Conserved properties of hydrogenosomal and mitochondrial ADP/ATP carriers: a common origin for both organelles

Mitochondria are one of the hallmarks of eukaryotic cells, exporting ATP in exchange for cytosolic ADP using ADP/ATP carriers (AAC) located in the inner mitochondrial membrane. In contrast, several evolutionarily important anaerobic eukaryotes lack mitochondria but contain hydrogenosomes, peculiar organelles of controversial ancestry that also supply ATP but, like some fermentative bacteria, make molecular hydrogen in the process. We have now identified genes from two species of the hydrogenosome-containing fungus Neocallimastix that have three-fold sequence repeats and signature motifs that, along with phylogenetic analysis, identify them as AACs. When expressed in a mitochondrial AAC- deficient yeast strain, the hydrogenosomal protein was correctly targeted to the yeast mitochondria inner membrane and yielded mitochondria able to perform ADP/ATP exchange. Characteristic inhibitors of mitochondrial AACs blocked adenine nucleotide exchange by the Neocallimastix protein. Thus, our data demonstrate that fungal hydrogenosomes and yeast mitochondria use the same pathway for ADP/ATP exchange. These experiments provide some of the strongest evidence yet that yeast mitochondria and Neocallimastix hydrogenosomes are but two manifestations of the same fundamental organelle.     

An experimental test of the relationship between temporal variability of feeding opportunities and baseline levels of corticosterone in a shorebird

In this study, we tested the hypothesis that baseline corticosterone levels increase with a change from constant to variable feeding schedules. Captive red knots, Calidris canutus, were presented with food that was either available during the same time each day (constant) or starting at variable times during the day. Food intake rates, frequency of aggressive interactions, and baseline levels of corticosterone were measured. In the majority of cases, red knots showed higher plasma corticosterone concentrations during feeding schedules that were irregular than when food was available at consistent times. These findings are supported by a previous study that showed that red knots take a long time to adjust to the newly offered, predictable conditions of their aviary environment. The frequency of conflicts in the different groups and (size-corrected) body mass were not correlated with average corticosterone level. The results are examined in the light of literature showing that increases in corticosterone in response to acute, unpredictable events mediate behavioral responses such as increased explorative behavior and memory. For red knots that have to find their food on the temperate-zone mudflats in Western Europe, an increased circulating corticosterone level may be adaptive during periods when the patchily distribution of buried bivalves and the burying behavior of such prey presents them with a variable and unpredictable food supply.     

Steroid sulfatase activity and expression in mammary myoepithelial cells

PURPOSE: This investigation examined mRNA expression and enzymatic activity of steroid sulfatase (STS) in human mammary myoepithelial cells (MMECs) and MCF-7 cells and assessed the effects of 17-beta estradiol on the activity of STS. METHODS: The mRNA level of STS in MMECs was determined by RT-PCR analysis using specific primers for STS. STS enzymatic activity prior to and after treatment with 17-beta estradiol was determined by measuring 3H-metabolites formed after exposure to [3H]estrone 3-sulfate (E1S) and [3H]dehydroepiandrosterone-sulfate (DHEA-S). RESULTS: Our data demonstrate the presence of STS in the MMECs. Based on RT-PCR analysis, MMECs had slightly lower levels of STS compared to MCF-7 cells. However, sulfatase activity was about 120 times greater in the MMECs than the MCF-7 cells (E1S V(max)=2640nmol/(mg DNAh) compared to 20.9nmol/(mg DNAh)). Exposure to 17-beta estradiol was associated with 70% reduction in E1S sulfatase activity in the MCF-7 cells and 9% increase in the MMECs after 6 days. DISCUSSION: Our studies indicate for the first time the presence of STS in MMECs. This is suggestive of a previously undetermined role for MMECs in converting precursor hormones into active steroid hormones within mammary tissue. In addition, differential response of the MMECs and the MCF-7 cells to estrogen demonstrates differences in hormone metabolism between these two cell types, perhaps related to the absence of estrogen receptors in the MMECs and their presence in the MCF-7 cells. The MMECs may have an important role in hormonal regulation within mammary tissue.     

GeneCards 2002: towards a complete,object-oriented,human gene compendium

MOTIVATION: In the post-genomic era, functional analysis of genes requires a sophisticated interdisciplinary arsenal. Comprehensive resources are challenged to provide consistently improving, state-of-the-art tools. RESULTS: GeneCards (Rebhan et al., 1998) has made innovative strides: (a). regular updates and enhancements incorporating new genes enriched with sequences, genomic locations, cDNA assemblies, orthologies, medical information, 3D protein structures, gene expression, and focused SNP summaries; (b). restructured software using object-oriented Perl, migration to schema-driven XML, and (c). pilot studies, introducing methods to produce cards for novel and predicted genes.     

Does any yeast mitochondrial carrier have a native uncoupling protein function?

In this study, we explore the hypothesis that some member of the mitochondrial carrier family has specific uncoupling activity that is responsible for the basal proton conductance of mitochondria. Twenty-seven of the 35 yeast mitochondrial carrier genes were independently disrupted in Saccharomyces cerevisiae. Six knockout strains did not grow on nonfermentable carbon sources such as lactate. Mitochondria were isolated from the remaining 21 strains, and their proton conductances were measured. None of the 21 carriers contributed significantly to the basal proton leak of yeast mitochondria. A possible exception was the succinate/fumarate carrier encoded by the Xc2 gene, but deletion of this gene also affected yeast growth and respiratory chain activity, suggesting a more general alteration in mitochondrial function. If a specific protein is responsible for the basal proton conductance of yeast mitochondria, its identity remains unknown.     

