Oxidation induces ClC-3-dependent anion channels in human leukaemia cells

To test for redox regulation of anion channels in erythroid cells, we exposed K562 cells to oxidants and measured changes in transmembrane Cl(-) currents using patch-clamp, and in intracellular Cl(-) content using the Cl(-) selective dye MQAE. Oxidation with tert-butylhydroperoxide or H(2)O(2) produced a plasma membrane anion permeability with a permselectivity of NO(3)(-)>lactate(-)>gluconate(-). The permeability increase was paralleled by insertion of ClC-3 protein into the plasma membrane as evident from immunofluorescence microscopy and surface biotinylation. Down-regulation of ClC-3 protein by RNA interference as assessed by immunoblotting decreased the oxidation-stimulated permeability. In conclusion, oxidation induces surface expression of ClC-3 and activation of a ClC-3-dependent anion permeability in K562 cells.     

Enhanced susceptibility to suicidal death of erythrocytes from transgenic mice overexpressing erythropoietin

Eryptosis, a suicidal death of mature erythrocytes, is characterized by decrease of cell volume, cell membrane blebbing, and breakdown of cell membrane asymmetry with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increased cytosolic Ca(2+) activity, which could result from activation of Ca(2+)-permeable cation channels. Ca(2+) triggers phosphatidylserine exposure and activates Ca(2+)-sensitive K(+) channels, leading to cellular K(+) loss and cell shrinkage. The cation channels and thus eryptosis are stimulated by Cl(-) removal and inhibited by erythropoietin. The present experiments explored eryptosis in transgenic mice overexpressing erythropoietin (tg6). Erythrocytes were drawn from tg6 mice and their wild-type littermates (WT). Phosphatidylserine exposure was estimated from annexin binding and cell volume from forward scatter in fluorescence-activated cell sorting (FACS) analysis. The percentage of annexin binding was significantly larger and forward scatter significantly smaller in tg6 than in WT erythrocytes. Transgenic erythrocytes were significantly more resistant to osmotic lysis than WT erythrocytes. Cl(-) removal and exposure to the Ca(2+) ionophore ionomycin (1 microM) increased annexin binding and decreased forward scatter, effects larger in tg6 than in WT erythrocytes. The K(+) ionophore valinomycin (10 nM) triggered eryptosis in both tg6 and WT erythrocytes and abrogated differences between genotypes. An increase of extracellular K(+) concentration to 125 mM blunted the difference between tg6 and WT erythrocytes. Fluo-3 fluorescence reflecting cytosolic Ca(2+) activity was larger in tg6 than in WT erythrocytes. In conclusion, circulating erythrocytes from tg6 mice are sensitized to triggers of eryptosis but more resistant to osmotic lysis, properties at least partially due to enhanced Ca(2+) entry and increased K(+) channel activity.     

Induction of suicidal erythrocyte death by listeriolysin from Listeria monocytogenes.

Listeriolysin, the secreted cytolysin of the facultative intracellular bacterium Listeria monocytogenes, is its major virulence factor. Previously, non-lytic concentrations of listeriolysin were shown to induce Ca2+-permeable nonselective cation channels in human embryonic kidney cells. In erythrocytes, Ca2+ entry is followed by activation of K+ channels resulting in K+-exit as well as by membrane scrambling resulting in phosphatidylserine exposure at the cell surface. Phosphatidylserine-exposing erythrocytes are recognized by macrophages, engulfed, degraded and thus cleared from circulating blood. Phosphatidylserine exposure is a key event of eryptosis, the suicidal death of erythrocytes. The present study utilized patch-clamp technique, Fluo3-fluorescence, and annexin V-binding in FACS analysis to determine the effect of listeriolysin on cell membrane conductance, cytosolic free Ca2+ concentration, and phosphatidylserine exposure, respectively. Within 30 minutes, exposure of human peripheral blood erythrocytes to low concentrations of listeriolysin (which were non-hemolytic for the majority of cells) induced a Ca2+-permeable cation conductance in the erythrocyte cell membrane, increased cytosolic Ca2+ concentration, and triggered annexin V-binding. Increase of extracellular K+ concentration blunted, but did not prevent, listeriolysin-induced annexin V-binding. In conclusion, listeriolysin triggers suicidal death of erythrocytes, an effect at least partially due to depletion of intracellular K+. Listeriolysin induced suicidal erythrocyte death could well contribute to the pathophysiology of L. monocytogenes infection.     

