Oxidative status of valinomycin-resistant normal, beta-thalassemia and sickle red blood cells

Exposure of red blood cells (RBC) to the K+ -ionophore valinomycin (val), causes loss of KCl and water, resulting in cell dehydration, manifested by increased cell density. While almost all normal val-treated RBC dehydrate, in sickle cell anemia (SCA) a portion of the RBC fail to dehydrate and maintain a light density, indicating the existence of val-resistant (val-res) RBC. In thalassemia and sickle cell disease (SCD), although the primary lesion is in the globin genes, damage to the RBC is partly mediated by oxidative stress. We previously showed that such RBC are under oxidative stress, having more reactive oxygen species (ROS) and less reduced glutathione than normal RBC. We now report a relationship between the phenomenon of val-res and the RBC oxidative status: Treatment with oxidants that increase ROS, also increased the frequency of val-res cells. Val-res cells had higher oxidative status than other RBC in the sample. Similar to SCA, thalassemic blood has more val-res cells than does normal blood. Val-res cells in thalassemic and sickle blood showed a higher oxidative status than normal val-res cells. Thus, oxidative stress might be involved in generation of val-res cells. Further studies are required to elucidate the origin and significance of these cells.     

No single irreplaceable acidic residues in the Escherichia coli secondary multidrug transporter MdfA

The largest family of solute transporters (major facilitator superfamily [MFS]) includes proton-motive-force-driven secondary transporters. Several characterized MFS transporters utilize essential acidic residues that play a critical role in the energy-coupling mechanism during transport. Surprisingly, we show here that no single acidic residue plays an irreplaceable role in the Escherichia coli secondary multidrug transporter MdfA.     

A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors

Endogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in cellular transformation, we identified miR-372 and miR-373, each permitting proliferation and tumorigenesis of primary human cells that harbor both oncogenic RAS and active wild-type p53. These miRNAs neutralize p53-mediated CDK inhibition, possibly through direct inhibition of the expression of the tumor-suppressor LATS2. We provide evidence that these miRNAs are potential novel oncogenes participating in the development of human testicular germ cell tumors by numbing the p53 pathway, thus allowing tumorigenic growth in the presence of wild-type p53.     

Refined, Circular Restriction Map of the Bacillus thuringiensis subsp. israelensis Plasmid Carrying the Mosquito Larvicidal Genes

All the genetic elements responsible for the mosquito larval toxicity of Bacillus thuringiensis subsp. israelensis are located on one of its largest plasmids, nicknamed pBtoxis. Two linkage groups (with sizes of about 75 and 55 kb) have previously been mapped partially with respect to SacI and BamHI restriction sites (Ben-Dov et al., 1996), but linking them to a single circular plasmid unambiguously was impossible with the available data. To finalize the plasmid map, another rare cutting restriction endonuclease, AlwNI, was used in addition. The two linkage groups and the fragments generated by AlwNI were aligned on the circular plasmid, and known insertion sequences were localized on the refined map. Pulsed-field electrophoresis revealed that the total size of pBtoxis (137 kb) was larger than thought before.     

Evidence for a Distinct D1Like Dopamine Receptor that Couples to Activation of Phosphoinositide Metabolism in Brain

Abstract: Dopamine and the D₁, receptor agonist SKF 38393 activate the phospholipase C-rnediated hydrolysis of phosphoinositides in brain slices. This action is selectively inhibited by SCH-23390, thus suggesting its mediation through the dopamine D₁ receptor. To determine if the dopamine receptor that mediates Phosphoinositide hydrolysis is the adenylyl, cyclase-linked D₁ receptor or a different subtype of the dopamine D₁ receptor, 20 benzazepine compounds that were previously characterized as selective dopamine D₁ receptor agonists were tested for stimulation of Phosphoinositide hydrolysis in rat striatal slices and for activation of adenylyl cyclase in rat striatal membranes. The compounds displayed a range of potencies and efficacies in stimulating adenylyl cyclase or Phosphoinositide hydrolysis. Compounds such as SKF 81427 and SKF 38393 were as efficacious as dopamine in stimulating Phosphoinositide hydrolysis, whereas other compounds, including SKF 85174 and SKF 86284, although showing high efficacy in stimulating cyclic AMP, failed to stimulate inositol phosphate formation. There was no correlation between the potencies (r= 0.016; p < 0.95) or efficacies (r=?0.294; p < 0.24) of the tested compounds in stimulating cyclic AMP formation and phosphoinositide hydrolysis. These observations indicate that the D₁-like dopamine receptor that mediates phosphoinositide hydrolysis is pharmacologically distinct from the classic D₁ receptor that is coupled to stimulation of cyclic AMP formation.     

