaction of 5-aminolevulinate synthase. Evidence for a rate-determining product release.
(61/16882)
5-Aminolevulinate synthase (ALAS) is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and the alpha-subclass of purple bacteria. The pyridoxal 5'-phosphate cofactor at the active site undergoes changes in absorptive properties during substrate binding and catalysis that have allowed us to study the kinetics of these reactions spectroscopically. Rapid scanning stopped-flow experiments of murine erythroid 5-aminolevulinate synthase demonstrate that reaction with glycine plus succinyl-CoA results in a pre-steady-state burst of quinonoid intermediate formation. Thus, a step following binding of substrates and initial quinonoid intermediate formation is rate-determining. The steady-state spectrum of the enzyme is similar to that formed in the presence of 5-aminolevulinate, suggesting that release of this product limits the overall rate. Reaction of either glycine or 5-aminolevulinate with ALAS is slow (kf = 0.15 s-1) and approximates kcat. The rate constant for reaction with glycine is increased at least 90-fold in the presence of succinyl-CoA and most likely represents a slow conformational change of the enzyme that is accelerated by succinyl-CoA. The slow rate of reaction of 5-aminolevulinate with ALAS is 5-aminolevulinate-independent, suggesting that it also represents a slow isomerization of the enzyme. Reaction of succinyl-CoA with the enzyme-glycine complex to form a quinonoid intermediate is a biphasic process and may be irreversible. Taken together, the data suggest that turnover is limited by release of 5-aminolevulinate or a conformational change associated with 5-aminolevulinate release. (+info)
Heparan sulfate proteoglycan expression is induced during early erythroid differentiation of multipotent hematopoietic stem cells.
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Heparan sulfate (HS) proteoglycans of bone marrow (BM) stromal cells and their extracellular matrix are important components of the microenvironment of hematopoietic tissues and are involved in the interaction of hematopoietic stem and stromal cells. Although previous studies have emphasized the role of HS proteoglycan synthesis by BM stromal cells, we have recently shown that the human hematopoietic progenitor cell line TF-1 also expressed an HS proteoglycan. Immunochemical, reverse transcriptase-polymerase chain reaction (RT-PCR), and Northern blot analysis of this HS proteoglycan showed that it was not related to the syndecan family of HS proteoglycans or to glypican. To answer the question of whether the expression of HS proteoglycans is associated with the differentiation state of hematopoietic progenitor cells, we have analyzed the proteoglycan synthesis of several murine and human hematopoietic progenitor cell lines. Proteoglycans were isolated from metabolically labeled cells and purified by several chromatographic steps. Isolation and characterization of proteoglycans from the cell lines HEL and ELM-D, which like TF-1 cells have an immature erythroid phenotype, showed that these cells synthesize the same HS proteoglycan, previously detected in TF-1 cells, as a major proteoglycan. In contrast, cell lines of the myeloid lineage, like the myeloblastic/promyelocytic cell lines B1 and B2, do not express HS proteoglycans. Taken together, our data strongly suggest that expression of this HS proteoglycan in hematopoietic progenitor cell lines is associated with the erythroid lineage. To prove this association we have analyzed the proteoglycan expression in the nonleukemic multipotent stem cell line FDCP-Mix-A4 after induction of erythroid or granulocytic differentiation. Our data show that HS proteoglycan expression is induced during early erythroid differentiation of multipotent hematopoietic stem cells. In contrast, during granulocytic differentiation, no expression of HS proteoglycans was observed. (+info)
Glycoprotein IIb-IIIa is expressed on avian multilineage hematopoietic progenitor cells.
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The fibrinogen receptor GPIIb-IIIa integrin is known to be expressed on cells of the megakaryocytic lineage, but its presence on hematopoietic progenitors has been a controversial issue. To resolve this ambiguity unequivocally, we performed clonogenic assays and intrathymic cell-transfer experiments in congenic animals. As the ontogeny of the avian hematopoietic system is well documented, we used this experimental model to trace GPIIb-IIIa expression during embryogenesis. Consequently, we now report that the GPIIb-IIIa integrin is expressed as early as embryonic day 3.5 (E3.5) to 4 in intraaortic hematopoietic clusters, the first site of intraembryonic hematopoietic progenitor emergence, and later in E6 paraaortic foci. Myeloid and erythroid progenitors were also detected within the GPIIb-IIIa+ CD45(+) population isolated from the E3.5 to 4 aortic area, while in embryonic and adult bone marrow, myeloid, erythroid, and T-cell progenitors were present in the GPIIb-IIIa+ c-kit+ population. Furthermore, we also provide the first evidence, that GPIIb-IIIa+ bone marrow cells can differentiate into T cells. Hence, GPIIb-IIIa can be used as a marker for multilineage hematopoietic progenitors, permitting identification of early intraembryonic sites of hematopoiesis, as well as the isolation of embryonic and adult hematopoietic progenitors. (+info)
Impairment of Plasmodium falciparum growth in thalassemic red blood cells: further evidence by using biotin labeling and flow cytometry.
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Certain red blood cell (RBC) disorders, including thalassemia, have been associated with an innate protection against malaria infection. However, many in vitro correlative studies have been inconclusive. To better understand the relationship between human RBCs with thalassemia hemoglobinopathies and susceptibility to in vitro infection, we used an in vitro coculture system that involved biotin labeling and flow cytometry to study the ability of normal and variant RBC populations in supporting the growth of Plasmodium falciparum malaria parasites. Results showed that both normal and thalassemic RBCs were susceptible to P falciparum invasion, but the parasite multiplication rates were significantly reduced in the thalassemic RBC populations. The growth inhibition was especially marked in RBCs from alpha-thalassemia patients (both alpha-thalassemia1/alpha-thalassemia2 and alpha-thalassemia1 heterozygote). Our observations support the contention that thalassemia confers protection against malaria and may explain why it is more prevalent in malaria endemic areas. (+info)