Synthesis and in vitro and in vivo antimycobacterial activity of isonicotinoyl hydrazones

The purpose of this study was to prepare various isoniazid derivatives by introducing the isoniazid pharmacophore into several molecules and screening for antimycobacterial activity. Ortho-hydroxy acetophenone reacts with isoniazid to form acid hydrazones. The C-Mannich bases of the above acid hydrazones were prepared by reacting them with formaldehyde and various secondary amines. The synthesized compounds were screened against M. tuberculosis H(37)R(v) using the alamar blue susceptibility test. The synthesized compounds inhibit Mycobacterium tuberculosis strain H(37)R(v) with minimum inhibitory concentrations ranging from 0.56 to 4.61 microM. Compound N'-{1-[2-hydroxy-3-(piperazin-1-ylmethyl)phenyl]ethylidene}isonicotinohydrazide 8 was found to be the most active compound with an MIC of 0.56 microM, and was more potent than isoniazid (MIC of 2.04 microM). After 10 days of treatment, compound 8 decreased the bacterial load in murine lung tissue by 3.7-log10 as compared to controls, which was equipotent to isoniazid. The results demonstrate the potential and importance of developing new isoniazid derivatives against mycobacterial infections.     

Production of congenic mouse strains carrying NOD-derived diabetogenic genetic intervals: An approach for the genetic dissection of complex traits

Insulin-dependent (Type 1) diabetes (IDD) in the NOD mouse is inherited as a complex polygenic trait making the identification of susceptibility genes difficult. Currently none of the non-MHC IDD susceptibility genes in NOD have been identified. In this paper we describe the congenic mouse approach that we are using for the dissection of complex traits, such as IDD. We produced a series of six congenic strains carrying NOD-derived diabetogenic genomic intervals, which were previously identified by linkage analysis, on a resistant background. These congenic strains were produced for the purpose of characterizing the function of each of these genes, alone and in combinations, in IDD pathogenesis and to allow fine mapping of the NOD IDD susceptibility genes. Histological examination of pancreata from 6 to 8-month-old congenic mice reveals that intervals on Chromosomes (Chrs) 1 and 17, but not 3, 6, and 11, contain NOD-derived genes that can increase the trafficking of mononuclear cells into the pancreas. Insulitis was observed only very rarely, even in older congenic mice, indicating that multiple genes are required for this phenotype. These results demonstrate the utility of this congenic approach for the study of complex genetic traits. Received: 1 September 1995 / Accepted: 20 December 1995     

Molecular modelling studies of kdr mutations in voltage gated sodium channel revealed significant conformational variations contributing to insecticide resistance

Voltage gated sodium channels (VGSC) of mosquito vectors are the primary targets of dichlorodiphenyltrichloroethane (DDT) and other synthetic pyrethroids used in public health programmes. The knockdown resistant (kdr) mutations in VGSC are associated with the insecticide resistance especially in Anophelines. The present study is aimed to emphasize and demarcate the impact of three kdr-mutations such as L1014S, L1014F and L1014H on insecticide resistance. The membrane model of sodium transport domain of VGSC (STD-VGSC) was constructed using de novo approach based on domain and trans-membrane predictions. The comparative molecular modelling studies of wild type and mutant models of STD-VGSC revealed that L1014F mutant was observed to be near native to the wild type model in all the respects, but, L1014S and L1014H mutations showed drastic variations in the energy levels, root mean square fluctuations (RMSF) that resulted in conformational variations. The predicted binding sites also showed variable cavity volumes and RMSF in L1014S and L1014H mutants. Further, DDT also found be bound in near native manner to wild type in L1014F mutant and with variable orientation and affinities in L1014S and L1014H mutants. The variations and fluctuations observed in mutant structures explained that each mutation has its specific impact on the conformation of VGSC and its binding with DDT. The study provides new insights into the structure–function-correlations of mutant STD-VGSC structures and demonstrates the role and effects of kdr mutations on insecticide resistance in mosquito vectors.     

Prevalence of African DNA RELP alleles in neotropical African honeybees

A few queens of the honeybee, Apis mellifera scutellata, were imported from Africa and released in Brazil in 1957. Progeny of these bees have now largely colonized the American tropics. Their imminent arrival in the United States poses a serious threat to the beekeeping industry and to agriculture dependent on honeybee pollination. African and European bees are morphologically very similar. DNA restriction fragment length polymorphisms are proving successful in distinguishing between the two. Several DNA markers specific to European honeybees have been described previously. Reported here are three cloned honeybee DNA probes that reveal polymorphisms that appear to be either African or European specific. Of fourteen alleles or haplotypes identified, five were present only in African and neotropical (Venezuelan and Mexican) African bees but absent in European-derived bees, two were present only in European-derived bees but absent in samples from South Africa. Another allele showed apparent frequency differences among populations. Such markers are useful in studying the genetics of neotropical African bee populations. Venezuelan and Mexican honeybee colonies show a preponderance of the African alleles with low levels of the European alleles. These observations of nuclear DNA, revealing limited paternal European introgression, together with previous mitochondrial DNA findings showing negligible European maternal gene flow into feral African populations, indicate that neotropical African bees are primarily African.     