Changes in LPLa and reverse cholesterol transport variables during 24-h postexercise period

We investigated the time course of exercise-induced lipoprotein lipase activity (LPLa) and reverse cholesterol transport (RCT) during the 24-h postexercise period. Subjects were 10 sedentary normolipidemic males [NTG; fasting triglyceride (TG) = 89.1 +/- 8.6 mg/dl] and 6 hyperlipidemic males (HTG; fasting TG = 296.8 +/- 64.0 mg/dl). Each subject performed a control trial (no exercise) and 4 exercise trials. In the exercise trials, a subject jogged on a treadmill at 60% of his maximal O(2) consumption for 1 h. Pre- and postheparin blood samples were taken before exercise (baseline) and at 4, 8, 12, and 24 h after exercise. There was no group difference in LPLa (P > 0.05) over the time points. When the LPLa data from the two groups were combined, LPLa at 24 h after exercise was higher than baseline or at 4, 8, 12 h after exercise (P < 0.05). Plasma TG and lecithin-cholesterol acyltransferase activity (LCATa) were higher in HTG than in NTG, and the total high-density lipoprotein-cholesterol (HDL(tot)-Chol) was lower in HTG than in NTG (P < 0.05). HDL(2)-Chol, LCATa, and cholesterol ester transfer protein activity did not differ during the 24-h postexercise period (P > 0.05). These results suggest that LPLa is still increasing 24 h after an acute aerobic exercise and that the magnitude of the increase in exercise-induced LPLa in HTG was similar to that in NTG. Furthermore, in the sedentary population with or without HTG, the variables related to RCT do not change during the 24-h period after exercise.     

Synergy between cationic lipid and co-lipid determines the macroscopic structure and transfection activity of lipoplexes

The large number of cytofectin and co-lipid combinations currently used for lipoplex-mediated gene delivery reflects the fact that the optimal cytofectin/co-lipid combination varies with the application. The effects of structural changes in both cytofectin and co-lipid were systematically examined to identify structure–activity relationships. Specifically, alkyl chain length, degree of unsaturation and the head group to which the alkyl side chain was attached were examined to determine their effect on lipoplex structure and biological activity. The macroscopic lipoplex structure was assessed using a dye-binding assay and the biological activity was examined using in vitro transfection in three diverse cell lines. Lipoplexes were formulated in three different vehicles currently in use for in vivo delivery of naked plasmid DNA (pDNA) and lipoplex formulations. The changes in dye accessibility were consistent with structural changes in the lipoplex, which correlated with alterations in the formulation. In contrast, transfection activity of different lipoplexes was cell type and vehicle dependent and did not correlate with dye accessibility. Overall, the results show a correlation between transfection and enhanced membrane fluidity in both the lipoplex and cellular membranes.     

Estrogens (E2) regulate expression and response of 1,25-dihydroxyvitamin D3 receptors in bone cells: changes with aging and hormone deprivation

Studies on the effect of estrogens (E(2)) on the expression of vitamin D receptor (VDR) and its bioresponse in bone have demonstrated that E(2) modulate activity and increase the number of VDRs in vitro; however, no in vivo studies have been pursued to assess this interaction. Our study identifies the changes in the number of VDR-expressing cells in bone of C57BL/6J young and old oophorectomized mice (4 and 24 months) with and without 17beta estradiol (E(2)) replacement. A total of 36 mice were sacrificed; both tibiae and femora were isolated and VDR expression was quantified by Northern blot, immunohistochemistry, immunofluorescence, and flow cytometry. Among the intact mice there was a significant difference in the number of VDR-expressing osteoblasts between young (68%) and old (56%) (p<0.04). In young oophorectomized mice the number of VDR-expressing osteoblasts decreased from 68% to 46% after oophorectomy and recovered to 72% after E(2) administration (p<0.02), while in the group of old mice, the number of VDR-expressing osteoblasts decreased from 56% to 48% after oophorectomy (p<0.01) and recovered to 85% after E(2) administration (p<0.001). Our results show that VDR expression in bone decreases with aging and estrogen deprivation but recovers after E(2) supplementation in both young and old mice with a more significant level of response in older bone. To evaluate the level of VDR bioresponse to E(2) we assessed the effect of E(2) supplementation to human osteoblasts (N-976) in vitro. Northern blot showed a significant up-regulation of VDR expression in E(2) treated cells as compared to non-treated cells (p<0.05). We also assessed the previously known anti-apoptotic effect of vitamin D in osteoblasts in vitro after serum deprivation by using either E(2), E(2)+1,25(OH)(2)D(3), or 1,25(OH)(2)D(3) alone. We found a lower number of apoptotic cells and longer cell survival after 48 h of treatment with 1,25(OH)(2)D(3)+E(2) as compared to 1,25(OH)(2)D(3) or E(2) alone (p<0.002). In summary, our results demonstrate that E(2) increases VDR expression in bone in vivo and potentiate the bioresponse of VDR in osteoblasts in vitro.