Biliary micellar cholesterol nucleates via the vesicular pathway

Biliary cholesterol nucleates primarily from phospholipid vesicles. In this study, we investigated the mode of nucleation of micellar cholesterol. Ten biles (four human and six model) were examined. The vesicular and micellar fractions of each bile were separated by gel chromatography. The whole biles and their isolated carriers were incubated at 37 degrees C until nucleation time. In whole human biles, the proportion of total cholesterol in vesicles rose throughout the incubation (from zero time to nucleation time) from 15.5 +/- 8.6% to 28.0 +/- 12.5%, and in model biles from 46.8 +/- 22.4% to 75.5 +/- 8.2%. The vesicular isolated fraction remained unchanged throughout incubation. In isolated micelles devoid of vesicles at zero time, new vesicles formed during incubation, carrying increasing proportions of cholesterol. At nucleation time, these vesicles contained 11.0% of originally micellar cholesterol in human biles, and 41.2% in model biles. The new vesicles formed in whole bile and in the micellar fraction were chromatographically and chemically similar to the vesicles originally present in bile. These data suggest that micellar cholesterol nucleates via the neoformation of phospholipid vesicles, which seem to be the final common pathway for cholesterol nucleation in bile.     

Cyt1Aa toxin: crystal structure reveals implications for its membrane-perforating function

During sporulation, Bacillus thuringiensis subsp. israelensis produces a mosquito larvicidal protein complex containing several crystalline and cytolytic (Cyt) toxins. Here, the activated monomeric form of Cyt1Aa, the most toxic Cyt family member, was isolated and crystallized, and its structure was determined for the first time at 2.2 Å resolution.Cyt1Aa adopts a typical cytolysin fold containing a β-sheet held by two surrounding α-helical layers. The absence of a β-strand (between residues V26 and I37) in the dimeric structure of Cyt2Aa led us to deduce that this is the only essential segment for dimer formation and that activation of the toxin occurs by proteolytic processing of its N-terminus. Based on the Cyt1Aa structure, we suggest that the toxicity of Cyt1Aa and other nonrelated proteins, all sharing a cytolysin fold, is correlated with their ability to undergo conformational changes that are necessary prior to their membrane insertion and perforation. This fold allows the α-helical layers to swing away, exposing the β-sheet to insert into the membrane. The identification of a putative lipid binding pocket between the β-sheet and the helical layer of Cyt1Aa supports this mechanism. Sequence-based structural analysis of Cyt1Aa revealed that the lack of activity of Cyt1Ca may be related to the latter's inability to undergo this conformational change due to its lack of flexibility. The pattern of the hemolytic activity of Cyt1Aa presented here (resembling that of pore-forming agents), while differing from that imposed by ionic and nonionic detergents, further supports the pore-forming model by which conformational changes occur prior to membrane insertion and perforation.     

Severe hepatopathy in geese and broilers associated with ochratoxin in their feed

The feeding of a shipment of imported corn was associated with a severe reduction in growth and increased mortality in geese, and increased mortality in broilers. Pathological examinations revealed hepatopathy, visceral gout and mild nephropathy in geese, and in broilers an hepatopathy, which was often severe, and ascites. Samples of feed from affected geese farms were examined for up to 24 mycotoxins, and ochratoxin was found in 6 of 15 samples at levels up to 930 ng/g. The syndrome was experimentally reproduced by feeding geese and broilers suspect feeds with the natural ochratoxin contamination. It is believed that another, unidentified, mycotoxin was the major cause of the hepatotoxicity, and that ochratoxin served in this case as an indicator of a multi-mycotoxin involvement. This revised version was published online in June 2006 with corrections to the Cover Date.     

The genetic history of Cochin Jews from India

Cochin Jews form a small and unique community on the Malabar coast in southwest India. While the arrival time of any putative Jewish ancestors of the community has been speculated to have taken place as far back as biblical times (King Solomon’s era), a Jewish community in the Malabar coast has been documented only since the 9th century CE. Here, we explore the genetic history of Cochin Jews by collecting and genotyping 21 community members and combining the data with that of 707 individuals from 72 other Indian, Jewish, and Pakistani populations, together with additional individuals from worldwide populations. We applied comprehensive genome-wide analyses based on principal component analysis, F _ST, ADMIXTURE, identity-by-descent sharing, admixture linkage disequilibrium decay, haplotype sharing, allele sharing autocorrelation decay and contrasting the X chromosome with the autosomes. We find that, as reported by several previous studies, the genetics of Cochin Jews resembles that of local Indian populations. However, we also identify considerable Jewish genetic ancestry that is not present in any other Indian or Pakistani populations (with the exception of the Jewish Bene Israel, which we characterized previously). Combined, Cochin Jews have both Jewish and Indian ancestry. Specifically, we detect a significant recent Jewish gene flow into this community 13–22 generations (~470–730 years) ago, with contributions from Yemenite, Sephardi, and Middle-Eastern Jews, in accordance with historical records. Genetic analyses also point to high endogamy and a recent population bottleneck in this population, which might explain the increased prevalence of some recessive diseases in Cochin Jews.