Biogenesis of endoplasmic reticulum transport vesicles transferring gastric apomucin from ER to Golgi.

Rough endoplasmic reticulum (RER) transport vesicles were generated from gastric mucous cell RER microsomes in the presence of labeled precursors of phospholipids. The vesicles contained 7-10% of their proteins in the form of apomucin (cargo), and 80% of de novo synthesized phosphatidylcholine (PC) was incorporated into the vesicular membrane. In the absence of choline and ethanolamine precursors or in the presence of 3 mM N-ethylmaleimide (NEM), an inhibitor of CTP:phosphocholine cytidylyltransferase, formation of the transport vesicles, their enrichment in the newly synthesized PC, and the total synthesis of PC decreased by 86%, whereas in the presence of 3 mM Zn2+, complete blockage of vesicle formation and PC synthesis was observed. Analysis of the mucin-transporting vesicles indicated that the CTP:phosphocholine cytidylyltransferase and 1,2-diacyl-sn-glycerol:CDP-choline phosphotransferase remained associated with transport vesicles released from ER. The enzymes and other proteins separated from the vesicle surface prior to vesicle fusion with Golgi and the process was induced by phosphorylation. Based on the results of this study, it is proposed that the formation of the ER transport vesicles of gastric mucosal cells is in concert with synthesis of phospholipids and thus in part is regulated by phospholipid-synthesizing enzymes that reside on the membrane during its biogenesis and dissociate from its surface once the task is completed.     

Mechanism of erythrocyte aggregate formation in presence of magnetic field and dextrans as analyzed by laser light scattering

The mechanism of erythrocyte aggregation has been studied in normal plasma, dextran 40 and dextran 70 suspensions in presence and absence of magnetic field at a concentration of 5 percent by laser light scattering. The inhomogeneous magnetic field enhances the aggregating tendency of normal erythrocytes. The growth of aggregates due to dextran 70 is enhanced in presence of magnetic field. On the other hand the disaggregating effect of dextran 40 is reduced due to this field. The induced changes due to magnetic field during the development of erythrocyte aggregates in these media are determined.     

Presence of a Ca2+-sensitive CDPdiglyceride-inositol transferase in canine cardiac sarcoplasmic reticulum

Sarcoplasmic reticulum (SR) and plasma membranes from canine left ventricle were used to evaluate the presence of the enzyme CDPdiglyceride-inositol transferase in these membranes. (K+,-Ca2+)-ATPase activity, a marker for SR, was 79.2 +/- 5.0 (SE) and 11.2 +/- 2.0 mumol.mg-1.h-1 in SR and plasma membrane preparations, respectively, and (Na+,K+)-ATPase activity, a marker for plasma membranes, was 5.6 +/- 1.2 and 99.2 +/- 8.0 mumol.mg-1.h-1, respectively. Contamination of SR and plasma membrane preparations by mitochondria was estimated to be 2% and 8%, respectively, and by Golgi membranes, 0.9% and 1.8%, respectively. Transferase activity, measured at pH 6.8, was 1.32 +/- 0.04 (SE) and 0.28 +/- 0.04 nmol of [3H]phosphatidylinositol ([3H]PtdIns).mg-1.min-1 in three SR and plasma membrane preparations, respectively. The transferase activity detected in the plasma membrane preparation could be accounted for largely, but not entirely, by contaminating SR membranes. The pH optimum for the SR transferase activity was between 8.0 and 9.0; little or no activity was detectable at pH 6.3 and 5.5, the lowest pH tested. Ca2+ inhibited the enzyme, half-maximal inhibition occurring at about 10 microM Ca2+; removal of the Ca2+ by addition of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid restored activity. No loss of [3H]PtdIns could be detected when membranes were incubated in the presence or absence of Ca2+. The Ca2+ inhibition of the transferase was noncompetitive with respect to CDP-dipalmitin while that with respect to myo-inositol was slightly noncompetitive at low [Ca2+] and became uncompetitive at higher [Ca2+].(ABSTRACT TRUNCATED AT 250 WORDS)